| Dairy cattle mastitis is one of the most common diseases and the most economically losses of dairy cattle diseases affecting dairy industries around the world. And dairy cattle mastitis has brought the latent harm to the human health. Nowadays, there were large numbers of pathogenic bacteria causing dairy cattle produce the resistance to pathogenic bacteria but also the residue of the antibiotics had been increasingly affecting the food sanitation security and the human health, et al. Utilizing the vaccines to preventing and control- ing dairy cattle mastitis was a kind of safe, economical and effective method. Over the past two years, researchers had made a lot work about epidemiology of clinical mastitis disseminated focused in Shandong Province, and had a conclusion that the main pathogens which caused the cow mastitis is Staph- ylococcus aureus. In this study, the genetically engineered proteinα-hemolys in and FnbpA of Staphylococcus aureus were purified and activity detected, and then prepared the engineered subunit vaccines respectively, the immunity effectiveness of those vaccine were evaluated on mouse models. This study was divided into four parts:Trial 1 Activity and quality comparison of the engineered protein Staphylococcus aureusα-hemolysin purified with different methods.Theα-hemolysin of Staphylococcus aureus, which was expressed in E.coli BL21(DE3) with recombinant pET32a+-α-HL plasmid, was purified with Gel filtration chromatography(GFC) and Ni-NTA Spin Columns respectively, and their quantity and biological characteristic were compaired. Firstly, the purified products were analyzed with SDS-PAGE, and there was expected protein band with molecular mass of 53 kD. Secondly, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC hemolysin yield was 14.04%, meanwhile the protein purified with the Ni-NTA Spin Columns hemolysin yield was 17.5%. The results showed that there was no significant difference in the quantity, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA Spin Columns and GFC, but lowed of the expenditure.Trial 2 Purification and activity detection of Staphylococcus aureusα-hemolysin and FnbpA from gene expression productSome investigation reports which were about the prevalence of toxin gene of Staphylococcus aureus showed thatα-hemolysin collaborate with coagulase could initiate mastitis in mouse. And the fibronectin-binding protein A of Staphylococcus aureus could recognize extracellular matrixes to assistance the bacteria adhere to host cells and then to multiplication. Those two patho- genic factors play very important roles in infection and pathogenesis of Staphylococcus aureus.In this studyα-hemolysin and fibronectin-binding protein A were purified from expression host strain by gel filtration chromatography and detected their biologic activity.Trial 3 Evaluation of the immunity effectiveness on the engineered subunit vaccine of Staphylococcus aureusα-hemolysinThe immunity effectiveness of the gene engineered vaccine which initiated from Staphylococcus aureusα-hemolysin was evaluated on mouse models. Antibody titers were evaluated on ELISA, and then challenged to gain the immunity protect index. There were specific antibodies acquired in blood-serum from mice after vaccined and the antibody titers rising until it had arrive the max, then following down; the antibody titers initiated by the protein vaccine go with regulation and the immunoprotection was satisfied.Trial 4 Evaluation of the immunity effectiveness on the engineered subunit vaccine of Staphylococcus aureus fibronectin-binding protein AThe immunity effectiveness of this vaccine which initiated from Staphylococcus aureus fibronectin-binding protein A was evaluated on mouse models. Firstly the evaluation of its competitive adhesion on MDBK cells againsted the adhesion of Staphylococcus aureus. Antibody titers were evaluated on ELISA, and then challenged to gain the immunity protect index. The results showed that the decreased Staphylococcus aureus was found to surround on MDBK cells pre-treated with the fusion protein compared with that on control MDBK cells. And there was specific antibody acquired in mice blood-serum after vaccined and the antibody titers rising until it had arrive the max, then following down, the antibody titers initiated by the protein vaccine gone with regulation and the immunoprotection was satisfied.
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