Ebp Pilus Gene Conservation Analysis And Polyclonal Antibody Preparation Of Animal-originated E.feacalis Isolates

E.faecalis are recognized as important pathogens in hospital and animal infections and pose serious threats to human and animal healths, food safety.The intestinal epidermal barrier is generally considered to be priority invision entrance of bacterial pathogens and the key step in the infection process is attachment and colonization to intestinal epithelial cells.Pilus plays a key role in infections from G+ and G- bacteria,but the carrying rate, conservation and pathogenicity of ebp pilus genes are unclear in animal-originated E.feacalis isolates.In this stud, we analyzed the distributions and conservations of ebp genes in animal-originated E.feacalis isolates.We also got the ebp pilus proteins by prokaryotic expression and prepared high titers specific polyclonal antibodies to analyze the pathogenicity and immunogenicity of ebp pilus.Which laid a good foundation for the prevention and treatment of E.faecalis infection.The ebp genes of fifty animal-originated E.feacalis isolates in henan region were detected by PCR method with the primer pairs described by Nallapareddy et al.The results of detection indicatesd that all fifty E.faecalis carried ebp ABC genes.Acording to the ebp genes sequences of E.faecalis OG1 RF in Gen Bank,the primer pairs of complete ebp ABC genes were designed by using Primer Premier 5.0 to amplify the coding genes of the ebp pilus proteins of ten E.faecalis.Productions were purified and recovered with gel extraction kit and connected with p MDl8-T vector,then transformed into competent cells of E.coli DH5α, and positive clones were selected for sequencing.The ebp genes sequences of ten E.faecalis were analysed by using DNASTAR software.The results of sequences analysis of ten E.faecalis indicatesd that the homologies of ebp ABC DNA sequences were respectively 98.0%-100%, 98.8%-100% and 99.0%-100%,and the homologies of Ebp ABC proteins amino acids sequences were respectively 98.4%-100%, 98.2%-100% and 98.7%-100%.Compared to E.faecalis OG1 RF, the homology of the ebp genes DNA sequences was 98.2%-99.7%,and the homology of the Ebp proteins amino acids sequences was 98.5%-99.8%.In conclusion,the ebp genes were ubiquitous and highly conserved among animal-originated E.feacalis isolates and were high homology with human E.faecalis.We predicted the epitopes of Ebp A protein and Ebp C protein in E.faecalis by using bioinformatics softwares.Accoding to the analysis results, the ebp A gene was divided into three fragments to express, named ebp A1, ebp A2 and ebp A3 respectively and the ebp C gene was divided into two fragments to express, named ebp C1 and ebp C2 respectively.Then, acording to the ebp A and ebp C genes sequences of E.faecalis OG1 RF in Gen Bank,five pairs of primers with reasonable restriction enzyme sites were designed to amplify ebp A1(1167bp), ebp A2(1098bp), ebp A3(963bp), ebp C1(981bp) and ebp C2(891bp) fragments of E.faecalis N9.The EF1092A(1233bp) gene of E.faecalis N9 was amplified by using the primer pairs described by Jouko Sillanpaa et al.Then we cloned these fragments into the p ET-28 a vector and successfully built the recombinant expression plasmids p ET-28a-ebp A1, p ET-28a-ebp A2,p ET-28a-ebp A3,p ET-28a-ebp C1,p ET-28a-ebp C2 and p ET-28a-ebp B(EF1092A).We transformed these recombinant plasmids into the expression strain BL21(DE3), successfully expressed proteins by IPTG induccing.These proteins approximately were 46.7 k Da, 45.0 k Da, 39.4 k Da, 39.3 k Da, 36.0 k Da and 50.0 k Da,which consisitented with the predicted fusion protein.By optimezing the expression condition,we got a large number of expressed recombinant proteins and purified recombinant proteins by urea and Ni agrose.We immunized the New Zealand white rabbits with the purified recombinant proteins and prepared six kinds of polyclonal antiserums.By using agar diffusion test method to detect positive antibodies titer,which were respectively 1:16, 1:8, 1:32, 1:32, 1:64 and 1:64. Western blotting exprement was done by using polyclonal antibodies and the results found that polyclonal antibodies could bind their recombinant proteins.In the experiment of biofilm production and the results showed that the biofilm production was the least in E.faecalis N9 when the final concentration of anti-Ebp A3 Ig G was 0.1875 mg /m L.Compared to the negative control, it was significantly different(P <0.05),which indicated that anti-Ebp A3 Ig G could inhibit biofilm generation of E. faecali N9.