Preliminary Study Of Anti-E.Coli Mechanism Of Porcine ? Defensin 2 In Vitro

Porcine defensins are antimicrobial peptides,which are widely found in pigs in recent years.They can protect hosts from a broad range of pathogens including bacteria,virus and fungu.Therefore,porcine defensins are proposed as new effective antimicrobial reagents for pig diseases.In all,the discovery of porcine ? defensin 2(pBD-2)is late,and natural pBD-2 are limited,so there is less research on its antibacterial mechanisms in vitro and in vivo.A large number of studies have shown that the special antibacterial mechanism of defensins is the reason that bacteria are hard to make resistance to it.Thus,in order to ascertain the antimicrobial mechanism of porcine ?defensin 2,the differential expression of endogenous gene in E.coli were detected when E.coli were co-cultured with pBD-2 in vitro,the results were as follows:1,The strain BL21-pET30a-pBD-2 was induced and crushed by sonication,the supernatant was collected and was applied on the Ni-NTA agarose column(His tag affinity column)to purify recombinant protein.By SDS-PAGE analysis,the purity is more than 95%.The antimicrobial activity of pBD-2 was evaluated by 96-well turbidimetric method after protein purification.It is showed that pBD-2 significantly inhibited the growth of E.coli.2,On the basis of the experiment above,150?g/ml pBD2 were selected to co-culture with E.Coli(E.Coli ATCC 25922 standard strain)in certain time(1h and 4 h).The total RNAs were extracted from the E.Coli cell,then differential expressed gene in E.coli were detected by the GeneFishing,then the differential expressed genes were cloned and sequenced.The results showed that 12 differential expressed bands were found when E.coli co-cultured with pBD-2 for 1h,and there were 7 fragments successfully cloned and sequenced;The results also showed that 18 differential expressed bands were found when E.coli co-cultured with pBD-2 for 4h,and there were 9 fragments successfully cloned and sequenced.3,These different genes were detected by semi-quantitative and real-time quantitative PCR.The final result showed that there were 3 differential expressed genes in E.coli when co-cultured with pBD-2 for 1h,there were 5 differential expressed genes in E.coli when co-cultured with pBD-2 for 4h.4,Differential expressed sequences were analysized by bioinformatics then detected by real-time quantitative PCR,the results showed that:the differential expressed fragments obtained in this study can be found highly homologous to known genes.The three different genes in E.coli which were co-cultured with pBD-2 for 1h were the transfer-messenger RNA(down),regulatory RNA CsrB(up)and Lactose operon repressor(down);The five different genes in E.coli which were co-cultured with pBD-2 for 4h were the Trehalose-6-phosphate phosphatase(down),a hypothetical protein(down),potassium transporting ATPase subunit B(up),phosphohistidine phosphatase(down)and another hypothetical protein(down)respectively.The different genes in E.coli which were co-cultured with pBD-2 for 1h mostly were regulatory genes,and the different genes in E.coli which were co-cultured with pBD-2 for 4h mostly were functional genes,which may invole in the procedures of pBD-2 inhibiting bacteria.The functions of These differential expressed genes,was that hinder the synthesis of proteins or enzymes,or involved in regulation of bacterial DNA or RNA,or enhance the permeability of the bacterial cell and hinder bacterial cell wall synthesis.Then subsequent trials required further verification.In this research,differential expressed genes were detected in E.coli when co-cultured with pBD-2 in different time.The study just preliminarily elucidated the antimicrobial mechanisms of porcine ? defensin 2,further study should be carried on.