real time bio-database teaching through internet

8/13/2019 Real Time Bio-Database Teaching Through Internet

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Department of iological Sciences and iotechnology

Experiment 9

Real time database teaching through

internet (Bioinformatics)




How to get gene information from

NCBI database

and construct a fusion protein

http://dict//key.0895DFE8DB67F9409DB285590D870EDD/fusion%20protein







outline

• NCBI introduction

• How to use NCBI to get gene info: Search paper , Nucleotide and protein

Blast

Alignment

Analysis of Chromosome Location

•

Construction of fusion protein




NCBI introduction

• National Center for

Biotechnology Information

• established on November 4,

1988, as a division of theNational Library of Medicine (NLM)

at the National Institutes of Health

(NIH).

• http://www.ncbi.nlm.nih.gov/

NCBI introduction

http://www.ncbi.nlm.nih.gov/






NCBI introduction

NCBI introduction




NCBI introduction


http://slidepdf.com/reader/full/real-time-bio-database-teaching-through-internet 7/55Department of iological Sciences and iotechnology

NCBI introduction



NCBI introduction



outline



Blast

Alignment


• Construction of fusion protein



Search paper , Nucleotide and protein

How to use NCBI



• Click on“Search”： PubMed search paper ；

Protein search protein； Nucleotide search sequence；

Structure；

Genome；

Gene

Search paper

How to use NCBI



For example 1 : mouse acid phosphatase

Choose PubMed

Input : mouse acid phosphatase

Search Nucleotide or protein

How to use NCBI

H t NCBI



For example 1 : mouse acid phosphatase

How to use NCBI

H t NCBI



For example 2 :protein of pincada fucata

How to use NCBI

H t NCBI




For example 3：download rice waxy gene

How to use NCBI

H t NCBI





How to use NCBI

How to use NCBI




Blast

How to use NCBI

How to use NCBI




How to use NCBI

How to use NCBI




Blast

How to use NCBI

How to use NCBI




Alignment

• One of the cornerstones of modern bioinformatics is the comparison

or alignment of protein sequences and DNA sequences. With the aid

of multiple sequence alignments, biologists are able to study the

sequence patterns conserved through evolution and the ancestral

relationships between different organisms.

• Sequences can be aligned across their entire length (global

alignment) or only in certain regions (local alignment).

• Common used software :clustal clustalw(http://www.ebi.ac.uk/clustalw/)

• Others: Multiple sequence alignment with the Clustal series of

programs. Nucleic Acids Research, 2003, Vol. 31, No. 13.• Bioedit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html

How to use NCBI

How to use NCBI

http://www.ebi.ac.uk/clustalw/

http://www.mbio.ncsu.edu/BioEdit/bioedit.html

http://www.mbio.ncsu.edu/BioEdit/bioedit.html

http://www.ebi.ac.uk/clustalw/




How to use NCBI

How to use NCBI




Analysis of Chromosome LocationExample 1小鼠染色体上定位SMCX基因

How to use NCBI

How to use NCBI




How to use NCBI




Example 2 小鼠染色体上定位SMCX基因

How to use NCBI




How to use NCBI




outline



Blast

Alignment


• Construction of fusion protein






• Fusion proteins, AKA chimeric proteins, are proteins created

through the joining of two or more genes which originally coded for

separate proteins.

• Translation of this fusion gene results in a single polypeptide with

functional properties derived from each of the original proteins.

• Recombinant fusion proteins are created artificially by recombinant

DNA technology for use in biological research or therapeutics.

• Chimeric mutant proteins occur naturally when a large-scale

mutation, typically a chromosomal translocation, creates a novel

coding sequence containing parts of the coding sequences from two

different genes.


http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Fusion_gene

http://en.wikipedia.org/wiki/Polypeptide

http://en.wikipedia.org/wiki/Recombinant_DNA_technology





http://en.wikipedia.org/wiki/Therapeutics


http://en.wikipedia.org/wiki/Mutation

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Translocation

http://en.wikipedia.org/wiki/Translocation

http://en.wikipedia.org/wiki/Chromosome

http://en.wikipedia.org/wiki/Mutation

http://en.wikipedia.org/wiki/Therapeutics


http://en.wikipedia.org/wiki/Polypeptide


http://en.wikipedia.org/wiki/Gene




Recombinant fusion proteins

• A recombinant fusion protein is a protein created through genetic

engineering of a fusion gene.

• This typically involves removing the stop codon from a cDNA

sequence coding for the first protein, then appending the cDNA

sequence of the second protein in frame through ligation or overlap

extension PCR.

• That DNA sequence will then be expressed by a cell as a single

protein. The protein can be engineered to include the full sequence

of both original proteins, or only a portion of either.

