expression of omp16 and l7/l12 brucella abortus protective antigens...

Indian Journal of Biotechnology

Vol 16, July 2017, pp 289-295

Expression of Omp16 and L7/L12 Brucella abortus protective antigens as

secretory fusion proteins in mammalian cells

Bikash R Prustya*

, Rizwana Tabassuma, Pallab Chaudhuri

b, Mohini Saini

c, V K Chaturvedi

d,

B P Mishraa, Praveen K Gupta

a*

aDivision of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, India bDivision of Bacteriology, Indian

Veterinary Research Institute, Izatnagar, India, cCenter for Wildlife, Indian Veterinary Research Institute, Izatnagar, India, dDivision of Biological Products, Indian Veterinary Research Institute, Izatnagar, India

Received 9 January 2015; revised 17 February 2016; accepted 22 February 2016

Brucella abortus is a facultative intracellular bacterial pathogen infecting animals and humans. This preliminary study

was designed to express two immunogenic genes of Brucella abortus as recombinant fusion proteins in mammalian cells for

being studied as a vaccine candidate in mammalian hosts, especially mice and cattle, in our next study. The complete open

reading frame sequences of two immune dominant genes of Brucella abortus namely, Omp16 and L7/L12 were fused with

an intermediate spacer along with N-terminal fusion with secretory signal sequence from immunoglobulin M. The complete

fusion gene sequence was codon optimized for expression in mammalian cells. For expression analysis, the codon optimized

synthetic gene was cloned in pDsRed-Express-N1 mammalian expression vector, with C-terminal fusion with red

fluorescent protein sequence. On transfection in MDBK and HEK-293 cells, appearance of red fluorescence in transfected

cells indicated expression of Omp16- L7/L12 fusion proteins along with RFP. The Omp16-L7/L12 fusion construct without

RFP sequence was also expressed in mammalian cells. The expressed Omp16-L7/L12 fusion proteins were confirmed

through both indirect fluorescence antibody test and western blot. This preliminary study suggested that the codon optimized

Omp16-L7/L12 fusion construct is ready to be studied in hosts like mice and cattle for its vaccine efficacy.

Keywords: Brucella abortus, Omp16, L7/L12, fusion gene, codon optimization, heterologous gene expression

Introduction Brucella abortus is a zoonotic bacterial pathogen

belonging to the genus Brucella. It causes abortion,

retention of placenta and infertility in ruminants and

undulant fever in humans. It is an intracellular pathogen

that multiplies in both non-phagocytic cells like

placental trophoblastic epithelial cells1 and phagocytic

cells like macrophages2. Like other intracellular bacterial

pathogens, the host resistance to B. abortus depends

mainly on acquired cell-mediated immune (CMI)

response. The interferon gamma cytokine is essential for

up regulating anti-Brucella activity of macrophages and

clearance of Brucella pathogens from the host3. The two

proteins, namely, outer membrane protein 16 (Omp16)

and ribosomal protein (L7/L12) have been identified as

protective immunodominant Brucella antigens and can

elicit strong CMI response4, 5, 6

. The Omp16 protein is

also endowed with self-adjuvant property and can

activate dendritic and macrophages7. Apart from CMI

response, antibody mediated humoral immune response

also affects course of infection with

B. abortus. The DNA vaccines encoding Omp16 and

L7/L12 proteins have been reported to produce both

humoral and CMI response and engender protection in

DNA vaccine immunized mice8. It was also reported

that DNA vaccine containing Omp

16-L7/L12 fusion sequence produces higher immune

response and provides higher protection in mice than

Omp16 and L7/L12 DNA vaccines do separately8. The

expression levels of heterologous genes have significant

influences on the immunogenicity of nucleic acid based

vaccines. Codon optimization of gene is an important

parameter for its improved expression in heterologous

hosts9, 10

. It is necessary to confirm the expression of

prokaryotic genes in mammalian cells in vitro before

study of immunogenicity and protectiveness of

prokaryotic genes based vaccine candidates in animal

models.

In this study, a fusion gene construct comprising

Omp16 and L7/L12 antigenic genes from B. abortus

was constructed and codon optimized for mammalian

cell expression.

