Expression Of Differential Gene Of Musca Domestica Larvae Induced By Escherichia Coli And Detection Of Antimicrobial Activities In Vitro

With the excessive use of clinical antibiotics, the emergence of resistant strains seriouslyaffect human health, the development of a new antibacterial drug has a unique mechanism ofaction to mitigate this issue has been imminent. People pay attention to the characteristics ofantibacterial peptide,it has a unique mechanism, non-toxic, no drug resistance and so on.It is thebest substitute of antibiotics. Musca domestica grew in the environment carrying many germs,but they didn’t get sick,due to the antibacterial active substance which is the body’s immunedefense system to produce. Therefore, study of Musca domestica antibacterial peptide by manydomestic and foreign scholars, has become the focus of antibacterial drugs.This study was based on the genes screened from suppression subtractive libraries (SSH) inMusca domestica larvae induced by E.coli. MdL1and three unknown functional genes of Muscadomestica(Md-UF1, Md-UF2, Md-UF3) were amplificated by RACE-PCR techniques, theproducts were sequenced and analyzed. Full-length PCR products were cloned into pMD18-TVector by T-A cloning technique. The Md-UF1, Md-UF2, Md-UF3,MdL1were subcloned intopET-32a (+) and pGEX-4T-1to construct a recombinant expression plasmid pET-32a-Md-UF1,pET-32a-Md-UF2,pGEX-4T-1-Md-UF2, pET-32a-Md-UF3, pGEX-4T-1-Md-UF3, pGEX-4T-1-MdL1and pET-32a-MdL1,transformed into E. coli BL21(DE3), induced by IPTG inductionand SDS-PAGE electrophoresis.The fusion protein was purified by nickel-metal chelate ionaffinity chromatography medium and antibacterial activity was analysed, the main results are asfollows:(1) Md-UF1, Md-UF2, Md-UF3and MdL1genes were amplified by rapid amplification ofcDNA ends (RACE), the full-length ORF sequence respectively is840bp,990bp,297bp,432bp.(2) Nucleotide and amino acid sequence analysis showed: Md-UF1, Md-UF2and Md-UF3genes have not yet found their homologous gene sequences; the mRNA sequence of MdL1genehas homology with AY344589.1was96%, there are5amino acid differences.The number ofMd-UF1gene encodes279amino acids, without signal peptide region, hydrophilicity,containsα-helix structure, the irregular coil and theβ-sheet structure; The number of Md-UF2geneencodes329amino acids, without signal peptide region, hydrophilicity,contains the irregularcoil and theβ-sheet structure; The number of Md-UF3gene encodes98amino acids, a signalpeptide region, hydrophilicity,contains α-helix structure, the irregular coil and theβ-sheet structure; The MdL1gene encodes143amino acids, a signal peptide region, hydrophilicity,contains α-helix structure, the irregular coil and theβ-sheet structure.(3) The prokaryotic expression plasmids of pET-32a-Md-UF1, pET-32a-Md-UF3, pGEX-4T-1-Md-UF3, pET-32a-MdL1, pGEX-4T-1-MdL1, pET-32a-Md-UF2and pGEX-4T-1-Md-UF2wereconstructed successfully. After induced by IPTG, pET-32a-Md-UF1, pET-32a-Md-UF3,pET-32a-MdL1and pGEX-4T-1-Md-UF2were successfully expressed in E.coli. Acquisitionsingle fusion protein by purification, the antibacterial experimental results show that theresistant strains of Escherichia coli isolated from clinical were inhibited by Trx-6His-Md-UF1,GST-Md-UF2、Trx-6His-MdL1fusion protein significantly; Fowl typhoid Salmonella resistantstrains isolated from clinical were inhibited by Trx-6His-Md-UF1,GST-Md-UF2fusionprotein;The four fusion protein did not have bacteriostatic action on Streptococcus suis.These results have lay the foundation for the further study of the mechanism aboutantibacterial activity of four fusion proteins and provided evidence to clinical search for newantimicrobial agents.