Escherichia coli producing heparinase, construction method and application thereof

A technology of Escherichia coli and its construction method, applied in the field of Escherichia coli producing heparanase and its construction, can solve the lack of systematic research on the production technology of heparinase and heparin drugs produced by recombinant bacteria And other issues

Active Publication Date: 2018-11-16
SHENZHEN HEPALINK PHARMA GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to the current situation, although my country is a major exporter of heparin raw materials, there has been a lack of systematic research on the production technology of heparin drugs. At present, my country's heparin drugs still need to rely on imports or production from joint ventures. Production research has important industrial application prospects
[0004] However, many studies on heparanase-producing microorganisms have been reported in the past ten years, generally about the heparanase produced by Flavobacterium heparinus. After verification by the NCBI database, so far, no heparanase gene has been used. heparanase

Method used

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  • Escherichia coli producing heparinase, construction method and application thereof
  • Escherichia coli producing heparinase, construction method and application thereof
  • Escherichia coli producing heparinase, construction method and application thereof

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preparation example Construction

[0080] In the present invention, the preparation method of the heparinase-producing Escherichia coli seed liquid preferably includes: inoculating the heparinase-producing Escherichia coli in a seed medium for shaking culture to OD 600 The value is 1 to 2. In the present invention, the conditions of the shaking culture include: the temperature of the shaking culture is preferably 35 to 37° C., and the rotation speed of the shaking culture is preferably 150 to 250 rpm, more preferably 180 to 220 rpm, and most preferably 200 rpm.

[0081] In the present invention, the seed culture medium preferably uses water as the solvent, and preferably includes: 10-15g tryptone, 5-10g yeast extract and 10-15g sodium chloride per liter; more preferably 11-14g tryptone , 6-9g yeast extract and 11-14g sodium chloride; most preferably including 12-13g tryptone, 7-8g yeast extract and 12-13g sodium chloride. In the present invention, the pH value of the seed culture medium is preferably 7.0-7.2.

[00...

Embodiment 1

[0093] Acquisition and preservation of engineered bacteria

[0094] 1. Construction of engineering bacteria

[0095] 1. Using Raoultella sp.NX-TZ-3-15 with the deposit number of CGMCC No. 13723, the bacterial genome DNA rapid extraction kit was used to extract the whole genome DNA of the bacteria.

[0096] 2. According to the sequence of the heparinase produced by the bacteria and the related gene sequence of the plasmid pBENT, according to the principle of seamless cloning, PCR primers are designed in the multiple cloning site region of the plasmid, including the upstream primer SEQ ID NO of the target gene. 1 and the downstream primer SEQ ID NO. 2 and the upstream primer SEQ ID NO. 3 and downstream primer SEQ ID NO. 4 of the plasmid.

[0097] 3. Use the PCR primers designed in step 2 for PCR amplification:

[0098] (1) PCR amplification of target gene: template target gene DNA 100ng / μL 1μL, primer SEQ ID NO.1 is 10μM 0.5μL, SEQ ID NO.2 is 10μM 0.5μL, PCR Premix (2x) enzyme 25μL, ddH ...

Embodiment 2

[0107] Application of engineering bacteria to produce heparinase

[0108] 1. Preparation of culture medium

[0109] Seed culture medium (pH7.0): Take 10g tryptone, 5g yeast extract and 10g sodium chloride, dissolve in water and dilute to 1L; sterilize at 121°C for 30min.

[0110] Fermentation medium (pH7.0): Take 10g tryptone, 5g yeast extract, 10g sodium chloride, dissolve in water and dilute to 1L; sterilize at 121°C for 30min.

[0111] 2. Application of engineered bacteria to produce heparinase

[0112] 1. Inoculate Escherichia coli BLR(DE3)-pBENT-H1 to the seed medium, and shake culture (200r / min) at 35℃ to OD 600nm =1, which is the seed liquid.

[0113] 2. Use a pipette to inoculate the seed solution of step 1 into 10 mL fermentation medium (50 mL shake flasks can be used) at an inoculum amount of 10% (v / v) to obtain OD 600nm =0.2 fermentation initial system; the fermentation process is as follows: first 37°C shaking culture (200r / min) for 2 hours, then add IPTG to the final con...

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Abstract

Belonging to the technical field of construction of genetic engineering bacteria, the invention provides a Escherichia coli BLR(DE3)-pBENT-H1 strain producing heparinase. The preservation number of the strain is CGMCC No.15819. The Escherichia coli producing heparinase provided by the invention can produce heparinase with enzyme activity of 2540U / L, the obtained heparinase is subjected to enzymolysis of heparin, the obtained ultra-low molecular weight heparin has a weight-average molecular weight of 1419Da, and the low molecular weight heparin has an Anti-FXa / Anti-FIIa titer ratio of 28.4.

Description

Technical field [0001] The invention belongs to the technical field of the construction of genetic engineering bacteria, and specifically relates to a strain of Escherichia coli producing heparinase and a construction method and application thereof. Background technique [0002] Heparin and its structural analogues Heparan sulfate is a long-chain polysaccharide composed of sulfated glucosamine and hexuronic acid, which is a kind of mucopolysaccharide. Since 1935, heparin and heparan sulfate have been formally used as clinical anticoagulants, becoming the second largest category of natural product drugs after insulin, with an annual global output value of 10 billion U.S. dollars, and they are widely used in the treatment of thromboembolism and fulminant flow. Brain, sepsis, nephritis, acute myocardial infarction, arteriosclerosis and other diseases. However, clinical applications have shown that heparin therapy is often accompanied by many different side effects, such as bleeding...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/56C12N15/70C12N9/24C12R1/19
CPCC12N9/2402C12Y302/01019
Inventor 赵丽青蒋莹子叶灿明杨海燕
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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