Nonreactive expression system based on bacillus subtilis and construction method

An expression system, bacillus technology, applied in biochemical equipment and methods, viruses/bacteriophages, using vectors to introduce foreign genetic material, etc., can solve problems such as degradation and slow folding of foreign proteins, and achieve the effect of low background expression

Active Publication Date: 2017-02-22
CHENGDU MYTECH BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Sec pathway has a regulatory mechanism that inhibits the secretion of incorrectly folded proteins, and foreign proteins generally fold slowly, and the folding process requires the participation of chaperone proteins, which are easily degraded by the Sec secretion pathway.

Method used

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  • Nonreactive expression system based on bacillus subtilis and construction method
  • Nonreactive expression system based on bacillus subtilis and construction method
  • Nonreactive expression system based on bacillus subtilis and construction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Cut the pBAV1K-T5-GFP plasmid with EcoR I and Apa I endonucleases, and perform homologous recombination with the synthesized MCS fragment to obtain the plasmid pBAV1K.

[0037] The endonuclease used in this experiment and follow-up experiments was the rapid endonuclease from Thermo Company, and the gel recovery kit (DE-02011) from Chengdu Fuji Biological Company was used for fragment recovery. The MCS fragment was synthesized by Jinweizhi Biotechnology Co., Ltd. (sequence As shown in SEQ ID NO.4), homologous recombination uses Tiangen’s EsayGeno rapid recombination cloning kit (VI201-02), the E. coli strain is top10, the preparation of competent cells uses the KCM method, and the plasmid extraction uses Fujibio’s Universal Plasmid Miniprep Kit (DE-01001).

[0038] 2. Design primers to amplify the pBAV1K fragment with synonymous mutation to delete the Nde I restriction site, connect the fragments by homologous recombination cloning, and obtain a plasmid named pBTS.

...

Embodiment 2

[0041] 1. Design primers to amplify the 602bp fragment wprA-F upstream of the wprA gene; design primers to amplify the 601bp fragment wprA-R downstream of wprA; synthesize xylR promoter and CDS and xylAB promoter region fragment xylR from the whole gene; synthesize T7RNA from the whole gene Polymerase fragment T7RP; vector pBTS-T7RP was constructed by homologous recombination.

[0042] The high-fidelity enzyme uses toyobo's KOD-Plus high-fidelity polymerase (KOD-201).

[0043] 2. Electrotransform pBTS-T7RP into Z12 strain to obtain strain Z12-pBTS-T7RP.

[0044] 3. Inoculate the bacterium Z12-pBTS-T7RP into 3ml LB medium, culture at 45°C, 180rpm for 24 hours; inoculate the bacterium Z12-pBTS as a control.

[0045] 4. Take 200ul of the bacterial solution in 3 and spread it on an LB plate containing 30mg / L kanamycin, and culture it overnight at 45°C. If Z12-pBTS has no colony growth and Z12-pBTS-T7RP has colony growth, the obtained strain has completed the first recombination ...

Embodiment 3

[0051] 1. For the strain transformation method, see the hyperosmolarity transformation method (High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis, 1999). Take a newly streaked single colony of the strain to be transformed, inoculate it into about 3ml of GM (LB+0.5M sorbitol) medium, and culture overnight at 37°C and 180rpm. The next morning, inoculate into 50ml GM medium at a ratio of 1:100, incubate at 37°C and 180rpm, and when the OD reaches 0.85-0.95, place the bacterium on ice to pre-cool for 10 minutes; at 4°C, Centrifuge at 5000g for 5min to remove the supernatant, resuspend the cells with an equal volume of pre-cooled EM (0.5M sorbitol + 0.5M mannitol + 10% glycerin aqueous solution), and centrifuge again at 4°C at 5000g for 5min to remove the supernatant. Clean, repeat washing 4 times in total; add about 1 / 40 volume of EM resuspended bacteria to ensure that the concentration of the b...

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Abstract

The invention belongs to the biological technical field and in particular relates to a nonreactive expression system expressed by utilizing bacillus subtilis and a construction method. Light background T7 RNA polymerase expressed by induction of a xylose operon is utilized, the T7 RNA polymerase specifically transcribes a T7 promoter, then a cascade amplification expression system of a target gene is transferred into a bacillus subtilis genome, and a selective marker gene used in an intermediate process is deleted. The construction method overcomes the defects that an antibiotic selective marker gene can drift easily, a plasmid expression system is unstable, expression can not be induced and expression quantity is not high, and a nonreactive expression system with light background, controllable induction and efficient expression is constructed. By combining the advantages that a bacillus subtilis expression system is low in fermentation cost, large scale production can be realized, fermentation products can be secreted into fermentation broth and endotoxin is not contained, a brand new expression system is provided for expression of target protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bacillus-based anti-resistance expression system and a construction method. Background technique [0002] Protein expression technology is one of the core technologies of modern biology. Protein expression can not only be used in biological research, but also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently commonly used expression systems include Escherichia coli, yeast, insect cells and mammalian cells, etc., but each of them has obvious advantages and disadvantages. Escherichia coli expression system is the most well-studied, and there are many options. The most commonly used is Novagen's pET expression system, which uses bacteriophage T7 RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Under optimal conditions, The target protein can reach more than 50% o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75
CPCC12N15/75C12N2800/101C12N2800/24
Inventor 任钧唐旭曹镜雷蕾樊超柴进凯曹富明孙楠范佳
Owner CHENGDU MYTECH BIOTECH
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