Expression And Activity Analysis Of Site-directed Mutangenesis Of Porcine β-defensin-2

Abstract:Defensins are a family of low-molecular weight peptides with resistance to microorganisms, which exist widely in nature. Mammal defensins have the broadest antibacterial spectrum and it is important for their applications. Porcine β-defensin-2 was secreted by pigs,and showed broad-spectrum antimicrobial activity,low toxic against porcine blood which made it a good candidate to prevent and cure pig diseases. Here different site-directed mutagenesis were designed based on the sequence of porcine β-defensin-2,and the cloning, expression and activity analysis of different muted proteins were studied.The details were as follows:1. The physicochemical property of the polypeptides with different mutational sites were analyzed and eight points were chose with higher net electrical charge,better amphipathic and lower instability index.2. The primers containing different mutant bases were designed and synthesized. Eight site-directed gene sequences(named for mpBD2-1～8) were amplified by PCR used porcine beta defensin-2 as templates. The PCR products were connected with pMD19-T vector and then were linked to pET30a plasmid after double digestion with restriction endonuclease Ncol and HindⅢ,respectively. The expression vectors were transformed into BL21(DE3)PlysS respectively, and the expression strains BL21-pET30a-mpBD2-1-8 were successfully constructed respectively.3. The strains BL21-pET30a-mpBD2-1-8 were induced and expressed respectively. The target protein accounted for 12.8% of total protein by SDS-PAGE analysis.Fusion proteins were released by sonication and purified by nickel ion affinity chromatography, their antibacterial activity and hemolytic activity were analyzed.The results showed different defensins with different muted site showed antimicrobial activity against Escherichia coli and Staphylococcus aureus.In additions, the defensin showed stronger activity against Staphylococcus aureus. Three new defensins mPBD2-1、mPBD2-2 and mPBD2-3 were better than PBD2,the hemolytica analysis indicated that the ratios of hemolysis were less than 5% for all the muted PBD2.In summary, eight mutant recombinant porcine β-defensin-2 genes mpBD2-1～8 were successfully cloned, and the corresponding recombination protein were purified and analyzed for their antibacterial activity. Three new defensins with relatively higher antimicrobial activity were selected, which was the base for the study of the mutational sites and the relationships between structure and function.