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 product

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
Comparison scheme
Effect test

Example Embodiment

[0039] Example 1

[0040] Construction of 9 knockout vectors

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

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

Example Embodiment

[0056] Example 2

[0057] Knockout of Bacillus subtilis

[0058] (1) Preparation of Bacillus subtilis competence: Pick a single colony of B.subtilis in 5ml LB medium and culture it with shaking at 37°C overnight; transfer the seed solution to 50ml B.subtilis electrotransformation growth medium with an inoculum of 5% , 37℃, 250r / min shaking culture for 2-3h to OD 600 For 0.85-0.95, ice bath the bacterial solution for 10min; centrifuge at 5000r / min for 5min 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 washing medium and aliquot to 1.5ml In the centrifuge tube, 100μl each tube is stored at -80℃, that is, competent cells. The above steps are all operated on ice.

[0059] (2) Electrotransformation: Take a tube of competent cells and add 5-10μl plasmid DNA (single knockout vector), mix gently, transfer to a pre-cooled electroporation cup (0.2cm), ice bath for 1-2min, use Elect...

Example Embodiment

[0064] Example 3

[0065] Engineering strain fermentation and fibrinolytic enzyme activity detection

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

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

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

[0069] (4) Plasminase activity detection: refer to patent CN103937830B, the specific operation is as follows: ①Pick up 1ml of fermentation broth in a cen...

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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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