_________

*Author for correspondence

Tel.: +91 581 2301584

[email protected]

INDIAN J BIOTECHNOL, JULY 2017

290

Materials and Methods

Cells Culture

Madin darby bovine kidney (MDBK) and

human embryonic kidney (HEK-293) cell lines,

used in this study, were maintained in Dulbecco’s

modified minimum essential medium (DMEM)

(Hyclone, Utah) supplemented with 10% fetal

bovine serum (FBS) (Hyclone, Utah), 2 g/l sodium

bicarbonate, 16.8 mM HEPES (Hyclone, Utah), 1 X

non-essential amino acids (NEAA) (Hyclone,

Utah), 0.25 µg/ml amphotericin B (Hyclone, Utah)

and 50 µg/ml of gentamicin (Sigma, USA) at 37°C

under 5% CO2.

In Silico Designing of Omp16 and L7/L12 Fusion Gene

Construct and Its Codon Optimization

The complete open reading frame (ORF)

sequences of Brucella abortus Omp16 (GenBank

accession no. L27996) and L7/L12 (GenBank

accession no. L19101) genes were retrieved from

GenBank of NCBI. The coding sequence for signal

peptide was excluded from ORF of Omp16 gene. The

Omp16 and L7/L12 ORF were joined with two serine

and four glycine spacer sequence. At the N- terminus

of the Omp16-L7/L12 fusion gene, kozak consensus

and immunoglobulin M (IgM) signal sequences (60

bp) were added. The final fusion gene sequence was

codon optimized for mammalian cell expression,

especially using ‘GenScript Optimum Gene’ codon

optimization analysis software and chemically

synthesized (GenScript, USA). For ease of cloning,

the NheI and BamHI restriction enzyme sites were

included at 5’- and 3’-end of the synthetic gene,

respectively (Fig. 1A).

Cloning of Omp16-L7/L12 Fusion Gene in a Mammalian

Expression Vector

The complete synthetic fusion gene was cloned

into pDsRed-Express-N1 mammalian expression

vector (Clontech, USA), in which multiple cloning

site (MCS) is in frame with red fluorescence protein

(RFP) reporter gene sequence at its C-terminus. Both

Omp16-L7/L12 fusion gene construct (insert) and

vector were digested with NheI and BamHI enzymes.

The restriction enzyme digested insert and vector

were ligated by T4 DNA ligase (Stratagene, USA) as

per manufacturer’s protocol. The newly constructed

recombinant plasmid was named as pDsRed-Omp16-

L7/L12-RFP as the Omp16-L7/L12 fusion gene

sequence was in frame with RFP sequence at its C-

terminus. The pDsRed-Omp16-L7/L12-RFP plasmid

was characterized through restriction enzyme analysis

by using EcoRI, PstI and PvuII enzymes. The

digested products were separated on 1% agarose gel

electrophoresis.

Expression of Omp16-L7/L12-RFP Fusion Gene Construct in

Mammalian Cells

The pDsRed-Omp16-L7/L12-RFP recombinant

plasmids were transfected in to MDBK cells seeded in

a 24 wells plate, by using Lipofectamine 2000 reagent

(Invitrogen, USA) following manufacturer’s

instructions. Briefly, the plasmid DNA-lipofectamine

complex was prepared by mixing 1.0 µg of pDsRed-

Omp16-L7/L12-RFP plasmid with 2.5 µl of

Lipofectamine 2000 reagent and incubated at room

temperature for 20 min. The plasmid DNA-

Lipofectamine complex was added to MDBK cell

monolayer of 90% confluence. At six hours post-

transfection, the complex was replaced with DMEM

containing 10% FBS and incubated for next 24 hours.

The transfected cells were observed under fluorescent

microscope to detect expressed Omp16-L7/L12-RFP

fusion proteins.

