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Fusion expression of heparin lyase in bacillus subtilis and application of heparin lyase

A technology of fusion expression and lyase, applied in the directions of lyase, carbon-oxygen lyase, application, etc., can solve the problems of low expression and limited application of heparin oligosaccharides, and achieve the effect of strong coagulation activity and high anticoagulant activity

Active Publication Date: 2021-12-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the expression level of existing heparin lyases is low, which limits its application in the preparation of heparin oligosaccharides

Method used

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  • Fusion expression of heparin lyase in bacillus subtilis and application of heparin lyase
  • Fusion expression of heparin lyase in bacillus subtilis and application of heparin lyase
  • Fusion expression of heparin lyase in bacillus subtilis and application of heparin lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Inducible expression of BhepI in Bacillus subtilis

[0045] Standard PCR amplification system and procedures were used, and the primers used are listed in Table 1. Using the plasmid pET28a-BhepI as a template, primers STOP-BhepI-F and STOP-BhepI-R were designed to amplify and obtain a DNA fragment containing the gene BhepI encoding heparin lyase I. The resulting gene was inserted into the P of plasmid pSTOP1622 by Gibson assembly xylADownstream of the xylose-inducible promoter, the inducible recombinant plasmid pSTOP-BhepI was obtained, and transformed into B. subtilis WB600 cells to obtain the recombinant strain BSBH, and the culture time and the addition amount of the inducer were optimized for the recombinant bacteria. The resulting recombinant strain was inoculated into 3 mL of fresh LB liquid medium containing 20 μg / mL tetracycline, cultivated at 37 ° C and 220 rpm for 8-10 h to the mid-logarithmic phase, and then transferred to a 50 mL In a 250mL Erle...

Embodiment 2

[0051] Example 2: Replacement of a constitutive promoter increases the constitutive expression level of BhepI

[0052] Selection of B. subtilis endogenous three promoters P spovG ,P lytR and P 43 , the P of the recombinant plasmid pSTOP-BhepI constructed in the above-mentioned Example 1 xylA The promoters were replaced by P spovG ,P lytR and P 43 Promoter. Design primer P 43 -F and P 43 -R, the primers used are shown in Table 1, and P 43 The gene sequence of the promoter, replacing the P of the pSTOP-BhepI plasmid by Gibson assembly xylA promoter, to obtain the recombinant plasmid P 43 -BhepI, obtain recombinant plasmid P in the same way spovG -BhepI and P lytR -Bhep I. And transferred to B. subtilis WB600 cells respectively to obtain recombinant strain BSBH / P spovG 、BSBH / P lytR and BSBH / P 43 . The resulting recombinant strain was inoculated into 3 mL of fresh LB liquid medium containing 20 μg / mL tetracycline, cultivated at 37 ° C and 220 rpm for 8-10 h to the...

Embodiment 3

[0054] Example 3: Optimizing the N-terminal sequence to improve the expression level of BhepI

[0055] On the basis of the recombinant plasmid constructed in Example 2 above, ten oligopeptide sequences were inserted into the N-terminus of the BhepI gene, and the N-terminal sequences are shown in Table 2. Using primers ybdD-F and ybdD-R to plasmid P spovG -BhepI is used as a template for full plasmid amplification to obtain recombinant plasmid P spovG -ybdD-BhepI, and so on, the primers used are shown in Table 1. Transform the constructed recombinant plasmids into B. subtilis WB600 cells to obtain 10 recombinant strains BSBH / P spovG -bltD, BSBH / P spovG -cspB, BSBH / P spovG -C4, BSBH / P spovG -yxjG, BSBH / P spovG -ydbD, BSBH / P spovG -valS, BSBH / P spovG -tufA, BSBH / P spovG -ybdD, BSBH / P spovG -yvyD and BSBH / P spovG -glnA. The resulting recombinant strain was inoculated into 3 mL of fresh LB liquid medium containing 20 μg / mL tetracycline, cultivated at 37 ° C and 220 rpm ...

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Abstract

The invention discloses fusion expression of heparin lyase in bacillus subtilis and application of heparin lyase, and belongs to the technical field of bioengineering. According to the invention, bacillus subtilis is used as a host for heterologous expression of heparin lyase I from Bacteroides thetaiotaomicron, an oligopeptide sequence, an optimized promoter and a 5'UTR sequence are fused at an N terminal, a gene FhepIII of heparin lyase III is connected to a C terminal of a BhepI gene by using a GGGS oligopeptide, a fusion enzyme BhepI-FhepIII is constructed, and through comparison of heparin sodium splitting capacities of the BhepI and the FhepIII, it is found that the molecular weight of heparin obtained by the fusion enzyme can be as low as 1300Da or below, and the heparin lyase has stronger anticoagulant activity, and lays a foundation for industrial production of the heparin lyase in bacillus subtilis and preparation of low-molecular-weight heparin.

Description

technical field [0001] The invention relates to fusion expression and application of heparin lyase in Bacillus subtilis, belonging to the technical field of bioengineering. Background technique [0002] Heparin, a complex, dispersed, highly sulfonated linear glycosaminoglycan, widely exists on the cell surface and in the extracellular matrix, and its sugar chain is composed of N-acetylglucosamine and D-glucuronic acid through 1-4 glycosidic bonds Composed of linked disaccharide units. Heparin is a biologically active molecule in the body for cell recognition, signal transmission, and anticoagulation. Side effects, such as affecting the stability of platelets and causing massive bleeding after surgery. Compared with unfractionated heparin, low molecular weight heparin (3000-8000Da) not only has strong anticoagulant activity, is easily absorbed and utilized by the body, has low side effects, but also has higher pharmacological activity predictability and higher bioavailabili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/21C12N15/75C12P19/26C12R1/125
CPCC12N9/88C12Y402/02007C12N15/75C12P19/26
Inventor 康振陈佳敏张琳王阳堵国成陈坚
Owner JIANGNAN UNIV
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