Expression Of AS61、AS566 And DefensinⅠof Musca Domestica And Analysis Of Antimicrobial Activities Of Products

With the irrational use of clinical antibiotics, the circumstance of antibiotic resistance is grimmer than before, which affects the health of human and animal seriously. The research of new pharmacy to substitute for traditional antibiotics is particularly important. Musca domestica and its larva possess the unique defense system to resist the infection of pathogenic bacteria so that they can survive in the dankish environment with bacterial. Antimicrobial peptides, the indispensable member of the defense system, can resist to variety of pathogenic bacteria, which hardly cause bacterial to produce drug resistance and produce drug-resistant bacteria and toxicity arousing interest from scholars both at home and abroad.This study was based on genes screened from suppression substactive libraries(SSH) in Musca domestica 3-day-old larvae induced by salmonella. AS61(an unknown functional gene of Musca domestica larva) was amplificatied by PCR, the sequence of amino acid and nucleic acid of AS61 was sequenced and analyzed. We constructed recombinant expression plasmid of AS61 pET-32 a, and made it express in the prokaryotic expression system. Based on the full length of the geng of AS566 and DefensinⅠwhich had been amplified. We constructed recombinant expression plasmid of pET-32 a, pGAPZaA-AS566 and pGAPZaA-DefensinⅠ, and made them express in the eukaryotic expression system. The antibacterial activity of expression production of all three genes would be tested by In vitro antibacterial test, the main results are as follows:1. AS61 genes were amplified by PCR, the full-length ORF sequence is 537 bp.The sequence of AS61 was analysed indicating that it encoded 179 amino acid, theoretical isoelectric point was 7.61, without transmembrane region.Secondary structure contained Alpha helix, extended strand, random coil, and few beta turn.2. The recombinant expression plasmid of pET-32 a was successfully expressed in E.coli under the condition of 0.4 mM of IPTG and 4h.The product is fusion, mainly existing in the supernatant. The recombinant expression plasmid of pGAPZαA-AS566 and pGAPZαA-Defensin were successfully expressed in P.pastoris at 72hⅠ, which was the optimal time to express.The products existed in the supernatant.3. The prokaryotic pET-32a-AS61 was purified and concentrated. The antibacterial activity of the production was testing by bacteriostatic experiment. The antibacterial experimental result showing that the fusion protein did not have bacteriostatic action on E.coli, Staphylococcus aureus and Salmonella.The eukaryotic expression products were concentrated by ultrafiltration device, the antibacterial experimental results showed that the resistant strains of E.coli isolated from clinic were inhibited by pGAPZaA-DefensinⅠ, but the production did not inhibit the growth of Staphylococcus aureus and Salmonella; the resistant strains of salmonella and Staphylococcus aureus isolated from clinical were inhibited by pGAPZaA-AS566, but the production did not inhibit the growth of E.coli.The results have laid foundation for further stud on mechanism concerning antibacterial activity of three proteins from Musca domestica Larva and povide theoretical evidence for the research of new agents.