Prokaryotic Expression And Immunological Characteristics Study Of The A And D Domain Of Fibronection-binding Protein A From Staphylococcus Aureus

Staphylococcus aureus is one of the most significant pathofens which causes the bovine mastitis. S. aureus adhesion and colonization in the mammary epithelial cells was identified as its main pathogenic mechanism. The effective atrategy for preventing infection may be blocking S. aureus adherension and colonization to the host cell surface.Fibronection-binding protein A(FnBPA) is considered to be the major virulence factors of S.aureus. The A domain of Fibronection-binding protein A (FnBPA-A) is major combined with fibrinogen(Fg), and the D domain of Fibronection-binding protein A (FnBPA-D) is major combined with frbronection(Fn). In this paper, FnBPA-A and FnBPA-D gene of S. aureus were obtained by the method of PCR. And the two DNA fragments were subcloned into the expression vector pET-32a(+) respectively. The two target fragment were induced expression by IPTQ and SDS-PAGE test results showed that the molecular weight of FnBPA-A was102KDa and the molecular weight of FnBPA-D was53KDa.To evaluate the immunogenicity of A domain and D domain of fibronection-binding protein A gene of Staphylococcus aureus, the purified recombinant prokaryotic expression of FnBPA-A and FnBPA-D protein was used to immunize New Zealand white rabbits. Then the serum antibody titers and the levels of cytokines were tested by the methods of ELISA. The whole bloods of vaccinated rabbits were used to opsonophagocytic assay. The cell immune efficacy was assessed through T lymphocyte proliferation test.The results showed that the serum antibody titer were up to1:12800(FnBPA-A) and1:25600(FnBPA-D) after three times immunization. Two groups were both improved the production of IL-2、IL-4、IL-10and IFN-γ, but FnBPA-D protein immune group was significantly higher than FnBPA-A protein immune group (p<0.05). T lymphocyte proliferation ability of adhesion FnBPA-D immune group was significantly higher than FnBPA-A immune group (p<0.05). The bactericidal capability of whole blood of rabbits immunized by FnBPA-A and FnBPA-D protein was significantly better than control immune group (p<0.05). In conclusion, FnBPA-A and FnBPA-D could induce both humoral immune response and specific cellular immune response, and FnBPA-D is more suitable than FnBPA-A as a candidate gene of genetic engineering subunit vaccine against mastitis of dairy cows infected by S.aureus.