A honeybee peptide expression vector and its construction method and application

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...

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