Preparation And Analysis On Inhibitive Bacterial Activity In Vitro Of Antibacterial Proteins In Musca Domestica

Antibiotics have played an important role in the treatment of human bacterial infectionsand prevention of livestock diseases. But the kind and the amount of drug-resistant strains havebeen increasing with large and irrational use of antibiotics, which have threatened the health ofanimal and human seriously. It is a currently problem to be solved that researching andscreening new antimicrobial agents to instead of the traditional antibiotics. It is a researchhotspot that antibacterial activity substances have features of low toxic and difficult to makebacteria produce drug-resistant in Musca domestica.Musca domestica and its larvae enduring expose to harsh environments chronically butrarely infected, suggesting that it has the ability to adapt to the harsh environment highly, thisability due to its antimicrobial substances present in the body[1-3]. The antibacterial substancesgenerally include antimicrobial peptides, lectins, chitin, alcaligenes faecalis, organiccompounds, allantoin and some proteins of unknown function in Musca domestica.This study was based on the genes screened from suppression subtractive library(SSH) inMusca domestica larvae induced by Mycoplasma hyopneumoniae. An unknown functional geneof Musca domestica (Md-UF1) was amplificated by RACE-PCR techniques, the product wassequenced and analyzed.Full-length PCR product was cloned into pMD18-T Vector by T-Acloning technique and the cloning plasmid Md-UF1-pMD18-T was constructed successfully.The Md-UF1, Md-UF2, MddⅠwere subcloned into pET-32a (+) to construct recombinantexpression plasmids Md-UF1-pET-32a, Md-UF2-pET-32a, MddⅠ-pET-32a, transformed intoE.coli BL21(DE3), induced by IPTG induction and SDS-PAGE electrophoresis. The fusionprotein was purified by nickel-metal chelate ion affinity chromatography medium (Ni-NTAAgarose)and antibacterial activity was analysed, the main results are as follows:1.The full-length ORF of Md-UF1sequence is531bp. Its hydrophobicity strong theoreticalmolecular weight is18.938ku, theoretical isoelectric point is8.23, without transmembraneregion, not a membrane or secreted protein, a signal peptide region, two α-helix structure, theirregular coil and the main structural elements of the chain extension are presented, smallamounts of β-sheet structure dispersed throughout the proteinby bioinformatics analysis.2.The recombinant expression plasmid of Md-UF1-pET-32a, Md-UF2-pET-32a,MddⅠ-pET-32a was expressed in E. coli. Protein mainly insoluble proteins in inclusion bodies. The best conditions for fusion protein expression were Md-UF1:IPTG concentration0.4mM,25℃induction4h;Md-UF2:IPTGconcentration0.4mM,30℃induction6h;Mdd Ⅰ:IPTG concen-tration0.4mM,30℃induction6h.3.Acquisition single fusion protein by purification, the antibacterial experimental resultsshow that the resistant strains of Streptococcus suis isolated from clinical were inhibited byMd-UF1-pET-32a, Md-UF2,-pET-32a MddⅠ-pET-32a fusion protein significantly; Escherichiacoli and fowl typhoid Salmonella resistant strains of chickens isolated from clinical wereinhibited by Md-UF1-pET-32a, Md-UF2,-pET-32a fusion; while Mycoplasma hyopneumoniaewere all inhibited by Md-UF1-pET-32a, Md-UF2-pET-32a, MddⅠ-pET-32a fusion protein.These results have lay the foundation for the further study of the mechanism aboutantibacterial activity of the three fusion proteins and provided evidences to clinical rsearch fornew antimicrobial agents.