• This technique is often used for identification and purification of

proteins, by fusing a GST protein, FLAG peptide, or a hexa-his

peptide (aka: a 6xhis-tag) which can be isolated using nickel or

cobalt resins (affinity chromatography).


http://en.wikipedia.org/wiki/Genetic_engineering










http://en.wikipedia.org/wiki/Codon

http://en.wikipedia.org/wiki/CDNA

http://en.wikipedia.org/wiki/Reading_frame

http://en.wikipedia.org/wiki/Ligase

http://en.wikipedia.org/wiki/PCR

http://en.wikipedia.org/wiki/Protein_expression

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/His-tag








http://en.wikipedia.org/wiki/Affinity_chromatography

http://en.wikipedia.org/wiki/Affinity_chromatography


http://en.wikipedia.org/wiki/FLAG-tag

http://en.wikipedia.org/wiki/GST-tag

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/Protein_expression

http://en.wikipedia.org/wiki/PCR

http://en.wikipedia.org/wiki/Ligase

http://en.wikipedia.org/wiki/Reading_frame

http://en.wikipedia.org/wiki/CDNA

http://en.wikipedia.org/wiki/Codon



http://en.wikipedia.org/wiki/Protein




Chimeric mutant proteins

• Several drugs made from chimeric proteins are

currently available for medical use. Several

chimeric protein drugs are TNFα blockers, such

as Etanercept, Infliximab, and Adalimumab andimmunosuppressants such as Daclizumab and

Basiliximab.


http://en.wikipedia.org/wiki/Drug

http://en.wikipedia.org/wiki/Tumor_necrosis_factor-alpha


http://en.wikipedia.org/wiki/Etanercept

http://en.wikipedia.org/wiki/Infliximab

http://en.wikipedia.org/wiki/Adalimumab

http://en.wikipedia.org/wiki/Immunosuppressants

http://en.wikipedia.org/wiki/Daclizumab

http://en.wikipedia.org/wiki/Basiliximab

http://en.wikipedia.org/wiki/Basiliximab

http://en.wikipedia.org/wiki/Daclizumab

http://en.wikipedia.org/wiki/Immunosuppressants

http://en.wikipedia.org/wiki/Adalimumab

http://en.wikipedia.org/wiki/Infliximab

http://en.wikipedia.org/wiki/Etanercept


http://en.wikipedia.org/wiki/Drug




Prokaryotic expression system

• In lab we commonly construct fusion expression plasmid with

GST tag or His tag in E. coli BL21, induced with IPTG and

finally purified the protein by the Ni column or the GST

column.

• Commonly used Prokaryotic expression plasmid

With His tag of pET series

（ Novagen:http://www.emdbiosciences.com/g.asp?f=NVG/pE

Ttable.html）

With the pGEX family of GST tag（Amersham Biosciences）





pET-30 and pGEX series vector





Eukaryotic expression system

• Construction of eukaryotic expression vector was

mainly used to study gene expression in targeted cells,

protein and protein interactions in cells, impact on

cell growth, cell differentiation and apoptosis, as wellas the construction of transgenic animals.

• The following is a list of three types of eukaryotic

cell expression vector

• pEGFP、pcDAN3.1、pCMV-Myc





Key points

• Select the appropriate expression vector;

• Choose protein coding sequences of the

gene, that is, the "cDS" regional of geneticinformation "FEATURES" from NCBI

For both prokaryotic cell expression vector and eukaryotic cell expression vector





• According to the position of the fusion protein

tag (such as His, GST) or other fusion protein

(such as fluorescent protein), consider the

fusion protein reading frame, design ofappropriate primers (adding several bases to

ensure a correct reading frame), connected

appropriate restriction sites at both ends toensure the correct translation of fusion protein;





• After primer synthesis, PCR, restriction enzyme digestion,

DNA recombination, and transformation process, finally

recombinant plasmid will be gotten. Then transformed to

appropriate cells for expression.

• In E. coli, in accordance with the protocol, usingappropriate conditions, induced the expression in E.

coli BL21, and finally detecting the fusion protein;

•

In eukaryotic cells, choose a suitable systemaccording to the experiment purpose;





• When the fusion protein used for antibody, the full-

length target gene expression product can be chosen,

and also the appropriate gene fragment (need to

consider right section of the target gene to buildfusion protein, the generally consider hydrophilicity

or hydrophobicity and also epitope prediction from

website)

http://dict//key.0895DFE8DB67F9409DB285590D870EDD/genetic%20fragment

http://dict//key.0895DFE8DB67F9409DB285590D870EDD/hydrophilicity

http://dict//key.0895DFE8DB67F9409DB285590D870EDD/hydrophilicity

http://dict//key.0895DFE8DB67F9409DB285590D870EDD/genetic%20fragment



Thank you!

real time bio-database teaching through internet

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