ClfA,EfB And FnBPA Antibody Inhibit Staphylococcus Aureus From Adhering To Bovine Mammary Epithelial Cells And Study On Immune Effect Of Secretory Protein EsxA In Mice

Staphylococcus aureus(S.aureus)caused of dairy cow mastitis as one of the most frequently occurred diseases of dairy cow,which was difficult to prevent and control and easy to recur,causing serious economic loss to cow industry and becoming a hazard to human health.S.aureus is one of main causative agent of bovine mastitis,Adhesins were one of the key factors when S.aureus infected the bovine mammary epithelial cells at the first stage of infection,Such as ClfA,EfB and FnBPA and so on.Which causes apoptosis of host epithelial cell EsxA was the main exocrine protein of the S.aureus,and has the character of immune evasion.This study focuses on comparing the different of adhesion inhibition of 3different adhesions antibodies immunized with ClfA,EfB and FnBPA.This study also expressed and purified EsxA recombinant protein of S.aureus and this was used to purified protein o immunize mice and immunological characteristics investigation of EsxA.This study that bovine mammary epithelial cells were isolated,purified and identified using mammary gland tissue culture and trypsin digestion method.Recombinant plasmids EfB and FnBPA were used in-combination with ClfA respectively to immunize new Zealand white rabbits and antibody levels were tested after immunization.The antibodies were used to inhibit the adhesion of 2 S.aureus isolates to bovine mammary epithelial cells in vitro.The plate counting method was used to quantitatively detect and compare the adhesive inhibition of antibodies immunized with different adhesins single or in-combination.The fluorescent staining were used to stain the cells and bacteria respectively and the adhesive inhibition of different antibodies were observed with fluorescent microscope.Our results indicated that bovine mammary epithelial cells were isolated and purified.Recombinant plasmids ClfA,FnBPA and EfB could be expressed in cells and induce specific antibodies after immunized the rabbits.The results of bacteria plate counting and fluorescent observation indicated that adhesins antibodies showed different adhesive inhibition ability to 2 different S.aureus isolates(GY278 and GY309)when immunized single or in-combination.ClfA showed the highest adhesive inhibition ability and FnBPA-A showed better inhibition ablity than that of FnBPA-D.The antibodies of rabbits that immunized with FnBPA+ClfA?EfB+ClfA had higher adhesive inhibition ability than that of single immunization group.The A domain of FnBPA showed greater enhancement to the adhesive inhibition of ClfA than t the D domain and A domain of FnBPA had no significant difference in adhesive inhibition enhancement to ClfA when compared with EfB.Conclusion that the in-combination immunization of FnBPA-A,FnBPA-D and EfB respectively with ClfA could affect the adhesive inhibition in various degree and this result provide experiment basis for the development ofbovine mastitis vaccine that target the S.aureus adhesins.In this study the EsxA was amplified using PCR and constructed prokaryotic expression plasmids pET28a-EsxA.The recombinant protein was induced and confirmed by SDS-PAGE and Western-blot analysis.Then the purified protein was used to immunize mice and the immune response,immune protection and histopathologic examination was investigated.Our results indicated that the recombinant expression plasmids pET-28a-EsxA was successfully constructed and after transforming and inducing it could expressed high level of EsxA protein.After immunizing mice with the purified protein the antibody titer of mice serum could reach 1:2700 and the mice protection rate was 75%.Bacteria counting result and histopathologic examination result indicated the bacteria loading of immune groups was significantly reduced than that of the control group.In addition EsxA+ClfA combination immunization could be helpful in reducing the bacteria loading and pathological damage of liver,spleen and kidney.This result laid experiment basis for the further studies on vaccine of bovine mastitis caused by S.aureus.