expression of f protein gene in maize for production of

Expression of F protein gene in maize for production of

edible vaccine against Newcastle disease virus

Naila Shahid

CENTRE OF EXCELLENCE IN MOLECULAR BIOLOGY,

UNIVERSITY OF THE PUNJAB, LAHORE,

PAKISTAN.

(2017)

Expression of F protein gene in maize for production of

edible vaccine against Newcastle disease virus

A THESIS SUBMITTED TO

THE

UNIVERSITY OF THE PUNJAB

In Partial Fulfillment of the requirement for the Degree of

DOCTOR OF PHILOSOPHY

In

MOLECULAR BIOLOGY

By

NAILA SHAHID

Supervisor:

Dr. ABDUL QAYYUM RAO

NATIONAL CENTRE OF EXCELLENCE IN MOLECULAR

BIOLOGY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN

2017

I

Expression of F protein gene in maize for production of edible vaccine against Newcastle disease virus

AUTHOR’S DECLARATION

I, Naila Shahid, here by state that my PhD thesis entitled “Expression of F protein

gene in maize for production of edible vaccine against Newcastle disease virus” is my own

work and has not been submitted previously by me for taking any degree as research work,

thesis or publication in any form University of Punjab, or anywhere else in country/world.

At any time, if my statement is found to be incorrect even after my graduation, the

university has the right to withdraw my Ph.D. degree.

_______________

Signature of Deponent

Naila Shahid

May, 2017

II


CERTIFICATE

This is to certify that the experimental work described in this thesis submitted by

Naila Shahid has been carried out under my direct supervision, and data/results reported in

this manuscript are duly recorded in the center’s official data book. I have personally gone

through the new data and certify the correctness/authenticity of all results reported here in. I

further certify that these data have not been used in part or full, in a manuscript already

submitted or in the process of submission in partial/complete fulfillment of the award of

another degree from any other institution at home or abroad. I also certify that the enclosed

manuscript has been prepared under my supervision and I endorse its evaluation for the

awards of Ph.D. degree through the official procedure of the centre/university. In accordance

with the rule of the centre, data book numbers 1171 will be kept in the center for a minimum

of three years from the data of the thesis defense examination.

Signature of the supervisor:

Name of the supervisor: Dr. Abdul Qayyum Rao

Designation: Assistant Professor

III


PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis titled “Expression of F

protein gene in maize for production of edible vaccine against Newcastle disease virus” is

solely my research work with no significant contribution from any other person. Small

contributions/help wherever taken, has been duly acknowledged and that complete thesis has

been written by me.

I understand the zero-tolerance policy of the HEC and university, “University of the

Punjab” towards plagiarism. Therefore, I as an author of the above titled thesis, declare that

no portion of my thesis has been plagiarized and any material used as reference is properly

referred/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis

even after award of the Ph.D. degree, the university reserves the rights to withdraw/revoke

my Ph.D. degree and that HEC and the university has the right to publish my name on the

HEC/university website on which names of students are placed who submitted plagiarized

thesis.

_______________

Signature of Deponent

Naila Shahid

May, 2017

IV


ACKNOWLEDGMENTS

In the name of Allah, the Most Gracious and the Most Merciful.

I am highly thankful to Almighty “ALLAH” whose bounteous blessing enabled me to

complete this research project as well as to write this thesis. He bestowed us with Holy Quran,

which is guidance for the use of reverent, a cure to the diseases and blessing for the believers,

and Prophet “MUHAMMAD” (Peace be upon Him), the most perfect and the best among and

of ever born on the surface of earth, who enlightens the hearts of believers in their life. I wish

to acknowledge several key figures that contributed much to my research endeavor.

I am grateful to Professor Dr. Tayyab Husnain, Acting Director, Centre of Excellence

in Molecular Biology, University of the Punjab, Lahore, for providing me the opportunity to

complete my research. His support has been of great value in this study.

It seems impossible to pay thanks to my kind and worthy supervisor Dr. Abdul

Qayyum Rao, Assistant Professor and incharge of Plant Biotechnology Lab, National Center

of Excellence in Molecular Biology, University of the Punjab, whose kind behavior, keen

interest and guidance throughout this study, helped me to do this task in-time.

I would like to express my sincere thanks to Dr. Ahmad Ali Shahid (Incharge

MPhil/PhD programme) for their inspiring guidance and support that I gained from him in

theory and practical research work.

I have been very lucky for having cooperative lab fellows whose accommodative and

friendly behavior made my work less laborious. Thanks to Dr. Bushra Tabusum, Dr. Bushra

Rashid, Dr. Sameera Hassan, Miss Ayesha Lateef, Miss Fatima Batool Miss Zahida, Miss

Saira Azam, Mr. Tahir Samiullah, Mr. Kamran Bajwa, Mr. M. Azam, Miss Amna. Miss Sidra,

Miss Ammara, Miss Zrnab and Ambreen.

I want to acknowledge HEC Pakistan for continues financial support during my PhD

and providing me an opportunity to visit foreign lab in USA to accomplish my PhD research.

I especially want to pay my best regard to my husband Mr. Aleem Faysal Atif who

injected professional confidence and encourage me on every step. His moral and material

support enabled me to reach over stairs of life smoothly and helped me to reach my

destination. I also want to express gratitude to my MOTHER for seeding character and strong

motivation. Her dedicated and strong will gave me the opportunity to travel along with my

V


dreams. I wish to pay my best regards to my sister Maryam. Finally, yet importantly, I am

enormously grateful for their assistance and continuous encouragement during the span of my

study.

_______________

Naila Shahid

VI


Dedicated to

My Family

VII


SUMMARY

The aim of present study is the cloning of F and HN gene in plant expression vector

to develop plant-based edible vaccine against Newcastle disease virus (NDV) of poultry.

NDV was collected from Veterinary Research Institute, Lahore, Pakistan on request. ~1662bp

F gene and ~1712bp HN gene were PCR amplified from cDNA of NDV that were TA cloned

and further evaluated through sequencing. BLAST results determined the specificity of these

genes in accordance with previously reported Mukteswar strain (Accession # GU182327).

Both F and HN genes were ligated into pET30a expression vector to produce

recombinant pET-F and pET-HN. Both recombinant constructs were transformed in E. coli

Rosetta cells to study the prokaryotic expression of immunogenic protein. Induction by adding

IPTG generated increased yield of protein and SDS polyacrylamide gel electrophoresis

confirmed protein on desired size (~67kDa for F and ~69kDa for HN). Further western blot

analysis, confirmed specificity of protein through antigen antibody reaction at proper size.

Protein purification using IMAC affinity chromatography was performed and the appearance

of single band of F protein at ~67kDa and HN protein at ~69kDa confirmed the specificity of

our desired immunogenic protein. 2D and 3D structural analysis of F and HN proteins through

Immune epitope database (IEDB) analysis resource tool revealed that more than 70% of its

sequence is antigenically active and the predicted protein regions behave as epitopes.

After prokaryotic expression of both F and HN genes, the next major objective of this

study was to construct pCAMBIA 1301 with both F and HN for plant transformation. After

confirming F and HN inserts, recombinant pCAMBIA-F+HN plasmid was electroporated into

Agrobacterium cells (LBA4404) using a Bio-Rad electroporation device. After confirming

the presence of F or HN genes in Agrobacterium, embryos from inbred lines of maize were

transformed with recombinant pCAMBIA-F+HN by Agrobacterium mediated nuclear

transformation.

Further, the presence of F and HN genes in transgenic maize plants were confirmed

through different molecular biological tools. The putative plants were confirmed through

VIII


polymerase chain reaction (PCR). PCR confirmed a short fragment of ~181bp and ~191 for

F and HN genes respectively.

RT-PCR analysis confirmed the expression of F and HN genes in corn leaves and

seeds respectively. The comparitive analysis of Ct values obtained by qRT-PCR revealed that

the expression of F gene increased from 2-7.1 fold. Similarly, comparison of Ct value by qRT-

PCR confirmed that expression of HN gene increased from 0.5 to 4 fold as compared to

negative control.

Protein expression of F and HN genes were confirmed through ELISA and western

blot by using gene specific antibodies. In ELISA, both F and HN proteins expression were

observed in putative plants. The maximum obtained concentration of F gene was in the range

of 0.15μg/ml to 0.166 μg/ml. Similarly, maximum obtained concentration of HN protein was

in the range of 0.195μg/ml 0.24μg/ml.

The plants with high mRNA expression of F and HN genes were confirmed through

western blot analysis. Transgenic plants produced a fragment of 67kDa for F protein and

69kDA for HN protein which confirmed the expression of transgene. Furthermore,

immuniztion of chicks with transgenic maize and immune response generated by ELISA

results showed production of anti-NDV antibodies in sera of chicks. On the other side ELISA

results from the sera of chicks having non-transgenic diet did not induce any significant

immune response.

This is a key achievement of this study, which can lead towards development of plant-

based edible vaccine against Newcastle disease virus of poultry.

IX


LIST OF ABBREVIATIONS

% percent

C Degree centigrade

µg micro gram

µl micro-liter

APS Ammonium per sulphate

BCIP 5-bromo-4-chloro-3'-indolyphosphate

bp base pair

CTB Cholera toxin subunit B

CV Column volume

cm centimeter

DAPI 4’, 6-diamidino-2-phenylindole

dH2O Distilled water

DMSO Dimethyl sulfoxide

DNA Deoxy-ribo Nucleic

dNTPs Dinucleotide Triphosphate

E. coli Eschericia coli

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme Linked Immune Sorbent Assay

G gram

H2O Water

HCl Hydro chloric acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HgCl2 Mercuric chloride

i.e; That is

IMAC Immobilized metal ion Affinity Chromatography

IL Interleukin

IPTG Isopropyl β-D-1-thiogalactopyranoside

kb kilo base

kg Kilo gram

http://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&ved=0CDEQFjAA&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FIsopropyl_%25CE%25B2-D-1-thiogalactopyranoside&ei=QrC6UdmrBMe7Pa_KgLgP&usg=AFQjCNHSCbamsKcxM_rhgtf8Yp1yVgm3Xw&sig2=VBWzbbnwVoxSqSvd8aynzg&bvm=bv.47883778,d.ZWU

X


kDA Kilo Daltons

KCl Potassium Chloride

LB Luria Broth

LTB Heat-labile enterotoxin B subunit

min minutes

mm milli metre

mg Milligram

ml Milliliter

mM milli molar

M Molar

NDV Newcastle disease virus

ND Newcastle disease

NaCl Sodium chloride

NaOH Sodium Hydroxide

No. Number

nm nanometer (wavelength)

ng Nano gram

NiCl Nickel chloride

OD Optical Density

PAGE polyacrylamide gel electrophoresis

PCR Polymerase Chain Reaction

PEG Polyethylene Glycol

pmol Pico moles

PMSF Phenylmethylsulfonyl Fluoride

pH Negative log of hydrogen ions

p.mol pico moles

PBS Phosphate buffer saline

RNA Ribo Nucleic Acid

rpm rotations per minute

RT-PCR Reverse Transcriptase Polymerase Chain Reaction

SDS Sodium Dodecyl Sulphate

http://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&ved=0CDgQFjAC&url=http%3A%2F%2Fwww.microbelibrary.org%2Fcomponent%2Fresource%2Flaboratory-test%2F3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol&ei=W7G6UcizNcLaOazSgegO&usg=AFQjCNEYzprSWxnGaXsin0Gj9hkerxDtDA&sig2=YZBFn0Z6IWHEbqf79EpjTA&bvm=bv.47883778,d.ZWU

XI


sec seconds

TAE Tris-acetate-EDTA

Taq Thermus aquaticus

TBE Tris-Borate-EDTA

TEMED Tetramethylethylenediamine

TGF-β Transforming growth factor-β

U unit

UV Ultra violet

V volts

X Gal 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside

β beta

http://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&ved=0CD8QFjAD&url=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fb4252%3Flang%3Den%26region%3DUS&ei=XrC6UdOtI4u3hAfoj4Fw&usg=AFQjCNGs4AhHCStNdPM08DwPbixGYaAFzw&sig2=97E1-eheX0Y-RMj6YIaqmA&bvm=bv.47883778,d.ZWU

XII


TABLE OF CONTENTS

AUTHOR’S DECLARATION I

CERTIFICATE II

PLAGIARISM UNDERTAKING III

ACKNOWLEDGMENTS IV

SUMMARY VII

LIST OF ABBREVIATIONS IX

LIST OF FIGURES XVIII

: INTRODUCTION 1

: REVIEW OF THE LITERATURE 5

2.1. Newcastle disease and its history 5

2.1.1. Symptoms of disease 5

2.1.2. Transmission of disease 6

2.2. NDV virus 6

2.2.1. Structure and function of NDV surface protein 7

2.3. NDV’S impact on poultry 8

2.4. The current status of NDV vaccines 9

2.4.1. Inactivated virus-based vaccines 9

2.4.2. Live virus-based vaccines 9

2.4.3. Advances in biotechnology and recombinant NDV vaccines 10

2.4.4. Vector vaccines against NDV 10

2.4.5. Oral vaccines against NDV 11

2.5. Plant-based vaccines: A system to produce low-cost NDV vaccine 11

2.6. Transformation procedures for edible vaccine production 13

XIII


2.6.1. Nuclear transformation 13

2.6.2. Chloroplast transformation 14

2.6.3. Plant viruses as vehicles for transformation 14

2.6.3. Plant virus-through Agrobacterium-mediated transformation 14

2.6.4. Cell suspension-based transformation 15

2.7. Strategies to enhance gene expression of edible vaccines 15

2.8. Attempts to produce edible vaccines against poultry diseases 16

2.9. Previous efforts to produce edible vaccines against NDV 19

2.10. Success with the world’s first approved plant-based NDV vaccines 22

2.11. Future prospects for plant-based NDV vaccines 22

2.12. Conclusion 22

: MATERIAL AND METHODS 24

3.1. Collection and Propagation of Virus 24

3.2. Complementary DNA (cDNA) Synthesis 24

3.2.1. Isolation of viral RNA 24

3.2.2. cDNA Synthesis 24

3.3. PCR amplification of F and HN genes from cDNA 25

3.4. TA cloning and transformation of F and HN genes 26

3.4.1. Gel purification of PCR product 26

3.4.2. Ligation of eluted product into the TA vector 27

3.4.3. Transformation of TA ligated product 27

3.4.5. Selection of positive clones 27

3.4.6. Plasmid DNA isolation from positive clones 28

3.4.7. Confirmation of positive clones 28

3.5. Sequencing of positive F and HN clones 29

XIV


3.6. Cloning of F and HN in prokaryotic expression vector 30

3.6.1. Restriction digestion 31

3.6.2. Ligation of Insert (F/HN) into the pET30a vector 31

3.6.3. Confirmation of F/HN genes in pET30a 32

3.7. Propagation of recombinant F/HN genes in the E. coli DE3 (BL-21) strain 32

3.7.1. Isolation and quantification of protein 33

3.7.2. SDS polyacrylamide gel electrophoresis (SDS-PAGE) 33

3.8. Protein purification through affinity chromatography 34

3.8.1. Biomass preparation 34

3.8.2. Column packing and protein purification 34

3.9. Identification of antigenic epitopes 35

3.9.1. Homology modeling using SWISS-MODEL 35

3.9.2. Antigenic epitope prediction using the Immune Epitope Database Analysis

Resource 35

3.10. Western blot analysis with anti-His-tag antibodies 36

3.11. Cloning of F and HN genes into pCAMBIA-1301under different promoters 36

3.11.1. Amplification of F gene 37


3.11.3. F gene ligation into pCAMBIA 38

3.11.4. Transformation of ligation 39

3.11.5. Selection of F ligated positive clones in pCAMBIA 39

3.11.6. Determination of F gene orientation into pCAMBIA 1301 39

3.11.7. Cloning of the HN gene in pCAMBIA 1301 under seed specific promoter

39

3.11.8. Cloning of the Zein promoter into pCAMBIA 1301 40

3.11.9. Ligation of the Zein promoter into pCAMBIA 1301 40

XV



3.11.11. Selection of positive clones with promoter 41

3.11.12. Cloning of the HN gene into pCAMBIA 41


3.11.14. Ligation of the HN gene into pCAMBIA 1301 42


3.11.16. Selection of HN ligated positives clones in pCAMBIA 42

3.11.17. Cloning of the NOS terminator into pCAMBIA1301 42

3.11.18. Ligation of the NOS terminator into pCAMBIA 1301 43


3.11.20. Selection of the NOS ligated positive clones in pCAMBIA 43

3.11.21. Determination of cassette (Promoter + HN + NOS) through restriction

analysis 44

3.12. Electroporation of recombinant plasmids into Agrobacterium 44

3.12.1. Determination of pCAMBIA (F+HN) in Agrobacterium 45

3.13. Transformation of maize 45

3.13.1. Collection of plant material and embryo isolation 45

3.13.2. MS medium preparation 45

3.13.3. Agrobacterium culture preparation 45

3.13.4. Sterilization of embryos 46

3.13.5. Agrobacterium-mediated transformation 46

3.13.6. Shifting of embryos to MS plates 46

3.13.7. Selection of transformed plants 46

3.13.8. Transfer of plants to soil 47

3.14. Molecular analysis 47

XVI


3.14.1. Isolation of genomic DNA 47

3.14.2. PCR Confirmation of Transgenic Plants 48

3.14.3. Expression studies of transgenic plants 48

3.14.4. Quantification of total soluble protein 49

3.14.5. Quantification of F and HN gene through Enzyme linked immunosorbent

assay (ELISA) 49

3.14.6. Calculation of F and HN percentage in total soluble protein 50

3.14.7. Western blot analysis for confirmation of F and HN gene expression 50

3.14.8. In vitro chicks feeding assay for immune response 51

: RESULTS 52

4.1. F and HN genes confirmation through PCR 52

(Negative control: Amplification without template DNA) 52

4.2. Confirmation of TA construct harboring F gene 53

4.3. Confirmation of TA vector harboring HN gene 54

4.4. Evaluation of F/HN genes through sequencing 55

4.5. Confirmation of F gene in prokaryotic expression vector 57

4.6. Confirmation of HN gene in prokaryotic expression vector 58

4.7. Confirmation of pET-F and pET HN in expression host 59

4.8. Expression analysis of F and HN by SDS-PAGE 60

4.9. Purification of F and HN proteins by IMAC 61

4.10. Confirmation of His-Tagged F/HN protein by western blotting 62

4.11. Structure Prediction of F and HN Proteins by homology modeling using SWISS-

MODEL 63

4.12. Prediction of antigenic epitope by Antibody epitope prediction and ElliPro 65

4.13. Confirmation F and HN in plant expression vector 74

4.13.1 F gene confirmation in pCAMBIA vector 74

XVII


4.13.2. Confirmation of correct orientation of F gene into pCAMBIA-F 75

4.13.3. HN gene confirmation under seed specific promoter and terminator 76

4.13.4. Confirmation of Cassette (Promoter + HN + NOS) in pCAMBIA-F + HN

78

4.14. Electroporation and confirmation of F and HN genes in Agrobacterium 79

4.15. Transformation of Maize 80

4.16. Molecular analysis 81

4.16.1. PCR Confirmation of Transgenic Plants 81

4.16.2. Quantitative studies of F and HN gene in transgenic maize through real

time PCR 83

4.16.3. Quantification of total soluble Protein 85

4.16.5. Quantification of F and HN protein through ELISA 86

4.16.7. Calculation of F and HN protein percentage in total soluble protein 88

4.16.8. Western Blot analysis of transgenic plants to confirm protein 89

4.16.9. Bird feeding assay 90

: DISCUSSION 95

CONCLUSION 99

REFERENCES 101

APPENDICES 113

XVIII


LIST OF FIGURES

Figure 2-1: Graph showing increasing demand for poultry .......................................................8

Figure 2-2: Diagram showing the process of edible vaccine product ......................................18

Figure 3-1: Map of the pET30a expression vector .................................................................30

Figure 3-2: F/HN gene cassette................................................................................................31

Figure 3-3: Map of pCAMBIA -1301. Source: http://www.cambia.org/ ................................37

Figure 3-4: Cassette having F gene under the CaMV35S promoter and HN gene under the

Zein promoter in pCAMBIA1301 ...........................................................................................37

Figure 4-1: Confirmation of F/HN genes from NDV cDNA through PCR amplification ......52

Figure 4-2: F gene confirmation of TA-F construct through PCR and restriction digestion. ..53

Figure 4-3: HN gene confirmation of TA-HN construct through PCR and restriction

digestion. ..................................................................................................................................54

Figure 4-4: Sequence analysis of F gene through ABI sequence scanner ...............................55

Figure 4-5: Sequence analysis of HN gene through ABI sequence scanner ............................56

Figure 4-6: F Gene Confirmation in pET-F through Restriction Digestion and PCR .............57

Figure 4-7: HN gene confirmation in pET30a through restriction digestion and PCR ...........58

Figure 4-8: pET-F and pET HN confirmation in DE3 (BL-21) cells through PCR ................59

Figure 4-9: Confirmation of F/HN Protein expression through SDS-PAGE ..........................60

Figure 4-10: SDS-PAGE analysis of eluted F and HN protein fractions after purification

through IMAC fast flow affinity chromatography...................................................................61

Figure 4-11: Confirmation of His-tagged F/HN proteins through western blotting ................62

Figure 4-12: Three-dimensional Swiss model of the fusion protein and its QMEAN score. ..63

Figure 4-13: Three-dimensional Swiss model of the HN protein and its QMEAN score .......64

Figure 4-14: Graphical representation of the occurrence frequency of F protein residues

based on individual score (Y-axis) and residue position (X-axis) ...........................................68

Figure 4-15: Graphical representation of the occurrence frequency of HN protein residues


Figure 4-16: JSmol-rendered 3D structures of continuous antigenic epitopes of the F protein

along with their PI values, as predicted by ElliPro ..................................................................72

XIX


Figure 4-17: JSmol-rendered 3D structures of continuous antigenic epitopes of the HN

protein along with their PI values, as predicted by ElliPro ......................................................73

Figure 4-18: Graphical representation of the occurrence frequency for F protein residues on

the basis of individual score (Y-axis) and residue position (X-axis) .......................................73

Figure 4-19: Graphical representation of the occurrence frequency for HN protein residues


Figure 4-20: Digestion of pCAMBIA and TA-F construct with Nco1 ....................................74

Figure 4-21: Confirmation of F gene integration into pCAMBIA-F .......................................75

Figure 4-22: Confirmation of F gene orientation in the pCAMBIA Vector ............................75

Figure 4-23: Confirmation of the Zein promoter in pCAMBIA -F .........................................77

Figure 4-24: Confirmation of the HN Gene in pCAMBIA -F .................................................77

Figure 4-25: Confirmation of the NOS Terminator in pCAMBIA-F ......................................77

Figure 4-26: Confirmation of Cassette (Zein + HN + NOS) in pCAMBIA-F + HN construct

..................................................................................................................................................78

Figure 4-27: Confirmation of F or HN in Agrobacterium .......................................................79

Figure 4-28: A schematic diagram showing transformation methodology .............................80

Figure 4-29: Transformation efficiency of maize ....................................................................80

Figure: 4-30. Confirmation of F and HN gene in transgenic plants ........................................82

Figure 4-31: Expression of F and HN gene through Real time PCR .......................................84

Figure 4-32: Standard curve for quantification of crude proteins ............................................85

Figure 4-33: Quantification of F and HN gene through ELISA ..............................................87

Figure 4-34: Confirmation of F/HN proteins through western blotting ..................................89

Figure 4-35: Induced immune response against F protein .......................................................92

Figure 4-36: Induced immune response against HN protein ...................................................94

XX


LIST OF TABLES

Table 2-1: Plant-based vaccines against NDV .........................................................................21

Table 4-1: Peptide length, sequence, start and end positions of predicted linear epitopes of the

F protein ...................................................................................................................................66

Table 4-2: Peptide length, sequence, start and end positions of predicted linear epitopes of the

HN protein ...............................................................................................................................67

Table 4-3: Peptide length, sequence, start and end positions, and PI score of predicted linear

epitopes of the F protein. .........................................................................................................70

Table 4-4: Peptide length, sequence, start and end positions and PI score of predicted linear

epitopes of the HN protein. ......................................................................................................71

Table 4-5: Quantification of Crude proteins ............................................................................86

Table 4-6: F and HN percentage in TSP ..................................................................................88

1


: INTRODUCTION

The demand for animal-based food has presumably increased with growing population

and production of livestock has played a key role to achieve this demand (Ali and Khan 2013).

Among livestock, the importance of poultry cannot be denied, as it makes up 33% of all meat

worldwide. Consumption of poultry meat is increasing as compared to red meat

(http://www.nationalchickencouncil.org/) because poultry is the most economical and

excellent source of high-quality animal protein (Mengesha 2012; Moreki et al. 2010). The

consumption of poultry has increased rapidly in the past 50 years and it is expected to increase

in the future due to factors such as health, price, and the expansion of poultry products.

According to the Food and Agriculture Organization (FAO) of the United Nations, the

global domestic chicken population was almost 18 billion in 2009. The poultry industry is

considered the largest animal stock in the world, encompassing commercial farms in

developed countries and backyard poultry raised extensively but in very small numbers in

developing countries. Backyard poultry may be improved or unimproved genetic stock but it

usually makes up 80% of all poultry in most developing countries (Conan et al. 2012; Sarwar

et al. 2015). Poultry is also a main source of income in rural areas and plays an important role

in lives of farmers. The average price per bird is equal to US$6.53 and is part of how farmers

meet their daily expenses (Moreki et al. 2010). Overall poultry creation has expanded quickly

up to 86 million tons and it is estimated to increase in future (Barbut 2012). The total world

market for chicken meat is almost US $16 billion; consumed eggs are 1.1 trillion and

approximately 90 million tons of meat are consumed each year, and United states and Asia

are major consumers in the international chicken market (Belova et al. 2013; Blake and

Tomley 2014).

