| The Staphylococcus aureus is the most important pathogen for the dairy cattle mastitis. the mastitis caused many troubles such as high treatment cost, the milk containing antibiotics and the emergency of drug resistant strains and so on, which influenced the development of cow farming industry and milk produceing industry seriously. The S. aureus adhesin ClfA and Cna which could mediated S. aureus adhere to the cell of host palyed an important role in the S. aureus infection. More and more domestic and foreign researchers selected adhesin as target antigen to research and develop the new mastitis vaccine.The surface proteins ClfA and Cna play an important role in S. aureus infections. In this study, a mouse mastitis model was applied to evaluate the antigenicity and protection of the protein vaccines against mastitis. The study were expected to lay a foundation for the development of subunit vaccine against S. aureus mastitis.The ligand-binding domain in fibronectin clumping factor A (ClfA) of S. aureus from bovine mastitis was ampilified, and cloned into PMD 18-T vector. The gene was subcloned into pET-28a(+) vector after identifing the PMD 18-T-ClfA exactly. The recombinant plasmid (pET-28a(+)-ClfA) was identified by enzyme restriction and sequencing. In addition, the recombinant plasmid (pET-28a(+)-Cna) had been constructed by my colleague. The two recombinant plasmids were converted into E.coli BL21, a 46 KD protein and a 55 KD protein were expressed highly in E.coli BL21 after IPTG induction. We obtained the high-purity goal protein after Ni2+-NTA His Bind Purification. Western blot was performed to confirm that the recombinant ClfA protein and Cna protein both had good immunogenicity with the rabbit anti-S. aureus immune serum and the mouse anti-recombinant proteins immune serum.The subunit vaccine and the S. aureus Inactivated vaccine, which were made by emulsifying the recombinant protein with the White oil adjuvant, Freund's incomplete adjuvant and Seppic ISA 206 adjuvant respectively, were injected to SPF BALB/c mice intraperitoneally. Challengeing and protection test and Evaluation of Immune Effects test which included antisera recognizing S. aureus test, neutrophil opsonophagocytic test, the concentration of IL-4 and IL-6, histopathologic slide detection were performed on mouse in White oil adjuvant which were detected having highest IgG titres. The result displayed that (1) the IgG level induced by the both subunit vaccines were high and higher than the inactivated vaccine; (2) compared with the control group, the capcities of binding to S. aureus by the antisera against both protein vaccines were enhanced; (3)the opsonophagocytic capcity of ClfA protein vaccine serum was enhanced when compared with the negative control group (P<0.05); (4) the CFUs of the both protein vaccine groups were lower than those of the inactivated vaccine group and the control group (P <0.05); (5) the ptotein vaccine could protect the mastitis structure intact and block the neutrophil immerging the gland alveolus.Above of all results indicated that the ligand binding protein of ClfA and the ligand binding protein of Cna had a good biological activity and immunogenicity and could protect the mouse against mastitis in some extent. Which showed that the ClfA gene and Cna gene would be used as an effective candidate antigen to prevent bovine mastitis, also showed that this research work could provide some theory foundation for the development of subunit vaccine against dairy cow matitis. |
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