Method for efficiently expressing Bombyx mori recombinant antibacterial peptides by using lactose culture medium

Abstract

The invention relates to a method for efficiently expressing Bombyx mori recombinant antibacterial peptides by using lactose culture medium, comprising the steps of (1), using intestinal tissues of five-instar seven-days-old silkworms as a material to extract total RNA, designing three pairs of specific primers, carrying out RT-PCR (reverse transcription-polymerase chain reaction) to amplify Bombyx mori antibacterial peptide cecropinB6, cecropinD and moricin genes, detecting PCR products by 2.5% agarose gel electrophoresis, and purifying BmcecropinB6, BmcecropinD and Bmmoricin by using a gel extraction kit; (2), subjecting three antibacterial peptide genes and pET-32a expression vector respectively double-enzyme digestion by using NcoI / XhoI to establish pET32a-BmcecropinB6, pET32a-BmcecropinD, pET32a-Bmmorincin expression vectors, and sequencing by dideoxy chain termination; (3), using a lactose medium method to induce the expression of BmcecropinB6, BmcecropinD and Bmmoricin antibacterial peptides. The expression yield of BmcecropinB6, BmcecropinD and Bmmoricin recombinant proteins is significantly increased, and new technical choices are provided for the application in the fields such as purification and activity identification for Bombyx mori peptides, and cosmetics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently expressing silkworm antibacterial peptides using lactose medium, and the antibacterial peptides include BmcecropinB6, BmcecropinD and Bmmoricin. Background technique [0002] Antimicrobial peptides exist in a large number of insect cells, and the content in normal tissues is very low. It is difficult to separate and purify, and the cost of chemical synthesis is very expensive. Therefore, antimicrobial peptides obtained through genetic engineering techniques have become the preferred method. The existing technology usually uses isopropyl-β-D-mercaptogalactoside (IPTG) to induce the expression of exogenous recombinant proteins and polypeptides, but due to the potential toxicity of IPTG, it has a certain inhibitory effect on the growth of bacteria and is expensive , and the expressed recombinant protein and polypeptide content is low. At present, th...

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Problems solved by technology

The existing technology usually uses isopropyl-β-D-mercaptogalactoside (IPTG) to induce the expression of exogenous recombinant proteins and polypeptides, but due to the potential toxicity of IPTG, it has a certain inhibitory effect on the growth of bacteria and is expensive , and the expressed recombinant protein and polypeptide content is low. At present, there have been studies at home and abroad that use lactose instead of IPTG as the inducer of the lactose promoter to induce the expression of recombinant protein, but there is no report on the expression of antimicrobial peptides in silkworm. At present, in silkworm More than 40 kinds of antimicrobial peptide genes have been found in , among which BmcecropinB6, BmcecropinD and Bmmoricin have extremely strong broad-spectrum antibacterial properties and can kill most Gram-positive and Gram-negative bacteria and fungi, indicating that these silkworms are antibacterial Peptides have broad application prospects

[0003] In the prior art, the yield of recombinant protein expression induced by IPTG as an inducer is generally not more than 30%. The reason is that IPTG has potential toxicity and has a certain inhibitory effect on the growth of bacteria, and the bacterial reproduction efficiency is low in this method, resulting in recombinant Low yield of protein expression

[0004] At present, people have not yet developed a technology to improve the expression yield of recombinant protein induced by IPTG as an inducer. Only by adjusting the concentration of IPTG, induction time, and induction temperature to improve the expression yield of recombinant protein, the effect is not good.

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