Pakistan is an agriculture-based country; agriculture makes up 26% of the national

GDP, and 55% of the agricultural sector is made up of livestock and poultry (Abidin and

Khatoon 2013). According to an economic survey of Pakistan (2013-2014), poultry is the

largest industry and it plays a large role in the country’s economy. Pakistan’s poultry sector

produces 987 million tons of meat, or 28% of all meat produced in the country (Bhatti et al.

2015; Shabbir et al. 2013b), as well as12857 million eggs annually, giving the poultry industry

2


great potential to generate income. Of the 180 million people that live in Pakistan, almost 1.5

million depend on the poultry sector directly or indirectly as an income source (Memon 2015).

The poultry industry’s 8-10% annual growth rate – the fastest in the country –has made it the

second largest industry in Pakistan (Ashraf and Shah 2014; Mukhtar et al. 2012).

The growth of the poultry industry is severely threatened by infectious and parasitic

diseases (Shabbir et al. 2013b). Newcastle disease (ND) is the most alarming viral infection

of poultry and can cause a loss of million dollars annually (Belova et al. 2013). Initially,

outbreaks of ND were reported in Indonesia but with time it became endemic to many other

countries as well (Dortmans et al. 2011; Kraneveld 1926). Total losses due to this disease are

difficult to predict because of limited surveys and poor disease control. Additionally, disease

control practices are uncommon among villagers because of their cost and restrictiveness,

making statistical data inadequate about mortality and morbidity caused by this disease (Aini

2013).

Poultry industries of Asia, Africa, Europe, Central and South America are facing severe

losses due to ND (Belova et al. 2013).Worldwide control measures have been in place since

1950, but ND remains one of most significant diseases in the world and causes substantial

losses to the world economy (Balachandran et al. 2014). This is because of the highly

evolutionary nature of NDV (Miller et al. 2010). ND exists in six of seven continents, and in

most countries it is enzootic in nature (Miller et al. 2010).

In Pakistan, ND is an endemic disease that causes big economic losses to poultry business.

In India and Pakistan, this disease is known as Ranikhait disease. NDV infects almost 240

species of birds, but poultry species are more susceptible to virulent strains (Dortmans et al.

2011; Shabbir et al. 2013a). ND is endemic in many developing countries (Ashraf and Shah

2014). It is caused by avian paramyxovirus type I (APMV-1), a serotype of genus Avulavirus

in the family Paramyxoviridae (Jang et al. 2010). It has linear, single-stranded, negative-

sense, filamentous RNA genome with a lipid membrane (Diel et al. 2012; Qiu et al. 2011;

Subramanian et al. 2009). There are nine different serotypes of the genus Avulavirus, which

are designated APMV-1 to APMV-9as classified by phylogenetic analysis and serological

testing (Beard and Hanson 1984).

The NDV genome is almost 15 kb long and has six open reading frames that encode a

matrix protein (M), nucleoprotein (NP), a phosphoprotein (P), a fusion protein (F),

3


haemagglutinin-neuraminidase (HN), and a large polymerase (L) (Pedersen et al. 2004;

Shabbir et al. 2013a). There are two possible non-structural proteins, V and W, that can be

generated by the transcription of P (Dortmans et al. 2011; Steward et al. 1993). NDV is

categorized into three different strains based on its pathogenicity: velogenic (highly

pathogenic), mesogenic (intermediately pathogenic), and lentogenic (mildly pathogenic)

(Seal et al. 1995). NDV can be categorized into lineages and sublineages on the basis of

phylogenetic study of the F gene nucleotide sequence (Fuller et al. 2007; Munir et al. 2012).

Another method of classification, which is based on genome size, divides NDV into two

classes: I and II. Class I consists of avirulent NDV strains with large genomes, whereas Class

II consists of NDV strains with short genomes and includes all velogenic, mesogenic, and

lentogenic strains. Class II can be further subdivided into 18 genotypes (I-XVIII) (Herczeg et

al. 1999; Miller et al. 2015; Munir et al. 2012).

Among six proteins of NDV genome, F and HN are the most important because they are

considered the major cause of virulence; they are responsible for membrane fusion and

attachment of cell surface. Both of these genes have the potential to induce immunity. The

cleavage site of F protein has been recognized as most important cause of NDV virulence (de

Leeuw et al. 2005; Glickman et al. 1988; Siddique et al. 2013).

Poultry is the second largest industry in Pakistan after textiles. Pakistan usually exports

its poultry products to Iran, Hong Kong and Afghanistan. In 2010-11, the total export from

Pakistan’s poultry sector was almost Rs 1.08 billion, but it decreased to Rs 365 million due

to the spread of NDV disease (Rasool et al. 2015).

In most developed countries, poultry diseases cause a 10 to 20% loss to the economy, and

this number is higher in developing countries. Vaccination is considered the solution to

overcome NDV problem of poultry. There have been many developments in the field of

vaccinology, such as subunit vaccines, live vaccines, and killed vaccines, aimed at controlling

the disease. The problem with these vaccines is that they require a complex system of

production and purification; they are expensive; they require a cold chain, which is difficult

to maintain; and skilled people are needed to administer them (Aswathi et al. 2014; Ferraro et

al. 2011; Nochi et al. 2007).

Developments in plant genetic engineering dating back to 1970 have made it easy to

develop plant-based, edible vaccines that can provide a new way to overcome these hurdles

4


(Mason et al. 2002; Walmsley and Arntzen 2003). An immunogenic gene is transformed into

a plant for production of recombinant plant-based vaccines; oral administration of this edible

plant promotes the production of antibodies against a particular disease (Lossl and Waheed

2011).

Edible vaccines have many advantages over traditional vaccines. They are economical

because of the low cost of production and purification. They do not need to be maintained at

cold temperatures because they are stable at room temperature. Their delivery via the oral

route is more comfortable for animals and poultry and multiple antigens can be delivered

through bioencapsulated edible vaccines. They are free of toxins and pathogens associated

with traditional vaccines; they can be easily scaled up, and they can induce higher immune

response than traditional vaccines. However, the main advantage of edible vaccines is that

they can induce a mucosal immune response, which is the first barrier against invading

pathogens (Shahriari et al. 2015).

The overall objective of this study is to transform immunogenic F and HN protein genes

into maize (Zea mays) plants and study their expression through different molecular biology

tools in resulting transgenic plants to produce an edible vaccine against Newcastle Disease.

5


: REVIEW OF THE LITERATURE

2.1. Newcastle disease and its history

ND is considered a major obstacle in establishment of poultry industry. In its virulent

form, NDV has destructive effects on poultry. As already stated (Alexander 1997), in spite of

control measures, it is difficult to protect a single commercial flock of poultry from ND;

(Sinkovics and Horvath 2000). ND has been declared as an alarming threat by World

Organization for Animal Health (OIE) to prevent the entry of virulent NDV into disease-free

countries (Aldous and Alexander 2008).

The first outbreak of the disease was reported by Doyle in 1926 in England. The name

was given to the disease because it was first reported on a farm near Newcastle-upon-

Tyne.The next outbreak was reported on the island of Java in Indonesia in March 1926, and

then in Korea and India in 1926 and 1927, respectively. These outbreaks caused 100%

mortality in susceptible birds. Many scientists speculate that the disease exsisted before this

time but remained unnoticed due to lack of expertise (Alexander 2012).

NDV is endemic to most of the countries such as Central and South America, Africa Asia

and the Middle East, and some sporadic outbreaks have also been found in the United States,

Canada, and many countries of the European Union. This worldwide spread of ND has made

it a significant poultry disease (Alexander et al. 2012). Mortality and morbidity rates depend

on factors like the surrounding environment, history of vaccination, and secondary infection.

All domestic and wild bird populations are major victims of NDV, but the rate of mortality

varies among different species. Chickens are more susceptible to the disease than any other

bird species. NDV also infects ducks and geese but causes mild disease symptoms in these

species. Poultry is big buisness, so birds are usually raised on large farms, and this living style

is also a major reason for the spread of disease (Gallili and Ben-Nathan 1998; Hanson 1965;

Lim 2014).

2.1.1. Symptoms of disease

General symptoms of NDV include a cough, flu, fever, less intake of feed and water,

and discharge from nose and eyes. Blindness has also been observed in some birds. Clinical

symptoms include loss of weight and appetite, secretions from nose and eyes, depression,

6


dullness, decreased egg production and water deficiency, which eventually causes death

(Pansota et al. 2013; Pazhanivel et al. 2002). Signs and symptoms of velogenic strains may

be different depending on the area of infection: neurotropic infections affect the brain and

nervous system; viscerotropic infections affect the digestive system; and pneumotropic

infections affect the respiratory system (Pansota et al. 2013; Shaheen et al. 2005; Sharif et al.

2014).

2.1.2. Transmission of disease

NDV infection is spread through direct contact between healthy and infected birds via oral

and respiratory routes. ND-infected birds shed virus from different body parts in feces

andnasal discharges, which are spread mechanically by the movements of rodents, dogs,

insects and humans. Air is also a major route of ND transmission. Due to its aerosol mode of

transmission, ND has the ability to infect all birds in a contained area of a farm (Li et al. 2009).

2.2. NDV virus

ND is an enveloped virus with negative-sense, single-stranded RNA that is 200-

300nm in diameter. Its genome is non-segmented and approximately 15 kb long. According

to the Baltimore system of classification, NDV belongs to the genus Avulavirus, family

paramyxoviridae subfamily Paramyxovirinae. On the basis of haemagglutination inhibition

assays, there are serotypes of genus Avulavirus, i.e. from APMV-1 to APMV-9. All strains of

NDV are categorized into serotype-1(Ganar et al. 2014).

The NDV genome consists of six major proteins (N), (M), (P), (F), (HN) and (L). Each

gene is separated from the next by a specific intergenic sequence (IGS) with specific signals

for the start and end of a protein. The nucleocapsid is a 55-kDa protein whose major role isto

form the capsid and the ribonucleoprotein complex (RNP). The RNP is a template for RNA

synthesis (Errington and Emmerson 1997). The phosphoprotein (P) is a 50-kDa protein that

controls replication and transcription of the virus (Dortmans et al. 2010; Ganar et al. 2014).

The matrix protein is almost 40 kDa in molecular weight. It is hydrophobic and can

localize to the nucleus without the help of any other protein. The M protein interacts with the

plasma membrane of the host cell and promotes the viral budding process. Moreover, it is also

important for viral assembly and maintains the shape of the nucleocapsid protein (Ganar et al.

2014; Mebatsion et al. 1999; Peeples and Bratt 1984).

7


The L protein is known as the large polymerase protein because of its large molecular

weight of approximately 250 kDa. The main function of the L protein is the synthesis of viral

messenger RNA, and it also functions in genomic RNA replication. It usually increases the

RNA synthesis rate during viral replication and controls the virulence of NDV (Rout and

Samal 2008).

2.2.1. Structure and function of NDV surface protein

The hemagglutinin neuraminidase (HN) fusion protein (F) and are two important envelope

glycoproteins that are the major cause of NDV infection. The HN protein aids in attachment

to the host cell and F allows the virus to penetrate into the host cell to form a syncytium

(Soñora et al. 2015). The integral membrane F protein of NDV is produced as inactive

precursor (Fo,66 kDa). Fo is cut into two active subunits, F1 at C-terminal (55 kDa) and F2 at

N-terminal (12.5 kDa), by the proteases of the host cell. This cleavage differs among strains

and depends on particular aminoacids at the cleavage site; the cleavage site of virulent strains

contains many basic aminoacids,whereas those of avirulent strains are usually monobasic or

dibasic. The cleavage site with lysines (K) and arginines (R) at (112R-R-Q-R/K-R116) and 117th

position of phenylalanine in F protein are major causes of virulence. These polybasic sites are

cleaved by proteases, resulted in F1 activation , which is the major contributor to neurological

effects (Ganar et al. 2014; Kattenbelt et al. 2006; Soñora et al. 2015).

The HN protein is a 74-kDa protein that activates the function of F. The HN protein in its

active form is tetrameric and there are specific factors in its stalk that help the F protein fusion

with the host cell.Any mutation in the stalk of HN can perturb its membrane fusion activity

(Melanson and Iorio 2006; Yuan et al. 2011). NDV targets the respiratory tract of Gallus spp.

and requires the two envelope glycoproteins F and HN. Binding to cell surface receptors is

mediated by HN, which promotes F protein penetration into host cells. These two proteins are

best targets to evoke animmune response in the host (Alexander 1997).

Mortality and morbidity rates vary among different bird species. There are three strains of

NDV on the basis of their pathogencity: velogenic, mesogenicand lentogenic.Velogenic

strains are further subdivided into three strains: neurotropic velogenic, viscerotropic

velogenic, and pleiotropic velogenic. All velogenic strains cause 100% mortality, with

hemorrhagic lesions in the intestine. Mortality can be 80 to 90% among adult birds.

Mesogenic strains are moderately virulent and cause mild symptoms like decreased egg

8


production. Lentogenic strains are considerably less virulent (Sharif et al. 2014; Susta et al.

2011).

2.3. NDV’S impact on poultry

The poultry industry acts as an essential sourse of protein and performs a major role

in a balanced diet. According to an estimate, more than 60% population of the developing

countries is facing the problem of protein deficiency; furthermore, the prerequisite of

aggregate protein for every individual is 103 g/day, out of which each individuals receives

only 69% of it form livestock meat. Red and white meat are important sources of animal

protein, and chicken and eggs are the cheapest sources of animal protein in most developing

countries; that is better approach to fathom protein deficiencies among people (Abedullah and

Bukhsh 2007; Ashraf and Shah 2014). However, NDV is a risk for the growing poultry

industry, and it is the only animal virus that has great potential to cause losses to the world

economy. Village chicken production is crucial in developing countries because most women

and children depend on village chicken for their dietary protein present in eggs and meat. In

countries with endemic NDV, NDV is a major limiting factor for their development of trade

and commerce (Lim 2014).

Figure 2-1: Graph showing increasing demand for poultry

9


2.4. The current status of NDV vaccines

Infectious diseases are major risk for the growing poultry industry, especially in

developing countries. Vaccination is a vital remidy in infectious diseases control (Loza-Rubio

and Rojas-Anaya 2010). Due to the rapid growth of NDV in the poultry industry, vaccination

has been used to prevent and control NDV. Since 1940, a variety of vaccines has been

developed, like inactive virus vaccines, attenuated virus vaccines and live virus vaccines

(Alexander 2012).

2.4.1. Inactivated virus-based vaccines

The inactivated viruses were part of the first vaccine-based strategy to control NDV

disease in which the virus has been inactivated by radiation, chemicals or heat treatment.

These vaccines donot need refrigeration and there is no risk of the virus mutating back to its

original disease-causing state. The major drawback of inactivated vaccine is weaker immune

response and multiples boosters are always required to produce sufficient level of systemic

immunity. Formalin-inactivated virus and β-propiolactone-inactivated virus are the most

important examples of inactive virus vaccines. These vaccines are produced by propagating

the virus in the allantoic fluid of chicken embryos, followed by inactivation with ß-

propiolactone or formalin. The administration of inactive vaccines is very laborious and

mostly delivers through subcutaneously or intramuscularly Inactivated vaccines don’t

produce very strong immunity, but in combination with live vaccines, they can produce better

immunity (Sharma 1999). This strategy has been used to control NDV in many poultry-

producing areas (Lim 2014; Senne et al. 2003).

2.4.2. Live virus-based vaccines

Inactivated vaccines failed to produce promising results in the control of NDV, which

led to the development of live virus-based vaccines. Live virus vaccines are prepared by live

and weak virus that is still able to cause infection. There are two types of live vaccines used

to control NDV: mesogenic and lentogenic. Lentogenic vaccines can be administered orally

or by the nasal route, but mesogenic vaccines require intramuscular injection. Live vaccines

can be given through drinking water or through aerosols. Most of them are prepared in

lyophilized form that and can be save at low temperature up to one year. Since that time, this

vaccination strategy is still in practice via drinking water or nasal route. The major drawback

regarding live vaccines is their retrieval virulence. For instance, Sabin strain reverted to

10


virulent virus by point mutation in live virus-based oral vaccine against polio (Shahid and

Daniell 2016). In addition, sprays or aerosols delivery modes are very common for live

vaccines can accidentally inoculate younger birds when they are used for birds of specific age

and eventually cause death as some of them are recommended for application of one-day-old

chicks and other vaccines for old chickens (Alexander et al. 2012; Zhang et al. 2010).

Moreover, it is quite unfeasible to perform injections for poultry reared in big farms (Shahid

and Daniell 2016).

2.4.3. Advances in biotechnology and recombinant NDV vaccines

Developments in genetic engineering dating back to 1970 have made it easy to develop

recombinant vaccines in E.coli expression system. Approved recombinant human insulin

developed in E.coli system introduced emerging trends in the field of recombinant

therapeutics. In E.coli-based expression system, an immunogenic gene is used to express and

purify instead of using whole virus system. Various immunogenic antigens against viral and

bacterial diseases of poultry have been successfully expressed through E.coli-based system

(Shahid and Daniell 2016). Yin et al. (2014) expressed EtIMP1 derived from C-terminal

against Coccidiosis in E.coli and reported positive immune response in chickens (Yin et al.

2014). Currently, a number of expression systems are commonly in practice such as yeast,

transgenic animals, insect cell and mammalian cell cultures. However, purification of

immunogenic protein and removal of host-derived contaminants are major contributing

factors towards cost of recombinant therapeutics (Richter and Kipp 2000). Many recombinant

vaccines have been developed for NDV and their promising results have led to the approval

in USA (King 1999). A recombinant plasmid DNA-based vaccine carrying both F and HN

genes has shown great protection against NDV (Loke et al. 2005).

2.4.4. Vector vaccines against NDV

Vector Vaccines are also engineered vaccines that use some Poxvirus like Vaccinia

virus, Fowlpox virus, Adenovirus and Flaviviruses to express immunogenic genes. These

viruses perform an abortive replication cycle like natural infection and triggers immune

response. Fowlpox viruses are more specific and restricted to avian species. There are some

problems linked with vector vaccines such as deletion of vectors gene, induction of non-

specific immune response and sometime decreased replication cycle does not attain require

11


immunity (Ryan and Walsh 2012). VECTORMUNE® ND is commercially available vector

vaccine of NDV (Esaki et al. 2013)

2.4.5. Oral vaccines against NDV

Different methods have been used to vaccinate chickens against NDV and researchers

have found that the best way to vaccinate chickens against NDV is the oral route. Spradbrow

and Samuel (1991) found that the immune response to the traditionally available V4

lentogenic strain is comparable to the immune response of orally vaccinated chickens, and

they concluded that the best mode to vaccinate village chickens is the oral route (Spradbrow

and Samuel 1991).

Rehmani et al. (1995) fed chickens with lactose-based vaccine pellets of the V4 virus and

found a protective immune response against virulent NDV. They concluded that oral vaccine

administration could produce high levels of antibodies, and it is a more convenient way to

vaccinate village chickens without having to store the vaccine (Rehmani et al. 1995). Zhao et

al. (2012) found a significant immune response after oral administration of nano-encapsulated

NDV vaccine as compared with live and inactivated NDV vaccines (Zhao et al. 2012).

2.5. Plant-based vaccines: A system to produce low-cost NDV vaccine

The production of plant-produced vaccines for veterinary application has received

extensive interest because of different health projects designed to decrease use of antibiotic

in farm animals and control the emergence of highly resistant strains, particularly of the

zoonotic and epidemic pathogens like NDV. These problems have encouraged the

development of most desired plant-produced vaccines to accomplish these requirements (Sack

et al. 2015).

NDV vaccines are easily accessible in the market and showing appropriate results. But,

they are often relatively expensive and require a cold storage, which is difficult to maintain;

and trained people are needed to deliver these vaccines. A subunit and whole cell vaccine is

costly, difficult production framework and always requires cool stockpiling. Monotonic

responses and safety issues are major concerns associated with DNA vaccines (Shahid and

Daniell 2016; Zhao et al. 2012).

The extensive research in the field of plant biotechnology was carried out in 1970 to

exploit the new ways to use plant rather than nutrition purpose. In 1990, Dr. Charles Arntzen

first introduced the idea of edible vaccines. Plant-based vaccines are recombinant vaccines

12


having immunogenic gene in plants; oral administration of transgenic plants to birds endorses

the production of antibodies against a particular disease (Aswathi et al. 2014; Lössl and

Waheed 2011). Plant-based vaccines can provide a new way to overcome these hurdles.

Veterinary vaccines needs some desirable characteristics and plant-based vaccines are best

substitute to fulfill these attributes (Shahid and Daniell 2016; Topp et al. 2016). The hopeful

results of plant-produced proteins make them novel contributor in the field of recombinant

vaccines (Stoger et al. 2002; Yusibov and Rabindran 2008).

Plant-based oral vaccines have many advantages over traditional vaccines. They are

cheaper because of their low cost of production and purification. They do not need to be

maintained at cold temperatures because they are stable at room temperature. Their delivery

via by the oral route is more comfortable for animals and poultry; multiple antigens can be

delivered through bioencapsulated plant-based vaccines; they are free of toxins and pathogens

associated with traditional vaccines; they can be easily scaled up in green house or field area,

which is a good substitute of fermenters. Moreover, lyophilized plant material expressing

vaccine antigen can be stored at room temperature for more than one year (Kwon and Daniell

2015; Shahid and Daniell 2016; Su et al. 2015). They can induce higher immune response

than traditional vaccines. They are more beneficial in animals as they can be given in the form

of feed by expressing vaccine antigen in fruit plants like maize, rice, soybean and potato after

oral priming with adjuvant. A number of bacterial and viral antigens have been expressed in

different plants against various diseases of animals. Moreover, another main advantage of

plant-produced vaccines is induction of mucosal resistance, which is the first barrier against

invading pathogens (Shahriari et al. 2015). Plant-produced vaccines have the same desired

characteristics as E.coli expressed protein except of their cheap production system (Daniell et

al. 2009). Plant-produced vaccines are post-translationally modified. They also wipe out the

question of retrieve virulence as in live vaccines (Clarke et al. 2013). Mucosal surface of host

is a primary barrier against encountering pathogens as it produces antibodies, which bind with

virus specific-antigens. Therefore, mucosal immune response is very important to prevent

various infections. Among many advantages of plant-based antigens, one of the significant

aspects is production of systemic and mucosal immunity. Orally delivered plant-based

antigens produce both mucosal and systemic immune response and protected against acidic

environment of host gastrointestinal tract. Moreover, plant-based antigens have ability to pass

13


through epithelial barrier and therefore provoke gut immune response. For example, antigens

expressed in angiosperms, such as rice, maize seeds offer better protection against specified

pathogen than purified product, and it could be further enhanced by expressing the antigen in

storage organelles like vacuoles, protein storage bodies and oil bodies. For poultry, the best

vaccine production systems are fodder or cereal crops; fodder and grains are a part of diet, are

easy to administer, and have minimal chances of rejection (Aswathi et al. 2014; Shahid and

Daniell 2016) . The foreign antigen is protected by cell wall of the plant then it is digested by

the residing microbes of gut to induce particular immune response (Kwon et al. 2013; Lugade

et al. 2010). Animals immunized with transgenic seeds, fruit or plants produce mucosal and

serum specific antibodies (Sack et al. 2015). Immunized animals with transgenic materials in

the form of fruits and plants activate GALT (Gut associated lymphoid tissue) to produce

specific immune response. M cells in the Peyer’s patches uptake the antigen from lumen and

stimulate B-lymphocytes with the assistance of Th cells. Ultimately, B-lymphocytes induce

specific IgA antibodies, transforming growth factor-β (TGF-β) and cytokines, such as

interleukin 4 (IL4), interleukin 10 (IL10).DCs also contribute to induce systemic immune

response. Cholera toxin subunit B (CTB) or heat-labile enterotoxin B subunit (LTB) directly

bind to GM-1 gangliosid receptors, after, but independently of, surviving the harsh

environments of the stomach (Chan and Daniell 2015; Guan et al. 2013; Lamichhane et al.

2014).

2.6. Transformation procedures for edible vaccine production

2.6.1. Nuclear transformation

Nuclear transformation is most commonly used method which introduces transgene in

nuclear genome following Mendelian law of inheritance for production of edible vaccines. In

this method, Agrobacterium tumefacien mediated transformation is used to express a foreign

antigen in the nuclear genome. Agrobacterium tumefacies, a natural soil bacterium that makes

a crown gall for transformation a desired gene into the host plant. Agrobacterium recognise

the specific phenolic secretions of injured plants and produces specific signals through

infections .The activated (Vir ) virulence genes by these signals produce specific Vir proteins.

After that, T-DNA molecules are produced by Ti plasmids that ultimatly forms T-DNA

complex with Vir proteins. A compex set of connections between T-DNA and Vir proteins

eventually transports the T-DNA into plant genome. The T-DNA method introduces desired

14


genes into the plants which eventually incorporate in nuclear genome, permitting the constant

production of desired protein (Kim and Yang 2010; Pitzschke 2013; Shahid and Daniell

2016). Nuclear transformation acts as an excellent system for the transformation of multiple

antigens. The post transtionally modified proteins is an other important characteristic of

nuclear transformation method same like protein produce in eukaryotic system. After that,

signal peptides stored these post-translationally modified proteins to various organelless or

secreted (Tremblay et al. 2010).

