Expression And Activity Analysis Of Differential Gene Md-D1, Md-D2 And MdL1 From Musca Domestica Larvae In P. Pastoris

The kind of clinical drug-resistant strains have been increasing with the wide and irrational use of antibiotics, which has brought huge economic loss to graziery as well as threatened the health of animal and human seriously, researching and screening new antimicrobial agents to replace the traditional antibiotics is a key to the solution of the current problem. Musca domestica living in a variety of pathogenic bacteria breeding environment carry and spread the bacteria but rarely infect themselves, due to an increasing number of its in vitro and in vivo antibacterial active substance. Therefore, more and more researchers focus on Musca domestica antimicrobial peptides in order to developing new antimicrobial drugs. Musca domestica can produce many differential genes which can express antibacterial protein after induced by microbial pathogens. The screening, expression and biological activity assay of differential genes through engineering technology is in progress to screening new antibacterial drugs from Musca domestica, which will lay foundations to solve drug-resistant problem.This study was based on three full-length differential genes screened and cloned from suppression subtractive library(SSH) in Musca domestica larvae induced by Mycoplasma hyopneumoniae and pathogenicity of Escherichia coli(differential gene 1 of Musca domestica(Md-D1), differential gene 2 of Musca domestica(Md-D2), lysozyme gene 1 of Musca domestica(MdL1)), PCR was performed to amplify the three full-length differential genes, the full-length differential genes were cloned into pMD18-T Vector by T-A cloning to construct positive cloned plasmid, Md-D1 was subcloned into pGAPZαA to construct the eukaryotic expression plasmid Md-D1- pGAPZαA; Md-D2 and MdL1 were subcloned into pPIC9 K to construct the eukaryotic expression plasmid Md-D2- pPIC9 K and MdL1-pPIC9 K. Recombinant plasmids were integrated into P.pastoris GS115 through electroporation; the antibacterial activities of expression product of Md-D2 and MdL1 were analyzed by cup-plate method and microdilution method. The main results are as follows:1. Md-D1, Md-D2 and Md L1 were successfully amplified by PCR and the cloned plasmid Md-D1-pMD-18 T, Md-D2-pMD-18 T and MdL1-pMD-18 T successfully constructed.2. Eukaryotic recombinant expression plasmids Md-D1-pGAPZαA, Md-D2-pPIC9 K and MdL1-pPIC9 K were successfully constructed, Md-D2 and MdL1 were expressed in P.pastoris GS115; the optimum inducing conditions of GS115/ Md-D2-pPIC9 K were as follows: optimum inducing time was 72 h, pH of medium was 6; the optimum inducing conditions of GS115/ MdL1-pPIC9 K were as follows: optimum inducing time was 72 h, pH of medium was 5.3. The antibacterial experimental results show that the purified expression product of MdL1 inhibits the resistant strains of Escherichia coli from clinical and possess weak antibacterial activity against Salmonella resistant strains; the purified expression product of Md-D2 that does not inhibit the strains of clinical isolates, is still a Musca domestica gene with unknown function.