Analysis of Expressed Omp16-L7/L12 -RFP Fusion Proteins

The expressed Omp16-L7/L12-RFP fusion

proteins were analysed by indirect fluorescence

antibody test (IFAT) and western blotting. The

localisation of expressed Omp16-L7/L12-RFP fusion

proteins in transfected MDBK cells were

demonstrated through IFAT. In brief, the MDBK cells

in a 24 wells plate were transfected with pDsRed-

Omp16-L7/L12-RFP plasmids using lipofectamine

2000 (Invitrogen, USA) as stated above. After 48

hours of transfection, the cells were fixed with chilled

80% acetone for 30 minutes. The blocking was done

with 5% skimmed milk powder (SMP) in 1X PBS for

1 hour. After washing with 1X PBS thrice, the cells

were allowed to react with hyper immune sera, raised

against purified recombinant L7/L12 protein in rabbit,

at 1:200 dilutions for 1 hour. The cells were washed

with 1X PBS and then reacted with FITC-labelled

goat anti-rabbit IgG conjugate (Invitrogen, USA) at

1:200 dilutions for 1 hour. Finally, the cells were

washed five times and counter stained with DAPI

(Invitrogen, USA). The expressed Omp16-L7/L12-

RFP fusion proteins were localised in fixed cells

under a fluorescence microscope.

For western blotting, the HEK-293 cells in a

25 cm2 flask were transfected with pDsRed-Omp16-

L7/L12-RFP plasmids by using lipofectamine 2000

PRUSTY et al: MAMMALIAN CELL EXPRESSION OF BRUCELLA ABORTUS GENES AS FUSION PROTEIN

291

reagent (Invitrogen, USA) as per manufacturer’s

instruction. The plasmid DNA-lipofectamine

complex was prepared by mixing 10 µg plasmid

DNA and 25 µl lipofectamine 2000. After 48 hours

of transfection, the transfected HEK-293 cells were

harvested and pelleted by centrifuging the tube at

500 × g for 5 minutes at room temperature. The cell

pellet was washed twice with 1X PBS. The cell

lysate prepared from the cell pellet was resolved in

12% SDS PAGE and subsequently electro-blotted

to a polyvinylidene fluoride (PVDF) membrane at

constant current of 0.8 mA/cm2 for 1 hour. Then

after, the membrane was incubated over night at 4°

C in 5% SMP prepared in 1X PBST (PBS + 0.05%

Tween 20) for blocking. The membrane was

washed thrice with 1X PBST, each wash for 5

minutes and then incubated with hyper immune sera

(1: 200 dilutions) raised against purified

recombinant L7/L12 proteins in rabbit, for 1 hour.

The membrane was washed thrice with 1X PBST

and incubated with horseradish peroxidase

(HRPO)-conjugated goat anti-rabbit IgG secondary

antibody (1: 2500 dilutions) for 1 hour. The

membrane was washed thrice with 1X PBST and

antigen antibody reaction was detected by

incubating the membrane with substrate 3’3’-

diamino benzidine (DAB) (5 mg/10 ml of 1X PBS)

in presence of 20 µl of 30% H2O2. The colour

reaction was terminated by washing the membrane

with distilled water.

Excision of RFP Sequence from pDsRed-Omp16-L7/L12-RFP

Plasmid

The coding sequence for RFP gene was removed

from pDsRed-Omp16-L7/L12-RFP plasmid by

digesting the plasmid with BamHI and NotI enzymes.

After digestion, resulted 5’ overhangs were made

blunt ended by using T4 DNA polymerase enzyme

(Fermentas, USA) as per manufacturer’s protocol.

The blunt ends of linear vector were allowed for self-

ligation by T4 DNA ligase (Stratagene, USA). The

resulted new recombinant plasmid pDsRed-Omp16-

L7/L12 was characterized through restriction enzyme

analysis by using PstI restriction endonuclease.

Analysis of Expressed Omp16-L7/L12 (without RFP) Proteins

The HEK-293 cells in a 25cm2

flask were

transfected with recombinant plasmid pDsRed-

Omp16-L7/L12 and processed for western blotting as

stated above.

Results and Discussion Species-specific disparities in codon usage are

frequently cited as the cause for failures in

recombinant gene expression by heterologous hosts.

Such failures include lack of expression or the

expression of protein that is non-functional or

insoluble or protein that is truncated owing to

proteolysis or premature termination of translation11

.