2.6.2. Chloroplast transformation

In chloroplast transformation, the desired gene is introduced into circular plastid of plant

genome by using particle gun (Daniell et al. 2002). In chloroplast transformation, gold

particles are coated with foreign DNA and plant tissue is bombarded with these particles to

deliver the DNA, which integrates into the plant genome after penetrating the cell wall. In

chloroplast genome, each cell in leaf has almost 1000 copies resulted in high expression of

introduced genes. Chloroplast transformation reduces the risk of gene silencing. In most of

the angiosperms, chloroplast transformation also prevents gene escape through pollination

because of maternal inheritance (Guan et al. 2013).

2.6.3. Plant viruses as vehicles for transformation

In this system, plant viruses such as bamboo mosaic virus, cauliflower mosaic virus

(CaMV), cowpea mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus serve as a

vector to introduce desired gene into the plants. In this method, engineered virus works under

the command of coat protein, which acts as best promoter for the transport of foreign genes.

The infectious nucleic acid of virus with multiple copies eventually transports the desired

gene into the plant cell. This whole process almost takes three weeks to infect the majority of

plant tissues (Gleba et al. 2007). The system has successfully produced the antibodies to treat

non-Hodgkin’s lymphoma, which is presently in clinical trials (Phase I) (Porta and

Lomonossoff 2002; Yusibov and Rabindran 2008). Plant viruses produce numerous copies of

recombinant protein in short time by self-mediated transcription and translation (Guan et al.

2013).

2.6.3. Plant virus-through Agrobacterium-mediated transformation

In this method, the engineered viral vector enters the plant through Agrobacterium and

produces numerous copies of engineered viral vector into the host plant. The Agrobacterium

15


transfers the desired gene either by vacuum infiltration or through stomata of the leaves. In

vacuum infiltration method, the applied vacuum assists to transport the desired gene via

Agrobacterium (Ling et al. 2010). Transcripts of engineered virus enter into the cytoplasm

and nucleus of the plant cell resulted the huge quantity of desired protein in short duration

(Yusibov and Rabindran 2008). Transient expression method has produced desired proteins

against , influenza virus, Yersinia pestis, and Bacillus anthracis (Chichester et al. 2007; Shoji

et al. 2008).

2.6.4. Cell suspension-based transformation

This system uses callus derived single cells or aggregate of cells. The separated cells

propagate and create a cell suspension. Transformed explants or individual callus cell produce

desired antigens using Agrobacterium co-cultivation method. This process can be magnified

in a fermenter. The world’s first officially accepted plant-based vaccine used to cure poultry

infection was produced in tobacco cell suspension culture (Yusibov and Rabindran 2008).

All transformation systems have advantages and disadvantages. Regardless of the system

used for transformation, once foreign DNA is inside the nucleus, it randomly integrates by

non-homologous recombination, resulting in the formation of a stably transformed plant

(Jacob et al. 2013).

2.7. Strategies to enhance gene expression of edible vaccines

Huge effeorts have been implemented to enhance the expression of transformed genes

and to create more immunogenic plant-based vaccines. The choice of appropriate promoter is

one of the most important approache to enhance the gene expression. The constitutive (CaMV

35S) promoter is commonly used promoter to express desired genes in majority of

dicotyledonous plants. Similarly, ubiquitin and the actin are most commonly used promoters

to express the desired genes in monocotyledonous and rice respectively. On the other hand,

tissue or seed-specific promoters have been used in several studies have to enhance expression

of foreign gene in particular organ. For example, the transformed tomatoes with CTB gene

under regulation of CaMV 35S promoter accumulate 0.2% to 0.4% of TSP as compared to

same transformed tomato plants with CTB gene under control of E8 promoter (tomato fruit-

specific promoter) showed the significant enhanced expression approximately 0.8% of TSP

(Guan et al. 2013; Jiang et al. 2002).

16


Likewise , the codon improvement by optimizin sequence is an extra way to boost the

expression of introduced foreign genes. This approach replaces the codons of desired gene

with ideal codons of host plant . Mason et al. (1998) observed that LTB gene after codon

optimization showe a high gene expression was almost 0.5-1mg as compared to native gene

(Mason et al. 1998).

Fusion of expressed gene with CTB or LTB as adjuvants or carriers enhances the

expression of foreign proteins.CTB and LTB can bind GM1 receptors in the intestinal mucosa;

when the CTB-fused antigen enters the gut, CTB directly translocates the antigen to GM1

receptors to stimulate mucosal (Okamura et al.) and systemic (IgG) immune responses(Guan

et al. 2013). Daniell et al. (2009) studied the expression of CTB-fused dual vaccines for

malaria and cholera in tobacco and lettuce, and their expression level was 8-10% of TSP in

tobacco and 4-9% of TSP in lettuce (Davoodi-Semiromi et al. 2009).

The use of an endoplasmic reticulum signal peptide stabilizes the expression of foreign

proteins and supports their maturation. (Guan et al. 2013).

Transformation of foreign genes into the chloroplast genome is another way to deal with

the high expression of foreign proteins; the high copy number of the chloroplast genome

means that there are as many as 10,000 copies of the transgene per cell.Transformation of

tobacco chloroplasts with CTB-fused tuberculosis antigens ESAT-6 and Mtb72F resulted in

high-level expression of CTB-ESAT-6 and CTB-Mtb72F, reaching approximately 7.5% and

1.2% of TSP, respectively (Lakshmi et al. 2013).

Sherman et al. (2014) expressed CTB-fused heavy chain and C2 antigens of coagulation

factor VIII in tobacco chloroplasts. The expression level of CTB-HC and CTB-C2 reached

almost 80 and 370µg/g, respectively, in fresh leaves (Sherman et al. 2014).

2.8. Attempts to produce edible vaccines against poultry diseases

There have been many attempts to produce edible vaccines against different poultry

diseases. Wu et al. (2004) developed an edible vaccine against infectious bursal disease (IBD).

The VP2 protein was introduced into Arabidopsis thalianaby A.tumefaciens-mediated

transformation. Oral administration of this plant-based vaccine resulted in IgG antibody

production that was comparable to the immune response elicited by commercially available

vaccines (Wu et al. 2009). Wu et al. (2007) developed transgenic rice (Oryza sativa)

17


expressing VP2. Chickens immunized with transgenic rice seed produced neutralizing

antibodies against a virulent viral challenge (Wu et al. 2007).

Taghavian and Schillberg (2013) produced transgenic tobacco expressing VP2 antigen

via Cauliflower mosaic virus transformation and found a protective immune response

(Taghavian and Schillberg 2013). Chen et al. (2012) also reported producing VP2 epitopes in

Chenopodium quinoausing the bamboomosaic virus transient expression system (Chen et al.

2012). Similarly, VP2 antigen was transiently expressed in tobacco plants. Feeding chicks

with three doses of 12µg protein produced an immuno-protective response in chickens

(Gómez et al. 2013). Zhou et al. (2003) produced edible vaccines against infectious bronchitis

disease virus of poultry. The S1 glycoprotein of IBV was transgenically expressed in potato.

Feeding mice and chickens with transgenic potatoes produced an immunoprotective response

after challege with lethel virus (Zhou et al. 2004).

Avian influenza virus is also a large threat to poultry. Although it infects poultry, it

can also cause serious infections in humans as well. Kalthoff et al. (2010) observed a

defensive immune response against a viral challenge after feeding chickens with transgenic

tobbaco expressing (rHA0) of HPAIV (H5N1influenza virus) (Kalthoff et al. 2010). Hwang

et al. (2012) genetically modified Arabidopsis thaliana by introducing HPAIV H5N1 of

influenza virus produce an edible vaccine against avian influenza virus (Aswathi et al. 2014).

Shoji et al. (2012) expressed the HA antigen of avian influenza virus in transgenic tobacco,

used it for oral immunization of mice, and found that mice produced specific IgG antibodies

against avian influenza virus (Shoji et al. 2012).

Sathish et al. (2012) expressed the EtMIC2 protein of E. tenella in in tobbaco via

Agrobacterium to produce a plant-produced vaccine against coccidiosis (Sathish et al. 2012).

Lacorte et al. (2007) produced a vaccine against chicken anemia disease by transforming

tobacco with genes encoding three structural proteins (Lacorte et al. 2007). Lu et al. (2011)

expressed the avian reovirus capsid protein in tobacco to develop an edible vaccine against

this virus (Lu et al. 2011).

18


Figure 2-2: Diagram showing the process of edible vaccine product

19


2.9. Previous efforts to produce edible vaccines against NDV

Untied struggles have been attempted to develop edible vaccines against NDV. The F

and HN are the two most important NDV glycoproteins and can induce a strong immune

response (Berinstein et al. 2005). In the F glycoprotein, amino acid residues 65 to 81 are the

most significant sites for antibody production, whereas, in HN glycoprotein residues 346 to

353 are the most important (Zhao and Hammond 2005).

F and HN in potato were expressed under regulated by the CaMV 35S promoter. These

accumulated leaf proteins in the range between 0.3 and 0.6µg/mg of total leaf protein. Mice

fed five times per month with transgenic leaves showed a high titer of IgA and a low titer of

IgG antibodies against NDV in their sera and intestinal fluid (Berinstein et al. 2005).

Genetically modified maize with F gene of NDV, under the regulation of the ubiquitin

promoter accumulated upto 1-3% of TSP. Feeding chickens with transgenic maize kernels

produced antibodies against a challenge virus (Guerrero-Andrade et al. 2006). Xing et al.

(2007) used Agrobacterium-mediated transformation to express the F protein in rice under the

regulation of two promoters: ubiquitin and glutelin. Feeding mice with crude protein extracts

from this rice elicited a specific immune response against NDV (Xing et al. 2007).

Zhao and Hammond (2005) used CMV (capsid protein of cucumber mosaic virus) to

express F and HN epitopes. They introduced modified recombinant CMV into tobacco plants

and the HN epitope could be recognized by anti-NDV sera. Expression of the HN epitope was

almost 1.2-1.5 mg in CMV virus particles (Zhao and Hammond 2005). Hahn et al. (2007)

transformed tobacco plants with HN gene by using Agrobacterium. Expression of the HN

gene was .069% of total soluble protein. Oral immunization of six-week old chickens with 2

g of lyophilized transgenic tobacco produced a slightly higher titer of HN-specific IgG, but

nasal immunization did not produce specific IgA or IgG against NDV (Hahn et al. 2007).

Gomez et al. (2008) expressed NDV HN and F genes in potatoes. These engineered plants

showed immune-defensive response in mice that had been fed transgenic leaves for one

month. ELISA of mucosal and intestinal washes showed that transgenic plants promoted the

production of specific IgA and IgG antibodies against NDV. Results also showed that F and

HN not only elicited the production of NDV-specific antibodies but also produced a cellular

immune response (Gómez et al. 2009).

20


Lai et al. (2012) used NDV strain (AF2240) to express the HN protein in the novel,

medicinally important plant Centella asiatica. They engineered a plant expression vector with

the HN gene under the regulation of the constitutive (CaMV 35S) promoter. The serum from

immunized chicken confirmed the expression of HN gene by dot blot test. All positive plants

and purified NDV showed positive signals, but non-transgenic plants did not show any signals

(Song Lai et al. 2012).

Lai et al. (2013) expressed the HN epitope of NDV in the Nicotiana tabacum L. cv. (Bright

Yellow-2 cell system. Transformation efficiency increased by using NtADH 5'–UTR (5'

untranslated region of the Nicotiana tobaccum alcohol dehydrogenase gene). The expression

of the HN ectodomain was in the range of 0.2-0.4% and produced a specific immune response

in a mouse model (Lai et al. 2013).

Shahriari et al. (2015) transformed genes encoding F and HN epitopes into hairy roots of

tobacco via transient expression to develop edible recombinant vaccines against NDV. Dot

blot assay confirmed the production of recombinant protein. Quantification of recombinant

protein expression through ELISA and real-time PCR showed very high protein levels that

were comparable to those of commercially available vaccines (Shahriari et al. 2015).

21


Table 2-1: Plant-based vaccines against NDV

Expressed

antigen of

NDV

Transformation

System/Promoter

Expression

Level

Productio

n System

Animal

Model

Immune

Response

Reference

F and HN Nuclear/CaMV

35S

0.3-0.6 µg/mg

total leaf

protein

Potato Mice NDV-specific

IgA and IgG

(Berinstei

n et al.

2005)

F gene Nuclear/ Ubiquitin 1-3% of TSP Maize Chicken NDV-specific

antibodies

(Guerrero-

Andrade et

al. 2006)

F gene Nuclear/ Ubiquitin/

glutelin

N/A Rice Mice NDV-specific

antibodies

(Nochi et

al. 2007)

F and HN

epitope

Transient

expression/ N/A

1.2-1.5 mg of

total leaf

protein

Tobacco N/A Response to

anti sera of

NDV

(Zhao and

Hammond

2005)

HN gene Nuclear/CAMV

35S

0.069% of TSP Tobacco Chicken anti-HN serum

IgG

(Hahn et

al. 2007)

F and HN Nuclear/CAMV

35S

0.3-0.6 µg/mg

of total leaf

protein

Potato Mice NDV-specific

IgA and IgG

(Gómez et

al. 2009)

HN gene Nuclear/CAMV

35S

3.6-4.0 µg/mg Centella

asiatica

N/A N/A (Song Lai

et al. 2012)

HN

ectodomain

Nuclear/CAMV

35S

0.2-0.4% of

total soluble

protein

Tobacco Mice NDV-specific

antibodies

(Lai et al.

2013)

F and HN

epitope

Transient

expression/ CAMV

35S

N/A Tobacco N/A N/A (Shahriari

et al. 2015)

22


2.10. Success with the world’s first approved plant-based NDV vaccines

In 2006 Dow AgroSciences officially received approval from the United States

Department of Agriculture (USDA) for first plant-made vaccine against NDV. Dow Agro

Sciences practiced cell suspension culture and developed a plant-made injectable vaccine

against NDV that showed almost 90% protection against a NDV virus challenge. This system

has huge capacity to produce recombinant protein within days. Although, it was a plant-based

but unfortunately, the company did not prefer to market this product because of its mode of

injectable commodity (Thomas et al. 2011; Yusibov et al. 2011).

2.11. Future prospects for plant-based NDV vaccines

Progress in edible vaccine production against poultry diseases is still in initial stages.

However, promising results suggest that plant-based vaccines could replace conventional

vaccines for poultry in the near future. There are some hurdles to overcome, including

standard dosing of expressed antigens, as well as vaccine formulation, but these are not

insurmountable challenges. Results reported so far show a low expression level, but with the

use of latest technologies, expression level can be improved (Aswathi et al. 2014). Reported

studies with virus challenge show that edible vaccines do confer enough immune response to

protect chickens from NDV (Table 2.1). Oral delivery would provide a new method to control

NDV and other poultry diseases and is distinct from the injection, spray, and drinking water

modes of administration (Lim 2014). Initially, scientists used tomato, tobacco, and potato to

express foreign genes. However, seed and cereal crops are preferable because of their high

levels of soluble proteins. Seed crops are also very suitable crops for storage. For production

and commercialization of edible veterinary vaccines, cereal crops are best because they

constitute a major part of the diet and do not require heat or pressure treatment (Daniell et al.

2001). Seeds are the most important source of high-level expression and long-term antigen

storage (Fiedler and Conrad 1995). Expressing antigens in cereal crops such as maize and rice

can resolve many issues, as cereals are a part of poultry feed. Kernels expressing high levels

of proteins can be stored for longer periods. Dry seeds can also be used as edible vaccines for

poultry diseases (Jacob et al. 2013).

2.12. Conclusion

Numerous efforts for edible vaccines and successful approval for certification of plant-

produced vaccine against NDV in 2006 create hopes toward commercialisation of plant-made

23


vaccines. Plant-based vaccines acquire all the appealing capabilities of vaccines such as low

temperature storage and stability and better efficiency at room temperature. Promising results

of edible vaccines against NDV show that edible vaccines are the best alternative to live and

killed vaccines.

24


: MATERIAL AND METHODS

3.1. Collection and Propagation of Virus

Newcastle disease virus (strain chicken/SPVC/Karachi/NDV/1/1974 fusion protein

gene, complete CDS, accession #GU182327) was collected from the Veterinary Research

Institute (Kolotilin et al.). The OIE classifies this as a virulent strain and it was first used as

a live vaccine in 1974. The virus was stored at -70°C in a Biosafety Level III Laboratory

(CEMB, Lahore, Pakistan) and in special containment facilities.

3.2. Complementary DNA (cDNA) Synthesis

3.2.1. Isolation of viral RNA

TRIzol direct RNA extraction method was done to isolate viral RNA (Chomczynski

and Sacchi 1987). A total of 0.7mL TRIzol reagent was added to 0.3 mL diluted virus and

homogenized through the vortex. Five min incubation was given to sample at room

temperature. Addition of 2mL chloroform was done followed by 15 seconds vortex. The upper

layer, which contain the RNAwas collected by Centrifugation (13000 × g) for 15 minutes at

25°C and precipitated with 500 µl of 100% isopropanol. The RNA pellet was taken after

second centrifugation at 13000× g at 4°C for 5 minutes. After washing with 70% ethanol and

air-drying the pellet, it was dissolved in 20µL RNAase-free water and RNA content was

quantified through Nanodrop (Thermo Scientific).

3.2.2. cDNA Synthesis

One-step RT-PCR was carried out with random hexamers using a RevertAid First

Strand cDNA Synthesis Kit (Thermo Scientific, K1622). Following reaction was prepared in

a 0.2-mL PCR tube and mixed via minifuge:

Reagent Quantity

Template RNA 1µg

Random Hexamers 1µL

Nuclease-Free water 10 µL

Total volume 12 µL

25


The tube with reaction mixture was undergone an incubation of 5 min in a thermocycler at

65°C in order to linearize the RNA, then kept on ice for 2 min. The ingredients were added in

linearized RNA in an order following the manufacturer instructions with continuous mixing

as follow:

Reagent Quantity

5X Reaction Buffer 4 µL

10 mM dNTP Mix 2 µL

RiboLockRNase Inhibitor 1 µL

RevertAid Reverse Transcriptase 1 µL

Total Volume 8 µL

The cDNA was prepared in a thermocycler in a single step reaction by placing it 25°C for 10

min, proceeding by 42°C for 60 min and 70°C for 5min. The resultant cDNA was collected

and preserved at -20°C.

3.3. PCR amplification of F and HN genes from cDNA

Polymerase chain reaction (PCR) was used to amplify ~1662bp F and ~1712bp HN

genes using cDNA of NDV as template. Primers were designed using sequences available on

NCBI (accession #GU182327).

Primers Used in PCR

Primer ID Sequence (5´- 3´) Annealing

temperature

Product

size

NDVF-F 5´CCAGTACCTCTAATGCTGACCATAC 3´ 61ºC 1662bp

NDVF-R 5´TCACATTTTTGTAACAGCTCTCATCT 3´

NDVHN-F 5´GACAGCGCAGTTAGCCAAGTT 3´ 61ºC 1712bp

NDVHN-R 5´TTAAACCCCACCATCCTTGAG 3´

The total 20µl reaction volume was used for amplification of both F and HN genes at

the following PCR conditions i.e. initial denaturation at 95˚C for 5min, followed by 30 cycles

26


of amplifications (denaturation at 94˚C for 1min, annealing at 61˚C for 1 min, extension at

72˚C for 1 min) and final extension was given at 72˚C for 10 min.

The amplified PCR product was analyzed on 0.8% agarose gel (Appendix II) provided

with 1.0µg/ml ethidium bromide and visualize under UV light. Desired PCR products of

~1662bp and ~1712bp for F and HN genes respectively were excised with a sharp surgical

blade under UV light by using safety glasses. Gel slices were placed into a pre-weighed 1.5-

mL tube.

3.4. TA cloning and transformation of F and HN genes

3.4.1. Gel purification of PCR product

PCR products were eluted from gel slices having F and HN genes using GeneJet™

Gel Extraction kit (Thermo Scientific Cat#k0692). Gel slices were dissolved in binding buffer

(1:1 v/w), the tubes were placed in water bath (55˚C) for 15-20 min and were periodically

inverted. Fully dissolved gel slices were poured into elution columns, followed by 1 min

centrifugation (12,000 rpm). After removal of column flow through, 500µL wash solution

was added to wash the column and centrifuged for 1 min. This step was repeated twice for

purification. Finally, elusion was done by adding 8-10 µL of elution buffer to each column

followed by centrifugation of 1 min. The eluted PCR product was collected in a separate tube

and quantified for ligation into the TA cloning vector.

Reagent Quantity

Template DNA (100ng/µL) 1µL

Forward Primer (10 pmol) 2µL

Reverse Primer (10 pmol) 2µL

10X PCR Buffer 2µL

MgCl2 (25mM) 2µL

dNTPs (2.0mM) 2µL

Taq Polymerase (5U/µL) 0.5µL

Nuclease Free water 8.5µL

Total Volume 20µL

27


3.4.2. Ligation of eluted product into the TA vector

Eluted PCR products of F and HN genes were directly cloned in TA cloning vector

(Invitrogen pCR®2.1) as per manufacturer’s protocol (Invitrogen, Cat # K 4500-01). TA

cloning is a single-step method for the direct ligation of foreign gene into a linearized vector

with a single 3´ deoxythymidine (T) residue. This type of cloning is based on the specific Taq

polymerase efficiency, which adds deoxyadenosine (A) to 3´ ends and enables ligation of the

insert at the 3´ deoxythymidine (T) residue of the vector. The below mentioned ligation

reaction was prepared in a separate PCR tube for each insert and overnight incubation was

done at 14ºC.

3.4.3. Transformation of TA ligated product

E. coli (Top 10) competent cells were used for transformation of TA ligated F and HN

gene products. For transformation, 2µL of each ligated product and competent cells were

mixed together and kept on ice for 30 min. The cells were given heat shock at 42ºC for 90

seconds. After heat shock, cells were immediately transferred onto ice for 5 min and then

revived in liquid LB media (800µl) (Appendix I.). Cells were kept on 37ºC incubator with

constant shaking for one hour. Plating was done by adding 100µl of incubated transformed

cells on LB agar plate (Appendix I) containing 20µg/µL IPTG and 40 mg/ml X-Gal followed

by spreading through glass spreader. Plates were incubated for 24 hours at 37ºC.

3.4.5. Selection of positive clones

Positive clones of F and HN genes were selected based on blue/white screening. White

colonies, which were considered positive clones, were selected and inoculated into 3 mL of

Reagent Quantity

pCR®2.1 Vector (25 ng/µL) 2 µL

10X Ligation Buffer 1 µL

Insert (25 ng/µL) 2 µL

T4 DNA Ligase (4.0 Weiss units) 1 µL

Sterile Water 4 µL

Total volume 10 µL

28


LB media containing tetracycline (12.5μg/ml) and kanamycin (50μg/ml) (Appendix III)

followed by incubation at 37ºC for 18 hours.

3.4.6. Plasmid DNA isolation from positive clones

Isolation of Plasmid DNA from positive clones was done by using Gene JET Plasmid

extraction kit (cat#K0503). After incubation, cells were pelleted down in 1.5-mL tubes at

12000 rpm for one min. Resuspension of Pellets was carried out in 250µL solution 1 by

vortexing the cells for 15 seconds. Then, 250µL of lysis solution (solution II) was added and

gently mixed until it became partially transparent. After that, 350 µL neutralization buffer

(solution III) was added and thoroughly mixed. Five min centrifugation was carried out to

pellet down chromosomal DNA and cell debris. The supernatant was shifted onto columns

provided with the kit. Centrifugation was done again for 1 min and the flow through was

thrown away. Later on, Columns were put back in collection tubes, and wash buffer 1 was

added onto the columns (500 µL), and centrifugation was done for 60 seconds. After

discarding the flow through washing step was repeated by addition of 500 µL wash solution.

Later, for removal of residual wash buffer, empty columns were centrifuged for 1 min. For

final elusion, columns were put back into nascent 1.5mL microfuge tubes and 50µL elution

buffer was put onto the center of each column membrane. Recovery of Plasmid DNA was

done by 2min centrifugation at 12000 rpm.

3.4.7. Confirmation of positive clones

TA ligated F and HN clones/inserts were confirmed through PCR and restriction

digestion. PCR was performed with gene-specific primers through same procedure as

mentioned in section 3.3. PCR products were observed on 0.8 % under UV light. The

confirmation of successful ligation of insert in TA vector was done through restriction

digestion with Eco.R1. The reaction mixtures of restriction digestion were prepared in PCR

tubes as follows by using the ingredients in the following order.

29


Reagent Quantity

TA plasmid containing F/HN gene 10 µL

EcoRI (10u/µL) 1 µL

10x EcoRI Buffer 1.5 µL

Injection water 2.5 µL

Total volume 15 µL

The digestion reaction in each tube was kept for one-hour incubation at 37 º C, resolved on

0.8% agarose gel and analyzed under UV light.

3.5. Sequencing of positive F and HN clones

Positive clones for both F and HN genes were further confirmed through sequencing.

Gene sequencing was performed using the Big Dye direct sequencing method. The following

reaction mixture was prepared for sequencing PCR:

Reagents Quantity

2.5X BigDye® 1.5µL

5X Sequencing Buffer 1.25µL

Plasmid 4µL

M13 primers (For/rev, 10 pmol) 1 µL

Injection water 2.25µL

Total volume 10µL

Sequencing PCR was done by using cycling conditions as follow: initial denaturation

at 96˚C for 1min, followed by 30 cycles of amplification (96˚C for 30 seconds, 50˚C for 15

seconds, and 60˚C for 4min) with final extension of 4 min at 60˚C. After the reaction, PCR

products for both genes were precipitated by using of 3M sodium acetate (2 µL), 125mM

EDTA (2 µL) and 25 µL of 100% ethanol. Tubes were placed for 20 min at room temperature.

