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Method for preparing prostaglandin e1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin e synthetase-1

A technology of genetic engineering and prostaglandin, applied in the field of preparation of prostaglandin E1, can solve the problems of reaction system pollution, large demand for fresh enzymes, excessive product impurities, etc.

Active Publication Date: 2021-08-20
CHANGCHUN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It solves the problems that the reaction system is easily polluted, the demand for fresh enzyme is too large, and the product has too many impurities in the industrial production of prostaglandin E1.

Method used

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  • Method for preparing prostaglandin e1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin e synthetase-1
  • Method for preparing prostaglandin e1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin e synthetase-1
  • Method for preparing prostaglandin e1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin e synthetase-1

Examples

Experimental program
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Effect test

preparation example Construction

[0036] The synthetic mechanism of the preparation method of prostaglandin E1 provided by the invention is:

[0037] Dihomo-γ-linolenic acid (DGLA) is catalyzed by cyclooxygenase-1 (COX-1) to generate PGG1, hydrogen atoms are extracted from C-9 and C-11 of dihomo-γ-linolenic acid, and O 2 Form a C-9 / C11 endoperoxide with the generated carbon radical, form a carbon-carbon double bond through cyclization between C-8 and C-12, and then add an O to the C-15 position again 2 , and then form PGG1. PGG1 can then be reduced by the peroxidase activity of cyclooxygenase (thus in the presence of a reducing agent such as glutathione) to form PGH1 (PGH1 is an extremely unstable prostaglandin species). PGH1 is converted into PGE1 by prostaglandin E synthase-1 (mPGES-1). The theoretical synthesis pathway of prostaglandin E1 is as follows: figure 1 shown. The specific synthetic route of the present invention is as figure 2 shown.

Embodiment 1

[0039] Example 1 Recombinant plasmid construction and identification

[0040] Obtain the coding regions of the sheep cyclooxygenase-1 and prostaglandin E synthase-1 genes by searching in NCBI. The gene ID of cyclooxygenase-1 in NCBI is 443551, and the amino acid reference sequence code is XP_027821574.1. Prostate Gene E synthetase-1 in NCBI has a gene ID of 9536 and an amino acid reference sequence code of NP_004869.1. Find the amino acids and their corresponding codons that cannot be expressed in E. coli, and then optimize the cyclooxygenase-1 and prostaglandin E synthase-1 genes to replace the codons that cannot be expressed in E. coli to ensure that the optimized genes can Expressed and active in E. coli. The optimized gene sequences of cyclooxygenase-1 and prostaglandin E synthase-1 are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0041] At the two ends of the optimized cyclooxygenase-1 and prostaglandin E synthetase-1 gene sequences, the restriction site sequences of HindⅢ e...

Embodiment 2

[0043] Example 2 Preparation and Identification of Recombinant Expression Strains

[0044] 1. Preparation of Escherichia coli BL21(DE3) Competent Cells

[0045] (1) Strain activation: Escherichia coli BL21(DE3) stored in an ultra-low temperature refrigerator at -180°C was inoculated into 5mL LB liquid medium at an inoculation ratio of 1:50, and the medium was placed in a constant temperature shaker at 37°C , adjust 160rpm / min shaking culture overnight (12-16 hours), to OD 600 = about 0.4.

[0046] (2) Strain expansion: Take 100 μl of the strain activated in the previous step, add it to 5 ml of sterilized LB liquid medium, and shake and cultivate in a constant temperature shaker at 37° C. and 160 rpm / min for 2 hours.

[0047] (3) Use a pipette gun to take 1ml of the bacterial solution amplified in the previous step, dispense it into 1.5ml EP tubes, cool in an ice bath for 30min, and collect the bacterial cells at 4°C and 4000rpm for 10min.

[0048] (4) Discard the supernatan...

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Abstract

The invention discloses a method for preparing prostaglandin E1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin E synthetase-1, belonging to the field of bioengineering. The method expresses the relevant enzymes required for the synthesis of prostaglandin E1 by means of prokaryotic expression, that is, cyclooxygenase-1 (COX-1) and prostaglandin E synthase-1 (mPGES-1), and utilizes enzymes and substrates reaction to synthesize prostaglandin E1. Through this method, prostaglandin synthase can be expressed in large quantities and prostaglandin E1 can be synthesized, which can avoid the problem of easy contamination of tissue enzymes taken from living bodies in industrial production. At the same time, the types of enzymes expressed by the prokaryotic are relatively single, and the concentration of the expressed enzymes is relatively high, and the organic matter impurities in the E. coli system are relatively small, and the product impurities after the enzymatic reaction are relatively small, which is conducive to the purification and utilization of prostaglandin E1. Synthetic prostaglandin E1 has opened up a new path.

Description

technical field [0001] The invention relates to the field of biological engineering, in particular to a method for preparing prostaglandin E1 by using genetic engineering cyclooxygenase-1 and genetic engineering prostaglandin E synthetase-1. Background technique [0002] Prostaglandins (PGs) are a group of physiologically active lipid compounds called eicosanoids that have various hormonal effects in animals. Prostaglandin E1 (Prostaglandin E1) is a member of the prostaglandin family, also known as alprostadil, is a biologically active substance widely present in the body, is a recognized endogenous physiologically active substance, and has obvious vasodilation effect. It can also inhibit platelet aggregation, reduce blood viscosity and red blood cell aggregation, and improve microcirculation. In addition, it has a protective effect on vascular endothelium, prevents the formation of atherosclerotic lipid plaques, and improves nerve damage. It is used clinically. It is widel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12P31/00C12N15/70C12N9/90C12N9/02C12N15/61C12N15/53
CPCC12N9/0083C12N9/90C12N15/70C12P31/00C12Y114/99001C12Y503/99003
Inventor 刘建姚震曹佩佩邢琦文小雪吉鑫马铭瑞
Owner CHANGCHUN UNIV OF SCI & TECH
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