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Preparation method of recombinant human acidic fibroblast growth factor (haFGF) protein

A fibroblast and growth factor technology, applied in the field of genetic engineering, can solve the problems of low haFGF content and unfavorable drug development, and achieve the effects of facilitating promotion, stable product quality, and simple preparation methods

Active Publication Date: 2014-06-25
杨霞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that the low content of natural haFGF is not conducive to drug development, the present invention provides a preparation method of recombinant human acidic fibroblast growth factor protein

Method used

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  • Preparation method of recombinant human acidic fibroblast growth factor (haFGF) protein
  • Preparation method of recombinant human acidic fibroblast growth factor (haFGF) protein
  • Preparation method of recombinant human acidic fibroblast growth factor (haFGF) protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Preparation method of recombinant human acidic fibroblast growth factor protein:

[0026] 1~2) Construct the backup vector of pET-26b-PDI-pp

[0027] Design primers for PDI, the corresponding nucleotide sequence is shown in SEQ ID NO.2, which needs to contain restriction sites, and the arrangement from 5`-3` is as follows:

[0028]

[0029] Primers were diluted to 50 pmol / μl, followed by the PCR step.

[0030] PCR reaction system 50ul:

[0031] wxya 2 O: 40ul

[0032] 10*PCR BUFFER 5ul

[0033] dNTP 1ul

[0034] Template: 1ul

[0035] Up / down stream primers: 1ul

[0036] pfu enzyme 1ul

[0037] PCR reaction conditions (Tag enzyme):

[0038] 94℃ 8min

[0039] 94°C 30s

[0040] 65℃ 30s

[0041] 72℃ 1min 30 cycles

[0042] 72°C 10min

[0043] Agarose gel electrophoresis detects that the PDI band is located at about 1600bp, and the target gene is recovered from the gel,...

Embodiment 2

[0071] The preparation method of recombinant human acidic fibroblast growth factor protein, its steps are:

[0072] ① According to the full-length sequence of human acidic fibroblast growth factor haFGF, primers were designed and cloned to obtain the original gene fragment, and the codons unfavorably expressed by Escherichia coli existed in it were replaced to obtain the optimized haFGF gene. The acid sequence is shown in SEQ ID NO.1; the original gene fragment of molecular chaperone PDI is obtained by cloning, and the codons unfavorably expressed by Escherichia coli present in it are replaced to obtain the optimized PDI gene, and the nucleoside The acid sequence is shown in SEQ ID NO.2;

[0073] ②Optimized haFGF gene 3' end to add HIS 6 , the gene fragment haFGF-HIS was constructed 6 ; Add the pp protease cleavage site sequence to the 5' end of the optimized PDI gene to construct the gene fragment pp-PDI; then connect the gene fragment pp-PDI to the gene fragment haFGF-HIS ...

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Abstract

The invention relates to a preparation method of a recombinant protein in the field of gene engineering and in particular relates to a preparation method of a recombinant human acidic fibroblast growth factor (haFGF) protein. The preparation method comprises the following steps: (1) designing primers according to the full-length sequence of the haFGF to obtain an original gene segment through cloning, and replacing a codon which exists in the original gene segment and is unfavourable to be expressed by escherichia coli, thus obtaining an optimized haFGF gene, wherein the nucleotide sequence of the optimized haFGF gene is shown in SEQ ID NO.1; cloning to obtain an original gene segment of a molecular chaperone PDI (protein disulfide isomerase), and replacing a codon which exists in the original gene segment and is unfavourable to be expressed by escherichia coli, thus obtaining an optimized PDI gene, wherein the nucleotide sequence of the optimized PDI gene is shown in SEQ ID NO.2; (2) linking and cloning the optimized haFGF gene, the optimized PDI gene, tag sequences and a protease cutting site sequence into a vector to obtain an efficient expression vector through construction; or firstly constructing standby vectors of part of the sequences, and then linking the remaining sequences on part of the sequences.

Description

technical field [0001] The invention relates to a method for preparing a recombinant protein in the field of genetic engineering, in particular to a method for preparing a recombinant human acidic fibroblast growth factor protein. Background technique [0002] In the 1930s, scientists discovered that there was an active substance that could promote the growth of fibroblasts in human brain and pituitary tissue extracts. By the 1970s, this substance was isolated and purified. In view of the role of this substance, it was named fibroblast growth factor (fibroblast growth factor, FGF). Later, these factors were found in various organs and tissues, collectively referred to as members of the FGFs family. Different FGFs play an important role in human embryonic development and differentiation as intercellular signaling molecules. So far, a total of about 23 human FGFs have been discovered. Most FGFs have signal peptides at their N-terminals to help mature proteins secrete extra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12C12N15/10
Inventor 杨霞
Owner 杨霞
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