After that, centrifugation was carried out at 15°C (3000 rpm) for 30 min. The pellets were

centrifuged for 15 min at 15°C (3000 rpm) after washing with 70% ethanol. Ethanol was

removed and tubes were spun again. Pellets were air dried at 50°C. Finally, 15 µl of

30


formamide was added to the pellet and preceded for 5 minutes denaturation at 95ºC.

Sequencing was carried using Sanger’s sequencing method at the ABI 3730 (Applied

Biosystems) sequencer facility at CEMB.

3.6. Cloning of F and HN in prokaryotic expression vector

The pET30a expression vector was obtained from the Plant Biotechnology

Laboratory, CEMB, Lahore. For expression studies, F and HN genes were ligated separately

into the pET30a vector. Map of the pET vector and expression cassettes for both genes are

shown below (Figure 3.1 and Figure 3.2)

Figure 3-1: Map of the pET30a expression vector

31


Figure 3-2: F/HN gene cassette

3.6.1. Restriction digestion

For cloning, TA plasmids of both genes and the pET expression vector were digested

with EcoR1 to produce overhangs. Reaction mixtures for restriction digestion were prepared

as follows:

Reagent Quantity Reagent Quantity

TA of F/HN gene 10 µL (1-3 µg) pET30a Plasmid 10 µL (1-3 µg)

EcoR1 1 µL EcoR1 1 µL

10x EcoR1 Buffer 1.5 µL 10x EcoR1 Buffer 1.5 µL

Injection water 2.5 µL Injection water 2.5 µL

Total volume 15 µL Total volume 15 µL

Reaction mixtures were incubated at 37ºC for one hour and separated on a 0.8 % gel.

The required bands of digested pET30a plasmid and F/HN genes were carefully taken from

the gel by cutting with a sterile blade under a UV transilluminator. The digested product was

eluted according to gel elution protocol explained in section 3.4.1 and its concentration was

quantified on a Nanodrop.

3.6.2. Ligation of Insert (F/HN) into the pET30a vector

Ligation of F and HN genes into the pET expression vector was carried out separately

by using a DNA ligation kit (Thermoscientific cat# K1422). The given ingredients were added

into the reaction mixture for the ligation of each gene into the pET expression vector in two

separate tubes and incubation was done at 22ºC for one hour.

32


Reagent Quantity

Vector (60ng/µl) 2 µL

Insert (70ng/µl) 1.8 µL

Buffer 4 µL

Ligase 1 µL


Total volume 20 µL

3.6.3. Confirmation of F/HN genes in pET30a

Ligated products of both genes were transformed in competent cells through heat

shock procedure as described in section 3.4.3. Plasmid miniprep and restriction digestion were

performed using colonies that appeared after plating of transformants on LB selection plates

as described in sections 3.4.6 and 3.6.1. The resolved digestion mixture on 0.8 % agarose gel

was analyzed under UV to confirm the presence of genes.

3.7. Propagation of recombinant F/HN genes in the E. coli DE3 (BL-21)

strain

For expression studies of both genes, the E. coli DE3 (BL-21) strain was obtained

from the CEMB culture collection lab. Two µL of the pET30 plasmid containing F or HN

genes was transformed separately into chemically competent DE3 (BL-21) cells. Cells were

kept on ice for half hours proceeded by heat shock for 90 seconds at 42ºC. After heat shock,

reaction mixture tubes were immediately placed on ice and cells were allowed to chill for 5

min. After cooling, cells were mixed with 800µL LB broth with constant shaking at 37ºC for

one hour. Transformed cells were spread on agar plates containing kanamycin (30µg/mL) and

chloramphenicol (12.5µg/mL) (Appendix III) and plates were kept overnight incubation at

37°C. Next day, transformants were picked and inoculated into 3mL LB broth with

chloramphenicol (12.5µg/mL) and kanamycin (30µg/mL) (Appendix III) selection. The

positive clones were further confirmed through PCR using gene-specific primers after plasmid

isolation, as described in section 3.3.

33


3.7.1. Isolation and quantification of protein

Transformed DE3 (BL-21) cells were grown for 19 hours at 37°C in LB liquid media

containing antibiotics chloramphenicol (12.5 µg/mL) and kanamycin (30 mg/mL) (Appendix

III) for selection. The next day, cultures were diluted (1:10 ratio) to attain an O.D.600 of 0.6-

0.8. The media was supplemented with 20% glucose and induced with 1mM IPTG at 30°C

for 16 hours with constant shaking in incubator. Cells were centrifuged (10,000 × g) for 10

min at 4°C. Pellets were dissolved in lysis buffer (1xPBS, 500mM NaCl, 1% Triton X-100,

6% glucose, 1mM PMSF, 4 mg/mL lysozyme). After centrifugation, the suspension was kept

at room temperature for 20-30 min followed by brief sonication of 6-8 times for 5 seconds

intervals with incubation on ice. After sonication, crude protein samples were centrifuged

(16,000× g) for 30 min at 4°C and supernatants were collected in a separate tube. Bradford

assay was used for quantification of proteins in 96 well plate. Bradford dye solution was made

by dissolving 5 ml Bradford protein dye in 20ml of 1XPBS. Five dilutions 0.8μg/μl, 0.4 μg/μl,

0.2 μg/μl, 0.1 μg/μl, 0.05 μg/μl of BSA (standard) was prepared. After addition of standards,

10μl of crude protein was added in duplicates along with water as blank. 200μl Bradford dye

was added in each well and absorbance was taken on ELISA plate reader (ELx 800) at 450

nm wavelength. (Bradford 1976). Concentration of protein was determined by plotting a

standard curve was prepared on excel sheet by selecting XY (scatter)" plot and option “add

trend line” and “Display equation on chart” ware selected to find linear regression and R2

values on graph. The linear region of graph directly quantified unknown protein concentration

with reference to known concentration of BSA (standard).

3.7.2. SDS polyacrylamide gel electrophoresis (SDS-PAGE)

The crude protein samples were analyzed through SDS PAGE in a Bio-Rad mini gel

apparatus according to procedures explained by Laemmli (Laemmli 1970). Fifteen

micrograms of protein from E. coli expressing F or HN genes and untransformed E. coli were

mixed with 1x protein loading dye (Appendix IV). The protein samples were boiled for 10

min. After boiling, the protein was incubated on ice and resolved on a 12 % SDS

polyacrylamide gel (Appendix IV). After completion of SDS-PAGE, gels were placed in

Coomassie blue staining solution (Appendix IV) with mild shaking for 30 to 40 min. After

that, the staining solution was removed and gels were treated with destain solution (Appendix

IV) with mild shaking for approximately 18 hours at room temperature.

34


3.8. Protein purification through affinity chromatography

3.8.1. Biomass preparation

For protein purification, cultures containing recombinant F or HN pET30a plasmids

were scaled up to 1L and induced with 1mM IPTG as discussed in section 3.7.1. Cells were

collected by centrifugation, and washed with 10 mL 1x PBS (w/v) and then resuspended in

lysis buffer for sonication as described in section 3.7.1. For cell disruption, protein samples

for both genes were divided into 1.5-mL microfuge vials and sonicated for 30 seconds in an

ice bath with a continuous10-KHz pulse. This process was repeated 10-15 times at 60-seconds

intervals until complete lysis occurred. Thirty min centrifugation (16,000 ×g) of disrupted

protein samples were carried for 30 minutes at to collect the supernatant, which was then

stored at 4°C.

3.8.2. Column packing and protein purification

A 16/20 XK column with a 20-mL bed volume and a 15-cm bed height packed with

chelating Fast Flow Sepharose adsorbent (GE Healthcare, cat. # 17-0575-01) was charged

with 2 column volumes (CV) of the 0.2M NiCl2 solution. Sepharose adsorbent was

equilibrated with 2 CV of the binding buffer (500mM NaCl, 20mM sodium phosphate buffer,

pH 7.4). All steps for purification were completed at 4°C inside a Kelvinator chromatography

refrigerator. Prior to protein loading, the supernatant was carefully cleaned from cellular

debris through a 0.45-µm filter assembly. After equilibration, the filtered supernatant was

loaded on column at a linear velocity of 150 cm/h. The time for completion of column loading

was calculated by first washing the column bed with 2 CV of wash solution1 (20mM

imidazole + binding buffer, pH 7.4) and then with wash solution 2 (40mM imidazole +

binding buffer, pH 7.4) to eliminate unbound solutes and protein contamination. His-tagged

F and HN proteins that remained bound to the adsorbent were separated by applying a step-

wise elution with increasing concentrations of imidazole. (Thermo Fisher Scientific, cat.

#AC30187-0010). Five concentrations of elution buffer were prepared (70mM to 150mM)

and five fractions (1 mL each) were collected from each elution and analyzed by Bradford

assay to check the concentration of protein as mention in section 3.7.1. Ten micrograms of

protein from E. coli expressing either F or HN genes was loaded and resolved with SDS-

PAGE to obtain a single band of purified protein. The concentration of isolated purified

proteins (F/HN) were quantified by Bradford assay (Bradford 1976).

35


3.9. Identification of antigenic epitopes

Studies regarding the immunogenicity of NDV surface proteins have been conducted

in wet labs using different immunological tests and experiments. In this research, the

immunogenicity of these proteins was analyzed in a dry lab using various tools from the

Immune Epitope Database Analysis Resource (http://tools.immuneepitope.org/main/), but

predicting their structures using SWISS-MODEL.

3.9.1. Homology modeling using SWISS-MODEL

Three-dimensional protein models have been generated successfully through online

available homology modeling databases. SWISS-MODEL, established 20 years ago by

Schwede et al (2003) is the first self-processing server for structural homology of proteins.

that has become more improved and advanced since its initial development (Schwede et al.

2003). The SWISS-MODEL web interface, available at http://swissmodel.expasy.org/, is

easily accessible from the web and is a fully automated protein structure homology-modeling

server. Input data were provided in the form of acid sequences of proteins of interest as

FASTA format which were retrieved from sequencing results. The server carried out multiple

sequence alignment of the target-template sequence. At this point, the template was selected

on the basis of maximum homology and a model was generated using modeler mode. For

each protein model generated, SWISS-MODEL provided model coordinates, a systematic

modeling log, ligands, cofactors and oligomeric state in the model, and the model was scored

according to QMEAN model quality estimation. Based on this scoring system, regions of

models were highlighted and visualized using Jmol. Protein Data Bank (PDB) files were

created and saved to predict antigenic epitopes.

3.9.2. Antigenic epitope prediction using the Immune Epitope Database

Analysis Resource

Linear continuous epitopes of the proteins were determined with the Antibody Epitope

Prediction tool using the Kolaskar and Tongaonkar Antigenicity algorithm. This method

analyzes the physicochemical properties and frequency of occurrence of amino acid residues

for antigenic prediction with 75% accuracy. The input amino acid sequence was provided in

FASTA format and the results were recorded.

http://tools.immuneepitope.org/main/

http://swissmodel.expasy.org/

36


Based on the protein’s 3D structure, the ElliPro tool was used to predict linear and

discontinuous epitopes. In this method, the protein’s shape is resolved into ellipsoids and each

ellipsoid is assigned a score known as a Protrusion Index (PI) value. Residues having a PI

score of 0.9 or more are associated with greater solvent accessibility. The PDB file of the

protein was provided as input to run the prediction. Predicted epitope structures were observed

and results were recorded.

3.10. Western blot analysis with anti-His-tag antibodies

For western blot, 15 µg protein from E. coli expressing either the F or HN gene was

resolved on separate 12 % SDS polyacrylamide gels (Appendix IV). Western blot uses

specific antibodies to identify proteins that have been resolved based on size by gel

electrophoresis. Transfer of protein from gels to membranes was performed in a Trans-Blot

Semi-Dry Transfer Cell (Bio-Rad) according to procedures stated by (Towbin et al. 1979).

Membrane and gel were sandwiched between two stacks of Whatman blotting paper to form

a direct connection with electrodes. This assembly was placed inside the Transblotter and an

electrical field of 17V was applied. After transfer, membranes blocking was achieved with

3% skim milk (Appendix IV) in 1xPBST (0.05%Tween-20 in 1xPBS) for 1 hour and

incubated with anti-His primary antibody (1: 10,000 dilutions, Santa Cruz) overnight with

mild shaking at 4ºC. Membranes were washed thrice with 1XPBST (0.05% Tween-20 in

1XPBS) and incubated in AP-conjugated secondary antibody (1: 5,000 dilutions, Santa Cruz)

overnight for 1 hour. After three washes, colour reaction was started by placing the membrane

in developed in the dark by flooding the membrane with BCIP/NBT (1-bromo-3-chloro-3-

indolyl phosphate/nitro blue tetrazolium) substrate in dark.

3.11. Cloning of F and HN genes into pCAMBIA-1301under different

promoters

The binary vector pCAMBIA-1301 was used as a plant expression vector, and F and

HN genes were cloned into it under the regulation of two different promoters. The F and HN

genes were cloned under the regulation of the CaMV35s and seed-specific promoter

respectively. The map of the vector is shown in Figure. 3.3.

37


Figure 3-3: Map of pCAMBIA -1301. Source: http://www.cambia.org/

Figure 3-4: Cassette having F gene under the CaMV35S promoter and HN gene under

the Zein promoter in pCAMBIA1301

3.11.1. Amplification of F gene

The F gene amplification from TA vector was done with primers that introduced Nco1

sites at 3' and 5' ends of the gene. The PCR product was excised, purified and cloned into the

38


TA vector with (pTZ57R/T) with specific Nco1 sites at both ends according to manufacturer

instruction (Thermo Scientific cat. # K1213).


The TA plasmid containing the F gene with Nco1 sites at both ends was digested with

Nco1 along with pCAMBIA1301. Digestion reactions were prepared as follows:


TA of F gene with Nco1 10 µL (1-3 µg) pCAMBIAPlasmid 10 µL (1-3 µg)

Nco1 1 µL Nco1 1 µL

10x Tango Buffer 1.5 µL 10x Tango Buffer 1.5 µL



Reactions mixture were kept at 37ºC for one hour and resolved on 0.8 % agarose gel.

The digested pCAMBIA plasmid and F gene were carefully taken from the resolved gel by

cutting with a sterile blade under a UV transilluminator. Digested products were eluted using

the gel elution protocol explained in section 3.4.1 and concentration was quantified on a

Nanodrop.

3.11.3. F gene ligation into pCAMBIA

Ligation of F gene was carried out with the plant expression vector pCAMBIA using

a rapid DNA ligation kit (Thermo Scientific cat. # K1422). Overhangs of both the pCAMBIA

vector and the F gene were ligated in 1:1 ratio with the help of T4 DNA ligase. The below

mentioned ingredients were carefully mixed in a PCR tube and allowed to be ligated at 22ºC

for one hour:

39


Reagent Quantity


Insert (F gene,120ng/µl) 1 µL

Buffer 4 µL

Ligase 1 µL

Injection water 12 µL

Total volume 20µL

3.11.4. Transformation of ligation

Ligated product containing the F gene was transformed into chemically competent

TOP10 cells with the same method described in section 3.4.3.

3.11.5. Selection of F ligated positive clones in pCAMBIA

Plasmid DNA was isolated from transformed bacterial colonies as described in section

3.4.6. The F gene’s insertion into pCAMBIA was confirmed by PCR and restriction digestion.

F gene (~1662bp) was amplified through PCR using the same procedure as stated in section

3.3. Restriction digestion was carried out with Nco1 as mentioned in section 3.6.1. The

resolved PCR and digestion mixtures on 0.8 % agarose gel were analyzed for the presence of

genes.

3.11.6. Determination of F gene orientation into pCAMBIA 1301

The accurate orientation of F gene in pCAMBIA was confirmed through digestion

analysis. One enzyme was selected at the 5’-end of the F gene insert and the other was selected

at the right border of the pCAMBIA vector. The resolved digested products on 0.8% agarose

gel were analyzed under UV find the right orientation of the F gene in pCAMBIA.

3.11.7. Cloning of the HN gene in pCAMBIA 1301 under seed specific

promoter

The HN gene was ligated into pCAMBIA vector carrying the F gene under the

regulation of the CaMV35S promoter. The HN gene was cloned under regulation of the Zea

mays zein promoter and Nos terminator.

40


3.11.8. Cloning of the Zein promoter into pCAMBIA 1301

The zein promoter is a specific promoter from maize that drives the expression of

exogenous genes in the endosperm of this crop. For cloning, a pUC57 vector harboring the

zein promoter and pCAMBIA carrying the F gene were digested with Sal1 and

Xba1.Digestion reactions were prepared as follows:


pUC57 with zein promoter 8 µL (1-3 µg) pCAMBIA Plasmid 8 µL (1-3 µg)

Sal1 1.5 µL Sal1 1.5 µL

Xba1 1.5 µL Xba1 1.5 µL

10x Tango Buffer 4 µL 10x Tango Buffer 4 µL

Injection water 5 µL Injection water 5 µL


3.11.9. Ligation of the Zein promoter into pCAMBIA 1301

The zein promoter was ligated into pCAMBIA (carrying the F gene) with a rapid DNA

ligation kit (Thermo Scientific cat. #1422). The overhangs of both pCAMBIA vector and zein

promoter gene were ligated in a 1:1 ratio with the help of T4 DNA ligase. The following

ingredients were mixed in 0.2ml PCR tube and ligated at 22ºC for one hour:

Reagent Quantity

Vector (50 ng/µl) 4 µL

Insert (HN gene,110 ng/µl) 1.8 µL

Buffer 4 µL

Ligase 1 µL


Total volume 20µL

41



Ligated product containing the zein promoters transformed into chemically competent

TOP10 cells with the same method as described in section 3.4.3.

3.11.11. Selection of positive clones with promoter

Plasmid DNA was isolated from transformed bacterial colonies as described in 3.4.6,

and the presence of the HN gene in the pCAMBIA vector was confirmed by restriction

digestion. Restriction digestion was performed with Sal1 and Xba1 as described in section

3.6.1. The resolved digestion mixture on a 0.8 % gel was analyzed under UV for the presence

of the zein promoter in pCAMBIA.

3.11.12. Cloning of the HN gene into pCAMBIA

The HN gene was amplified from the TA vector with primers that introduced Xba1 at

the 5' and BamH1 at 3' of the HN gene. The PCR product was excised, purified and cloned

into the TA vector (pTZ57R/T) according to the manufacturer protocol (Thermo Scientific

cat# K1213).


The TA plasmid containing HN gene with Xba1 and BamH1 sites at 5'- and 3'-ends

and pCAMBIA1301 carrying the F gene under the CaMV35S and zein promoters at the MCS

were digested with Xba1 and BamH1 for production of overhangs in the pCAMBIA vector

and the HN gene. Digestion reactions were prepared as follows:


TA of HN gene 8 µL (1-3 µg) pCAMBIA Plasmid 8 µL (1-3 µg)

BamH1 1 µL BamH1 1 µL

Xba1 1 µL Xba1 1 µL

10x Tango Buffer 1.5 µL 10x Tango Buffer 1.5 µL



42


3.11.14. Ligation of the HN gene into pCAMBIA 1301

Ligation of HN gene into pCAMBIA (carrying the F gene) was carried out with a rapid

DNA ligation kit (Thermo Scientific Cat#. 1422). The overhangs of both pCAMBIA and the

HN gene were ligated in a 1:1 ratio using T4 DNA ligase. The following ingredients were

mixed in 0.2ml PCR tube and ligated at 22ºC for one hour:

Reagent Quantity


Insert (HN gene, 130ng/µl) 1.7 µL

Buffer 4 µL

Ligase 1 µL


Total volume 20µL


The ligated HN gene was transformed into competent cells (TOP 10) by the same

method as mentioned in section 3.4.3.

3.11.16. Selection of HN ligated positives clones in pCAMBIA

Plasmid DNA was isolated from transformed bacterial colonies as described in

section3.4.6 and the existence of HN gene in pCAMBIA was confirmed through PCR and

restriction digestion. ~1712bp HN gene fragment was amplified through PCR using the same

procedure as stated in section 3.3. The digestion reaction was done with BamH1 and Xba1 as

described in section 3.6.1. The resolved PCR and digested products on 0.8 % agarose gel were

analyzed for the presence of HN.

3.11.17. Cloning of the NOS terminator into pCAMBIA1301

To clone the NOS terminator, pUC57 and pCAMBIA carrying F, the zein promoter

and the HN gene were digested with BamH1 and Kpn1. Digestion reaction mixtures were

prepared as follows:

43



pUC57 with NOS terminator 10 µL (1-3

µg)

pCAMBIA plasmid 10 µL (1-3 µg)

BamH1 1 µL BamH1 1µL

Kpn1 2 µL Kpn1 2 µL

10x BamH1 buffer 2 µL 10x BamH1 buffer 4 µL

Injection water 5 µL Injection water 5 µL


3.11.18. Ligation of the NOS terminator into pCAMBIA 1301

The NOS terminator was ligated into pCAMBIA to complete the cassette (zein

promoter, HN and NOS terminator) using a rapid DNA ligation kit (Thermo

ScientificCat#1422). Overhangs from both the pCAMBIA vector and the NOS terminator

were ligated in a 3:1 ratio using T4 DNA ligase. The following ingredients were carefully

mixed in 0.2ml PCR tube and ligated at 22ºC for one hour:

:

Reagent Quantity

Vector (60 ng/µl) 1 µL

Insert (NOS terminator,80 ng/µl) 2.3 µL

Buffer 4 µL

Ligase 1 µL


Total volume 20µL


The ligated NOS terminator product was transformed into competent cells (TOP 1 by

same method as mentioned in section 3.4.3.

3.11.20. Selection of the NOS ligated positive clones in pCAMBIA

Plasmid DNA was isolated from transformed bacterial colonies as described in section

3.4.6 and the presence of the NOS terminator in pCAMBIA was confirmed through restriction

44


digestion. Restriction digestion was performed with BamH1 and Kpn1 as described in section

3.6.1. The resolved digested product on 0.8 % agarose gel was analyzed for presence of the

NOS terminator in pCAMBIA.

3.11.21. Determination of cassette (Promoter + HN + NOS) through

restriction analysis

Restriction digestion was carried out to excise the 3.3-kb fragment from pCAMBIA.

Because the promoter was cloned into the Sal1 site and the NOS terminator was cloned into

the Kpn1 site, Sal1 and Kpn1 were used to digest out the cloned cassette (Promoter + HN +

NOS) from pCAMBIA. The digestion reaction mixture was prepared as follows:

Reagent Quantity

pCAMBIA 10 µL(1-3µg)

Sal1 2 µL

Kpn1 2 µL

10x BamH1 buffer 2 µL

Injection water 4µL

Total volume 20 µL

3.12. Electroporation of recombinant plasmids into Agrobacterium

Two recombinant plasmids, pCAMBIA F and pCAMBIA F+HN, were electroporated

separately into competent Agrobacterium cells (LBA4404) using a Bio-Rad electroporation

device (# 165-2105). Two microliters of each plasmid was mixed with 100 µl of competent

cells. The electroporation was performed at 25μF capacitance, 2.2 kV voltage and 200 ohms’

resistance. After electroporation, cells were mixed separately in 1 ml of LB media (Appendix

I) and incubated at 30ºC for 2 hours followed by plating of 100µl of transformed cultures for

both plasmids on YEP agar (Appendix I) plates having 50 µg/ml kanamycin (Appendix III)

and kept at 30ºC incubation for 24 hours.

45


3.12.1. Determination of pCAMBIA (F+HN) in Agrobacterium

Colony PCR was performed to confirm the presence of genes in Agrobacterium.

Transformed Agrobacterium colonies for both genes were picked with a sterile tip and mixed

with 30 µl of dH2O in a PCR tube. The PCR tubes placed at 96ºC for 10 min and centrifuged

(13K) for 2 min. Total 2 µl of supernatant from each tube was used as a template DNA. The

PCR was performed with F and HN-specific primers according to same method as described

in section 3.3. The resolved PCR product on 0.8 % agarose gel was observed under UV light

for presence of both (F and HN) genes in Agrobacterium. For long-term storage, glycerol

stocks for both F and HN genes were prepared and preserved at -70ºC.

3.13. Transformation of maize

3.13.1. Collection of plant material and embryo isolation

Inbred lines of Maize plants selected for transformation were planted in the

greenhouse of the Centre of Excellence in Molecular Biology (CEMB) for immature embryos.

Ears containing immature embryos were harvested from the field. Selection of ears from the

field is the most critical step, as immature embryos of 1 mm or 1.2 mm in length along the

axis are best for transformation.

3.13.2. MS medium preparation

MS (Murashige and Skoog 1962) broth (liquid) and solid media (Appendix I) were

prepared for culturing of transformed zygotic embryos by adding ingredients in an order

mentioned in Appendix I followed by autoclavation at 121ºC and 15 PSI for 20 minutes. MS

media was poured in petri plates and glass tubes after addition of 50 µg/mL kanamycin

(Appendix III) for selection of transformants.

3.13.3. Agrobacterium culture preparation

Agrobacterium strain LBA4404 containing plasmid pCAMBIA F+HN were

inoculated in 20 ml of YEP broth (Appendix I) with 50 µg/ml kanamycin (Appendix III)

selection. Cultures were kept for overnight incubation at 28ºC with constant 10 min. The

supernatant was thrown away and pellets of both cultures were resuspended in 5 mL MS liquid

media.

