Immunological Study Of Subunit Vaccine And Nucleic Acid Vaccine Of Fibronectin-binding Protein Of Staphylococcus Aureus

The cow mastitis is an important disease in the development of dairy industry, it has a high incidence of infection, and the danger is great. Mastitis can be caused by many factors, of which Staphylococcus aureus is the most important contagious pathogen. The adhesion to the host is the most critical aspect of the role of bacterial infect the host , which links with the bacteria-specific protein expression, and it can identify and adhesion to the host extracellular matrix.The surface of Staphylococcus aureus matrix contains many elements to identify the microbial surface components, such as fibronectin-binding protein (FnBP), collagen-binding protein (Cna) and agglutination factor (Clf). Studies have shown that resistance of Staphylococcus aureus surface protein FnBP antibodies can reduce the toxicity of the bacteria, so the protein component can be used as vaccines to prevent infections caused by Staphylococcus aureus mastitis in dairy cows. The vast majority of Staphylococcus aureus are the two fibronectin-binding protein: FnBPA and FnBPB, this experimental study is FnBA.First, the genome of Staphylococcus aureus was extracted as template, specific primers were designed and synthesized, the FnBA gene of Staphylococcus aureus was amplified by PCR. Then the purified fragment was connected to the pMD18-T vector, positive plasmid was sequenced after double digestion, the result showed that the complete genome sequence in length was 1735bp as anticipation. Comparison of the identity of FnBA by NCBI Blast showed that nucleotide sequence of the cloned gene was very conservative with sequences which GenBank + EMBL + DDBJ + PDB indexed; Identity was more than 99%.The pET-28a(+) was chosen as prokaryotic expression vector, pMD18T-FnBA and pET-28a vectors were double-digested by BamHI and HindIII, respectively, then the two fragments were connected with T4-DNA ligase, and transformed into E.coli BL21 for digestion identification. Bacteria was induced with IPTG,protein expression was analyzed by SDS-PAGE, and the band at 85kD could be observed. Western blot analysis showed that the expression product contained His-tag fusion peptide,which could specifically response with anti-His-tag monoclonal antibody, and it was the expressed product of exogenous gene under the control of T7 promoter of pET-28a (+) vector. The results demonstrated that the exogenous gene was successfully expressed.The pVAX1 was also chosen as a eukaryotic expression vector, pMD18-FnBA and pVAX1 were double-digested by BamHI and HindIII, then the two fragments were connected with T4-DNA ligase, and transformed into E.coli JM109 for digestion identification. Positive plasmid transfected HeLa cells, indirect immunofluorescence assay (IFA) results showed that the constructed plasmid DNA vaccine can express the purpose of the correct protein in eukaryotic cells.During the process of developing the subunit vaccine, the optimized conditions for the protein expression were determined by SDS-PAGE: Induction temperature, 37℃; Time, 5h; IPTG concentration, 0.8 mM. Under the optimized conditions, the highest expression quantity of the interest protein (0.28mg/mL) accounts 18.86% of the total bacterial proteins. The results of bacterial decomposition indicated that the interest protein mainly existed in a dissolving form, however, the level of inclusion body was pretty low, which was convenient to the purification process. It was purified by the Ni2+metal chelate chromatography because of the existence of His-tag in the interest protein. The results of SDS-PAGE and gel thin-layer scanning analysis showed that the protein purity was more than 95% after the purification process. Large quantity of recombination protein was obtained, and mice were inoculated with the subunit vaccine. Determination results of ELISA antibody indicated that specific antibody was detected in mice serum of the experimental group, and the titer was inclined to increase; Determination results of the Th1/Th2 type cytokines in the serum of vaccinated mice by kits showed that the cytokines content was higher compared with control group; Spleen lymphocytes were stimulated with specific antigens and ConA, determination results of proliferation capacity of these cells showed that recombination protein vaccine could elicit the proliferation of T-lymphocytes to certain extent.DNA vaccine was inoculated into mice after the processes of extraction and purification, determination results of ELISA antibody indicated that the serum antibody titer of vaccinated mice presented a significant increasing trend, and a boost of the titer was observed after day 21 (first blood after the second vaccination); Determination results of the Th1/Th2 type cytokines in the serum of vaccinated mice by kits showed that the cytokines content significantly raised (p<0.05); Spleen lymphocytes were stimulated with specific antigens and ConA, determination results of proliferation capacity of these cells showed that constructed DNA vaccine, to some extent, could induce the proliferation of T-lymphocytes, mainly a cellular immune-based immune response.Through the experimental research,it has laid a solid foundation to prevention and treatment of Staphylococcus aureus mastitis of dairy cows better for the future.