| Cow mastitis is an important factor that restricting the development of the dairy industry and harming the health of consumers. Domestic and international data show that the number of various types of mastitis, especially hidden mastitis is about lactation cows1/3, and the incidence rate showed a rising trend recent years.Mastitis caused by Escherichia coli and Staphylococcus aureus primarily, the latter more dangerous.Antibiotics is used long-term and blindly in cow farming, leading Staphylococcus aureus to produce drug-resistant characteristics, making the prevention and control of mastitis more and more difficult.Bacteria need bound to adsorption and cell colonization of tissues and organs.The FnbpA (fibronectin binding protein A) and ClfA (clumping factor A) play a key role in Staphylococcus aureus colonization process, which FnbpA can bind fibronectin (Fn),fibrinogen (Fg) and elastin (En). The aim of this study is to proceed in the way of Staphylococcus aureus infection of animals, preventing the adsorption of bacteria on the matrix and cells so as to prevent bacterial infection.In this study, primers were designed base on the conserved region of FnbpA gene sequence that published in GenBank. Part of FnbpA gene was amplified by PCR using Staphylococcus aureus genomic DNA as template. the PCR product was connected to pMD-18T Simple vector and sequenced.The results show that the homology of obtained FnbpA gene fragments with domestic and international is from77%to98%.According A subdomain of FnbpA,the primers were designed, PCR products and PGEX-4T-2were digested by double endonuclease respectively, the PGEX-Fn prokaryotic expression plasmid was constructed. The plasmid was transformed in BL21E. coli to induce the protein expression and purified the expression product by GST agarsugar gel,the protein bands appeared nearby66kD by SDS-PAGE and the results is in keeping with the expected molecular size. the purified protein with Freund adjuvant was injected to the rabbits by subcutaneous immunization to prepare hyperimmune serum, antibody titer was detected by indirect ELISA,sera titer to106.18after third immunization.The signal peptide sequence in IFN-y gene of Holstein cows was added to the5’end of target gene by extension PCR, and the PCR product was inserted into the eukaryotic expression vector PVAX-1to construct the PV-SFn. The successfully constructed plasmid was sequenced and analyzed by bioinformatics software and Western Blot test. The result showed that the expression product has a typical signal peptide sequence and can be successful secreted in BHK-21cells, and the antigenicity will not changed.100μg/only endotoxin free recombinant plasmid was injected to the C57female healthy mice,25g or so, intramuscularly immunized with chitosan as an adjuvant, the second immunization was three weeks later. At the same time the control group is PBS and pVAX1empty plasmid. Blood samples were collected one week after each immunization to do ELISA assay. The mouse spleen lymphocytes were separated to practice MTT trail3months after the second immunization, detecting the lymphocyte proliferation. The other mice attacked the drug test at the same time, calculated the rate of protection. ELISA results showed that the antibody titer increased slightly after the first immunization, after the second immunization, antibody titers further improved; The MTT assay results showed that the experimental group had activated cellular immune system in mice according to mice spleen SI values is higher than the control group, indicating that the DNA vaccine had the ability to activate a certain extent immune response; Animal experiment results showed the protection rate of the control group was0, and the experimental group was60%.This research laid the foundation for epidemiological investigation and prevention of mastitis in Xinjiang as well as application of nucleic acid vaccines,providing a new approach for the treatment of cow mastitis. |
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