Preliminary Study Of Anti-S.aureus Mechanism Of Porcine ?-Defensin-2 In Vitro

Mammalian defensins attract wide attentions because of their strong and broad antimicrobial spectrum,and pathogenic microorganisms having little resistance to them.Therefore,defensins were proposed as a new effective antimicrobial agents instead of traditional antibiotics in practice..porcine ? defensin 2(p BD2)is found late,and natural p BD2 islimited.so its antibacterial mechanisms is little sudied in vitro and in vivo.In order to explore the antimicrobial mechanism of porcine ? defensin 2,the differentially expressed endogenous genes were detected in S.aureus which were co-cultured with p BD2 in vitro,the results were as follows:1.pBD2 were induced and purified based on engineering strain BL21-pET30a-pBD2 in our laboratory.The results showed that p BD2 could inhibit the growth of S.aureus(109 CFU/ml)and the antimicrobial activity incresed with times and concentrations.2.S.aureus were co-culture with different concentrations of pBD2(0 ?g/ml ? 37.5?g/ml?75 ?g/m and 150 ?g/ml)for 1h and 4 h.then the total RNAs were extracted from the S.aureus and differentially expressed genes in S.aureus were detected by the Gene Fishing.All the differentially expressed genes were cloned and sequenced.The results showed that 11 differentially expressed bands were found when S.aureus were co-cultured with pBD2 for 1h,and 12 differential expressed bands were found for 4h.3.by blast,the differentially expressed genes associated with cell membrane synthesisand transport are gluconate transporter,membrane protein,Na(+)H(+)ant Iporter subunit D,gamma-aminobutyrate permease,bacitracin ABC transporter permease,heme ABC transporter permease,MFS transporter,membrane protein,hypothetical protein and molybdenum cofactor biosynthesis protein A.Genes associated with DNA or RNA synthesisare Excinuclease ABC subunit B,DNA mismatch repair protein Mut L,23 S r RNA,replication-associated protein,DNA topoisomerase III,translation initiation factor 2,translation initiation factor 3.Genes associated with metabolism and protein synthesis are6-phospho-beta-galactosidase,long-chain fatty acid-Co A synthetase,thiamine-phosphate pyrophosphorylase,Zn-dependent hydrolase,quinol oxidase polypeptide II,Ara Cfamily transcriptional regulator.Totally,the differentialgenes in S.aureus are mostly permease,transporter and phosphatase.4.The flourescence quantitative analysis showed most of differentially expressed genes were down expressed which are related to cell membrane composition and transportation.these differentially expressed genes could inhibt the S.aureus by enhancing the cell permeability,hindering the cell wall or membrane synthesis.