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Apidaecin expression vector as well as construction method and application thereof

An expression vector and a construction method technology, applied in the field of expression vector construction, can solve the problems of low expression amount of honeybee peptide expression system, complicated operation of honeybee peptide extraction process, etc., and achieve easy control of culture conditions, fast reproduction speed and low production cost. Effect

Active Publication Date: 2016-06-15
ZHANGYE OLYMBEL BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a honeybee peptide expression vector and its construction method and application, which are used to solve the technical defects of the prior art, such as the low expression level of the honeybee peptide expression system, the complex operation of the honeybee peptide extraction process, etc.

Method used

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  • Apidaecin expression vector as well as construction method and application thereof
  • Apidaecin expression vector as well as construction method and application thereof
  • Apidaecin expression vector as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] 1. Artificially synthesized expression operon gene encoding TEV protease

[0038] The commercial company uses professional software to optimize the expression operon gene of TEV protease to the best codon gene of Bacillus subtilis, wherein:

[0039] The nucleotide sequence of the artificially synthesized expression operon gene encoding TEV protease is shown in SEQ ID NO: 1, and the size of the gene fragment is 726bp.

[0040] 2. Artificially synthesized fusion protein encoding xylanase and honeybee peptide to express operon gene

[0041] The commercial company uses professional software to optimize the expression operon gene containing the fusion protein encoding xylanase and honeybee peptide to the best codon gene of Bacillus subtilis, in which:

[0042] The nucleotide sequence of the synthetic expression operon gene containing the fusion protein encoding xylanase and honeybee peptide is shown in SEQ ID NO: 2, and the size of the gene fragment is 1566bp.

[0043] 3. ...

Embodiment 2

[0076] 1. Transformation of honey bee peptide expression vector pNF11-AP

[0077] Mix competent Bacillus subtilis 1A751 and plasmid pZF11-AP, transfer to a 0.2cm electroporation cuvette, electric shock at 2500V, 5ms, immediately add 800ul pre-cooled Bacillus subtilis electroporation recovery medium LBSPG, resume culture at 37°C, 200rpm for 1h , Centrifuge at 4500rpm to collect bacteria and spread on LB (contains final concentration of 50ug / ml spectinomycin) plate, culture at 37°C until a single colony appears, pick positive transformants, culture and extract plasmids for EcoRI, SacI double enzyme digestion and PCR identification ; Clones with 1566 bp amplified were identified as positive transformants of pZF11-AP1, pZF11-AP2, and pZF11-AP3.

[0078] 2. Secretory expression of recombinant Bacillus subtilis

[0079] Inoculate the positive transformants of pZF11-AP1, pZF11-AP2, and pZF11-AP3 that were screened into 25ml of LB medium, shake and culture at 37°C and 250r / min for 22...

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Abstract

The invention provides an apidaecin expression vector as well as a construction method and application thereof. The construction method comprises the following steps: 1) synthesizing an expression operon gene containing encoding NTEV protease; 2) synthesizing a fusion protein expression operon gene containing encoding xylanase and apidaecin; 3) conducting double-enzyme digestion on the expression operon genes obtained in the steps 1) and 2) in a separated mode, and constructing into a vector pBluescript II SK(+) so as to obtain a recombinant plasmid; 4) extracting a genome DNA of bacillus subtilis 168, amplifying a promoter p43, cloning by virtue of a T vector so as to obtain a plasmid containing a promoter gene, and constructing into a plasmid pGJ103 so as to obtain a plasmid pGJ284; conducting reverse PCR (polymerase chain reaction) so as to obtain an efficient promoter sequence gene containing partial gene sequences of the p43 promoter, and constructing into the plasmid pGJ284 so as to obtain a plasmid pGJ288; and 5) conducting double-enzyme digestion on the recombinant plasmid and plasmid pGJ288 in a separated mode, recovering enzyme digestion products and linking so as to obtain an apidaecin expression vector. The expression vector is high in expression amount; and the apidaecin extraction method is simple and convenient, and is suitable for industrial large-scale preparation.

Description

technical field [0001] The invention relates to a method for constructing an expression vector, in particular to a honeybee peptide expression vector and its construction method and application. Background technique [0002] Honey bee peptide (apidaecin, referred to as AP) is a kind of proline-rich polypeptide derived from Hymenoptera insects. It was first isolated from the lymph fluid of honeybees, generally containing 16-18 amino acid residues, and it is effective for a variety of leather Lamb-negative bacteria, especially Enterobacteriaceae, have a strong killing effect, but have no effect on Gram-positive bacteria including Lactococcus lactis, and have no toxic effect on eukaryotic cells, so they are used in food and It has broad application prospects in feed additives, pharmaceutical industry and plant antibacterial disease genetic engineering. [0003] Due to the low content of honeybee peptide in insects, the cost of directly purifying protein from insect lymph is hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/66C12N1/21C07K7/08C12R1/125
Inventor 武刚祝发明刘文庆朱剑
Owner ZHANGYE OLYMBEL BIO TECH
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