Prokaryotic Expression And Antibacterial Activity Analysis Of Differential Gene Of Musca Domestica Larvae Induced By Pathogenic

Antimicrobial peptides(AMPs),as the first line to defend outer pathogenic bacteria,constitutes an important part of body’s innate immune system.AMPs also performs direct and effective defense to invasive pathogens without immune memory.Recently,a variety of active AMPs have been isolated from plants,amphibians,insects or even human body.Musca domestica,living in environment full of pathogenic microorganism and carring pathogenic microorganisms,is widely recognized as mechanical vectors to spread pathogenic bacteria to human and animals but rarely get infected,which can be attributed to their unique immune defense mechanism.It was reported that housefly antimicrobial peptide/protein(AMPs)played a major role in its body immunity,and had a strong inhibitory effect on bacteria,viruses,fungi,and cancer cells.Therefore,antimicrobial peptides,as potential antimicrobial agents,have received increasing focus of attention.Due to the limited natural housefly antibacterial peptides,chemical synthesis and genetic engineering have become the major methods to obtain AMPs.In the present study,three full-length gene(housefly unknown function gene MD-UF672,housefly unknown function MD-UF21 and Musca domestica larva antibacterial proteins MLAP)were screened from M.domestica larva suppression subtractive library(SSH)induced by Pasteurella multocida,avian Escherichia coli and Samonella.The full-length gene were amplified by PCR and then inserted into a pMD18-T vector.The MD-UF672(digested by BamH?/Xho?)was linked to pET-32a(+)expression vector,MD-UF21(digested by Eco R?/Xho?)and MLAP fragment were connected to pGEX-4T vector and pET-32a(+)expression vector respectively,to construct prokaryotic recombinant expression plasmid pET-32a(+)-MD-UF672,pET-32a(+)-MLAP and pGEX-4T-MD-UF21.The recombinant plasmids were transformed into E.coli BL21(DE3).Then,the recombinant protein pET-32a(+)-MD-UF672,pET-32a(+)-MLAP were purified using Ni-NTA affinity chromatography,and pGEX-4T-MD-UF21 using GST affinity chromatography.Antimicrobial activity of MD-UF672,MD-UF21 and MLAP purification products were tested by Oxford plate assay system.Growth inhibition assay and the minimal inhibitory concentration(MIC)of the recombinant protein pET-32a(+)-MLAP to Escherichia coli,Samonella and Staphylococcus aureus were detected,and the main results were as follows:1.The MD-UF672,MD-UF21 and MLAP full-length gene were amplified byPCR and cloned into pMD18-T successfully.2.Prokaryotic recombinant expression plasmid pET-32a(+)-MD-UF672,pET-32a(+)-MLAP and pGEX-4T-MD-UF21 were successfully constructed and expressed in E.coli BL21 expression system.3.Antibacterial activity analysis of MD-UF672,MD-UF21 and MLAP purification product showed that the recombinant protein pET-32a(+)-MLAP could inhibit clinical isolated E.coli,Salmonella and Staphylococcus aureus.The recombinant protein pET-32a(+)-MD-UF672 and pGEX-4T-MD-UF21 which have no antibacterial activity are housefly protein with unknown function.4.Growth inhibition assay and minimal inhibitory concentration(MIC)of the recombinant protein pET-32a(+)-MLAP to E.coli,Salmonella and Staphylococcus aureus were detected and growth inhibition curve was drawn.The MIC values of MLAP protein against E.coli,Salmonella and Staphylococcus aureus were 0.012μg/ml,0.003μg/μl and 0.025μg/μl,respectively.