Eukaryotic Expression Of MDAP-2,Md-UF4 And Md-UF21 Gene From Musca Domestica And The Study Of Antimicrobial Activities Of Products

Antimicrobial peptide is a type of cationic peptide which is produced by animal innate immune system against microbial invasion. It can effectively prevent the invasion of bacteria and has a broad spectrum of antimicrobial activity. Compared to the traditional antibiotics, it has lower possibility of causing the problem of bacterial resistance. Therefore, in recent years,the research of antimicrobial peptide has been highly valued by domestic and foreign scholars.Musca domestica, living in a variety of pathogenic bacteria breeding environment, can carry and spread the bacteria yet rarely get infected themselves. This is owing to its in vitro and vivo antibacterial active substance. As one type of the most effective antibacterial active substances,antimicrobial peptide of Musca domestica can inhibit a variety of bacteria, fungi, parasites,viruses, cancer cells and have no damaging effect on normal cells. In the field of antimicrobial peptides, recent research is mainly focused on the screening, expression and biological activity of antimicrobial peptides.In this study, three full-length differentially expressed genes(MDAP-2, Md-UF4,Md-UF21), screened from the SSH library of Musca domestica induced by high pathogenicity of Escherichia coli and Salmonella pullorum, were chosen and developed using PCR to them.Followed by first cloning the full-length differentially expressed genes into p MD18-T vector by T-A cloning, and then subcloning them into eukaryotic expression vector to construct recombinant eukaryotic expression plasmids(p PIC9K-MDAP-2, p PIC9K-Md-UF4,p PIC9K-Md-UF21). Futhermore, the recombinant plasmid was transformed into the Pichia pastoris GS115 cells through electroporation. PCR-confirmed, positive recombinants was expressed by induction of methanol. The expression was detected using SDS-PAGE or Tricine-SDS-PAGE. Optimization was conducted on the temperature and p H environment of fermentation broth of the recombinant protein. The recombinant protein was purified using Ni-NTA His Trap FF crude column chromatography, followed by assessment of its bacteriostatic activity via tube plate method. The main results are as follows:(1) Three full-length differentially expressed genes, MDAP-2, Md-UF4, Md-UF21 were developed by PCR. The three recombinant cloning plasmids were successfully constructed through cloning the three genes into p MD-18 T vector.(2) Eukaryotic recombinant expression of plasmids pPIC9K-MDAP-2, pPIC9K-Md-UF4,p PIC9K-Md-UF21 was successfully constructed. MDAP-2 and Md-UF4 were expressed in Pichia pastoris expression system. The optimal induction conditions of GS115-MDAP-2 were as follows: the induction time was 96 h and the p H value of fermentation broth was7. The optimal induction conditions of GS115-Md-UF4 were as follows: the induction time was 72 h and the p H value of fermentation broth was 7.(3) The recombinant proteins were purified using Ni-NTA His Trap FF crude column chromatography. MDAP-2 had in vitro antibacterial activity against Escherichia coli and Salmonella pullorum. Md-UF4 didn’t inhibit the three strains of clinical isolates still remains a Musca domestica gene with unknown function.