46


3.13.4. Sterilization of embryos

Ears were dehusked and the surface of each cob was sterilized with sodium

hypochlorite with 0.1% Tween-20 for 15 min. After that, each cob was rinsed 4 to 5 times

with sterilized distilled water. Kernels were removed from the cob using a sterile scalpel and

blade and immature zygotic embryos were removed from kernels using a spatula. Isolated

embryos were washed with water mixed with 0.01% Tween-20 followed by five washes with

simple autoclaved water. The embryos were sterilized by immersed in 0.05 % HgCl2 with

continuous shaking for 20 min. After that, embryos were properly washed with autoclaved

water. Later on, embryos were placed on moist filter paper to prevent them from drying.

3.13.5. Agrobacterium-mediated transformation

Isolated embryos were transformed using CEMB’s modified shoot and cut method.

Isolated embryos were cut at specific points with a sharp blade under a light microscope. After

cutting, the embryos were transferred to an Agrobacterium suspension F+HN genes for

cocultivation. Almost 5000 embryos were used for transformation of F+HN genes. through

Agrobacterium. Suspension culture of F+HN genes were incubated in a shaker at 28ºC for 1

hour.

3.13.6. Shifting of embryos to MS plates

Embryo suspensions were transferred to empty Petri plates. The suspension was

discarded and embryos were kept on autoclaved filter paper. After that, embryos were shifted

to fresh MS plates supplemented with auxin (1mg/ml) and kinetin (1mg/ml). Embryos were

placed in a way that the scutellum was pointing upward. Plates were sealed with Parafilm and

kept in a growth room at 25ºC for cocultivation.

3.13.7. Selection of transformed plants

After 3-4 days, emerging plants were shifted to selection media containing 50 µg/ml

kanamycin (Appendix III). The media was also supplemented with B5 vitamin complex

(Appendix I) and different hormones, such as kinetin (1 mg/ml) and cytokinin (1 mg/ml), for

shooting and rooting. Plants were subculture 3-4 times on new selection media, and based on

their survival rate; their transformation efficiency was calculated 10 weeks after

transformation.

47


3.13.8. Transfer of plants to soil

A soil mixture composed of equal quantities of clay and sand was prepared and

autoclaved to transfer the transformed plants. Transformed plants were removed from glass

tubes using long forceps. After that, washed roots with autoclaved water were sprayed with

fungicides and moved to small pots containing autoclaved soil. After that, Plants with

polythene covers were kept in a culture room at 30ºC. After 2 to 3 days, plants were

acclimatized and their covers were removed after each one hour. After one week, the time

interval was increased, and three weeks later, when plants were able to survive without covers,

they were shifted to the greenhouse.

3.14. Molecular analysis

3.14.1. Isolation of genomic DNA

Extraction of genomic DNA was done from both transgenic and control plant samples

as well as corn seeds according to the protocol described by (Saha et al. 1997). Fresh leaves

from transgenic and non-transgenic maize were collected from the greenhouse in liquid

nitrogen. Leaves were finely ground to a powdered form in liquid nitrogen by using pestle

and mortar. Powder was scraped with a clean spatula and 1 mL of pre-heat extraction buffer

(Appendix IV) was added to 1 g of fresh leaves. Samples were undergone a short vortex and

centrifugation at 3000 x g for 15 min. After discarding the supernatant, the pellet was

resuspended in 700 µl of lysis buffer. Preceded by a vortex, an incubation at 65ºC for one

hour was given. After incubation, equal volumes of chloroform and isoamyl alcohol were

mixed by vortexing. Samples were centrifuged (13k rpm) for 15 min and the upper layer was

carefully shifted to a new 1.5ml tube. A volume of isopropanol equal to 0.6 volumes of

supernatant was added. Samples were placed at room temperature for half an hour.

Furthermore, centrifugation (13 k) was given for 15 min and supernatant was removed to get

the pellet. Later on, washing with 70% ethanol was done and pellet was dissolved in 200 µl

of double distilled water.50 µl of 3M sodium acetate and two volumes of 100% ethanol and

incubated on ice for one hour. Supernatant was discarded after centrifugation and the

recovered DNA pellet was dissolved in 30µL water after washing with 70% ethanol. The

resolved DNA was analyzed on a 0.8% gel under UV.

48


3.14.2. PCR Confirmation of Transgenic Plants

PCR was performed with gene specific detection primers using isolated DNA from

transgenic corn leaves and grains for F (~1662bp) and HN (~1712bp) genes detection. DNA

from non-transgenic plant was considered as negative control. The procedure was carried out

by same method as described in section 3.3. Resolved PCR products on 0.8% gel was

observed under UV to confirm the presence of F and HN genes.

Primer ID Sequence (5´- 3´) Annealing

temperature

Product

size

NDV IF-F 5´AAGCACAACCGAAGGATTTG3´ 59ºC 191bp

NDVIF-R 5´GCCGCTCAAACAGGAATAAA3´

NDVIHN-F 5´ATATCCCGCAGTCGCATAAC3´ 58ºC 172bp

NDVIHN-

R

5´TTAAACCCCACCATCCTTGA3´

3.14.3. Expression studies of transgenic plants

Total RNA contents were isolated from transgenic plants leaves and grains by

modified CTAB method (Hou et al. 2011). cDNA was synthesized from both transgenic corn

plants leaves and seeds by using a Revert Aid First Strand cDNA Synthesis Kit (Thermo

Scientific, K1622) as described in section 3.2. Real-time (RT) PCR were carried out in Piko

Real Real-Time PCR (Thermo scientific) with micro 96-well plate by using the Maxima

SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific, K0221). Glyceraldehyde 3-

phosphate dehydrogenase (GAPDH), housekeeping gene was used as comparative control to

normalize data. Two hundred ng of cDNA was used as template in RT- PCR. Below given

pairs of primers were used for comparative quantification through RT-PCR for both F and

HN gene.

49


Primers used in real time PCR

Primer ID Sequence (5´- 3´) Annealing temperature

Product

size

NDV IF-F 5ÁAGCACAACCGAAGGATTTG3´ 59ºC 191bp

NDVIF-R 5´GCCGCTCAAACAGGAATAAA3´

NDVIHN-F 5ÁTATCCCGCAGTCGCATAAC3´ 58ºC 172bp

NDVIHN-R 5´TTAAACCCCACCATCCTTGA3´

GAPDH-F 5ÁGGAAGAGCTGCTTCGTTCA3´ 56ºC 162bp

GAPDH-R 5ĆCGCCTTAATAGCAGCTTTG3´

RT-PCR was done by using the given cycling conditions: initial denaturation at 96˚C

for 4 min, followed by 35 cycles of amplification (96˚C for 30 seconds, 59˚C for 45 seconds,

and 72˚C for 45 seconds). The CT values of genes obtained from different transgenic plants

were statistically analyzed by using Piko Real software normalized with housekeeping

GAPDH relative control

3.14.4. Quantification of total soluble protein

Two hundred mg transgenic corn leaves and seeds were ground in liquid nitrogen for

quantification of total soluble protein. Total 400µl of freshly prepared ice-cold plant

extraction buffer (Appendix IV) was added in each tube containing transgenic material.

Samples were homogenized for 5 min by incubating the samples on ice to prevent degradation

of proteins by overheating. Homogenized samples were allowed to centrifuge (13000 g) at 4̊C

for 15 min. Supernatant from both leaves and seeds samples were collected. Total soluble

protein (TSP) extracted from both leaves and corn seeds was quantified by using bovine serum

albumin (BSA) Standards through Bradford assay as mention in section 3.7 (Bradford 1976).

3.14.5. Quantification of F and HN protein through Enzyme linked

immunosorbent assay (ELISA)

ELISA is highly sensitive and specific assay which is used to determine the

concentration of protein. The 100 µl crude protein extracted from transgenic corn plants

leaves and seeds were added to ELISA plate wells and were placed at 4 ̊C for 24 hours. Next

50


day the plate was washed with 100µl of PBST. After washing, the plate was blocked with

PBST having 3 % skim milk powder and incubated for two hours. After blocking the plate

was washed three times with PBST followed by d H2O to remove excess solution. Total 100

µl of gene specific Anti-Newcastle Disease virus antibody (100µg/ml; Abcam 34404) and

monoclonal HN antibody (200µg/ml: Santacruze) diluted in PBST were added on ELISA

plate at 4 ̊ C for 4 hours. Next day the plate was washed with 100µl of PBST. Later on, the

plate was kept at 37 ̊ C for two hours. After incubation, plate was washed with PBST and

100μl secondary antibody anti-chick IgY (2.5:10000) and anti-mouse IgG with (1:10000)

diluted in PBST was added in each well for F and HN gene detection respectively. After that,

the plate was incubated at 37 ̊ C followed by incubation in BCP/NBIT substrate for as

development of colour. At the end reaction was stopped by adding HCL. ELISA plate was

read on ELISA plate reader at 450nm with reference to different 0.05μg/ml, 0.1μg/ml,

2.5μg/ml, 5μg/ml standards of known concentration.

3.14.6. Calculation of F and HN percentage in total soluble protein

F and HN protein were calculated from total soluble protein extracted from transgenic

corn plant leaves and seeds with the help of following formula.

% of TSB= TP/TSPx100

TP= protein concentration obtained from ELISA in section 3.14.5, TSP = protein

concentration obtained from Bradford assay in section 3.14.4.

3.14.7. Western blot analysis for confirmation of F and HN gene

expression

Hundred µg of freshly extracted protein from both leaves and seed were analyzed on

12% SDS PAGE. Initially gel was run at 85 volts preceded by increased up to 110 volts after

transfer of proteins to resolving gel. After completion, the isolated proteins were shifted to

nitrocellulose membrane for purpose of proteins detection through western blot analysis

according to same protocol as described in section 3.10. The specific F protein on blot was

detected through specific Anti-Newcastle Disease virus primary antibody (100 µg/ml)

(Abcam 34404) and anti-chick IgY secondary antibody (200µg/ml). The HN protein on blot

was detected through monoclonal HN primary antibody (100µg/ml) and anti-mouse IgG

secondary antibody.

51


3.14.8. In vitro chicks feeding assay for immune response

The plant with higher expression for both F and HN genes was multiply to study the

immune response in chickens. Broiler Chickens aged upto 15 days were purchased from

hatcheries and reared in animal house of CEMB (Center of Excellence in Molecular Biology)

University of Punjab Lahore. Chickens were categorized into three groups with 5 chickens in

each group. Group 1 was a control group in which chickens were fed with non-transgenic

maize. Group 2 chicken were fed with transgenic corn seeds with 200ug of F and HN protein

once in a week. Group 3 chickens were fed with transgenic corn and each chicken in this

group was boosted with complete and incomplete adjuvant. All groups of chickens were feed

ad libitum. Blood was collected after every ten days of feeding and isolated serum was

checked for the presence of NDV specific IgG up to 60 days. The blood was incubated at 25 ͦ

C for 45 minutes, centrifuged (5000xg) at 4 ͦ C for 15 minutes. The collected sera at different

times were preserved at -20 ͦ C and specific immune response was determined through specific

immunoglobulin based ELISA by some modification of protocol as described by (Lai et al.

2013) by coating plate wells with purified F and HN protein obtained after affinity

chromatography. The commercial HRP conjugated goat to anti-chick IgY (ab 6877) was used

as secondary antibody and specific absorbance values were recorded at 450nm wavelength to

find out IgG level in collected serum.

52


: RESULTS

4.1. F and HN genes confirmation through PCR

Amplification of F (~1662bp) and HN (~1712bp) gene fragments were obtained by

using NDV cDNA as a template. Gene-specific primers amplified required fragments for both

F and HN genes. Amplified ~1662bp fragment by PCR analysis confirmed the presence of F

gene (Figure 4.1A). Similarly, amplification of ~1712bp PCR product confirmed the presence

of HN gene (Figure 4.1 B). A bright sharp fragment was obtained with out any impurity for

both gene fragments.

Figure 4-1: Confirmation of F/HN genes from NDV cDNA through PCR amplification

(A): Amplification of F gene (B) Amplification of HN gene

Lane 1: 1-kb DNA ladder Lane 1: 1-kb DNA ladder

Lanes 2-7: Amplified F gene Lane 2: Positive control (commercial vaccine)

Lane 8: Positive control (vaccine) Lanes 3-11: Amplified HN gene

Lane 9: Negative control Lane 12: Negative control

(Negative control: Amplification without template DNA)

53


4.2. Confirmation of TA construct harboring F gene

Amplified PCR product for F gene was ligated into the Invitrogen TA vector

(pCR®2.1). A recombinant TA vector (TA-F) for F gene was confirmed through PCR and

restriction digestion. Amplification and appearance of digested ~1662bp F gene fragment with

EcoR1 from TA-F construct confirmed the successful ligation of F gene in TA vector (Figure

4.2A&B).

Figure 4-2: F gene confirmation of TA-F construct through PCR and restriction

digestion.

(A) Amplification of F gene from TA-F (B) Restriction digestion TA-F plasmid

Lane 1: Amplified TA-F as Positive control Lane 1: Amplified TA-F as Positive control

Lanes 2-14: Transformed clones Lane 2: 1-kb DNA Ladder

Lane 15: 1-kb DNA Ladder Lanes 3-5: Restriction digestion of TA-F

Lane 16: Negative control (Amplified product without template)

54


4.3. Confirmation of TA vector harboring HN gene

Amplified PCR product for HN gene was ligated into the Invitrogen TA vector

(pCR®2.1). A recombinant TA vector (TA-HN) for HN gene was confirmed through PCR and

restriction digestion. Amplification and appearance of digested ~1712bp HN gene fragment

with EcoR1 form TA-HN construct confirmed the successful ligation of HN gene in TA vector

whereas Lane 2 and 5 in Fig 4.2B were rejected on the basis of undigested plasmids and

samples with non-specific bands were rejected (Figure 4.2A&B).

Figure 4-3: HN gene confirmation of TA-HN construct through PCR and restriction

digestion.

(A)Amplification of HN from TA-HN (B) Restriction digestion of TA-HN

Lane 1-5: Transformed clones Lane: 1-kb DNA ladder

Lane 6: 1-kb ladder Lanes 2-7: Restriction digestion of TA-HN

Lane7: Negative control (Amplification Lane 8: Amplified TA-HN as Positive

Without template) control

55


4.4. Evaluation of F/HN genes through sequencing

Recombinant TA clones harboring F and HN gene were sequenced through M13

primers. Gene sequencing was carried out using Sanger’s sequencing method on an ABI 3730

Sequence reader (Applied Biosystems). Sequencing confirmed that the ~1662bp F gene

fragment (Figure 4.4) and the ~1712bp HN gene fragment (Figure 4.5) was 100% identical to

those of a Newcastle disease virus isolate (Accession # GU182327). The sequences results

were analyzed using sequencing scanner software 2.

>NDV1_M13F sequence exported from NDV1_M13F.ab1

CGGATTTCCACTTTACCAGTGCCATTGGAAAGATCCCGCAATTTTTAATGCCCAGTTAAATACACAGTCCAAGAGTTGGACT

GATAAAAATTCCACACCAGGTTGGTGTAGAACTCAACTTATACTTAACTGAATTGACTACAGTGTTCGGGCCACAAATCAT

TTCCCCTGCCTTAACTCAGTTGACTGTTCAGGCTCTTTACAATCTGGCTGGTGGTAATGTAGATTACTTGTTGACTAAGTTAG

GTGTAGGGAACAACCAGCTCAGCTCATTGATTGGTAGCGGCTTGATCACCGGTAACCCTATTTTTTACGACTCACAGACTCA

ACTCTTGGGTATACAGGTGACTTTACCCTCAGTCGGGAACCTAAATAATATGCGTGCCACCTACTTGGAGACCTTGTCTGTA

AGCACAACGAAGGGATTTGCCTCAGCACTTGTCCCAAAAGTAGTGACACAGGTCGGTTCTGTGATAGAAGAGCTTGACACC

TCATACTGTATAGAAGCTGATTTGGATTTATATTGTACAAGAATAGTGACATTCCCTATGTCCCCTGGTATTTATTCCTGTTT

GAGCGGCAATACATCGGCTTGCATGTATTCAAAGACTGAAGGCGCACTTACTACACCATACATGACTCTCAAAGGCTCAGT

TGTTGCCAATTGCCAGATGACAACATGTAGATGTGCAGACCCCCCGGGTATCATATCACAAAATTATGGAGAGGCTGTGTC

TCTAATAGATAAGCACTCATGCAATGTCGTATCCTTAGACGGGATAACTTTGAGGCTCAGTGGGGAATTTGATGCAACCTA

TCAAAAGAATATCTCAATATTAGATTCTCAAGTACTAGTGACAGGCAATCTCGATATCTCAACTGAGCTTGGGAATGTCAA

CCACTCAATAAGTAATGCTTTGGATAAGTTAGAGGAAAGCAACAGCAAACTAGACAAAGTCAATGTCAAACTGACCAGCA

CATCTGCTCTCATTACCTATATTGTTTTAACTGTCATATCTCTTGTTCTTGGTATGCTTAGCCTGGTTCTAGCATGCTATCTGA

TGTACAAGCAAAAAGCGCAACGAAAGACCTTGTTGTGGCTTGGAAATAATACCCTAGATCAGATGAGAGCTGTTAAGCCG

AATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTTCGCCCCTATATATTCATCCC

Figure 4-4: Sequence analysis of F gene through ABI sequence scanner

56


>30-Naila-R sequence exported from 30-Naila-R-3130xl-2015-05-13.ab1

TACTAGTAACGGGCATTCTGATTCCCCGAGATGGTATTGGATATTTCTGCATGCTGAGGCATAGGTTTTATTGGTCTTGACA

ACTTTAAAAACACGTTGATGTCGTGTATGCTGCCCTGGTACTGCTTGAACTTACCCGAGTTATGCGACTGCGGGATATGTTA

TCAAATACTGCAGATACAGGGTTGAGTCTTGCTTGTTCGTTATCAAGCATTGTTCCGAATACCCCTCGCAAGGTGTGGTTCC

TATGGAAGACTAAGGGATATGGATCAGTATAGACTCCGGTAACACATGAGTTGGGGCATCTTGCTGAAGCCTGGCAAGGG

ACACTACCTGGCCGAGTGAAAGCATTGAATGTATAAGGATTATGAAGAGTGGCTGTTTTGTTGTTGACAGTCATAGGGTAT

AACAAAGCGGGAGAGAAGTATGATGACCCTCGCTGATATAAGAAATGAGATGTCCCCACTGTGAGAACTCTGCCTTCGGCC

CCATAAGTGCGACCGTATTAGCGGTACAGTCAACACCGGGTCCTCACCCAAGGATGTTGACACTTGATGGATAGGATGGCT

GCTGTACGCGTTTCCACCAAACCGCCAGGCTTATACGATGACTTAGCATCGATCTGGTATCTTGCTCATCGGCATGTGTCAT

TGTTCGCTTGTATTATACGTATCTCCTTCTTGTGCATGTCACAAGCAGCTGGATTAGCCCCGTAACGGAACATACCCGTGTT

AATAAAAACACCTCTCTATCTCTGGTATTGCACCAGTCTTATATATTGGCTTGGACGCTTAAGCCTTATGTTGGCGCTACCTT

ACTCCATAACACCAATTGGGATACAGATAACCTTCCCACGCTCTGTTAGAGCCACGCAAATCACACTAAGAAGTGCTCATA

CTCGAACTCCGATTGGCTCTCATTGAACACGAGATTAGGAATTCCTTTGTGATGCGCACAAGCACTTGGGTGGATTGCCAAC

CTG

Figure 4-5: Sequence analysis of HN gene through ABI sequence scanner

57


4.5. Confirmation of F gene in prokaryotic expression vector

Determination of functional protein is very important while dealing with edible

vaccine therefore, availability of pure F protein was achieved through expression in pET30a

vector with a His tag. F gene was ligated in pET30a vector to obtain recombinant pET-F

clones. The successful ligation of F gene in the pET30a was confirmed through PCR and

restriction digestion with EcoR1. Amplified PCR product and released ~1662bp fragment for

F gene confirmed successful ligation of F gene in pET30 a vector (Figure 4.6 A&B).

Figure 4-6: F Gene Confirmation in pET-F through Restriction Digestion and PCR

(A): Restriction digestion of pET-F (B): Amplification of F gene from pET-F

Lane 1: Negative control (undigested Lane 1: Positive control

Plasmid)

Lane 2: Amplified TA-F Positive control Lane 2:1-kb DNA ladder

Lane 3: 1-kb DNA ladder Lanes 3-4: Transformed clones

Lanes 4-12: Restriction digestion pET-F Lane 5: Negative control (Amplification

Clones without template

58


4.6. Confirmation of HN gene in prokaryotic expression vector

Similarly, HN gene was ligated in pET30a vector to obtain recombinant pET-HN

clones. The successful ligation of HN gene in the pET30a was confirmed through PCR and

restriction digestion with EcoR1. Amplified PCR product and released ~1712bp fragment for

HN gene confirmed successful ligation of HN gene in pET30a vector. (Figure 4.7 A&B).

Figure 4-7: HN gene confirmation in pET30a through restriction digestion and PCR

(A) . Restriction digestion of pET-HN (B) Amplification of HN from pET-HN

Lane1: Negative control (undigested Lane 1: Negative control (amplification

plasmid) without template

Lane 2: 1-kb ladder Lanes 2: 1kb ladder

Lane 3&4: Restriction digestion pET-HN Lanes 3-5: Transformed clones

clones

59


4.7. Confirmation of pET-F and pET HN in expression host

Recombinant clones pET-F and pET-HN genes were transformed separately into E.

coli expression host DE3 (BL21) cells through heat shock method. F and HN genes in DE3

(BL21) cells were confirmed through PCR with gene-specific primers. The PCR analysis

confirmed the presence of the F gene (~1662bp) and the HN gene (~1712bp) in the DE3 (BL-

21) expression host (Figure 4.8).

Figure 4-8: pET-F and pET HN confirmation in DE3 (BL-21) cells through PCR

Lane 1: Positive control for HN gene

Lanes 2-5: PCR amplification of pET-HN gene in DE3 (BL-21) cells

Lane 6: 1-kb DNA ladder

Lane 7: Negative control (amplification without template)

Lane 8: Positive control for F gene

Lanes 9-12: PCR amplification of pET- F gene in DE3 (BL-21) cells

Lane 13: 1-kb DNA ladder

Lane 14: Negative control

60


4.8. Expression analysis of F and HN by SDS-PAGE

F and HN protein expression was determined both in the soluble and inclusion bodies

fractions upon induction with 1mM Isopropyl Thio--D-Galactoside (IPTG). The obtained

fractions were analyzed on SDS-PAGE. Majority of the obtained fractions in soluble form

revealed a sharp band of 67kDa (F protein) and 69kDa (HN protein) that were clearly visible

on a 10% SDS PAGE with Coomassie blue staining. Expressed proteins were fusions of the

desired protein and a His tag (Figure 4.9).

Figure 4-9: Confirmation of F/HN Protein expression through SDS-PAGE

(A) SDS PAGE of crude F protein sample

Lane 1: Negative control (Untransformed Rosetta cells)

Lanes 2&3: Transformed Rosetta cells with F protein gene

Lane 4: Pre-stained protein marker (Fermentas)

(B) SDS PAGE of crude HN protein sample

Lanes 1&2: Negative control (Untransformed Rosetta cells)


Lanes 4&5: Transformed Rosetta with HN protein gene

61


4.9. Purification of F and HN proteins by IMAC

Immobilized metal affinity chromatography (IMAC) allows the purification of a

specific protein based on the function of metals (e.g., Ni, Co, Cu, and Fe). Metal ions act as

ligands for binding and purification of the target protein. The bound F and HN proteins to

IMAC resins at low imidazole concentrations (10-25 mM) were eluted from the IMAC resin

at higher imidazole concentrations. After Coomassie blue staining of SDS-PAGE, eluted

proteins were confirmed on it. At 125mM imidazole, a single 67-kDa band of F protein was

observed, whereas at 100mM imidazole, a single 69-kDa band of HN protein was detected

(Figure 4.10 A&B). Concentrations of purified proteins were quantified with reference to

known concentration of BSA (standard). The protein concentration of F gene and HN genes

were 32ng/μl and 34.1 ng/μl respectively (Bradford 1976).

Figure 4-10: SDS-PAGE analysis of eluted F and HN protein fractions after purification

through IMAC fast flow affinity chromatography

(A) F protein fractions at different imidazole concentrations

Lane 1: Wash solution

Lane 2: Pre-stained protein marker.

Lanes3-6: Fractions obtained at 100mM, 125mM, 150mM and 200mM imidazole.

(B) HN protein fractions at different imidazole concentrations

Lane 1: Pre-stained protein marker.

Lanes 2-4: Fractions obtained at 100mM imidazole

62


4.10. Confirmation of His-Tagged F/HN protein by western blotting

Identification of His-tagged F/HN protein was done through by western blot analysis

on nitrocellulose membranes (HyBond C, Amersham) by using His-specific primary

antibodies and AP-conjugated secondary antibodies. Colour development with BCIP/NBT

substrate confirmed the E. coli expression of 67kDA F protein and 69kDa HN protein (Figure

4.11).

Figure 4-11: Confirmation of His-tagged F/HN proteins through western blotting

(A) Western Blot for F protein

Lanes 1&2: F-transformed in Rosetta cells



(B) Western Blot for HN protein


Lanes 2-5: HN-Transformed in Rosetta cells.

