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Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I

A technology of Bacillus subtilis and engineering bacteria, applied in the direction of microorganism-based methods, bacteria, lyase, etc., can solve the problem of no successful expression of heparanase Ⅰ, and achieve high activity and yield

Active Publication Date: 2012-07-04
SHANDONG UNIV
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AI Technical Summary

Problems solved by technology

A large number of protein drugs have been successfully expressed in the Bacillus subtilis expression system, but so far there has been no report on the successful expression of heparanase Ⅰ in this system

Method used

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  • Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I
  • Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I
  • Strain of bacillus subtilis engineering bacteria and application thereof in producing heparinase I

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Embodiment 1

[0030] Example 1 Construction of Bacillus subtilis WB600 (pBE2-S-H), an engineering bacterium of Bacillus subtilis expressing recombinant heparanase I.

[0031] 1. Acquisition of heparanase Ⅰ gene from Flavobacterium heparinus

[0032] Extract the genomic DNA of Flavobacterium heparinus DSMZ 2366, design a pair of primers H1 and H2 according to the gene sequence of heparinase Ⅰ, conduct conventional polymerase chain reaction, amplify the gene sequence of Flavobacterium heparinus heparinase Ⅰ, and obtain a DNA fragment of 1100bp (See figure 2 ), the product was cloned into pGEM after amplification and purification -T Easy vector, constructed into recombinant plasmid HepA / pGEM -T Easy, the sequence determination results show that the obtained heparanase I gene sequence and the GenBank number are EU541216.1 The nucleotide sequence of the 1st-1089th base of the 5'-end (shown in SEQ ID NO.2) totally agree. The two primers are:

[0033] H1: 5′CG GGATCC CAGCAAAAAAAAATCCGGTA...

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Abstract

The invention discloses bacillus subtilis engineering bacteria expressing recombinant flavobacteriun heparinum heparinase I, wherein the class name is bacillus subtilis which is preserved in the CGMCC (China General Microbiological Culture Collection Center) of CCCCM (China Committee for Culture Collection of Microorganisms) on 15th February, 2012, with preservation number CGMCC No.5757. A construction method comprises the following steps: transferring the coding gene of the flavobacteriun heparinum heparinase I into the bacillus subtilis WB600 to obtain the engineering bacteria capable of secreting and expressing heparinase. The bacillus subtilis engineering bacterial disclosed by the invention can stably and efficiently express the recombinant heparinase I; and the heparinase I can specifically degrade heparin and heparan glucosidic bond, and can be applied to the industrial production of low-molecular heparin, research of heparin and clinical medicine.

Description

technical field [0001] The invention relates to a strain of Bacillus subtilis engineering bacteria, its construction method, and its application in producing heparanase I. Background technique [0002] Heparin is a long-chain glycosaminoglycan composed of sulfated glucosamine and hexuronic acid connected by β-1,4 glycosidic bonds. It is mainly used clinically to prevent thrombosis or as an anticoagulant drug in vitro. Low molecular weight heparin (LMWH) is a low molecular weight fragment isolated from unfractionated heparin or produced by splitting heparin, with a molecular weight of about 4000Da to 6000Da. Compared with unfractionated heparin, LMWH has more advantages, such as high bioavailability, long plasma half-life, easy oral absorption, significantly enhanced antithrombotic effect, and significantly weakened hemorrhage-induced effect, so it is widely used in the treatment of thromboembolic diseases. Prevention and treatment. Although LMWH has many advantages, its pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/88C12P19/04C12R1/125
Inventor 王凤山李潇孙永福李娜周帅
Owner SHANDONG UNIV
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