Studies On Activities Of Cloning And Expression Of Antibacterial Peptide Attacin Gene Isolated From Musca Domestica

With resistance to antibiotics, more and more in-depth study of drug side effects, development of new natural antimicrobial agents has become the hotspot of antibiotics. Antimicrobial peptides as a new category of natural peptide antibiotics, can kill resistant strains, and the bactericidal mechanism of resistance to pathogenic mutation is not easy, which has broad application prospects. However, the main source of antimicrobial peptides was natural extracts, which costs relatively high. Using molecular biology methods, the target gene which was transformed into the bacterial expression, thereby reducing the technical difficulty and cost of purification.This paper focused on Musca domestica Md-Cec gene expression in E. coli, and purification of the expressed product and its activity detection. Total RNA of the housefly-pupae as template, obtained Md-Cec gene from Musca domestica by RT-PCR. The target gene was inserted into plasmid pMAL-c2x and reorganized into the expression strain induced for expression. The induced protein after extracting and purificating was pure. Finally, a preliminary determination of antibacterial activity of the purified protein was done.The results showed that:The total RNA of house fly pupae as template, gene from Musca domestica Md-Cec obtained by RT-PCR. was successfully constructed into cloning vector pMAL-Md-Cec, and Md-Cec gene sequencing resulted consistent with the Genebank dates. According to the nucleic acid sequence encoding amino acid sequence of the Musca domestica predicts the chemical properties and structure of the protein.BL21(DE3) PlysS was the best expression strain to the recombinant plasmid pMAL-Md-Cec. IPTG concentration of0.5mM,induction time of4hours were the best inducing conditions. Extracted by crushing the expression cell after purification with amylose resin column, expression protein was high purity and high concentration (control protein pMAL (P) content was:7.565mg/mL; recombinant pMAL-Cec (A) content of:6.170mg/mL). Compared to the control strain bacteriostatic growth curve of the recombinant strains had a certain inhibitory effect on its own; purified protein results showed that there were some antibacterial activities to Bacillus subtilis and Salmonella.