Lane 6: Pre-stained Protein marker (Fermentas)

63


4.11. Structure Prediction of F and HN Proteins by homology modeling

using SWISS-MODEL

SWISS-MODEL predicted three models for F and HN proteins based on the principle

of structure homology modeling. The chosen model for the F protein had a maximum amino

acid sequence coverage of over 95% and a high GMQE score of 0.66. Normalized QMEAN

score as well as Z score for the predicted protein model with non-redundant PDB structures according

to protein size clearly confirmed that the QMEAN score is greater than 0.5 and that the model is stable.

Its three-dimensional Swiss model captured at different angles using Pymol showed the head,

neck and stalk regions of the fusion protein oriented at 90° angles in a spherical mesh

conformation. The structure shows the three polypeptide chains highlighted with different

colours. The axial channel runs in the center from the head region to the neck region (Figure

4.12).

Figure 4-12: Three-dimensional Swiss model of the fusion protein and its QMEAN

score.

The chosen model for the HN protein had a maximum amino acid sequence coverage

of over 95% and a GMQE score of 0.77, which was higher than that of the F protein. The

QMEAN score is greater than 0.5 and the model has a stable conformation. A three-

dimensional Swiss model of the HN protein shows its homo-tetramer structure containing 4

polypeptide chains in ribbon conformation (Figure 4.13).

64


Figure 4-13: Three-dimensional Swiss model of the HN protein and its QMEAN score

65


4.12. Prediction of antigenic epitope by Antibody epitope prediction and

ElliPro

Determination of the B cell linear epitopes of F and HN query proteins by the Kolaskar

and Tongaonkar Antigenicity algorithm predicted 24 antigenic peptides in the F protein. The

smallest peptide contains 6 residues, whereas the largest peptide consists of 46 residues (Table

4.1). Similarly, 24 linear antigenic peptides were predicted in HN, with the smallest peptide

containing 7 residues and the largest peptide containing 32 residues (Table 4.2). These regions

are expected to function as epitopes to induce B cell response.

66


Table 4-1: Peptide length, sequence, start and end positions of predicted linear epitopes

of the F protein

No. Start End Peptide Sequence Length

1 8 31 RIPVPLMLTIRITLALSYVRLTSS 24

2 34 47 GRPLAAAGIVVTRD 14

3 49 55 AVNIYTS 7

4 59 68 GSIIVKLLPN 10

5 73 83 KEACAKAPLEA 11

6 89 97 TTLLTPLGD 9

7 118 143 IGAIIGSVALGVATAAQITAASASIQ 26

8 151 157 ILRLKES 7

9 165 184 VHEVTGGLSQLAVAVGKMQQ 20

10 196 241 ELDCIKITQQVGVELNLYLTELTTVFGPQITSPALTQLTVQALYNL 46

11 243 253 GGNVDYLLTKL 11

12 260 267 LSSLIGSG 8

13 275 293 FYDSQTQLLGIQVTLPSVG 19

14 302 309 YLETLSVS 8

15 315 332 ASALVPKVVTQVGSVIEE 18

16 335 354 TSYCIEADLDLYCTRIVTFP 20

17 356 365 SPGIYSCLSG 10

18 380 385 TTPYMT 6

19 387 394 KGSVVANC 8

20 396 410 MTTCRCADPPGIISQ 15

21 413 437 GEAVSLIDKHSCNVVSLDGITLRLS 25

22 447 460 NISILDSQVLVTGN 14

23 488 528 LDKVNVKLTSTSALITYIVLTVISLVLGMLSLVLACYLMYK 41

24 534 539 KTLLWL 6

67


Table 4-2: Peptide length, sequence, start and end positions of predicted linear

epitopes of the HN protein

No. Start End Peptide Length

1 4 11 AVSQVALE 8

2 21 47 WRLVFRIATLLLMVITLAVSAVALAYS 27

3 53 65 PGDLVSIPTAIYR 13

4 77 100 NQDVVDRIYKQVALESPLALLNTE 24

5 108 115 TSLSYQIN 8

6 123 131 CGAPVHDPD 9

7 138 145 KELIVDDT 8

8 148 166 VTSFYPSAFQEHLNFIPAP 19

9 183 213 THYCYTHNVILSGCRDHSHSHQYLALGVLRT 31

10 216 224 TGRVFFSTL 9

11 235 254 RKSCSVSATPLGCDMLCSKI 20

12 262 270 YKSVIPTSM 9

13 283 291 EKDLDVTTL 9

14 296 302 VANYPGV 7

15 313 323 WFPVYGGLKPS 11

16 332 338 GRYVIYK 7

17 369 381 VQQAILSIKVSTS 13

18 384 398 EDPVLTVPPNTVALT 15

19 402 429 GRVLTVGTSHFLYQRGSSYFSPALLYPM 28

20 450 481 PGSVPCQASARCPNSCVTGVYTDPYPLVFHRN 32

21 499 508 LCNPVSAVFDN 10

22 514 520 ITRVSSS 7

23 527 547 TTSTCFKVVKTNKTYCLSIAE 21

24 555 566 EFRIVPLLVEIL 12

68


The frequency of occurrence of each residue in the predicted epitopes of F and HN

proteins was graphically plotted based on their individual score (Y-axis) and residue position

(X-axis). With a window size of 7, the average calculated score for F protein epitopes is 1.046,

with 1.223 as the maximum score and 0.902 as the minimum score (Figure 4.14). For HN, the

average calculated score is 1.039 with 1.194 as the maximum score and 0.870 as the minimum

score (Figure 4.15). These residues are more likely to be part of the 24 predicted epitopes

shown in Tables 4.1 and 4.2.

Figure 4-14: Graphical representation of the occurrence frequency of F protein

residues based on individual score (Y-axis) and residue position (X-axis)

The yellow highlighted portion indicates residues with greater scores.

69


Figure 4-15: Graphical representation of the occurrence frequency of HN protein



Based on 3D protein antigen structure, 9 linear epitopes of the F protein and 17 linear

epitopes of the HN protein were predicted using ElliPro, as listed in Table 4.3 and Table 4.4.

By calculating the residue’s center of mass and solvent accessibility, ElliPro assigned a

protrusion index value (PI) to these residues. JSmol-rendered 3D structures of continuous

antigenic epitopes of F or HN proteins were built along with their PI values (Figure 4.16 &

Figure 4.17). The smaller the PI value, the greater is its ability to be a stable epitope. For the

F protein, the lowest PI value is 0.542, which indicates that 54% of residues are inside and

46% are outside the globular surface to make antigen-antibody interactions. The lowest PI

value of the HN protein is 0.514, indicating higher interactive accessibility as compared to

the F protein.

70


Table 4-3: Peptide length, sequence, start and end positions, and PI score of predicted

linear epitopes of the F protein.

No. Start End Peptide Number of

residues

Score

1 459 493 GNLDISTELGNVNNSISNALNKLEESNSKLDKVNV 35 0.849

2 135 181 ITAASALIQANQNAANILRLKESIAATNEAVHEVTDGLSQIAVAVGK 47 0.781

3 93 125 TPLGDSIRRKQESVTTSGGRRQRRFIGAIIGSV 33 0.778

4 390 441 VIANCKMTTCRCADPPGIISQNYGEAVSLIDRHSCNVLSLDGITLRLSG

EFD

52 0.753

5 306 316 LSVSTTKGFAS 11 0.702

6 364 383 SGNTSACMYSKTEGALTTPY 20 0.658

7 274 281 ILYDSQTQ 8 0.6

8 34 41 GRPLAAAG 8 0.544

9 326 330 VGSVI 5 0.542

71


Table 4-4: Peptide length, sequence, start and end positions and PI score of predicted

linear epitopes of the HN protein.

No. Chain Start End Peptide Number

of

residues

Score

1 A 564 571 EILKDGGV 8 0.824

2 A 321 357 KPSSPSDTAQEGRYVIYKRYNDTCPDEQDYQIRMAKS 37 0.813

3 A 533 539 KVVKTNK 7 0.799

4 A 164 172 PAPTTGSGC 9 0.748

5 A 124 135 GAPVHDPDYIGG 12 0.734

6 A 377 386 KVSTSLGEDP 10 0.733

7 A 549 557 SNTLFGEFR 9 0.718

8 A 212 234 RTSATGRVFFSTLRSINLDDAQN 23 0.706

9 A 365 368 GGKR 4 0.683

10 A 276 296 GFDGQYHEKDLDVTTLFRDWV 21 0.68

11 A 140 161 LIVDDTSDVTSFYPSAFQEHLN 22 0.675

12 A 478 485 FHRNHTLR 8 0.662

13 A 254 268 ITETEEEDYKSVIPT 15 0.65

14 A 192 203 ILSGCRDHSHSH 12 0.636

15 A 508 524 NISRSRITRVSSSSTRA 17 0.619

16 A 451 465 GSVPCQASARCPNSC 15 0.588

17 A 493 500 DDKQARLN 8 0.514

72


Figure 4-16: JSmol-rendered 3D structures of continuous antigenic epitopes of the F

protein along with their PI values, as predicted by ElliPro

73


Figure 4-17: JSmol-rendered 3D structures of continuous antigenic epitopes of the HN

protein along with their PI values, as predicted by ElliPro

Figure 4-18: Graphical representation of the occurrence frequency for F protein

residues on the basis of individual score (Y-axis) and residue position (X-axis)


Figure 4-19: Graphical representation of the occurrence frequency for HN protein



74


4.13. Confirmation F and HN in plant expression vector

4.13.1 F gene confirmation in pCAMBIA vector

After finding F and HN gene expression in pET vectors, the next major concern of

present study was to construct pCAMBIA 1301 with both F and HN for plant transformation.

For this purpose, the F protein gene was cloned into pCAMBIA 1301 at Nco1 site under

CaMV35s promoter to obtained pCAMBIA-F construct. (Figure 4.20). A restriction digestion

reaction was carried out with Nco1 to release the F gene fragment from the pCAMBIA-F. Out

of fifteen clones, six were positive for the F gene (Figure 4.21).These were further analyzed

for correct orientation because they were the result of a single enzyme ligation into the

pCAMBIA vector.

Figure 4-20: Digestion of pCAMBIA and TA-F construct with Nco1

Lanes 1&2: pCAMBIA vector digested with Nco1

Lane 3: 1-kb Ladder

Lanes 4-6: TA-F construct digested with Nco1

75


Figure 4-21: Confirmation of F gene integration into pCAMBIA-F

Lanes1: 1-kb Ladder

Lanes 2-16: Restriction digestion of pCAMBIA-F clones to verify ligation

4.13.2. Confirmation of correct orientation of F gene into pCAMBIA-F

The accurate F gene orientation in pCAMBIA-F was confirmed through restriction

digestion analysis. The accurate orientation was confirmed by released fragment of ~1588bp

upon restriction digestion. Out of six positive clones, four showed the correct orientation of

the F gene (Figure 4. 22).

Figure 4-22: Confirmation of F gene orientation in the pCAMBIA Vector

Lane 1: 1-kb ladder

Lanes 3, 4, 5, 7: Confirmation of positive orientation

Lanes 2&6: Negative orientation of F gene

76


4.13.3. HN gene confirmation under seed specific promoter and

terminator

The HN gene with the zein promoter (seed specific promoter) and NOS terminator

was cloned in pCAMBIA-F to obtain final pCAMBIA-F+HN construct that was confirmed

through restriction digestion. Restriction digestion with Sal1 and Xba1 released a ~1425bp

fragment from pCAMBIA-F, confirmed the prescence of the seed specific promoter in

pCAMBIA-F. Five clones were selected, and all were positive for the zein promoter (Figure

4.23). The presence of HN gene in pCAMBIA-F carring promoter was confirmed by digestion

reaction with Xba1 and BamH1, which released the ~1712bp HN gene fragment from the

vector. Out of twelve clones, four were positive for the HN gene (Fiure 4.24). The presense

of NOS terminator in pCAMBIA-F carring Promoter and HN gene was confirmed by

digestion reaction with BamH1 and Kpn1. Restriction digestion released a ~218bp NOS

terminator fragment from pCAMBIA-F. Eight clones were selected, and five were positive

for the NOS terminator (Figure 4.25)

77


Figure 4-23: Confirmation of the Zein promoter in pCAMBIA -F

Lanes 1-5: Restriction digestion of pCAMBIA-F to confirm zain ligation

Lane 6: 1-kb ladder

Figure 4-24: Confirmation of the HN Gene in pCAMBIA -F

Lanes 1-10: Restriction digestion of pCAMBIA-F to confirm HN ligation

Lane 11: 1-kb ladder

Figure 4-25: Confirmation of the NOS Terminator in pCAMBIA-F

Lanes 1-8: Restriction digestion of pCAMBIA-F to confirm Nos terminator ligation

Lane 9: 1-kb ladder

78


4.13.4. Confirmation of Cassette (Promoter + HN + NOS) in pCAMBIA-F

+ HN

The whole cassete (promoter + HN + NOS) was confirmed through restriction

digestion. Digestion of final pCAMBIA-F + HN construct was carried out to excise the 3.3-

kb fragment. Because the promoter was cloned into the Sal1 site and the NOS terminator was

cloned into the Kpn1 site, Sal1 and Kpn1 were used to digest out the cloned cassette (Promoter

+ HN+ NOS) from the vector. The released 3.3-kb fragment confirmed the presence of the

cassette in pCAMBIA-F and HN construct (Figure 4.26).

Figure 4-26: Confirmation of Cassette (Zein + HN + NOS) in pCAMBIA-F + HN

construct

Lane 1: 1-kb Ladder

Lanes 2-9: Restriction digestion of selected pCAMBIA-F+HN clones to confirm cassette

79


4.14. Electroporation and confirmation of F and HN genes in

Agrobacterium

After confirming F and HN gene, recombinant pCAMBIA-F+HN plasmid was

electroporated into competent Agrobacterium cells (LBA4404). After transformation, four

colonies were selected and confirmed through PCR for detection of F and HN gene. Amplified

PCR products of ~1662bp and ~1712bp with gene-specific primers confirmed the presence

of F or HN genes in Agrobacterium (Figure 4.27).

Figure 4-27: Confirmation of F or HN in Agrobacterium

Lane 1: Negative control for F gene

Lane 2-5: Positive clone for F gene

Lane 6: Positive control for F gene

Lane 7: 1kb ladder

Lane 8: Negative control for HN

Lane 9-12: Positive clone for HN gene

Lane 13: Positive control for HN gene

80


4.15. Transformation of Maize

Agrobacterium mediated nuclear transformation was used to transform inbred lines of

maize. Almost 5000 embryos from these lines were transformed with F and HN gene by

Agrobacterium mediated nuclear transformation. Out of 5000, 1200 plants were regenerated

on MS media after 5-6 weeks of 50mg/ml kanamycin selection (Appendix III). Out of total

1200, 64 plants were shifted in pots. Only 10 plants namely as CNP1, CNP2, CNP3, CNP4,

CNP5, CNP6, CNP7, CNP8, CNP9, and CNP10 were able to survive in pots and shifted to

greenhouse (Figure 4.28).

Figure 4-28: A schematic diagram showing transformation methodology

Figure 4-29: Transformation efficiency of maize

81


4.16. Molecular analysis

4.16.1. PCR Confirmation of Transgenic Plants

The presence of F and HN genes in putative transgenic Maize (Zea mays) plants

namely CNP1, CNP2, CNP3, CNP4, CNP5, CNP6, CNP7, CNP8, CNP9, CNP10 were

confirmed through Polymerase Chain reaction. Short detection primer for amplification of

~184bp fragment for F gene and ~191 for HN gene were used. Amplification of ~184bp of F

gene was obtained in seven out of ten survived plants, namely CNP1, CNP3, CNP4, CNP5,

CNP6, CNP8, CNP9 and CNP10. CNP2 and CNP7 were not amplified for F gene. Out of ten

plants, six plants namely CNP2, CNP3, CNP6, CNP8, CNP9 and CNP10 were matured to

develop maize cob. The seeds from these transgenic cobs were analyzed for the presence of

HN gene. A short fragment of ~194bp was detected in CNP3, CNP6, CNP8, CNP9, and

CNP10, which were positive for the HN gene while CNP2 were not amplified for HN gene.

It was found that out of ten plants namely CNP1, CNP2, CNP3, CNP4, CNP5, CNP6, CNP7,

CNP8, CNP9, CNP10 five plants namely CNP3, CNP6, CNP8, CNP9, and CNP10 were PCR

positive for both F and HN gene (Figure: 4.30).

82


Figure: 4-30. Confirmation of F and HN gene in transgenic plants

(A) F gene confirmation in transgenic (B) HN gene confirmation in transgenic corn

leaves seeds

Lanes1: Positive Control Lane 1: Positive control

Lane 2-11: CNP1- CNP10 Lane2:Negative control (Non-transgenic

Lane12: Negative Control plant)

(Non-transgenic Plant) Lane 3: 50bp ladder

Lane 13: 50bp ladder Lanes 5-10: CNP2, CNP3, CNP6, CNP8,

CNP9,CNP10

83


4.16.2. Quantitative studies of F and HN gene in transgenic maize through

real time PCR

Quantitative real time PCR is the best technique to study mRNA expression of

transformed genes. mRNA transcripts of both F and HN genes were reverse transcrbied into

cDNA. cDNA of eight plants namely CNP1, CNP3, CNP4, CNP5, CNP6, CNP8, CNP9,

CNP10 were used for quantification of F gene. CNP1 and CNP3 showed 4 fold CNP4, CNP6

and CNP9 showed five fold while CNP5 and CNP8 showed seven fold increase in expression

of F gene (Figure 4.31A). Similarly, five plants namely CNP3, CNP6, CNP8, CNP9, CNP10

were analysed for qunantifcation of HN gene as seeds were obtained from these plants only.

CNP6 and CNP9 showed almost three to four fold , CNP8 and CNP10 are the plants showing

almost 4 to 5 fold and CNP5 and CNP8 showed five fold increase in expression of HN genes

in seeds ( 4.31B) . While it is clear from the figure 4.31A and 4.31B that CNP8 plant is the

best plant showing maximum expression of both F and HN gene.

84


Figure 4-31: Expression of F and HN gene through Real time PCR

(A) Relative expression of F gene (B) Relative expression of HN gene

Lanes1: Negative control Lane 1: Negative control

Lane 2-9: CNP1, CNP3, CNP4, CNP5, Lane 2-5: CNP3, CNP6, CNP8, CNP9, CNP6,

CNP8, CNP9, CNP10 ` CNP10

85


4.16.3. Quantification of total soluble Protein

A total crude protein from leaves and seeds of transgenic plants namely CNP1, CNP3,

CNP4, CNP5, CNP6, CNP8, CNP9 and CNP10 was extracted. BSA standards were used and

standard curve was plotted to quantify total soluble protein as mention in section 3.7.1. and

4.7 (Bradford 1976) (Figure 4.32). The estimated crude protein was measured in mg/ml as

mention in table 4.5.

Figure 4-32: Standard curve for quantification of crude proteins

86


Table 4-5: Quantification of Crude proteins

4.16.5. Quantification of F and HN protein through ELISA

The F and HN proteins of transgenic maize plants were also quantified through

ELISA. F and HN proteins were quantified from crude protein isolated from transgenic corn

leaves and seeds respectively. Total extracted protein from transgenic leaves and seeds

namely CNP1, CNP3, CNP4, CNP5, CNP6, CNP8, CNP9, CNP10 were coated and treated

with specific antibodies on ELISA plate for quantification of F and HN protein. It was found

that that plants namely CNP5 and CNP8 and showed the maximum concentration of F gene

almost 0.15μg/ml and 0.166μg./ml respectively (Figure: 4.33A). Similarly the plant namely

CNP8 and CNP9 showed the maximum concentration of HN protein 0.24μg/ml and 0.195

μg/ml respectively. (Figure: 4.33B).

Plant

Name

Optical density/ Protein

Concentration from leaves

(mg/ml)

Optical density/ Protein

Concentration from seeds

(mg/ml)

CNP1 0.301/0.25 didn’t produce cob

CNP3 0.300/0.25 0.20/0.169

CNP4 0.317/0.27 didn’t produce cob

CNP5 0.314/0.27/ didn’t produce cob

CNP-6 0.290/0.25 0.233/.198

CNP-8 0.319/0.271 0.100/0.08

CNP-9 0.260/0.22 0.18/.212

CNP-10 0.26/0.22 0.247/0.291

87


Figure 4-33: Quantification of F and HN gene through ELISA

(A). Quantification of F protein (B) Quantification of HN gene

Lanes1: Negative Control Lane 1: Negative Control

Lane 2-9: CNP1, CNP3, CNP4, CNP5, Lane 2-6: CNP3, CNP6, CNP8, CNP9,

CNP6, CNP8, CNP9, CNP10 CNP10

88


4.16.7. Calculation of F and HN protein percentage in total soluble protein

F and HN protein concentration was calculated in total soluble protein with formula

mention in section 3.14.6. The concentration of F and HN protein was 0.1 to 0.6 % of TSP. It

was found that plant CNP8 was the best plant showing highest concentration of F and HN

protein among all other plants (Table 4.6).

Table 4-6: F and HN percentage in TSP

Plant

Name

Percentage of F protein

in TSP

Percentage of HN protein in

TSP

CNP1 0.02% of TSP Cob were not matured

CNP3 0.02% of TSP 0.04% of TSP



CNP-6 0.06% of TSP 0.1% of TSP

CNP-8 0.19% of TSP 0.24% of TSP

CNP-9 0.01% of TSP 0.1%of TSP

CNP-10 0.01% of TSP 0.04 % of TSP

89


4.16.8. Western Blot analysis of transgenic plants to confirm protein

expression

Three plants with namely CNP5, CNP6, and CNP8 which were showing higher mRNA

expression of the F gene were also evaluated through western blot analysis on nitrocellulose

membranes (HyBond C, Amersham) through specific primary and AP-conjugated secondary

antibodies. . The presence of F protein at their respective sizes of 67kDa confirmed the

successful expression of F protein in transgenic maize plants (Figure 4.34A). Similarly four

plants namely CNP6, CNP8, CNP9 and CNP10 with high mRNA expression for HN gene

were also subjected to western blot analysis for determination of HN protein on nitrocellulose

membrane at its specific size with the help of protein ladder. BCIP/NBT was used as substrate

for colour development. The presence of HN protein at their respective sizes 69 kDa

confirmed the successful expression HN protein in transgenic corn seeds (Figure 4.34B).

Figure 4-34: Confirmation of F/HN proteins through western blotting

(A) Western Blot for F protein

Lanes 1&3: CNP5, CNP6, CNP8


Lane 5: Negative control (wild type plant)

(B) Western Blot for HN protein

Lane 1: Protein marker

Lanes 2-5: CNP6, CNP8, CNP9, CNP10

Lane 6: Negative control (wild type plant)

90


4.16.9. Bird feeding assay

Immunized chickens were bleed after every ten days and sera were examined for the

specific IgG level against F and HN protein. The immunized chicks showed rise in humoral

response with each booster. The highest antibodies level was recorded at 50 days and almost

remain same at 60 days. Similarly, the chickens receiving transgenic corn along with complete

and incomplete adjuvants in group-3 also showed a little rise in anti IgG antibodies against F

and HN as compared to chicks in group-2 receiving only transgenic corn. The chickens in

group-1, receiving non-transgenic corn didn’t show any significant increase level of IgG.

Figure 4.35&4.36).

