Expression And Analysis Of Multipoint Mutagenesis Of Porcine β Defensin 2

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,which are important for their applications. Porcine β defensin 2 (PBD2) showed broad-spectrum antimicrobial activity,which made microorganisms hard to resist. In addition, pBD2 showed low toxic against porcine blood,which made it attention. On the basis of preliminary experiments in our lab, we designed different multipoint mutagenesis based on the sequence of optimized pBD2 gene.The cloning, expression and analysis of different muted proteins were studied.The details were as follows:1. According to the codon preference in E. coli, PBD2 gene was optimized which was in order to improve the expression in E. coli. The antimicrobial activities were similar with non-optimized PBD2.2.On the basis of the previous experimental results, We structured multipoint mutagenesis ofpBD2.The primers containing different multipoint mutation were designed and synthesized. Six multipoint mutagenesis (named for M1-M6) were amplified by PCR useing optimized pBD2 as templates. The PCR products were connected with pMD18-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-M1-M6 were successfully constructed respectively.3. The strains BL21-pET30a-Ml-M6 were induced and expressed respectively. SDS-PAGE analysis showed that the molecular weight of target protein were consistent with the expection.The purity of protein can reach more than 90% after purified by nickelion affinity chromatography.The antibacterial activity, hemolytic activity and thermal stability were analyzed as well.The results showed M1-M6 had obvious inhibitory effect on S.aureus2952 and E.Coli29522. and the hemolysis were less than 10%. PBD2 showed thermal stability after heat treatment at 80℃ for 30 min.In conclusion, In high concentration, M1, M4, M5,M6 had higher antibacterial activity than PBD2, which was the base for further study of the relationship between structure and function of pBD2.