Bacillus subtilis xylose induced exocrine expression vector

A technology of Bacillus subtilis and expression vector, which is applied in the direction of introducing foreign genetic material, DNA/RNA fragments, recombinant DNA technology using vectors, etc., can solve the problem of no expression vector, etc., and achieve the effect of wide application prospect.

Inactive Publication Date: 2012-03-21
FUZHOU UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Many studies have been done on the development and use of Bacillus subtilis as an expression system at home and abroad, but so far there is still no set of commercially efficient expression vectors, and the research and development of expression vectors for Bacillus subtilis has become one of the focuses of people's research. one

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  • Bacillus subtilis xylose induced exocrine expression vector
  • Bacillus subtilis xylose induced exocrine expression vector

Examples

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Embodiment 1

[0035] Example 1 Construction of Bacillus subtilis secretion expression vector pXYL-amyQ

[0036] 1. The clone containing the xylose-induced promoter and its repressor gene xylose cassette

[0037] Using pAX01 as template, with primer pair:

[0038] xyl+: 5'GCAT GAGCTC CTAACTTATAGGGGTAAC 3' (with SacⅠ restriction site)

[0039] xyl-: 5' GCAT GGATCC CATTTCCCCCCTTTGATTT 3' (including BamHI restriction site) (the underlined part is the restriction site)

[0040] The xylose-induced promoter and its repressor gene were amplified. The PCR reaction conditions were: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 2 minutes, and 30 cycles; extension at 72°C for 10 minutes. After gel recovery of the amplified product, double digestion with BamH1 and Sac I, the 1440bp target gene fragment was recovered with a gel recovery kit to obtain the xylose cassette of the xylose promoter of Bacillus subtilis. ...

Embodiment 2

[0049] Secreted expression of embodiment 2 cellulases

[0050] To assess the secreted expression level of this expression vector, cellulase was used as the reporter gene in this example.

[0051] 1. Construction of recombinant expression vector containing cellulase gene

[0052] Using the Bacillus subtilis168 genome as a template, use the primer pair:

[0053] cel+: 5’GCAT GGATCC ATGAAACGGTCAATCTCT 3' (with BamH1 restriction site)

[0054] cel-:5'GCAT CCCGGG CTAATTTGGTTCTGTTCC 3' (with SmaⅠ restriction site)

[0055] A 1500bp cellulase gene was amplified. The PCR reaction conditions were: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 59.5°C for 30 s, extension at 72°C for 2 min, and 30 cycles; extension at 72°C for 10 min. The target fragment was recovered, BamH1 and SmaI double-enzyme digested and gel recovered, and the same enzyme double-enzyme digested pXYL-amyQ carrier gel recovered. The two recovered fragments were connected under the...

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Abstract

The invention provides a bacillus subtilis xylose induced secretion expression vector pXYL-amyQ. The expression vector is constructed by using bacillus subtilis plasmids pHT43 as starting plasmids, removing original promoters and a repressor gene sequence thereof and inserting the bacillus subtilis plasmids into an expression box of bacillus subtilis xylose promoters and a signal peptide sequence. The xylose promoter expression box comprises a xylose induced promoter sequence and a repressor gene sequence thereof. The constructed expression vector pXYL-amyQ can secrete expression homogenous and heterogenous proteins in bacillus subtilis. The host bacillus subtilis of the vector is a generally recognized as safe (GRAS) host, and the adopted xylose serving as an inducer is nontoxic, so the expression vector has biological safety and plays an important role in protein expression, particularly enzyme and antibody preparation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression vector pXYL-amyQ capable of secreting and expressing exogenous genes in Bacillus subtilis. Background technique [0002] Bacillus subtilis ( Bacillus subtilis ) is a Gram-positive bacterium whose cell wall does not contain endotoxin, can form spores, and has strong protein secretion ability. Later, with the invention of DNA recombination technology, especially the discovery that Staphylococcus aureus (Staphylococcus aureus) with resistance marker plasmid can be used as its carrier, the work of Bacillus subtilis genetic engineering developed rapidly. [0003] As an attractive host for exogenous gene expression, Bacillus subtilis has the following advantages: ① Bacillus subtilis is a non-pathogenic soil microorganism that does not produce endotoxin. Bacillus is listed as a microbial GRAS (generally recognized as safe) host that is considered safe in princip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/113
Inventor 吕暾黄文斌
Owner FUZHOU UNIV
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