Thrombolytic enzyme gene, recombinant expression vector and recombinant bacteria comprising same and application

A recombinant carrier and thrombolytic enzyme technology, applied in the biological field, can solve the problems of low output of thrombolytic enzyme, high production cost, low enzyme activity, etc., and achieve the effects of reducing cost, increasing solubility and activity, and good practicability.

Active Publication Date: 2015-09-23
湖北真福医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the defects of low yield, low enzyme activity and high production cost of existing thrombolytic enzymes, and provide a thrombo

Method used

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  • Thrombolytic enzyme gene, recombinant expression vector and recombinant bacteria comprising same and application
  • Thrombolytic enzyme gene, recombinant expression vector and recombinant bacteria comprising same and application
  • Thrombolytic enzyme gene, recombinant expression vector and recombinant bacteria comprising same and application

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Experimental program
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Effect test

Embodiment 1

[0033] The preparation process of embodiment 1 Bacillus subtilis QK-02

[0034] Put the rice leaves at 85°C for heat treatment for 10 minutes, then wrap the sterilized soybeans with them, cultivate them at 42°C for 24 hours, and then soak the treated soybeans with the culture solution, and use the soaked soybean culture solution for 10 -2 ~10 -6Diluted, spread on nutrient agar plate, and cultured upside down at 37°C. The obtained colonies were respectively inoculated into 5 mL of culture solution, cultured at 37° C. at 250 rpm / min for 24 hours, and then centrifuged at 6000 rpm / min for 5 minutes. Take 20 μl of the supernatant and measure the thrombolytic enzyme activity on the fibrin plate, and then select the strain with the highest enzyme activity.

Embodiment 2

[0035] Embodiment 2 PCR amplification and sequence optimization of gene qk encoding thrombolytic enzyme

[0036] 2.1 PCR amplification and cloning of gene qk encoding Bacillus subtilis thrombolyticase

[0037] Genomic DNA extraction of Bacillus subtilis QK-02 was performed according to a conventional method (Saito, H. et al, 1963, Biochim. Biophys. Acta 72:619-629).

[0038] Primer design for PCR amplification:

[0039] Primer P1: 5'ACGCGTCGACATGGCGCAATCTGTTCCTTACGGC

[0040] Primer P2: 3'CCGGAATTCTAGCTATTATTTGTGCAGCTGC

[0041] PCR amplification reaction system 50ul system: 5μl Bacillus subtilis genomic DNA as a template, 1μl each of primers P1 and P2 (10μM), 5μl of 10×PCR buffer, 5μl of dNTP (2mmol), 0.5μl of KOD enzyme, and finally use sterile water Make up to 50ul. The reaction program is: 94°C, 5min; 94°C, 30s; 56°C, 10s; 74°C, 30s; 74°C, 6min, a total of 35 cycles. After the amplification was completed, the PCR product was detected by 1% agarose gel electrophoresis,...

Embodiment 3

[0048] Embodiment 3 Construction of Thrombolytic Enzyme Recombinant Expression Vector

[0049] The optimized qk gene fragment and pET40b vector (see figure 2 ) were both digested by Sal I and EcoR I, and the fragments were recovered by gel and ligated with the carrier at 16°C overnight, and then passed through CaCl 2 Transformed into DH5α cells by the method, spread the transformed product on LB agar plate containing kanamycin (Kan) resistance, and incubate upside down at 37°C for 14-16h. After the positive clones were screened out, the plasmids were extracted for double-enzyme digestion identification, such as image 3 (M: Marker, 1: vector fragment before digestion, 2: vector after SalI / EcoRI double digestion), and the positive recombinants were sent to the company for sequencing.

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Abstract

The invention discloses a thrombolytic enzyme gene, a recombinant expression vector and recombinant bacteria structured by the thrombolytic enzyme gene and application of the thrombolytic enzyme gene. A qk sequence obtained by optimization through the overlap extension of PCR (polymerase chain reaction) can be identified by an escherichia coli expression strain DE 3 better, meanwhile, the vector pET-40b for improving protein solubility is selected to be structured with the optimized qk so as to form the new recombinant expression vector and the recombinant bacteria for preparing expression proteins, functional extracellular proteases can be secreted into periplasm so as to improve solubility and activity of fusion proteins, and finally bacillus subtilis thrombolytic enzyme high in expression quantity and enzyme activity is obtained. The method does not include operations of solubilization of inclusion bodies and protein renaturation, so that cost is lowered, and better practicality is achieved.

Description

technical field [0001] The invention relates to a thrombolytic enzyme gene, a recombinant expression vector containing the gene, a recombinant bacterium and its application, and belongs to the field of biotechnology. Background technique [0002] Bacillus subtilis QK02 was screened by detecting the ability to degrade fibrin. Studies have shown that the enzyme has strong fibrinolytic activity and is a potential thrombolytic drug. It not only has a good thrombolytic effect, but also can achieve the purpose of fibrinolysis through oral administration, which is more advantageous than the current clinically used thrombolytic drugs (such as streptokinase, urokinase, etc.). The inventors have also found that the enzyme has antagonism in vivo and in vitro and in vascular endothelial cells produced by heme, NaNO 2 and H 2 o 2 ability to inflict damage. At present, the enzyme mainly comes from natto and fermented soya bean fermented by wild Bacillus subtilis, but the flavor of na...

Claims

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Application Information

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IPC IPC(8): C12N15/57A61K38/48A61P7/02C12N9/56C12N15/72C12N1/21C12R1/19
Inventor 王业富马立新高丽舒敏王亚平周康平张展
Owner 湖北真福医药有限公司
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