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Recombination human alkaline fiber forming cell growth factor gene and its nonfusion expression product, production method and application

A fibroblast, growth factor technology, applied in the field of genetic engineering applications, can solve the problems of non-expression, fast mRNA translation, slow mRNA translation, etc., and achieve the effects of high translation initiation efficiency, high expression, and improved expression rate.

Inactive Publication Date: 2004-07-21
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when a foreign gene is expressed in a certain expression system, the mRNA with a high proportion of codons preferred by the expression system will be translated faster, while the mRNA with a high proportion of rare codons will be translated slowly
However, when there are many rare codons in the mRNA, the expression efficiency of the foreign gene is often low or the serious consequences are not expressed.

Method used

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  • Recombination human alkaline fiber forming cell growth factor gene and its nonfusion expression product, production method and application
  • Recombination human alkaline fiber forming cell growth factor gene and its nonfusion expression product, production method and application
  • Recombination human alkaline fiber forming cell growth factor gene and its nonfusion expression product, production method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Preparation of hbFGF gene modified in TIR region

[0057] Design upstream primers as follows:

[0058] F1: GAA GCT GCT AGT ATT ACG ACA CTG CCG GCT CTG CCG GAA GAT GGT

[0059] GGT AGC GGT GCT

[0060] F2: GAA GCT GCT GGT AGT ATT ACG ACG CTG CCG GCC CTG CCG GAA GAT

[0061] GGT GGT AGT GGT GCA

[0062] F3: GAA GCT GCT GGT AGT ATT ACT ACA CTG CCG GCC CTG CCG GAA GAT GGT

[0063] GTT AGC GGT GCG

[0064] F4: GAA GCT GCT GGT AGT ATT ACT ACG CTG CCG GCT CTG CCG GAA GAT GGT

[0065] GGT AGT GGT GCG

[0066] F5: GAA GCT GCT GGT AGC ATT ACA ACT CTG CCG GCA CTG CCG GAA GAT

[0067] GGT GGT AGT GGT GCC

[0068] F6: GAA GCT GCT GGT AGT ATT ACA ACT CTG CCG GCA CTG CCG GAA GAT

[0069] GGT GGT AGT GGT GCA

[0070] F7: GAA GCT GCT GGT AGC ATT ACG ACC CTG CCG GCG CTG CCG GAA GAT

[0071] GGT GAT AGT GGT GCT

[0072] Design downstream primers as follows:

[0073] R: GACA TTA TCA GCT CTT AGC AGA CAT TGG

[0074] The base sequence in the box is th...

Embodiment 2

[0081] Example 2: Construction of pET-JN and digestion of recombinants

[0082] The digested and purified DNA fragments of vector plasmid DNA pET-3c / Nde I / BamH I and hbFGF / Nde I / Bgl II were ligated in a constant temperature water bath at 16°C for 30 minutes according to conventional methods, and when the ligation reaction was completed, , to transform DH5α competent cells, take 100 μL of the transformation solution and apply it to AMP-resistant LB plate, and culture it in a 37°C incubator for 12-16h. See the build process figure 1

[0083] Use a sterile toothpick to pick 3-5 colonies grown on each resistant plate, inoculate them into test tubes of 5 mL liquid LB medium with 100 μg / mL AMP, and culture with shaking at 37°C and 200 rpm / min for 12-16 hours Afterwards, use the alkali method to extract a small amount of plasmids, extract the plasmids for BamH I+Nde I double enzyme digestion, identify by electrophoresis, and select recombinants.

[0084] It can be seen from the f...

Embodiment 3

[0085] Example 3: Expression analysis of non-fusion hbFGF protein

[0086] The DH5α cells containing the recombinant plasmid pET-JN series identified by enzyme digestion were amplified in 5 mL LB liquid medium containing 100 μg / ml AMP, the plasmid was extracted by the alkaline method, and transformed into BL21(DE3)pLysS cells. Spread on LB culture plates containing 100 μg / ml AMP and chloramphenicol (Chloromycetin, hereinafter referred to as CHL) double antibody for culture, and culture overnight at 37°C. Pick positive colonies and inoculate them into 50ml LB test tubes with 100μg / ml AMP and 100μg / ml CHL for expression test, culture at 37°C until OD 600 was 0.8, IPTG (isopropyl-β-D-thiogalactoside) was added for induction for 4 hours.

[0087] In order to analyze the more detailed situation of the obtained bacterial strain expressing non-fusion hbFGF, the obtained bacterial strain with expression was further expanded and cultured, and after induction, the bacterial cells were ...

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Abstract

A gene for the non-fusion recombination of human alkaline fibroblast growth factor, its carrier and prokaryotic expression system, said non-fusion recombined human alkaline fibroblast growth factor and the application of said factor in preparing medicines for treating nervous system injury are disclosed.

Description

technical field [0001] The invention belongs to the application field of genetic engineering. The invention relates to a modified human basic fibroblast growth factor gene, an expression carrier and a prokaryotic expression system containing the gene. The invention also relates to the non-fusion expression product of the modified gene and the application of the product in the preparation of medicines for treating nervous system damage and other diseases. Background technique [0002] Basic Fibroblast Growth Factor (bFGF) is one of the fibroblast factor family. Fibroblast growth factor (FGF) was originally isolated from bovine pituitary gland tissue as a factor having growth-promoting activity on balb / c3T3 cells (D. Gospodarrowicz, Nature 249: 123, 1974). The factor is sensitive to acid and temperature, has a high (basic) isoelectric point, pi=9.3-9.6. [0003] bFGF can selectively stimulate biological responses in many cells including endo...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61P9/10A61P25/02A61P27/16C07H21/04C07K14/50C12N15/12C12N15/70C12P19/34
Inventor 陈小佳孙奋勇林剑洪岸谢秋玲张玲汪炬李志英
Owner JINAN UNIVERSITY
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