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Genetic engineering bacteria and application thereof, method for producing prostaglandin E2

A prostaglandin and coding gene technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of cumbersome operation, many impurities and by-products, and inconvenient acquisition of process materials, and achieve high efficiency and preparation The effect of simple method

Active Publication Date: 2020-07-10
HEFEI KNATURE BIO PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process material is inconvenient to obtain, high cost, cumbersome operation, low conversion rate, many impurities and by-products, and subsequent purification is complicated

Method used

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  • Genetic engineering bacteria and application thereof, method for producing prostaglandin E2
  • Genetic engineering bacteria and application thereof, method for producing prostaglandin E2
  • Genetic engineering bacteria and application thereof, method for producing prostaglandin E2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 prostaglandin H synthetase gene and prostaglandin E synthetase gene synthesis

[0026] According to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), the inventors published the prostaglandin H synthase of Gracilaria genus Gracilaria true, the human prostaglandin E synthase, and the prostaglandin E synthase of cynomolgus monkey. The nucleotide sequence of prostaglandin E synthase was codon-optimized for the Escherichia coli expression system to obtain the corresponding optimized nucleotide sequence, and entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to perform gene synthesis; the optimized nucleotide sequences were respectively for:

[0027] The nucleotide sequence of the coding gene of the prostaglandin H synthase (referred to as the PGHS coding gene for short) of the optimized Gracilaria genus after optimization is shown in SEQ ID No.1, and its amino acid sequence is:

[0028]

[0029] The nucleotide sequence of the gene encoding human prostagland...

Embodiment 2

[0033] The construction of embodiment 2 prostaglandin H synthase PGHS and prostaglandin E synthase PGES expression plasmid

[0034] 1 Construction of plasmid pET28a-PGHS

[0035] 1.1 Using the PGHS coding gene synthesized in Example 1 as a template, amplify using the PGHS-F / PGHS-R primer pair to obtain a PGHS fragment with a size of 1738bp;

[0036] 1.2 Take the expression vector pET28a, and use restriction endonucleases NcoI and BamHI to digest to obtain the vector fragment 1; use the gel recovery and purification kit to perform gel recovery and purification on the vector fragment 1 and PGHS fragment, and use the GBclonart seamless cloning kit without Slit cloning recombination, transformation into competent cells of Escherichia coli TG1 strain, and finally construction of plasmid pET28a-PGHS;

[0037] 2 Construction of co-expression plasmid pET28a-PGHS-mPGES1 and co-expression plasmid pET28a-PGHS-mPGES2

[0038] 2.1 Using the mPGES1 encoding gene synthesized in Example 1 a...

Embodiment 3

[0046] The acquisition of embodiment 3 recombinant strains

[0047] 1 Preparation of BL21(DE3) competent cells:

[0048] Pick a loop of Escherichia coli BL21 (DE3) from a glycerol tube, streak and purify it on an LB plate, place it in a 37°C incubator, and culture it for 16 hours. extract 5g / L, sodium chloride 10g / L) in a test tube, cultured at 37°C and 250rpm for 16h, then transferred to a shaker flask containing 50mL LB medium according to the transfer amount of 2%, at 37°C Grow to OD 600 = 0.8, collect the bacteria and put them on ice to pre-cool, centrifuge to remove the supernatant, and use 0.1MCaCl in ice bath 2 Wash the bacteria twice with the solution, and finally use 2ml of pre-cooled 0.1M CaCl containing 15% glycerol 2 Resuspend the bacteria in the solution, aliquot and place on ice to obtain Escherichia coli BL21(DE3) competent cells;

[0049] 2 Preparation of recombinant Escherichia coli (Escherichia coli) strain BL21(DE3) / pET28a-PGHS-mPGES1:

[0050] Take 100...

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Abstract

The present invention discloses recombinant escherichia coli. The recombinant escherichia coli over-expresses an encoding gene of prostaglandin H synthase and an encoding gene of prostaglandin E synthase. The present invention also discloses an application of the recombinant escherichia coli in production of prostaglandin E2. The present invention also discloses a method for producing the prostaglandin E2. Arachidonic acid is used as a substrate and the recombinant escherichia coli is used to produce the prostaglandin E2. The escherichia coli strain capable of expressing the prostaglandin H synthase and the prostaglandin E2 synthetase is constructed and the expressed enzymes can directly catalyze conversion of the arachidonic acid into the prostaglandin E2.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered bacterium and its application, and a method for producing prostaglandin E2. Background technique [0002] Prostaglandin E2 (PGE2) is a metabolite of arachidonic acid cyclooxygenase, a 20-carbon unsaturated fatty acid, and a kind of prostaglandin (PG), with the molecular formula C 20 h 32 o 5 , molecular weight 352, white crystal, melting point 68-69 ℃, soluble in organic solvents such as ethyl acetate, acetone, ether methanol and ethanol, insoluble in water. PGE2 is an important cell growth and regulatory factor. Its physiological functions are mainly manifested in: dilating blood vessels, increasing organ blood flow, reducing peripheral resistance of blood vessels, and lowering blood pressure; relaxing bronchial smooth muscle, reducing ventilation resistance; inhibiting gastric acid secretion, promoting gastric Intestinal smooth muscle peristalsis; has im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P31/00C12R1/19
CPCC12N9/0083C12N9/90C12P31/00C12Y114/99001C12Y503/99003
Inventor 王伟王康林袁哲诚金永红王颀彤贾和平胡志浩
Owner HEFEI KNATURE BIO PHARM CO LTD
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