Expression Of The Site-directed Mutangenesis Porcine-β Defensin-2 And Its Preliminary Antibacterial Activity Analysis

Porcineβ-defensin-2 has attracted increasing attention for its characteristics such as efficient broad-spectrum antimicrobial activity,difficult to make microbes resistant to it and weak cytotoxicity. With the understanding of antimicrobial peptides’structure,activity,antibacterial mechanism and regulation of gene expression,it’s entirely feasible to design a kind of antimicrobial peptide which is efficient and good for human health to replace antibiotics. In our study,we forecast the protein structure of porcineβ-defensin-2 by bioinformatics tools and make site-directed mutagenesis selectively in the amino acid sequence ,only to acquire the construction of porcineβ-defensin-2 mutant called mpBD2.We make one or more point mutant in the protein to exploit new-type porcineβ-defensin-2 with more biological activity. The main research contents are as follows:1. Tthe Laboratory has been constructed pBD2 gene as template, by designing PCR primers to introduce the different sites of seven different mutations, and their respective connected to the PMD19-T vector, after PCR, restriction enzyme digestion and end sequencing Demonstrate the different mutations were inserted into the expression vector PET30a in the successful construction of the seven different prokaryotic expression vector PET30a-mpBD2-1 ~ 7, the final transformed into E. coli BL21 (DE3) PlysE, the obtained expression strain BL21-PET30a seven -mpBD2-1 ~ 7.2. Recombinant strains with mutations in BL21-PET30-mpBD2-1~7 were induced for expression, the expressed product by nickel ion affinity chromatography purification, the initial purpose of the purified protein.3. Detection of antibacterial activity of seven recombinant proteins. Antibacterial results show that the minimum inhibitory concentration 20μg/mL take samples of harmful strains were cultured with three to observe the dynamic growth trend, the results show 24h time, mpBD2-4, 5,7 of the recombinant protein on the inhibition of three bacteria Activity was higher than mutation pBD2; mpBD2-1,2,3,6 of different strains were lower than the corresponding control pBD2 antibacterial results.4. Detection of hemolytic activity of seven of the recombinant protein. The results showed that 1. the experimental group mpBD2 hemolytic rate in all conditions, the final concentration was lower than the control group pBD2. 2.recombinant protein mpBD2 maximum hemolysis rate of 10%. 3. mpBD2-3 hemolytic rate in the comparison data, hemolysis was the lowest,only 5%. In summary, we successfully cloned seven mutant recombinant porcineβ-defensin-2 gene mpBD2-1 ~ 7, and with and without mutations pBD2 antibacterial activity compared to the initial access to the reorganization of antibacterial activity than pBD2 Protein mpBD2-4, 5,7, in order to further study the biological activity and inhibition mechanism of the foundation.