91


Day 10

GroupG-1

G-2

G-3

0.00

0.05

0.10

0.15

0.20

OD

s 4

50n

m

* *

20 days

G-1

G-2

G-3

0.00

0.05

0.10

0.15

0.20

OD

s 4

50n

m

Groups

** **

30 days

GroupsG-1

G-2

G-3

0.0

0.1

0.2

0.3

0.4

0.5

OD

s 4

50n

m

*****

GroupsG-1

G-2

G-3

0.0

0.1

0.2

0.3

0.4

0.5

OD

s 4

50n

m

** ***

40 days 50 days

GroupsG-1

G-2

G-3

0.0

0.2

0.4

0.6

OD

s 4

50n

m

** **

60 days

GroupsG-1

G-2

G-3

0.0

0.2

0.4

0.6

OD

s 4

50n

m

*** ***

GroupG-1

G-2

G-3

0.04

0.05

0.06

0.07

0.08

OD

s 4

50n

mns ns

day 0

BA

DC

FE

G

92


Figure 4-35: Induced immune response against F protein

A: Sera samples collected at day 0 B: Sera samples collected at day 10

C: Sera samples collected at day 20 D: Sera samples collected at day 30

E: Sera samples collected at day 40 F: Sera samples collected at day 50

G: Sera samples collected at day 60

Results on the basis of one-way ANOVA showed significant differences between groups (P

< 0.05) and paired t-test comparisons between groups showed significant differences between

specific treatment groups and respective control group. (*P < 0. 05, **P < 0.005, ***P <

0.0005, NS not significant)

(G1: Chickens feds with non-transgenic maize; G2: Chickens fed with transgenic corn; G3:

Chickens fed with transgenic corn along with complete and incomplete adjuvants)

93


day 0

GroupsG

-1G

-2G

-3

0.05

0.06

0.07

0.08

0.09

OD

s 4

50

ns ns

10 days

GroupsG

-1G

-2G

-3

0.00

0.05

0.10

0.15

0.20

OD

s 4

50

ns ns

20 days

G-1

G-2

G-3

0.00

0.05

0.10

0.15

0.20

OD

s 4

50

Groups

* *

30 days

GroupsG

-1G

-2G

-3

0.0

0.1

0.2

0.3

0.4

0.5

OD

s 4

50

** **

40 days

G-1

G-2

G-3

0.0

0.2

0.4

0.6

0.8

OD

s 4

50

Groups

** **

50 days

G-1

G-2

G-3

0.0

0.2

0.4

0.6

0.8

OD

s 4

50

Groups

** ***

60 days

G-1

G-2

G-3

0.0

0.2

0.4

0.6

0.8

1.0

OD

s 4

50

Groups

*** ***

E

A

DC

B

F

G

94


Figure 4-36: Induced immune response against HN protein

A: Sera samples collected at day 0 B: Sera samples collected at day 10

C: Sera samples collected at day 20 D: Sera samples collected at day 30

E: Sera samples collected at day 40 F: Sera samples collected at day 50

G: Sera samples collected at day 60

Results on the basis of one-way ANOVA showed significant differences between groups (P

< 0.05) and Paired t-test comparisons between groups showed significant differences between

specific treatment groups and respective control group. (*P < 0. 05, **P < 0.005, ***P <

0.0005, NS not significant

NOTE:

(G1: Chickens feds with non-transgenic maize; G2: Chickens fed with transgenic corn; G3:

Chickens fed with transgenic corn along with complete and incomplete adjuvants)

95


: DISCUSSION

Vaccination is a major tool to prevent and control infectious diseases as it is a good

alternative of antibiotic and reduces the chances of secondary infections. No doubt,

Vaccination has improved the animals health and reduced the risk of zoonotic infections in

humans by food contamination (Shahid and Daniell 2016). A variety of vaccines like

attenuated virus vaccines, inactive virus vaccines, live virus vaccines, subunit vaccines and

vector vaccines have been used successfuly to control and prevent major viral diseases of the

poultry (Alexander 2012). These vaccines require a complex system of production and

purification; they are expensive; they require a cold chain, which is difficult to maintain; and

trained manpower are needed to administer them (Aswathi et al. 2014)

Furthermore, numerous outbreaks of NDV in poultry farms have raised the question

against the protection of the available vaccines. Plant based vaccines provide more better and

best option to control infectious diseases of poultry and outbreaks in farms by providing large

number of vaccines in short period of time (Guerrero-Andrade et al. 2006). The current study

is an effort to produce plant based vaccines by isolation, characterization of immunogenic F

and HN gene of NDV for possible control of this disease at farmer farms by some

modifications of protocols as described by (Guerrero-Andrade et al. 2006; Hahn et al. 2007;

Lai et al. 2013; Shahriari et al. 2015). Amplified ~1662bp F gene and ~1712bp HN gene were

TA ligated and evaluated through sequencing. The Blast results determined the specificity of

these genes belonging to Mukteswar strain. Similar type of results were obtained by Paldurai

et al., (2014) while confirming the N terminal sequence of that virus (Paldurai et al. 2014).

Determination of functional protein is very important while dealing with edible vaccine

therefore, availability of pure F and HN protein was achieved through prokaryotic expression.

F and HN gene were ligated in pET30a vector to obtain recombinant pET-F and pET HN

constructs. The choice of a suitable expression vector is a matter of great concern for gaining

high yield protein product. To obtain an increased expression, pET30a expression vector was

used with His-tag as selection marker .Similar types of results were obtained by (Nallaiyan et

al. 2010). Expression studies of F and HN gene was done in E. coli expression host, BL21-

DE3 specifically for protein expression as suggested by (Nallaiyan et al. 2010). The

96


expression of full length F and HN proteins was a challenging task. The capsid proteins of

paramyxoviridae are major determinants of infection in host cells (Morrison 2003) as they

support attachment and fusion with the host’s membrane. Yusoff and Tan (2001) had found

that transmembrane domains of capsid proteins fix tightly through a C terminal hydrophobic

segment on the virus surface. Thses tight interactions makes very difficult to purify these

capsid proteins (Yusoff and Tan 2001). Solubility of expressed proteins was successfully

improved by inducing the host cells containing the pET30a-F and pET30a-HN recombinant

construct at 30°C because growth at 37°C results inclusion body formation. Consequently,

the F and HN protein was obtained in soluble fractions. Similar methadoligies had been

adopted to obtaine higher expression of proteins in soluble fraction (Frozandeh-Moghadam et

al. 2009; Pearce et al. 2015).

Induction by adding IPTG was done to generate increased yield of protein and its

presence and size was confirmed through SDS polyacrylamide gel electrophoresis.The choice

of lysis buffer was also a matter of great concern because a standard lysis buffer containing

only 1XPBS was not suitable for hydrophobic viral proteins (Aghaeepoor et al. 2011). Yap et

al.( 2013) also reported similar results for purification of NP protein from NDV (Yap et al.

2013). Furthermore, transferring the proteins on nitrocellulose membrane in western blotting,

confirmed the specificity of His-tagged protein through antigen antibody reaction at the

expected size of 67kDa for F and 69kDa for HN. These finding are in accordance with Yap

et al. (2013) to detect 53kDa NP protein by NDV specific antibdies (Yap et al. 2013). Protein

purification using nickel metal affinity chromatography was performed by preparing the

protein sample in buffer containing lysozyme and non-ionic detergent triton X-100 to

minimize protein precipitation and folding. The addition of lysozyme also improved

sonication. Frosting and overheating during sonication was avoided to prevent protein

denaturation and co purification of host proteins. In this way, the problem of protein

aggregation and refolding was solved. Protein purification using nickel metal for ligand

binding proved to be a success. Tan et al. (2006) also used Immobilized metal affinity

chromatography (IMAC) to purify recombinant nucleoprotein (NP) from NDV and recoverd

5.6% (NP) protein (Tan et al. 2006). Step-wise elution with increasing imidazole

concentration from 70mM to 300mM provided easy detection of purified His-tag protein. The

purified protein product was further investigated through SDS-PAGE. The appearance of

97


single band of F protein at 67kDa and HN protein at 69kDa confirmed the specificity of our

desired immunogenic protein. These results are in accordance with purification of histidine

tagged protein by IMAC (Iram et al. 2014; Nallaiyan et al. 2010). The purified form of F and

HN proteins is a suitable agent for various molecular and diagnostic studies. As a diagnostic

agent they can be used to develop specific antibodies for detection of strain specific NDV by

ELISA(Joseph et al. 2016) .

Bioinformatics tools such as Immune epitope database (IEDB) analysis resource were

applied for 2D and 3D structural analysis of F and HN protein. These tools predicted that

more than 70% of its sequence is antigenically active and the predicted protein regions behave

as epitopes that can be readily recognized in a B cell immune response to generate specific

antibodies inside the host and build host immunity. This is in accordance with Motamedi who

found out conserved epitopic region of Fusion (F) and hemagglutinin–neuraminidase (HN)

protein gene by in silico analysis against Newcastle disease virus (Motamedi et al. 2014;

Pradhan et al. 2012).

After F and HN protein expression study in E. coli, the next main objective of this

study was to transform both F and HN gene in maize for development of plant-made vaccines.

The F and HN genes were cloned under the influence of constitutive and seed specific

promoter respectively. Berinstein et al. (2005) transformed two immunogenic F and HN genes

in potato under the influence of constitutive promoter for production of plant-based vaccine

against NDV (Berinstein et al. 2005).

Henn et al. (2007) cloned ~1716bp fragment of HN gene in plant expression vector

p221 under regulation of CMV 35S promoter and produced genetically modified tobacco

expressing HN gene of NDV (Hahn et al. 2007). Shahriari et al. (2015) cloned epitopes of

haemagglutinin-neuraminidase (HN) and fusion (F) gene in plant expression vector pBI121

to produce a plant-based vaccine in tobacco leaf discs (Shahriari et al. 2015).

After Agrobacterium mediated nuclear transformation of maize, putative transgenic

maize plants were subjected to different molecular analysis such as PCR, ELISA, Real time

PCR, Western blot analysis. Polymerase Chain reaction confirmed the presence of F gene in

seven putative transgenic maize plants and HN gene in five plants It was found that out of

total ten plants only five plants namely CNP3, CNP6, CNP8, CNP9, and CNP10 were PCR

positive for both F and HN gene. Similarly, other scientist also confirmed the presence of

98


NDV antigens in different plants through PCR (Berinstein et al. 2005; Gómez et al. 2013;

Gómez et al. 2009; Hahn et al. 2007; Shahriari et al. 2015).

Quantitative real time PCR is the golden tool to study mRNA expression of transgenic

plants. The mRNA transcripts of both F and HN genes were reverse transcrbied into cDNA.

RT-PCR analysis confirmed the expression of F gene in leaves of different plants namely

namely CNP1, CNP3, CNP4, CNP5, CNP6, CNP8, CNP9, CNP10. The comparitive analysis

of Ct values obtained by qRT-PCR revealed that the expression of F gene. varied from 2-7.1

fold. Transgenic plants namely, CNP5 and CNP8 were selected as best the plants showing

maximum expression of F gene mRNA with six to seven fold higher as compared to non-

transgenic plants. Similarly, comparison of Ct value by qRT-PCR of four plants namely

CNP6, CNP8, CNP9, CNP10 confirmed that the expression of HN gene increased from 0.5

to 4 fold as compared to negative control. Two plants namely CNP6 and CNP8 are the plants

showed almost 4 to 5 fold higher expression of HN genes in seeds as comparded to non-

transgenic plants. CNP8 plant was selected as the best plant showing maximum expression of

both F and HN gene. Shahriari et al. (2015) also quantified F and HN mRNA expression in

secondry roots of tobacco plants and found 8-10 fold increased expression of transgenes in

roots of tobacco plants (Shahriari et al. 2015). Lai et al. (2013) also observed almost four to

five fold increases in the mRNA expression of HN ectodomain in transgenic tobacco. He also

found a positive propotional relationship between mRNA transcripts levels of the eHN and

HN protein produced (Lai et al. 2013)

The activity of recombinant F and HN proteins were quantified through ELIZA. The

ELISA results showed that plants namely CNP5 and CNP8 were the best plants showing

maximum concentration of F gene almost 0.15μg/ml and 0.166μg/ml respectively. Similarly,

the plant namely CNP8 and CNP9 were best plant showing good of HN protein 0.24μg/ml

and 0.195μg/ml respectively. These results are in accordance with the findings that there is a

direct correlation between mRNA expression and amount of protein produced (Lai et al. 2013)

The total protein concentration for both F and HN protein was in the range of 0.02%

to 0.2% of TSP. These results are considerably better than the 0.06% of total soluble protein

produced as stated by Hehn (Hahn et al. 2007). Lai et al. (2013) also expressed HN

ectodomain in tobacco cells and quantified 0.2 to 0.4% of TSP (Lai et al. 2013). The expressed

F and HN antigens of NDV had been reported between ranges of 0.06-3% of TSP depending

99


on choice of promoter, transformation system and plant in which interested antigens had been

transformed (Berinstein et al. 2005; Guerrero-Andrade et al. 2006; Lai et al. 2013).

The final objective of this study was to immunized chick with transgenic maize and

studies the immune response generated by chicks. ELISA results showed the production of

anti-NDV antibodies in sera of chicks after ingestion of transgenic corn. On the other side,

ELISA results from the sera of chicks having non-transgenic diet did not induce any

significant immune response. The maximum antibodies level was recorded upto 50 days and

almost remain constant at 60 days. Even though, in group-3 received incomplete and complete

adjuvants also showed a little rise in anti-IgG antibodies against F and HN as compared to

chicks in group-2 receiving only transgenic corn. These results are in accordance when

immunized mice with eHN protein from tobacco cells showed gradual increase in immune

response in the presence of incomplete adjuvant (Lai et al. 2013). The induce immune

response in immunized chicks demonstrated that transformed genes were producing active

protein as stated by Lai (Lai et al. 2013). The induced immune response was also reported in

immunized chicks with transgenic maize expressing fusion protein (Guerrero-Andrade et al.

2006). Furthermore, rise in immune response was greater against HN protein as compared to

F protein. This is supposed to be the use of seed specific promoter for increased expression

of HN gene in seeds. For example, the transformed tomatoes with CTB gene under regulation

of CaMV 35S promoter accumulate 0.2% to 0.4% of TSP as compared to same transformed

tomato plants with CTB gene under control of E8 promoter (tomato fruit-specific promoter)

showed the significant enhanced expression approximately 0.8% of TSP (Guan et al. 2013).

Conclusion

Plants are gaining increasingly acceptance for production of recombinant proteins.

The reason for this is their ability to carry out posttranslational protein modifications in a

similar if not identical way as mammalian cells. Plants are also well known to carry out

human-like complex glycosylation. These outstanding results have placed plants in a

satisfactory position compared to other eukaryotic expression systems (Strasser et al.

2014). In this study, development of immune response in this study is the key success that can

lead towards plant-based vaccine production in future studies. The results of current study

will help in reducing the overall usage of heat liable live truncated attenuated vaccines

100


providing better protection against lethal effects of NDV in chicken. The transformed maize

expressing F and HN protein have a potential to be a source of plant-based oral vaccine to

protect chicken against NDV. This was the first report regarding plant-based vaccine

production against NDV in Pakistan therefore, modified experiments to enhanced protein

expression and modified animal study would be essential in future to increase immune

response in sera of immunized animals to protect them against viral challenge.

Bird feeding assays by using E. coli produced NDV F and H proteins along with

market available injectable vaccine in comparison to plant based oral vaccine developed in

this study are planned with collaborators in near future. The efficacy of NDV oral vaccine

bird feeding assay as planned with collaborators will open new horizon to produce plant based

immunogenic proteins against other notorious diseases of poultry in future.

101


REFERENCES

Abedullah M, Bukhsh K. (2007). Issues and economics of poultry production: A case study of

faisalabad pakistan. Pakistan Veterinary Journal. 27(1):25-28.

Abidin Z, Khatoon A. (2013). Heat stress in poultry and the beneficial effects of ascorbic acid

(vitamin c) supplementation during periods of heat stress. World's Poultry Science

Journal. 69(01):135-152.

Aghaeepoor M, Mozafari S, Shahraki M, Tabandeh F, Bambai B. (2011). High level of

extracellular fermentation and alternative purification of escherichia coli asparaginase

ii. Biharean Biol. 5(2):96-101.

Diseases in rural family chickens in south-east asia. INFPDE-CONFERENCE; 2013.

Aldous E, Alexander D. (2008). Newcastle disease in pheasants (phasianus colchicus): A

review. The Veterinary Journal. 175(2):181-185.

Alexander D. (1997). Newcastle disease and other avian paramyxovirus infections. In.

Diseases of poultry. Iowa State University Press, Ames, Iowa. 50014:541-569.

Alexander DJ. 2012. Newcastle disease. Springer Science & Business Media.

Alexander DJ, Aldous EW, Fuller CM. (2012). The long view: A selective review of 40 years

of newcastle disease research. Avian pathology. 41(4):329-335.

Ali A, Khan M. (2013). Livestock ownership in ensuring rural household food security in

pakistan. The J of Animal & Plant Sci. 23(1):313-318.

Ashraf A, Shah M. (2014). Newcastle disease: Present status and future challenges for

developing countries. African Journal of Microbiology Research. 8(5):411-416.

Aswathi PB, Bhanja SK, Yadav AS, Rekha V, John JK, Gopinath D, Sadanandan GV, Shinde

A, Jacob A. (2014). Plant based edible vaccines against poultry diseases: A review.

Balachandran P, Srinivasan P, Sivaseelan S, Balasubramanlam G, Muthy TGK. (2014).

Isolation and characterization of newcastle disease virus from vaccinated commercial

layer chicken. Vet World. 7(7):457-462.

Barbut S. (2012). Convenience breaded poultry meat products–new developments. Trends in

Food Science & Technology. 26(1):14-20.

Beard C, Hanson R. (1984). Newcastle disease.

Belova AV, Smutka L, Rosochatecká E. (2013). World chicken meat market–its development

and current status. Acta universitatis agriculturae et silviculturae mendelianae

brunensis. 60(4):15-30.

102


Berinstein A, Vazquez-Rovere C, Asurmendi S, Gómez E, Zanetti F, Zabal O, Tozzini A,

Grand DC, Taboga O, Calamante G. (2005). Mucosal and systemic immunization

elicited by newcastle disease virus (ndv) transgenic plants as antigens. Vaccine.

23(48):5583-5589.

Bhatti SS, Javed MT, Kausar R, Deeba F, Akhtar F, Rasool A. (2015). Urea and copper

sulphate in different combinations alter haematology and some serum proteins in

broilers. Pak J Agri Sci. 52(2):539-544.

Blake DP, Tomley FM. (2014). Securing poultry production from the ever-present eimeria

challenge. Trends in parasitology. 30(1):12-19.

Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analytical

biochemistry. 72(1-2):248-254.

Chan HT, Daniell H. (2015). Plant‐made oral vaccines against human infectious diseases—are

we there yet? Plant biotechnology journal. 13(8):1056-1070.

Chen T-H, Chen T-H, Hu C-C, Liao J-T, Lee C-W, Liao J-W, Lin M-Y, Liu H-J, Wang M-Y,

Lin N-S. (2012). Induction of protective immunity in chickens immunized with plant-

made chimeric bamboo mosaic virus particles expressing very virulent infectious

bursal disease virus antigen. Virus research. 166(1):109-115.

Chichester JA, Musiychuk K, de la Rosa P, Horsey A, Stevenson N, Ugulava N, Rabindran S,

Palmer GA, Mett V, Yusibov V. (2007). Immunogenicity of a subunit vaccine against

bacillus anthracis. Vaccine. 25(16):3111-3114.

Chomczynski P, Sacchi N. (1987). Single-step method of rna isolation by acid guanidinium

thiocyanate-phenol-chloroform extraction. Analytical biochemistry. 162(1):156-159.

Clarke JL, Waheed MT, Lössl AG, Martinussen I, Daniell H. (2013). How can plant genetic

engineering contribute to cost-effective fish vaccine development for promoting

sustainable aquaculture? Plant molecular biology. 83(1-2):33-40.

Conan A, Goutard FL, Sorn S, Vong S. (2012). Biosecurity measures for backyard poultry in

developing countries: A systematic review. BMC veterinary research. 8(1):240.

Daniell H, Khan MS, Allison L. (2002). Milestones in chloroplast genetic engineering: An

environmentally friendly era in biotechnology. Trends in plant science. 7(2):84-91.

Daniell H, Singh ND, Mason H, Streatfield SJ. (2009). Plant-made vaccine antigens and

biopharmaceuticals. Trends in plant science. 14(12):669-679.

Daniell H, Streatfield SJ, Wycoff K. (2001). Medical molecular farming: Production of

antibodies, biopharmaceuticals and edible vaccines in plants. Trends in plant science.

6(5):219-226.

Davoodi-Semiromi A, Samson N, Daniell H. (2009). The green vaccine: A global strategy to

combat infectious and autoimmune diseases. Human vaccines. 5(7):488-493.

103


de Leeuw OS, Koch G, Hartog L, Ravenshorst N, Peeters BP. (2005). Virulence of newcastle

disease virus is determined by the cleavage site of the fusion protein and by both the

stem region and globular head of the haemagglutinin–neuraminidase protein. Journal

of General Virology. 86(6):1759-1769.

Diel DG, Miller PJ, Wolf PC, Mickley RM, Musante AR, Emanueli DC, Shively KJ, Pedersen

K, Afonso CL. (2012). Characterization of newcastle disease viruses isolated from

cormorant and gull species in the united states in 2010. Avian diseases. 56(1):128-133.

Dortmans J, Koch G, Rottier P, Peeters B. (2011). Virulence of newcastle disease virus: What

is known so far. Vet Res. 42(1):122.

Dortmans J, Rottier P, Koch G, Peeters B. (2010). The viral replication complex is associated

with the virulence of newcastle disease virus. Journal of virology. 84(19):10113-10120.

Errington W, Emmerson PT. (1997). Assembly of recombinant newcastle disease virus

nucleocapsid protein into nucleocapsid-like structures is inhibited by the

phosphoprotein. Journal of general virology. 78(9):2335-2339.

Esaki M, Godoy A, Rosenberger JK, Rosenberger SC, Gardin Y, Yasuda A, Dorsey KM.

(2013). Protection and antibody response caused by turkey herpesvirus vector

newcastle disease vaccine. Avian diseases. 57(4):750-755.

Ferraro B, Morrow MP, Hutnick NA, Shin TH, Lucke CE, Weiner DB. (2011). Clinical

applications of DNA vaccines: Current progress. Clinical infectious diseases.

53(3):296-302.

Fiedler U, Conrad U. (1995). High-level production and long-term storage of engineered

antibodies in transgenic tobacco seeds. Nature Biotechnology. 13(10):1090-1093.

Frozandeh-Moghadam M, R M, Dehghani M, Mosavi S, Pourbakhsh S, Golchinfar F. (2009).

Cloning & expression of f protein gene (hr1 region) of newcastle disease virus nr43

isolate from iran in e. Coli. Iranian Journal of virology. 3(3):16-22.

Fuller C, Collins M, Easton AJ, Alexander D. (2007). Partial characterisation of five cloned

viruses differing in pathogenicity, obtained from a single isolate of pigeon

paramyxovirus type 1 (ppmv-1) following passage in fowls’ eggs. Archives of

virology. 152(8):1575-1582.

Gallili GE, Ben-Nathan D. (1998). Newcastle disease vaccines. Biotechnology advances.

16(2):343-366.

Ganar K, Das M, Sinha S, Kumar S. (2014). Newcastle disease virus: Current status and our

understanding. Virus research. 184:71-81.

Gleba Y, Klimyuk V, Marillonnet S. (2007). Viral vectors for the expression of proteins in

plants. Current Opinion in Biotechnology. 18(2):134-141.

104


Glickman RL, Syddall RJ, Iorio RM, Sheehan JP, Bratt MA. (1988). Quantitative basic residue

requirements in the cleavage-activation site of the fusion glycoprotein as a determinant

of virulence for newcastle disease virus. Journal of Virology. 62(1):354-356.

Gómez E, Lucero MS, Zoth SC, Carballeda JM, Gravisaco MJ, Berinstein A. (2013). Transient

expression of vp2 in nicotiana benthamiana and its use as a plant-based vaccine against

infectious bursal disease virus. Vaccine. 31(23):2623-2627.

Gómez E, Zoth SC, Berinstein A. (2009). Plant-based vaccines for potential human

application. Human vaccines. 5(11):738-744.

Guan Z-j, Guo B, Huo Y-l, Guan Z-p, Dai J-k, Wei Y-h. (2013). Recent advances and safety

issues of transgenic plant-derived vaccines. Applied microbiology and biotechnology.

97(7):2817-2840.

Guerrero-Andrade O, Loza-Rubio E, Olivera-Flores T, Fehérvári-Bone T, Gómez-Lim MA.

(2006). Expression of the newcastle disease virus fusion protein in transgenic maize

and immunological studies. Transgenic Research. 15(4):455-463.

Hahn B-S, Jeon I-S, Jung Y-J, Kim J-B, Park J-S, Ha S-H, Kim K-H, Kim H-M, Yang J-S,

Kim Y-H. (2007). Expression of hemagglutinin-neuraminidase protein of newcastle

disease virus in transgenic tobacco. Plant Biotechnology Reports. 1(2):85-92.

Hanson RP. (1965). Newcastle disease virus. The American Journal of the Medical Sciences.

249(5):618.

Herczeg J, Wehmann E, Bragg R, Dias PT, Hadjiev G, Werner O, Lomniczi B. (1999). Two

novel genetic groups (viib and viii) responsible for recent newcastle disease outbreaks

in southern africa, one (viib) of which reached southern europe. Archives of virology.

144(11):2087-2099.

Hou P, Xie Z, Zhang L, Song Z, Mi J, He Y, Li Y. (2011). Comparison of three different

methods for total rna extraction from fritillaria unibracteata, a rare chinese medicinal

plant. Journal of Medicinal Plants Research. 5(13):2835-2839.

Iram N, Shah MS, Ismat F, Habib M, Iqbal M, Hasnain SS, Rahman M. (2014). Heterologous

expression, characterization and evaluation of the matrix protein from newcastle

disease virus as a target for antiviral therapies. Applied microbiology and

biotechnology. 98(4):1691-1701.

Jacob SS, Cherian S, Sumithra T, Raina O, Sankar M. (2013). Edible vaccines against

veterinary parasitic diseases—current status and future prospects. Vaccine.

31(15):1879-1885.

Jang J, Hong S-H, Choi D, Choi K-S, Kang S, Kim I-H. (2010). Overexpression of newcastle

disease virus (ndv) v protein enhances ndv production kinetics in chicken embryo

fibroblasts. Applied microbiology and biotechnology. 85(5):1509-1520.

105


Jiang P, Qin S, Tseng C. (2002). Expression of hepatitis b surface antigen gene (hbsag) in

laminaria japonica (laminariales, phaeophyta). Chinese Science Bulletin. 47(17):1438-

1440.

Joseph N, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS. (2016). Production of the virus‐like

particles of nipah virus matrix protein in pichia pastoris as diagnostic reagents.

Biotechnology progress. 32(4):1038-1045.

Kalthoff D, Giritch A, Geisler K, Bettmann U, Klimyuk V, Hehnen H-R, Gleba Y, Beer M.

(2010). Immunization with plant-expressed hemagglutinin protects chickens from

lethal highly pathogenic avian influenza virus h5n1 challenge infection. Journal of

virology. 84(22):12002-12010.

Kattenbelt JA, Stevens MP, Gould AR. (2006). Sequence variation in the newcastle disease

virus genome. Virus research. 116(1):168-184.

Kim T-G, Yang M-S. (2010). Current trends in edible vaccine development using transgenic

plants. Biotechnology and Bioprocess Engineering. 15(1):61-65.

