real time bio-database teaching through internet
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Experiment 9
Real time database teaching through
internet (Bioinformatics)
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How to get gene information from
NCBI database
and construct a fusion protein
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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
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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
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NCBI introduction
NCBI introduction
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NCBI introduction
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NCBI introduction
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NCBI introduction
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NCBI introduction
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NCBI introduction
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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
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Search paper , Nucleotide and protein
How to use NCBI
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• Click on“Search”: PubMed search paper ;
Protein search protein; Nucleotide search sequence;
Structure;
Genome;
Gene
Search paper
How to use NCBI
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For example 1 : mouse acid phosphatase
Choose PubMed
Input : mouse acid phosphatase
Search Nucleotide or protein
How to use NCBI
H t NCBI
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For example 1 : mouse acid phosphatase
How to use NCBI
H t NCBI
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For example 2 :protein of pincada fucata
How to use NCBI
H t NCBI
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For example 2 :protein of pincada fucata
How to use NCBI
H t NCBI
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For example 3:download rice waxy gene
How to use NCBI
H t NCBI
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For example 3:download rice waxy gene
How to use NCBI
How to use NCBI
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For example 3:download rice waxy gene
How to use NCBI
How to use NCBI
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For example 3:download rice waxy gene
How to use NCBI
How to use NCBI
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For example 3:download rice waxy gene
How to use NCBI
How to use NCBI
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Blast
How to use NCBI
How to use NCBI
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Blast
How to use NCBI
How to use NCBI
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Blast
How to use NCBI
How to use NCBI
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Blast
How to use NCBI
How to use NCBI
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How to use NCBI
How to use NCBI
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Blast
How to use NCBI
How to use NCBI
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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
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How to use NCBI
How to use NCBI
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How to use NCBI
How to use NCBI
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How to use NCBI
How to use NCBI
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Analysis of Chromosome LocationExample 1小鼠染色体上定位SMCX基因
How to use NCBI
How to use NCBI
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How to use NCBI
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Example 2 小鼠染色体上定位SMCX基因
How to use NCBI
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How to use NCBI
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How to use NCBI
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How to use NCBI
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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
Construction of fusion protein
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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.
Construction of fusion protein
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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).
Construction of fusion protein
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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.
Construction of fusion protein
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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)
Construction of fusion protein
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pET-30 and pGEX series vector
Construction of fusion protein
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Construction of fusion protein
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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
Construction of fusion protein
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Construction of fusion protein
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Construction of fusion protein
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Construction of fusion protein
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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
Construction of fusion protein
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• 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;
Construction of fusion protein
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• 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;
Construction of fusion protein
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• 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)