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Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis

A Bacillus subtilis and exogenous protein technology, applied in the biological field, can solve the problems of difficult separation and purification, low expression of exogenous protein, and poor secretion, and achieve the effects of efficient secretion and expression, simple culture conditions, and reduced production costs

Active Publication Date: 2014-09-10
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the shortcomings of low expression, poor secretion, and difficult separation and purification of exogenous proteins, the present invention provides a plasmid, expression equipment and gene for expressing exogenous proteins in Bacillus subtilis cells that can be secreted and expressed, and are easy to operate Engineering bacteria, which can efficiently secrete and express heterologous proteins from bacteria, etc.

Method used

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  • Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis
  • Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis
  • Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 , Construction of Bacillus subtilis expression vector pBNS2

[0049] 1.1 Cloning of P43 constitutive promoter

[0050] The P43 promoter is a constitutive promoter derived from Bacillus subtilis, which contains two promoters and are separated by σ A and σ B factor identification. According to the P43 promoter gene sequence of Bacillus subtilis in GenBank, primers P43F and P43R were designed and synthesized, and their sequences are as follows:

[0051] P43F: 5'-CGC GAATTC TGATAGGTGGTATGTTTT-3' (SEQ ID NO: 6)

[0052] P43R: 5'-CGC TCTAGA TTCATGTGTACATTCCTC-3' (SEQ ID NO: 7)

[0053] Among them, an EcoRI restriction site was introduced into the primer P43F, and an XbaI restriction site was introduced into the primer P43R.

[0054] Using the total DNA of Bacillus subtilis AS.168 as a template, PCR amplification was performed using the P43F and P43R primer pair, and the obtained PCR product was cloned into the pMD19-T vector, and the recombinant plasmid w...

Embodiment 2

[0070] Embodiment 2, the transformation system optimization of Bacillus subtilis

[0071] 2.1. Chemical transformation of Bacillus subtilis

[0072] 2.1.1. Preparation of Bacillus subtilis competent cells

[0073] The glycerol-preserved strain of Bacillus subtilis was added to the GMI solution at an inoculum of 1%, and cultured overnight at 30°C on a slow shaker (100rpm). On the next day, transfer 10% of the inoculum to fresh GMI, and culture on a fast shaker (200 rpm) at 37°C for 3.5 hours. Then transfer it to GMII with 10% inoculum amount, cultivate it on a slow shaker at 37°C for 90 minutes, and collect the bacteria by centrifugation at 4°C. Use 1 / 10 of the volume of the supernatant to suspend the bacteria, and add sterile glycerol to a final concentration of 10%, mix well and distribute into sterile centrifuge tubes, and then store at -40°C.

[0074] 2.1.2. Chemical transformation of Bacillus subtilis competent cells

[0075] When transforming, take out the Bacillus su...

Embodiment 3

[0081] Example 3, expression of green fluorescent protein using Bacillus subtilis expression equipment

[0082] 3.1. Construction of Bacillus subtilis engineering bacteria for expressing green fluorescent protein

[0083] In order to detect the expression efficiency of the foreign gene of the expression vector pBNS2 constructed in Example 1.3, the green fluorescent protein coding region was cloned into the constructed expression vector as a reporter gene.

[0084] First, design and synthesize primers pBNS2-gfp-U / pBNS2-gfp-D for amplifying the gfp gene, and its sequence is as follows:

[0085] pBNS2-gfp-U:5'-GCC GGATCC AGTAAAGGAGAAGAA-3' (SEQ ID NO: 14)

[0086] pBNS2-gfp-D:5'-GCC CTCGAG TTTGTATAGTTCAT-3' (SEQ ID NO: 15)

[0087] Among them, BamHI and XhoI restriction sites were respectively introduced on the primers pBNS2-gfp-U and pBNS2-gfp-D.

[0088] The plasmid pTM117 was used as a template, and the pBNS2-gfp-U and pBNS2-gfp-D primer pairs were used for PCR amplific...

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Abstract

The invention discloses an expression apparatus used for the secretory expression of exogenous proteins in Bacillus subtilis. The apparatus sequentially comprises a P43 promoter, an SD sequence, a signal peptide, a multiple cloning site and a protein purifying label. The Bacillus subtillis expression apparatus can realize the high-efficiency secretory expression of exogenous proteins in Bacillus subtillis. The culturing conditions of Bacillus subtilis are simple, and bacteria are safe and are convenient to ferment and produce, so the Bacillus subtilis is suitable for solid culturing and liquid depth fermentation, generate no endotoxin or thermogenic lipopolysaccharides, and accords with international food safety authentication. The above Bacillus subtilis genetic engineering strain can be used for preparing protein products comprising industrial enzyme preparations, feed enzymes, protein drugs and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression device for secreting and expressing foreign proteins in Bacillus subtilis. Background technique [0002] Gram-positive bacteria are widely known for their contributions to agriculture, medical and food biotechnology and recombinant protein production, among others. Among all Gram-positive bacteria, Bacillus vectors are particularly attractive due to their excellent properties. The Bacillus expression system started from Bacillus subtilis, also known as Bacillus subtilis, in the 1970s, and gradually expanded to other species. Bacillus subtilis is second only to Escherichia coli, and is a kind of prokaryote that has been studied in detail in terms of genetics, physiology and biochemistry. The ability of Bacillus subtilis to develop into the first genetically engineered expression system in Bacillus is closely related to its early genetic work. Since Spizize...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/75C12N1/21C12P21/00C12R1/125
Inventor 魏巍魏东芝郭苏唐嘉婕马静
Owner EAST CHINA UNIV OF SCI & TECH
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