Isolation, Identification Of Bovine S. Aureus And Cloning, Expressiong Of FnBP And ClfA Gene

Bovine mastitis, an inflammation of the mammary gland, is the most important factor contributing to economic losses in the dairy industry. Although researchers work hard all over the world,there is no good method to control it. The most control method is using antibiotics. It is necessary to create new method in order to control mastitis, because a ntibiotics not only have side effect, also it can decrease milk quality. Certainly, vaccine prevention is the best way for controlling. Pathogenic survey showed that Staphylococcus aureus (S.A) is caused cow mastitis the main pathogenic factor, But till today, only E.coli form J5 vaccine is successfully development , its protection ratio is more than 85%. However , the Gram-positive vaccine rare, especially Staphylococcus, therefore, for Staphylococcus aureus vaccine research is an important value. The traditional inactivated vaccine and low virulence vaccine are not effective, this study used methods of genetic engineering, cloning and expression of the original cow mastitis major pathogenic Staphylococcus aureus antigen gene - the adhesion factor clfA,FnBP, for further study cows Mastitis Staphylococcus aureus in the breast tissue (milk duct) of the adhesion, growth and colonization FnBP and clfA two genes together synergies of livestock infected with the molecular mechanism provides a theoretical basis, but also for the development of genetic engineering cows mastitis vaccine. And the development of adjuvant to provide a new way of thinking and direction.Sources from the animals (cows) was isolated MRSA and highlights from the grassroots veterinary treatment of a variety of diseases caused by bacteria administration focus, express the gene encoding FnBP,clfA of Staphylococcus aureus and provide target protein for further immunological study, at the same time to study treatment of mastitis in dairy cows provides the theoretical basis for vaccine and drug research and development, such as providing direction. Recovery strain by conventional methods, with coagulase test reverification SA, KB, agar screening method, apparatus Identification Act (VITEK32), the polymerase chain reaction method. The conventional identification of Staphylococcus 105strains, Staphylococcus aureus (Staphylococcus aureus, SA)62 strains, MRSA 13 strains detection rate of 20.10%,Blurred 2 strains. Amplify FnBP/ clfA gene from the genomic (C2694)DNA of standard Staphylococcus aureus strain C2694 by PCR and insert into pGM -T vector by T/A cloning.Digest the recombinant plasmid with restriction endonuclease BamH I/ Nco I and Xho I and clone the obtained gene fragments into pET-28a(+) vector.Transform the constructed recombinant plasmid pET-28a(+)-FnBP/ clfA into BL21(DE3)/pET system for expression of Trix- FnBP/clfA fusion protein.The expressed product was identified by SDS-PAGE and Western blot. The homology of amplified FnBP/clfA gene sequence to that of FnBP/ clfA of Staphylococcus aureus (J04151/ AB245457)reported in GenBank was 100%/95.69%. SDS-PAGE showed a protein band with relative molecular weight of 58kDa/45kDa.The activity of recombinant protein was analyzed by Western blot. Part of the reclamation area in Xinjiang dairy cattle mastitis has been kind of MRSA was spreading trend in the sick livestock suffering from the disease were detected in milk samples MRSA detection rate of 12.38%. Staphylococcus aureus FnBP/ clfA fusion protein was successful expressed.It laid a foundation of study on role of FnBP/ clfA in pathopoiesis of bacteria and development of relevant vaccine.