Prokaryotic Expression And Assay Of The Antisera Bioactivity Of Staphylococcus Aureus Adhesin And Eukaryotic Coexpression With Bovine Interleukin 18

Mastitis of dairy cows was caused by bacterium, which can decrease the quality and quantity of milk, even deprived the milk secretion capability of dairy cows. Staphylococcus aureus (S. aureus) is the major cause of mastitis in cows. In some farms, over 50% mastitis was caused by S. aureus. Adhesin is the surface protein of S. aureus, which has close interaction with its virulence. So adhesin of S. aureus is a hot spot in prevention mastitis of S. aureus. Interleukin-18 has various bioactivities in immunoregulation, anti-infection and chronic inflammation. So this study was forced on adhesin of S. aureus and bovine Interleukin-18.Test 1 Amplified and Prokaryotic Expression of Staphylococcus aureus ClfAGene of clumping factor A (ClfA) from Staphylococcus aureu(sS. aureus)Shandong isolate ZFB1 amplified by polymerase chain reaction (PCR) was cloned into pGEX-6p-1 getting the recombinant pGEX/ClfA, and the ClfA was fused and coexpressed with GST. The SDS-PAGE and Western-blotting analysis indicated that the fusion protein was 67kDa in molecular weight and had immunological activity. This assay indicated that ClfA gene of S. aureus was only one different with NEWMAN in 41st nucleotide, which a T changed into a C. The protein sequence of ClfA was the same with NEWMAN. And densitometric scanning showed the expressed fusion protein was about 24.2% of total bacterial protein of BL21. The study provides theoretical base for the prevention of bovine S. aureus mastitis.Test 2 Assay of the Antisera Bioactivity of Staphylococcus aureus ClfA In this test, the fused protein expressed by E. coli. BL21 in test 1 was injected subcutaneously (s.c.) in mice at a dose of 20μg of protein in PBS for five times as two weeks internals. And the antiserum was collected after 10 days of the last injected. Adherence inhibition assay was used to detect the bioactivity of antiserum. The result showed that 2%,1%,0.5% and 0.2% antiserum of ClfA protein can inhibited 65%,60%,46% and 41% S. aureus adhere to the cell surface (p<0.001). But 0.1% antiserum did not showed difference in this assay (p=0.35). This test testified that ClfA protein was a protective antigen, which can contribute to protect animals from S. aureus's invasion, planting and infection.Test 3 Construction of Eukaryotic Co-expression Plasmid of pBUDCE/ClfA/BoIL-18 and Its ExpressionTo detect the co-expression of Clumping factor A (ClfA) gene and Bovine Interleukin-18 (BoIL-18) gene, the recombinant co-expressed vector of pBUDCE/ClfA/BoIL-18 was constructed. ClfA gene and BoIL-18 gene were amplified by two pairs of specific primers and cloned into pBUDCE4.1 at the downstream of promoter CMV and EF-1α. The recombinant vector was transfected into 239T cell by Cellfection reagent. Indirect immunofluorescence assay (IFA) showed that the recombinant vector co-expressed ClfA protein and BoIL-18 protein. The construction of this recombinant plasmid lays foundation to further study the effect of BoIL-18's immunologic activity and represents a new type of vaccine against S.aureus infection.Test 4 Construction of Eukaryotic Co-expression Plasmid of pBUDCE/FnbpA/BoIL-18 and Its ExpressionTo detect the co-expression of FnbpA gene and Bovine Interleukin-18 (BoIL-18) gene, the recombinant co-expressed vector of pBUDCE/FnbpA/ BoIL-18 was constructed. FnbpA gene and BoIL-18 gene were amplified by two pairs of specific primers and cloned into pBUDCE4.1 at the downstream of promoter CMV and EF-1α. The recombinant vector was transfected into 239T cell by Cellfection reagent. Indirect immunofluorescence assay (IFA) showed that the recombinant vector co-expressed FnbpA protein and BoIL-18 protein. The construction of this recombinant plasmid lays foundation to further study the effect of BoIL-18's immunologic activity and represents a new type of vaccine against S.aureus infection.