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Bacillus subtilis engineering bacteria for efficiently expressing plasmin and preparation method thereof

A high-efficiency expression technology of Bacillus subtilis, which is applied in the field of high-efficiency expression of Bacillus subtilis engineering bacteria and preparation of plasmin, can solve the problems of plasmin production and enzyme activity defects, etc., and achieve the effect of clear route and good practicability

Active Publication Date: 2018-08-17
WUHAN ZHENFU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the defects of existing high-yield plasminase strains in terms of plasminase production and enzyme activity, and provide a Bacillus subtilis engineering bacterium that expresses plasminase efficiently and its preparation method

Method used

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  • Bacillus subtilis engineering bacteria for efficiently expressing plasmin and preparation method thereof
  • Bacillus subtilis engineering bacteria for efficiently expressing plasmin and preparation method thereof
  • Bacillus subtilis engineering bacteria for efficiently expressing plasmin and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Construction of 9 Knockout Vectors

[0041] The Bacillus subtilis strain (B. subtilis) comes from the China Industrial Microorganism Culture Collection Center (CICC), and the preservation number is No. 20637.

[0042](1) Genomic DNA extraction of Bacillus subtilis: pick a single colony of B. subtilis in 5ml LB liquid medium, culture overnight at 37°C with shaking, collect the bacteria by centrifugation at 12000r / min for 5min; wash the bacteria once with STE; add 200μl Solution I solution and 20μl 100mg / ml lysozyme, resuspended bacteria, placed at 37°C for 2-3h or placed in a refrigerator at 4°C overnight; add 200μl 10% SDS, 50-60°C water bath for 30min until clarified; add 100μl or 200μl 5mol / L NaCl, mix well and centrifuge at 12000r / min for 5min, take the supernatant into a clean centrifuge tube; extract twice with equal volume of phenol / chloroform, take the supernatant into a clean centrifuge tube; add 2 times the volume of absolute ethanol Precipitate at -20°C for 2...

Embodiment 2

[0057] Gene knockout in Bacillus subtilis

[0058] (1) Competent preparation of Bacillus subtilis: pick a single colony of B.subtilis in 5ml LB medium, and culture overnight at 37°C with shaking; the seed solution is transferred to 50ml B.subtilis electroporation growth medium with 5% inoculum , 37 ° C, 250r / min shaking culture 2-3h to OD 600 If the temperature is 0.85-0.95, put the bacterial solution in ice bath for 10 minutes; centrifuge at 5000r / min for 5 minutes to collect the bacterial cells, wash the bacterial cells with B. subtilis electroporation washing medium for 4 times, and finally resuspend the cells with 1ml of washing medium, and aliquot to 1.5ml In centrifuge tubes, 100 μl per tube was stored at -80°C, namely competent cells, and the above steps were performed on ice.

[0059] (2) Electroporation: Take a tube of competent cells and add 5-10 μl plasmid DNA (single knockout vector), mix gently and transfer to a pre-cooled electroporation cup (0.2 cm), ice bath f...

Embodiment 3

[0065] Fermentation of engineering strains and detection of plasmin activity

[0066] (1) Strain activation: Pick the B.subtilis 20637 and B.subtilis 20637Δ9 strains preserved in glycerol tubes and streak them on the LB plate, culture at 37°C for 12 hours, pick a single colony and streak it on the LB plate, and culture at 37°C 12h;

[0067] (2) Seed culture: Pick a single colony from the LB solid medium and inoculate it into 50 ml of liquid seed medium at a rotation speed of 220 r / min, shake and culture at 37°C for 12 hours, and use it as a fermented seed liquid;

[0068] (3) Fermentation culture: a 3L fermenter was used, the liquid volume was 1.5L, the inoculum size was 2%, and the fermentation temperature was set at 37°C. The stirring speed is 800r / min, the ventilation rate is 2L / min, the pH is controlled at 6.8-7.2, and the fermentation is 48h.

[0069] (4) Detection of plasmin enzyme activity: refer to patent CN103937830B, the specific operation is as follows: ① draw 1ml...

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Abstract

The invention discloses a Bacillus subtilis engineering bacteria for efficiently expressing plasmin and a preparation method thereof. An existing Bacillus subtilis strain is transformed by gene knocking technology to obtain the Bacillus subtilis engineering bacteria for efficiently expressing the plasmin. The method is clear in route, simple and effective, can be applied to a variety of Bacillus subtilis, and has good practicability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Bacillus subtilis engineering bacterium for highly expressing plasmin and a preparation method thereof. Background technique [0002] Plasmin is a proteolytic enzyme that can specifically degrade fibrin gel. It has the functions of dissolving thrombus, reducing blood viscosity, improving blood circulation, softening and increasing blood vessel elasticity. In recent years, nattokinase (NK for short), as a new efficient and safe plasminase, is becoming the focus of current thrombolytic drug research. [0003] The enzyme is a serine protease produced by Bacillus subtilis natto during the fermentation process of natto. Since the first report on natto and plasmin in my country in 1995, many related articles have been published so far. The thrombolytic mechanism of lysozyme has been elucidated, including ①direct thrombolysis, activation of cross-linked fibrin, and degradation of fibrin d...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N9/54C12R1/125
CPCC07K14/32C12N9/2411C12N9/54C12N9/93C12N15/75C12Y304/21062C12Y603/02
Inventor 王业富张媛张磊胡定邦温佳文何改粉
Owner WUHAN ZHENFU PHARMA CO LTD
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