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Fusion gene MBP-H1 expressing heparinase, recombinant plasmid of fusion gene MBP-H1 and application of recombinant plasmid

A technology of MBP-H1 and fusion gene, which is applied in the fusion gene MBP-H1 expressing heparanase and its recombinant plasmid and application field, which can solve the problems of high cost of heparanase preparation and limited source of heparanase

Inactive Publication Date: 2020-06-02
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, although there are many research reports on the preparation of low molecular weight heparin by heparinase cleavage, there are still many limiting factors, such as the narrow source of heparinase, and many heparinases used in the preparation of low molecular weight heparin are basically derived from heparin Flavobacterium, in addition, the preparation cost of heparanase is relatively high

Method used

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  • Fusion gene MBP-H1 expressing heparinase, recombinant plasmid of fusion gene MBP-H1 and application of recombinant plasmid
  • Fusion gene MBP-H1 expressing heparinase, recombinant plasmid of fusion gene MBP-H1 and application of recombinant plasmid
  • Fusion gene MBP-H1 expressing heparinase, recombinant plasmid of fusion gene MBP-H1 and application of recombinant plasmid

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Effect test

Embodiment 1

[0056] 1.11 Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the MBP-H1 sequence (as shown in SEQ ID NO. 1).

[0057] 1.12 Culture the strain E.coli TOP10 pET-28a, extract the plasmid and double-enzyme digestion The above-purified PCR product and the vector plasmid pET-28a were double-enzyme digestion with XbaI and XhoI restriction endonucleases, and the double-enzyme digestion conditions were: : 37°C digestion for 2h, the double digestion reaction system (50μL) is shown in Table 4:

[0058] Table 4. Composition of double-enzyme digestion system

[0059]

[0060] Agarose gel electrophoresis was performed on the enzyme-digested product of the above-mentioned target gene and the vector plasmid, followed by gel cutting and recovery. The result is as figure 1 As shown, lane M is DL10000 DNAMarker; lane 1 is the double-enzyme digestion product of the target gene; lane 2 is the plasmid double-enzyme digestion product; the target gene and plasmid band after doubl...

Embodiment 2

[0084] 2.1 Experimental method

[0085] 2.1.1 Optimization of IPTG concentration of inducer

[0086] Take 50 μL of E.coli BL21(DE3)pET-28a-MBP-H1-2 stored in a glycerol tube, put it into 5 mL of LB medium containing 50 μg / mL kanamycin, and culture at 37°C, 200 rpm, overnight. Take 100 μL of seed solution and add it to 10 mL of LB medium (containing 50 μg / mL kanamycin), cultivate at 37 ° C and 200 rpm until the OD600 is about 0.7, and add IPTG with final concentrations of 0.1, 0.25, 0.5, 0.75, 1mM, each group. Do three parallel controls, 37 ℃, 200rpm, induction 10h. Add 200 μL / well to 96-well plate, measure OD with a microplate reader 600 , and then crushed the cells to extract the crude enzyme solution to measure the enzyme activity.

[0087] Induction results such as Figure 4 shown, where -●- represents OD 600 ;-■- represents enzyme activity (U / L); with the increase of IPTG concentration, the general direction of the enzyme activity curve is a gradual decrease, and its ...

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Abstract

The invention provides a fusion gene MBP-H1 expressing heparinase, a recombinant plasmid of the fusion gene MBP-H1 and an application of the recombinant plasmid, and relates to the technical field ofheparinase production. The nucleotide sequence of the fusion gene MBP-H1 is shown as SEQ ID NO.1. The fusion gene MBP-H1 is utilized to construct a pET-28a-MBP-H1 fusion protein recombinant plasmid, and finally primary induction expression condition optimization is carried out on the pET-28a-MBP-H1 to determine that the optimal induction expression condition is as follows: when culture is carriedout at 37 DEG C at a condition of 200 rpm to a condition that the OD600 of bacterial liquid is about 0.7, isopropyl-beta-d-thiogalactoside (IPTG) (100 mM) is added until the final concentration is 0.1mM; and induction is carried out at 30 DEG C at a condition of 200 rpm for 9 hours, wherein highest activity of the heparinase can reach 4512 U / L.

Description

technical field [0001] The invention belongs to the technical field of heparinase production, and in particular relates to a fusion gene MBP-H1 expressing heparinase and its recombinant plasmid and application. Background technique [0002] At present, most of the heparin drugs on the market are prepared by chemical methods. This method is too intense, which can easily lead to the loss of biological activity of heparin, so that the low molecular weight heparin prepared by this method has microscopic heterogeneity, polydispersity and Disadvantages such as large structural variation. In contrast, the reaction conditions of enzymatic preparation are milder, no toxic substances are introduced into the product, the reaction efficiency is high, and the heparinase and the product are easy to separate. At the same time, the use of immobilized enzyme technology can also achieve continuous production. Cost saving, is an industrialized way to produce low molecular weight heparin. So ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/56C12N15/70C12N1/21C12N9/24C12R1/19
CPCC07K2319/24C12N9/2402C12N15/70C12Y302/01019
Inventor 赵丽青李茵茵
Owner SHENZHEN UNIV
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