The translational efficiency of heterologous genes can

often be improved by optimizing synonymous codon

usage to better match the host organism9. In this

study, we reported codon optimization of B. abortus

Omp16 and L7/L12 gene sequences and fusion of

both gene sequences for expression as a single protein

in mammalian host cells. The ORF sequences for

Omp16 and L7/L12 genes were fused with an

intermediate spacer sequence and also having kozak

sequence and IgM signal sequence at 5’ end. The

whole fusion construct sequence was codon optimized

for expression in mammalian cells (hosts), especially

in cattle. About 60% of codons in original sequence

were changed after codon optimization (Fig. 1B). The

codon optimized gene sequences were translated into

amino acid sequences in silico by using Lasergene

software (DNASTAR) and then compared with amino

acid sequences of original sequences (Fig. 1C). The

amino acid sequence remained same in both cases.

The codon-optimized fusion gene construct was

synthesized chemically and cloned into pDsRed-

Express-N1 mammalian expression vector in such

way that the RFP gene sequence of vector remained in

frame with C-terminus of cloned fusion gene

construct in order to express fusion gene construct

and RFP as a single polypeptide. The recombinant

plasmid pDsRed-Omp16-L7/L12-RFP (Fig. 2A) was

characterized by using restriction endonucleases (Fig.

2B). The EcoRI produced a single fragment of

5513bp (Lane 2, Fig. 2B), PstI produced two

fragments of sizes 999 bp and 4514 bp with (Lane 3,

Fig. 2B) and PvuII produced three fragments of sizes

618 bp, 969 bp and 3318 bp (Lane 4, Fig. 2B). These

restriction fragments were corroborated with

fragments predicted in silico through SeqBuilder

software (DNASTAR) (Fig. 2A).


292

Now, the pDsRed-Omp16-L7/L12-RFP

recombinant plasmid presented the fusion construct

IgM signal- Omp16-L7/L12 sequence (887bp) in

fusion with RFP sequence at C-terminal end as a

single ORF. The whole ORF was under the control of

human CMV immediate early promoter at 5’end and

having SV40 polyA tail at 3’ end (Fig. 2A). The

human CMV immediate early promoter and SV40

polyA tail are parts of the expression vector. The

sufficient long serine-glycine spacer sequence (18 bp)

in between Omp16 and L7/L12 ORF allows folding

of both proteins separately and thus maintains distinct

Fig. 1 — : (a) Organization of coding sequence of Omp-16-L7/L12 fusion gene. (b) The coding nucleotide sequence was optimized for

expression in mammalian cells and compared with original sequence. The sequences in red are optimized sequences. (c) The translated

amino acid sequence yielded similarity with original sequence. The sequences in bold and underlined are signal sequence. The sequences

in bold and italic are spacer sequence.


293

functional domains of both proteins. The serine

residues may facilitate solubility of the fusion proteins

and thus enhances immunogenicity of expressed

fusion proteins. The glycine residue being having a

short side chain prevents steric hindrance during

folding of separate proteins in the fusion construct12

.

After 24 hours of transfection of pDsRed-Omp16-

L7/L12-RFP plasmids into MDBK cells, red

fluorescence as secretory vesicles in transfected cells

were observed under fluorescent microscope,

indicated expression of Omp16-L7/L12-RFP as a

single polypeptide (Fig. 3 A, B). Secretion of fusion

proteins are more effective when fused with IgM

signal sequence13, 14

. In this study, addition of IgM

signal sequence at the

N-terminus of the fusion construct directed the

expressed proteins to cell surface as secretory vesicles

through secretory pathways. The expressed Omp16-

L7/L12-RFP proteins in transfected MDBK cells were

localised through IFAT. As indicated in Figure 3B,

the transfected MDBK cells demonstrated both green

(Fig. 3C) and red (Fig. 3D) fluorescence indicating

the presence of L7/L12 and RFP, respectively, in

same cells. The green fluorescence resulted after

tagging of expressed proteins with FITC-labelled anti-

rabbit antibody. The nuclei of transfected cells took

DAPI stains and appeared blue. In western blot

analysis, the expressed proteins produced a band of 57

kDa as expected, along with some additional bands of

smaller sizes. Expressed protein degradation during

cell lysate preparation and use of polyclonal hyper

immune serum could be reasons for additional bands

on membrane.

Fig. 2 — : (a) Vector map of pDsRed-Omp16-L7/L12-RFP

plasmid. (b) Restriction enzyme analysis of pDsRed-Omp16-

L7/L12-RFP with EcoRI (lane 2), PstI (lane 3) and PvuII (lane 4).

Lane 1: High range Gene ruler, lane 5: Uncut plasmid.

