| Bovine mastitis is an inflammation reflection in mammary glands that was stimulated by the factors. There are many kinds of pathogens, such as bacteria, fungi, virus and mycoplasma. Staphylococcus aureus is considered as one of the most important pathogens which causes bovine mastitis. The major virulent factors of S. aureus are adhesions, toxin, biofilm, capsular polysaccharide(CP) and enzyme. Adhesion and colonization in the mammary epithelial cells were considered as the first stage of infection. Blocking S. aureus adherence to cell and colonization of the mucosal surface may be the most effective strategy for preventing infection. Fibronection-binding protein B(FnBPB) and clumping factor A(ClfA) are important adherension factors. The D domain of Fibronection-binding protein B(FnBPB-D) and the A domain of clumping factor A(ClfA-A) are main function domains about adherension. And capsular polysaccharide is also one of mainly virulence factors at the first stage of infection.Fc receptor(FcRn), is considered as the receptor that specifically binding to immunoglobulin G Fc fragment. FcRn could mediate to transport IgG by binding to IgG Fc, then FcRn could mediate transcytosis to transport antigen across mucous membrane to act antigen submission ruction to induce special local immunity of mammary gland which could prevent infection. IgG Fc gene from wistar rat was fused to FnBPB-ClfA gene by SOE-PCR and cloned into pET-32a(+) vector to express the recombinant protein of rFc-FnBPB-ClfA in E. coli. The target fragment was induced expression by IPTG, and SDS-PAGE showed the molecular weight of rFc-FnBPB-ClfA was 78 KDa. Western Blot indicated fusion recombination protein(rFc-FnBPB-ClfA) have good reactionogenicity, Fc, FnBPB and ClfA all have protein activity. Then the purified CP8, EDC as the couplant, binds to Fc-FnBPB-ClfA with 1.89:1 ratio into conjugation CP8+Fc-FnBPB-ClfA successfully. At last, lacting rats were immuned with rFnBPB-ClfA, rFc-FnBPB-ClfA, CP8+FnBPB-ClfA and CP8+Fc-FnBPB-ClfA separately by muscle and teat canal(the forth on the right, R4), alum and E. coli heat-labile toxin B subunit(LTB) as different adjuvants, at the same time rats were immuned by rFc-FnBPB-ClfA first by muscle injection, and teat canal as boost immune with LTB. Except that, the rat anti-FnBPB-ClfA serum which was obtained before, mixed with rFnBPB-ClfA and rFc-FnBPB-ClfA separately to different antigen-antibody complex, then rats were immuned with complex by LTB. According to the immunization above, the experiments were carried out to evaluate the effects of these groups.To evaluate the immunogenicity of these immune groups, the serum and whey antibody titers, IgGl/IgG2a and the levels of Th1/Th2 cytokines in serum and whey were tested by the methods of ELISA. The cell immune efficacy was tested by T lymphocyte proliferation test. The whole bloods of vaccinated rats were assessed through opsonophagocytic assay. And antigen in rat mammary gland specifically binds to rabbit anti-FnBPB-ClfA serum by immunohistochemistry. The rat toxicity was tested through the R4 teat canal by injecting S. aureus. The results of group rFc-FnBPB-ClfA (M-TC/LTB) showed that antibody titer in serum was up to 1:102400 and the whey antibody titer was up to 1:1600 after boost immunization 7 days. Then the IgGl/IgG2a productions of group rFc-FnBPB-ClfA(M-TC/LTB) higher than others, the production of IgGl was the mainly part. The production of IL-4 and IFN-y of rFc-FnBPB-ClfA(M-TC/LTB) is significantly higher than other groups. T lymphocyte proliferation ability of rFc-FnBPB-ClfA(M-TC/LTB) group was markedly higher than others (p<0.05). The bactericidal capability of whole blood of rats immunized by rFc-FnBPB-ClfA(M-TC/LTB) was observably better than saline immune group (p<0.05). Rat mammary gland immunohistochemistry of rFc-FnBPB-ClfA(M-TC/LTB) immune group was colored. And the results of rat toxicity test by teat canal injection showed the counts of rFc-FnBPB-ClfA(M-TC/LTB) immune group was significantly lower than other groups (p <0.05). In conclusion, rFc-FnBPB-ClfA(M-TC/LTB) which was immuned first by muscle injection, and teat canal as boost immune with LTB could induce both humoral immune response and cellular immune response and could improve effective mammary gland local response. The effects of rFc-FnBPB-ClfA(M-TC/LTB) was the best in immune groups which provide evidences for engineering vaccine against mastitis of dairy cows infected by S. aureus. |
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