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Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene

A hydroxysteroid and codon optimization technology, applied in the field of 7β-hydroxysteroid dehydrogenase (7β-HSDH) gene, can solve the problems of low yield and complicated chemical synthesis process, and achieve the effect of simple and fast purification method

Inactive Publication Date: 2012-12-19
SHANGHAI KAIBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the process of chemical synthesis is complex and the yield is low

Method used

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  • Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene
  • Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene
  • Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Optimum Design and Synthesis of 7β-Hydroxysteroid Dehydrogenase Gene

[0014] Analysis of rare codons: rare codons are mainly analyzed by the software E.coli Codon Usage Analysis2.0 (Morris Maduro, reference: Analysis and Predictions from Escherichia coli sequences in: Escherichia coli and Salmonella, Vol.2, Ch.114:2047- 2066, 1996, Neidhardt FC ed., ASM press, Washington, D.C.) analyzed the codons whose usage frequency in Escherichia coli was lower than the threshold (10‰).

[0015] Optimize rare codons: optimize the codons whose usage frequency is lower than the threshold according to the usage frequency of different codons in E. coli. The optimization results are as follows:

[0016] Optimized codons used in the present invention

[0017] before optimization

Optimized

amino acid

before optimization

Optimized

amino acid

AAG

AAA

Lys

CTAs

CTG

Leu

ACA

ACC

Thr

CTT

CTG

Leu

...

Embodiment 2

[0020] Example 2: Construction of the recombinant expression vector pGEX-6p-1-7β-HSDH of the optimized 7β-hydroxysteroid dehydrogenase gene and the acquisition of Escherichia coli containing the recombinant expression vector

[0021] ⑴ strains and plasmids

[0022] Escherichia coli: BL21 strain, purchased from Tianjin Bomeike Biotechnology Co., Ltd.

[0023] Expression vector pGEX-6p-1: purchased from GE (GE Healthcare).

[0024] (2) Construction of recombinant expression vector and acquisition of Escherichia coli containing the recombinant expression vector

[0025] Use restriction endonuclease BamHI / NotI to double digest the optimized gene and recover the target gene fragment; use restriction endonuclease BamHI / NotI to double digest the prokaryotic expression vector pGEX-6p-1 and recover the target vector fragment; ligate, Escherichia coli was transformed by heat shock method; positive clones were screened by PCR, and then verified by sequencing to obtain recombinant expre...

Embodiment 3

[0026] Example 3: Expression of optimized 7β-hydroxysteroid dehydrogenase gene in Escherichia coli

[0027] The successfully transferred E. coli recombinant strains were added to the LB liquid medium at an addition rate of 5%, and cultured at 220 rpm and 37°C. When the OD600 is 0.5, induce with IPTG at 16°C for 12 hours to make Escherichia coli produce GST fusion protein.

[0028] Centrifuge the induced Escherichia coli at 10,000rpm for 3min to collect the bacterial cells, and ultrasonically break the cell wall at 300W for 5min to clarify; centrifuge at 14,000rpm at 4°C for 15min to collect the supernatant; medium (purchased from GE healthcare company) for 2 hours at 4°C to bind the GST fusion protein to the glutathione agarose gel, and then wash three times with pre-cooled five-fold volume of 0.25% Tween 20 in PBS (100mM) , washed three times with pre-cooled five-fold volume of PBS (100mM). The glutathione agarose gel containing the GST fusion protein was directly digested ...

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Abstract

The invention discloses a codon-optimized 7 beta-hydroxy steroid dehydrogenase gene, a recombination expression vector Pgex-6P-1-7 beta-HSDH containing an optimized gene and escherichia coli containing the recombination expression vector. Glutathione S-transferase (GST) fusion expression of the codon-optimized 7 beta-hydroxy steroid dehydrogenase gene and a prokaryotic expression vector Pgex-6P-1 in the escherichia coli is adopted, so that a large quantity of active target proteins with an integral structure can be generated in a short time; and a method for purifying the GST fusion expression protein is simple and quick, so that the active target proteins can be quickly obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 7β-hydroxysteroid dehydrogenase (7β-HSDH) gene optimized according to the codon preference of Escherichia coli. Background technique [0002] Bear bile is a precious Chinese medicinal material, which is used to treat gallstone disease and various acute and chronic liver diseases, and has a good therapeutic effect. Ursodeoxycholic acid (3α-7β-dihydroxy-5β-cholanic acid, hereinafter referred to as UDCA) and taurine / glycined ursodeoxycholic acid (T / GUDCA) are the main active ingredients of the traditional Chinese medicine bear bile. However, since the traditional Chinese medicine bear bile powder is mainly obtained by "draining live bears for bile extraction", this violates the Wildlife Protection Law. And it is considered a very inhumane way of obtaining it. In addition, the chemical synthesis process is complex and the yield is low. However, there are many bile acids in animal bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 王伯初娄德帅谭君祝连彩季庆治岑小惜刘宜善
Owner SHANGHAI KAIBAO PHARMA
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