Fig. 3 — : (A & B) Expression of Omp16-L7/L12 as RFP fusion

protein in transfected-MDBK cells showing expressed proteins in

secretory vesicles. Localisation of Omp16-L7/L12-RFP fusion

proteins in transfected MDBK cells by indirect

immunofluorescence test (IFAT). (C) The pDsRed-Omp16-

L7/L12-RFP-transfected MDBK cells were fixed with 80%

acetone probed with hyperimmune sera against L7/L12 and

detected using FITC labelled anti-rabbit antibody. (D) The cells

with expression of Omp16-L7/L12-RFP were analysed with

fluorescent microscope after counter staining with DAPI. Arrow

indicated cell expressing Omp16-L7/L12-RFP.


294

To express Omp16-L7/L12 fusion protein without

RFP reporter gene, the 693 bp fragment encoding

RFP was removed from pDsRed-Omp16-L7/L12-RFP

plasmid (Fig. 4) by using BamHI and HindIII

restriction enzymes. The newly generated pDsRed-

Omp16-L7/L12 plasmid, on characterization with PstI

restriction enzyme produced a linear fragment of 4820

bp (Fig. 4B) as expected. Reduced length of the new

recombinant plasmid confirmed deletion of RFP

sequence (Fig 4A). To confirm the expression of

Omp-16-L7/L12 without RFP, the pDsRed-Omp16-

L7/L12 plasmids were transfected into MDBK cells.

Now, no red fluorescence in transfected MDBK cells

(data not shown) were observed indicated absence of

RFP expression. However, expressed Omp16-L7/L12

proteins were detected in transfected HEK-293 cell

lysate through western blot (Fig 5). The HEK-293 cell

line was chosen considering its higher transfection

efficiency. The expressed Omp16-L7/L12 fusion

proteins showed immuno-reactivity with hyper-

immune sera raised against L7/L12 proteins and

produced a band of approximately 32 kDa

(Lane L1, Fig. 5) corresponded to in silico predicted

molecular mass of Omp16-L7/L12 fusion proteins

(without RFP). The expressed proteins (without RFP) of

reduced size i.e. 32 kDa (Lane 1, Fig. 5) as compared to

previously expressed proteins (with RFP) of 57 kDa

(Lane 4, Fig. 5) further confirmed expression of

transfected Omp16-L7/L12 fusion construct even after

removal of RFP sequence from the expression vector. All

the results demonstrated expression of correct fusion

proteins in mammalian cells.

In conclusion, the present study reported

optimization of B. abortus Omp16 and L7/L12 genes

for mammalian expression as fusion proteins. The

expressed fusion proteins were detected as secretory

vesicles inside cells, confirming expression of

proteins as secretory by utilizing IgM secretory signal

sequence fused to Omp16-L7L12 construct at 5’ end.

Now, the Omp16-L7/L12 fusion construct is ready to

be experimented as a vaccine candidate in mammalian

hosts like mice and cattle.

Acknowledgements The authors thank the Director, Indian Veterinary

Research Institute, Izatnagar for providing necessary

Fig.5 — : Western blot analysis of expressed Omp16-L7/L12

protein with and without RFP fusion. The HEK-293 cells were

transfected with either pDsRed-Omp16-L7/L12-RFP or pDsRed-

Omp16-L7/L12-Stop plasmid and cell lysate was probed with

hyperimmune sera against L7/L12 and detected using HRPO

labelled anti-rabbit antibody. Lane 1: Lysate with pDsRed-

Omp16-L7/L12-Stop plasmid; Lane 2: Pre-stained protein

molecular weight marker; Lane 3: cell control; Lane 4: Lysate

with pDsRed-Omp16-L7/L12-RFP plasmid.

Fig. 4 — : (a) Vector map of pDsRed-Omp16-L7/L12 plasmid

without RFP fragment. (b) Restriction endonuclease analysis of

pDsRed-Omp16-L7/L12-stop with PstI (lane 2). Lane 1: λ DNA

double digested with EcoRI and HindIII as marker.


295

facilities to carry out this work. This study was

supported by National Fund for Basic, Strategic and

Frontier Application Research in Agriculture

(NFBSFARA), Indian Council of Agricultural

Research (ICAR), Government of India.

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