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7alpha-hydroxyl steroid dehydrogenase gene optimized by codon

A hydroxysteroid and codon optimization technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve the problems of low yield and complex 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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  • 7alpha-hydroxyl steroid dehydrogenase gene optimized by codon
  • 7alpha-hydroxyl steroid dehydrogenase gene optimized by codon
  • 7alpha-hydroxyl steroid dehydrogenase gene optimized by codon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Optimum Design and Synthesis of 7α-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

CTC

CTG

Leu

...

Embodiment 2

[0020] Example 2: Construction of the recombinant expression vector pGEX-6p-1-7α-HSDH of the optimized 7α-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] Utilize restriction endonuclease BamHI / NotI to double digest the optimized gene and reclaim the target gene fragment; use restriction endonuclease BamHI / NotI to double digest the prokaryotic expression vector pGEX-6p-1 and reclaim the target vector fragment; connect, Escherichia coli was transformed by heat shock method; positive clones were screened by PCR and then verified by sequencing to obtain the recombina...

Embodiment 3

[0026] Example 3: Expression of optimized 7α-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 3 minutes to collect the cells, and ultrasonically break the wall at 300W for 5 minutes to clarify; centrifuge at 14,000rpm at 4°C for 15 minutes to collect the supernatant; medium (purchased from GE healthcare company) at 4°C for 2 hours to bind the GST fusion protein to the glutathione agarose gel, and then washed three times with pre-cooled five times the volume of 0.25% Tween 20 in PBS (100mM). Wash three times with five volumes of pre-cooled PBS (100mM). The glutathione agarose gel containing the GST fusion protein was directly digested ...

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Abstract

The invention discloses 7alpha-hydroxyl steroid dehydrogenase gene optimized by a codon, a recombinant expression vector pGEX-6p-1-7alpha-HSDH containing the optimized gene, and escherichia coli containing the recombinant expression vector. The 7alpha-hydroxyl steroid dehydrogenase gene optimized by the codon is used to carry out GST (glutathione S-transferase) fusion expression with a prokaryotic expression vector pGEX-6p-1 in the escherichia coli. A lot of target proteins with activity and intact structure can be generated within a short time, and the purification method of GST fused protein is simple and fast, thereby rapidly obtaining the target protein with activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 7α-hydroxysteroid dehydrogenase (7α-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绍勇
Owner SHANGHAI KAIBAO PHARMA
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