King D. (1999). A comparison of the onset of protection induced by newcastle disease virus

strain b1 and a fowl poxvirus recombinant newcastle disease vaccine to a viscerotropic

velogenic newcastle disease virus challenge. Avian diseases.745-755.

Kolotilin I, Topp E, Cox E, Devriendt B, Conrad U, Joensuu J, Stöger E, Warzecha H,

McAllister T, Potter A. (2014). Plant-based solutions for veterinary

immunotherapeutics and prophylactics. Veterinary research. 45(1):1.

Kraneveld F. (1926). A poultry disease in the dutch east indies. Ned Indisch Bl Diergeneeskd.

38:448-450.

Kwon K-C, Verma D, Singh ND, Herzog R, Daniell H. (2013). Oral delivery of human

biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.

Advanced drug delivery reviews. 65(6):782-799.

Kwon KC, Daniell H. (2015). Low‐cost oral delivery of protein drugs bioencapsulated in plant

cells. Plant biotechnology journal. 13(8):1017-1022.

Lacorte C, Lohuis H, Goldbach R, Prins M. (2007). Assessing the expression of chicken

anemia virus proteins in plants. Virus research. 129(2):80-86.

Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of

bacteriophage t4. nature. 227:680-685.

Lai KS, Yusoff K, Mahmood M. (2013). Functional ectodomain of the hemagglutinin-

neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine

against newcastle disease virus. Plant Cell, Tissue and Organ Culture (PCTOC).

112(1):117-121.

106


Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H. (2013). Low cost tuberculosis vaccine

antigens in capsules: Expression in chloroplasts, bio-encapsulation, stability and

functional evaluation in vitro. PLoS One. 8(1):e54708.

Lamichhane A, Azegami T, Kiyono H. (2014). The mucosal immune system for vaccine

development. Vaccine. 32(49):6711-6723.

Li X, Qiu Y, Yu A, Chai T, Zhang X, Liu J, Wang D, Wang H, Wang Z, Song C. (2009).

Degenerate primers based rt-pcr for rapid detection and differentiation of airborne

chicken newcastle disease virus in chicken houses. Journal of virological methods.

158(1):1-5.

Lim MAG. 2014. Newcastle disease vaccines. Commercial plant-produced recombinant

protein products. Springer. p. 179-195.

Ling H-Y, Pelosi A, Walmsley AM. (2010). Current status of plant-made vaccines for

veterinary purposes.

Loke C, Omar AR, Raha A, Yusoff K. (2005). Improved protection from velogenic newcastle

disease virus challenge following multiple immunizations with plasmid DNA encoding

for f and hn genes. Veterinary immunology and immunopathology. 106(3):259-267.

Lossl AG, Waheed MT. (2011). Chloroplast‐derived vaccines against human diseases:

Achievements, challenges and scopes. Plant Biotechnology Journal. 9(5):527-539.

Lössl AG, Waheed MT. (2011). Chloroplast‐derived vaccines against human diseases:

Achievements, challenges and scopes. Plant Biotechnology Journal. 9(5):527-539.

Loza-Rubio E, Rojas-Anaya E. (2010). Vaccine production in plant systems—an aid to the

control of viral diseases in domestic animals: A review. Acta Veterinaria Hungarica.

58(4):511-522.

Lu S-W, Wang K-C, Liu H-J, Chang C-D, Huang H-J, Chang C-C. (2011). Expression of avian

reovirus minor capsid protein in plants. Journal of virological methods. 173(2):287-

293.

Lugade AA, Kalathil S, Heald JL, Thanavala Y. (2010). Transgenic plant-based oral vaccines.

Immunological investigations. 39(4-5):468-482.

Mason HS, Haq TA, Clements JD, Arntzen CJ. (1998). Edible vaccine protects mice against

escherichia coli heat-labile enterotoxin (lt): Potatoes expressing a synthetic lt-b gene.

Vaccine. 16(13):1336-1343.

Mason HS, Warzecha H, Mor T, Arntzen CJ. (2002). Edible plant vaccines: Applications for

prophylactic and therapeutic molecular medicine. Trends in molecular medicine.

8(7):324-329.

Mebatsion T, Weiland F, Conzelmann K-K. (1999). Matrix protein of rabies virus is

responsible for the assembly and budding of bullet-shaped particles and interacts with

the transmembrane spike glycoprotein g. Journal of virology. 73(1):242-250.

107


Melanson VR, Iorio RM. (2006). Addition of n-glycans in the stalk of the newcastle disease

virus hn protein blocks its interaction with the f protein and prevents fusion. Journal of

virology. 80(2):623-633.

Memon IN. (2015). Economic analysis of poultry egg production in quetta district balochistan

pakistan. Journal of Marketing and Consumer Research. 14:5-12.

Mengesha M. (2012). The issue of feed-food competition and chicken production for the

demands of foods of animal origin. Asian Journal of Poultry Science. 6(2):31-43.

Miller PJ, Decanini EL, Afonso CL. (2010). Newcastle disease: Evolution of genotypes and

the related diagnostic challenges. Infection, genetics and evolution. 10(1):26-35.

Miller PJ, Haddas R, Simanov L, Lublin A, Rehmani SF, Wajid A, Bibi T, Khan TA, Yaqub

T, Setiyaningsih S. (2015). Identification of new sub-genotypes of virulent newcastle

disease virus with potential panzootic features. Infection, Genetics and Evolution.

29:216-229.

Moreki J, Dikeme R, Poroga B. (2010). The role of village poultry in food security and hiv/aids

mitigation in chobe district of botswana. Livest Res Rural Dev. 22(3).

Morrison TG. (2003). Structure and function of a paramyxovirus fusion protein. Biochimica

et Biophysica Acta (BBA)-Biomembranes. 1614(1):73-84.

Motamedi MJ, Amani J, Shahsavandi S, Salmanian AH. (2014). In silico design of multimeric

hn-f antigen as a highly immunogenic peptide vaccine against newcastle disease virus.

International Journal of Peptide Research and Therapeutics. 20(2):179-194.

Mukhtar N, Khan S, Khan R. (2012). Structural profile and emerging constraints of developing

poultry meat industry in pakistan. World's Poultry Science Journal. 68(04):749-757.

Munir M, Cortey M, Abbas M, Afzal F, Shabbir MZ, Khan MT, Ahmed S, Ahmad S, Baule

C, Ståhl K. (2012). Biological characterization and phylogenetic analysis of a novel

genetic group of newcastle disease virus isolated from outbreaks in commercial poultry

and from backyard poultry flocks in pakistan. Infection, Genetics and Evolution.

12(5):1010-1019.

Murashige T, Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco

tissue cultures. Physiologia plantarum. 15(3):473-497.

Nallaiyan S, Abbadorai RSAJ, Sundaramoorthy S, Nelson J, Sanyasi SVV. (2010). Production

and application of recombinant haemagglutinin neuraminidase of newcastle disease

virus. Asian Pacific Journal of Tropical Medicine. 3(8):629-632.

Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi U, Matsumura A,

Uozumi A, Hiroi T. (2007). Rice-based mucosal vaccine as a global strategy for cold-

chain-and needle-free vaccination. Proceedings of the National Academy of Sciences.

104(26):10986-10991.

108


Okamura K-i, Matsuda Y, Igari K, Kato K, Asao H, Matusi T, Takita E, Sawada K, Fukuda H,

Murase H. (2014). Effects of plant cultivation density and light intensity on the

production of a vaccine against swine edema disease in transgenic lettuce.

Environmental Control in Biology. 51(4):207-213.

Paldurai A, Xiao S, Kim S-H, Kumar S, Nayak B, Samal S, Collins PL, Samal SK. (2014).

Effects of naturally occurring six-and twelve-nucleotide inserts on newcastle disease

virus replication and pathogenesis.

Pansota FM, Rizvi F, Sharif A, Javed MT, Muhammad G, Khan A, Khan MZ. (2013). Use of

hyperimmune serum for passive immunization of chicks experimentally infected with

newcastle disease virus. Pak J Agri Sci. 50(2):279-288.

Pazhanivel N, Balsubramaniam G, GEORGE VT, Mohan B. (2002). Study of natural outbreak

of newcastle disease in and around namakkal. Indian veterinary journal. 79(3):293-294.

Pearce LA, Yu M, Waddington LJ, Barr JA, Scoble JA, Crameri GS, McKinstry WJ. (2015).

Structural characterization by transmission electron microscopy and immunoreactivity

of recombinant hendra virus nucleocapsid protein expressed and purified from

escherichia coli. Protein expression and purification. 116:19-29.

Pedersen JC, Senne DA, Woolcock PR, Kinde H, King DJ, Wise MG, Panigrahy B, Seal BS.

(2004). Phylogenetic relationships among virulent newcastle disease virus isolates

from the 2002-2003 outbreak in california and other recent outbreaks in north america.

Journal of clinical microbiology. 42(5):2329-2334.

Peeples M, Bratt M. (1984). Mutation in the matrix protein of newcastle disease virus can

result in decreased fusion glycoprotein incorporation into particles and decreased

infectivity. Journal of virology. 51(1):81-90.

Pitzschke A. (2013). Agrobacterium infection and plant defense—transformation success

hangs by a thread. Frontiers in plant science. 4.

Porta C, Lomonossoff GP. (2002). Viruses as vectors for the expression of foreign sequences

in plants. Biotechnology and Genetic Engineering Reviews. 19(1):245-292.

Pradhan SN, Prince PR, Madhumathi J, Roy P, Narayanan RB, Antony U. (2012). Protective

immune responses of recombinant vp2 subunit antigen of infectious bursal disease

virus in chickens. Veterinary immunology and immunopathology. 148(3):293-301.

Qiu X, Sun Q, Wu S, Dong L, Hu S, Meng C, Wu Y, Liu X. (2011). Entire genome sequence

analysis of genotype ix newcastle disease viruses reveals their early-genotype

phylogenetic position and recent-genotype genome size. Virol J. 8:117.

Rasool MH, Afzal AM, Baker A, Tahir MF. (2015). Antiviral activity of viro care gz-08 against

newcastle disease virus in poultry and its in-vitro cytotoxicity assay. Pakistan J Agric

Res Vol. 28(2).

109


Rehmani SF, Spradbrow P, West R. (1995). Lactose pellets—a new approach to oral

vaccination of village chickens against newcastle disease. Veterinary microbiology.

46(1):47-53.

Richter L, Kipp P. 2000. Transgenic plants as edible vaccines. Plant biotechnology. Springer.

p. 159-176.

Rout SN, Samal SK. (2008). The large polymerase protein is associated with the virulence of

newcastle disease virus. Journal of virology. 82(16):7828-7836.

Ryan MP, Walsh G. (2012). Veterinary-based biopharmaceuticals. Trends in biotechnology.

30(12):615.

Sack M, Hofbauer A, Fischer R, Stoger E. (2015). The increasing value of plant-made proteins.

Current opinion in biotechnology. 32:163-170.

Saha S, Callahan F, Dollar D, Creech J. (1997). Lyophilization of cotton tissue on quality of

extractable DNA, rna, and protein. Journal of cotton science.

Sarwar F, Usman M, Umar S, Hassan M, Rehman A, Rashid A. (2015). Some aspects of

backyard poultry management practices in rural areas of district rawalpindi, pakistan.

International Journal of Livestock Research. 5(5):14-20.

Sathish K, Sriraman R, Subramanian BM, Rao NH, Kasa B, Donikeni J, Narasu ML,

Srinivasan V. (2012). Plant expressed coccidial antigens as potential vaccine

candidates in protecting chicken against coccidiosis. Vaccine. 30(30):4460-4464.

Schwede T, Kopp J, Guex N, Peitsch MC. (2003). Swiss-model: An automated protein

homology-modeling server. Nucleic Acids Res. 31(13):3381-3385.

Seal BS, King DJ, Bennett JD. (1995). Characterization of newcastle disease virus isolates by

reverse transcription pcr coupled to direct nucleotide sequencing and development of

sequence database for pathotype prediction and molecular epidemiological analysis.

Journal of clinical microbiology. 33(10):2624-2630.

Senne D, King D, Kapczynski D. (2003). Control of newcastle disease by vaccination.

Developments in biologicals. 119:165-170.

Shabbir MZ, Abbas M, Yaqub T, Mukhtar N, Subhani A, Habib H, Sohail MU, Munir M.

(2013a). Genetic analysis of newcastle disease virus from punjab, pakistan. Virus

genes. 46(2):309-315.

Shabbir MZ, Zohari S, Yaqub T, Nazir J, Shabbir MAB, Mukhtar N, Shafee M, Sajid M, Anees

M, Abbas M. (2013b). Genetic diversity of newcastle disease virus in pakistan: A

countrywide perspective. Virology journal. 10(1):170.

Shaheen S, Anjum A, Rizvi F. (2005). Clinico-pathological observations of pigeons (columba

livia) suffering from newcastle disease. Liver. 2:7.14.

110


Shahid N, Daniell H. (2016). Plant based oral vaccines against zoonotic and non‐zoonotic

diseases. Plant Biotechnology Journal.

Shahriari AG, Bagheri A, Bassami MR, Malekzadeh Shafaroudi S, Afsharifar AR. (2015).

Cloning and expression of fusion (f) and haemagglutinin-neuraminidase (hn) epitopes

in hairy roots of tobacco (nicotiana tabaccum) as a step toward developing a candidate

recombinant vaccine against newcastle disease. Journal of Cell and Molecular

Research. 7(1):11-18.

Sharif A, Ahmad T, Umer M, Rehman A, Hussain Z. (2014). Prevention and control of

newcastle disease. International Journal of Agriculture Innovations and Research.

3(2):454-460.

Sharma J. (1999). Introduction to poultry vaccines and immunity. Advances in veterinary

medicine. 41:481-494.

Sherman A, Su J, Lin S, Wang X, Herzog RW, Daniell H. (2014). Suppression of inhibitor

formation against factor viii in hemophilia a mice by oral delivery of antigens

bioencapsulated in plant cells. Blood. 124:1659-1668.

Shoji Y, Chichester JA, Bi H, Musiychuk K, de la Rosa P, Goldschmidt L, Horsey A, Ugulava

N, Palmer GA, Mett V. (2008). Plant-expressed ha as a seasonal influenza vaccine

candidate. Vaccine. 26(23):2930-2934.

Shoji Y, Farrance CE, Bautista J, Bi H, Musiychuk K, Horsey A, Park H, Jaje J, Green BJ,

Shamloul M. (2012). A plant‐based system for rapid production of influenza vaccine

antigens. Influenza and other respiratory viruses. 6(3):204-210.

Siddique N, Naeem K, Abbas MA, Malik AA, Rashid F, Rafique S, Ghafar A, Rehman A.

(2013). Sequence and phylogenetic analysis of virulent newcastle disease virus isolates

from pakistan during 2009–2013 reveals circulation of new sub genotype. Virology.

444(1):37-40.

Sinkovics JG, Horvath JC. (2000). Newcastle disease virus (ndv): Brief history of its oncolytic

strains. Journal of clinical virology. 16(1):1-15.

Song Lai K, Yusoff K, Mahmood M. (2012). Heterologous expression of hemagglutinin-

neuraminidase protein from newcastle disease virus strain af2240 in centella asiatica.

Acta Biologica Cracoviensia Series Botanica. 54(1):142-147.

Soñora M, Moreno P, Echeverría N, Fischer S, Comas V, Fajardo A, Cristina J. (2015). An

evolutionary insight into newcastle disease viruses isolated in antarctica. Archives of

virology.1-8.

Spradbrow P, Samuel J. (1991). Oral newcastle disease vaccination with v4 virus in chickens:

Comparison with other routes. Australian Veterinary Journal. 68(3):114-115.

Steward M, Vipond IB, Millar NS, Emmerson PT. (1993). Rna editing in newcastle disease

virus. Journal of General Virology. 74(12):2539-2548.

111


Stoger E, Sack M, Perrin Y, Vaquero C, Torres E, Twyman RM, Christou P, Fischer R. (2002).

Practical considerations for pharmaceutical antibody production in different crop

systems. Molecular Breeding. 9(3):149-158.

Strasser R, Altmann F, Steinkellner H. (2014). Controlled glycosylation of plant-produced

recombinant proteins. Current opinion in biotechnology. 30:95-100.

Su J, Zhu L, Sherman A, Wang X, Lin S, Kamesh A, Norikane JH, Streatfield SJ, Herzog RW,

Daniell H. (2015). Low cost industrial production of coagulation factor ix

bioencapsulated in lettuce cells for oral tolerance induction in hemophilia b.

Biomaterials. 70:84-93.

Subramanian SK, Tey BT, Hamid M, Tan WS. (2009). Production of the matrix protein of

nipah virus in escherichia coli: Virus-like particles and possible application for

diagnosis. Journal of virological methods. 162(1):179-183.

Susta L, Miller P, Afonso C, Brown C. (2011). Clinicopathological characterization in poultry

of three strains of newcastle disease virus isolated from recent outbreaks. Veterinary

Pathology Online. 48(2):349-360.

Taghavian O, Schillberg HDS. 2013. Expression and characterization of infectious bursal

disease virus protein for poultry vaccine development and application in

nanotechnology. Hochschulbibliothek der Rheinisch-Westfälischen Technischen

Hochschule Aachen.

Tan YP, Ling TC, Tan WS, Yusoff K, Tey BT. (2006). Recovery of histidine-tagged

nucleocapsid protein of newcastle disease virus using immobilised metal affinity

chromatography. Process biochemistry. 41(4):874-881.

Thomas DR, Penney CA, Majumder A, Walmsley AM. (2011). Evolution of plant-made

pharmaceuticals. International journal of molecular sciences. 12(5):3220-3236.

Topp E, Irwin R, McAllister T, Lessard M, Joensuu JJ, Kolotilin I, Conrad U, Stöger E, Mor

T, Warzecha H. (2016). The case for plant-made veterinary immunotherapeutics.

Biotechnology advances.

Towbin H, Staehelin T, Gordon J. (1979). Electrophoretic transfer of proteins from

polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.

Proceedings of the National Academy of Sciences. 76(9):4350-4354.

Tremblay R, Wang D, Jevnikar AM, Ma S. (2010). Tobacco, a highly efficient green bioreactor

for production of therapeutic proteins. Biotechnology Advances. 28(2):214-221.

Walmsley AM, Arntzen CJ. (2003). Plant cell factories and mucosal vaccines. Current opinion

in biotechnology. 14(2):145-150.

Wu H, Scissum-Gunn K, Singh NK, Giambrone JJ. (2009). Toward the development of a plant-

based vaccine against reovirus. Avian diseases. 53(3):376-381.

112


Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X. (2007). Oral immunization with transgenic rice seeds

expressing vp2 protein of infectious bursal disease virus induces protective immune

responses in chickens. Plant Biotechnology Journal. 5(5):570-578.

Xing Y, Yang Q, Ji Q, Luo Y, Zhang Y, Wang KGD. (2007). Optimization of agrobacterium-

mediated transformation parameters for sweet potato embryogenic callus using [beta]-

glucuronidase (gus) as a reporter. African Journal of Biotechnology. 6(22):2578.

Yap CF, Tan WS, Sieo CC, Tey BT. (2013). Purification of long helical capsid of newcastle

disease virus from escherichia coli using anion exchange chromatography.

Biotechnology progress. 29(2):564-567.

Yin G, Lin Q, Wei W, Qin M, Liu X, Suo X, Huang Z. (2014). Protective immunity against

eimeria tenella infection in chickens induced by immunization with a recombinant c-

terminal derivative of etimp1. Veterinary immunology and immunopathology.

162(3):117-121.

Yuan P, Swanson KA, Leser GP, Paterson RG, Lamb RA, Jardetzky TS. (2011). Structure of

the newcastle disease virus hemagglutinin-neuraminidase (hn) ectodomain reveals a

four-helix bundle stalk. Proceedings of the National Academy of Sciences.

108(36):14920-14925.

Yusibov V, Rabindran S. (2008). Recent progress in the development of plant derived

vaccines.

Yusibov V, Streatfield SJ, Kushnir N. (2011). Clinical development of plant-produced

recombinant pharmaceuticals: Vaccines, antibodies and beyond. Human vaccines.

7(3):313-321.

Yusoff K, Tan WS. (2001). Newcastle disease virus: Macromolecules and opportunities. Avian

Pathology. 30(5):439-455.

Zhang R, Wang X, Su J, Zhao J, Zhang G. (2010). Isolation and analysis of two naturally-

occurring multi-recombination newcastle disease viruses in china. Virus research.

151(1):45-53.

Zhao K, Chen G, Shi X-m, Gao T-t, Li W, Zhao Y, Zhang F-q, Wu J, Cui X, Wang Y-F.

(2012). Preparation and efficacy of a live newcastle disease virus vaccine encapsulated

in chitosan nanoparticles.

Zhao Y, Hammond RW. (2005). Development of a candidate vaccine for newcastle disease

virus by epitope display in the cucumber mosaic virus capsid protein. Biotechnology

letters. 27(6):375-382.

Zhou J-Y, Cheng L-Q, Zheng X-J, Wu J-X, Shang S-B, Wang J-Y, Chen J-G. (2004).

Generation of the transgenic potato expressing full-length spike protein of infectious

bronchitis virus. Journal of biotechnology. 111(2):121-130.

113


APPENDICES

Appendix I

LB Media,pH 7.5

Chemicals For 1L

Trypton 10g

Yeast extract 5g

NaCl 10g

Agar 15g

LB Broth, pH 7.5

Chemicals For 1L

Trypton 10g

Yeast extract 5g

NaCl 10g

YEP Liquid, pH 7.5

Chemicals For 1L

Bactopeptone 5g

Yeast extract 10g

NaCl 10g

YEP Agar, pH 7.5

Chemicals For 1L

Bactopeptone 5g

Yeast extract 10g

NaCl

BactoAgar

10g

15g

MS Medium (Murashige and Skoog, 1962) pH 5.6

Chemicals For 1L

MS Salt 5g

sucrose 30g

114


B5 Vitamins

Chemicals For 50mL

10mMNicotinic Acid 40.6 µl/

10mM Pyridoxine HCL 24.3 µl

10mM Thymine HCL 148.25 µl

10mM Myo-inositol 280 µl/

Appendix II

50x Tris –Acetate (TAE)

Chemicals For 1L

Tris base 242g

Glacial Acetic Acid 57.1mL

0.5M EDTA 100mL

0.8% Agarose gel

Chemicals For 100mL

1X TAE Buffer 100mL

Agarose 0.8g

Appendix III

Chloramphenicol drug

Chemicals For 10mL

Chloramphenicol 0.3g

70% Ethanol 10mL

Ampicilin drug

Chemicals For 10mL

Ampicilin 1gm

Auto H2O 10mL

Tetracycline

Chemicals For 10mL

Tetracycline 1gm

70% Ethanol 10mL

115


Rifampicin

Chemicals For 10mL

Rifampicin 12.5mg

70% Ethanol 10mL

Kanamycin

Chemicals For 10mL

Kanamycin 0.5gm

Auto H2O 10mL

30% Polyacrylamide

Chemicals For 100mL

Acrylamide 29.2g

Bisacrylamide 0.8g

Auto H2O 100mL

Appendix IV

1.5M Tris, pH 8.8

Chemicals For 1L

Tris base 181.65g

Water 700mL

Conc. HCl 120mL

1M Tris, pH 6.8

Chemicals For 1L

Tris base 121.1g

Water 700mL

Conc. HCl 70mL

10% SDS

Chemicals For 100mL

SDS 10g

Auto H2O 100mL

10% Ammonium Persulfate

Chemicals For 1mL

APS 0.1g

Auto H2O 1mL

116


10X SDS running buffer

Chemicals For 1L

Tris base 30.3g

Glycine 144g

SDS 10g

Staining solution

Chemicals For 1L

Coomassie blue 0.025g

Methanol 400mL

Glacial acetic acid 70mL

Auto H2O 530mL

10% Resolving gel

Chemicals For 10mL

Auto H2O 4.15mL

30% Acrylamide 3.3mL

1.5M Tris 2.5mL

10% SDS 150µL

10% APS 150µL

TEMED 5µL

5% Stacking gel

Chemicals For 5mL

Auto H2O 3.05mL

30% Acrylamide 0.67mL

1M Tris 1.25mL

10% SDS 25µL

10% APS 25µL

TEMED 5µL

Destaining solution

Chemicals For 1L

Methanol 450mL

Glacial acetic acid 100mL

Auto H2O 450mL

Western blot transfer buffer

117


Chemicals For 500mL

Trizma base 2.91g

Glycine 1.46g

Methanol 100mL

Auto H2O 400mL

Skimmed milk

Chemicals For 50mL

1X PBS 50mL

Skimmed milk 2.5g

5X SDS protein loading buffer

Chemicals For 10mL

Glycerol 4mL

Tris-Cl pH 6.8 3.5mL

Bromophenol Blue 0.02g

SDS 1.2g

600mM β-Mercaptoethanol 250µL

Genomic DNA extraction buffer

Chemicals For 50ml

10%CTAB 10ml

100% β Merceptoethanol 0.5mL

5MNaCL 14ml

10%PVP 10ml

1M Tris pH 8 5mL

Auto H2O 8.5mL

Protein extraction buffer for Plants

Chemicals For 45mL

5MNaCl 900μl

0.5M EDTA 900μl

IM sucrose 9mL

10%SDS 450μl

1M Tris pH 8 9mL

100mMPMSF 900

MDTT 4.5mL

118


Tween 20 30 μl

Auto H2O 19.275mL

expression of f protein gene in maize for production of

Documents