Cloning and expression of fnbB gene of Staphylococcus aureus in E.coli
金黄葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

Staphylococcus aureus was found to be the most common cause of cow matitis.Binding of cells of Staphylococcus aureus to fibronectin,which may represent a mechanism of host tissue adherence,involves a fibronectin-receptor protein present on the bacterial surface.The ability of Staphylococcus aureus to adhere to platelets and to induce aggregation of platelets is considered to be a critical factor in S.aureus-associated infective matitis.Fibronectin-binding proteins present on the surface of S.aureus mediate internalization into nonphagocytic cells.Fibronectin-binding proteins was considered to important proteins in the vaccine development of cow matitis The gene encoding fnbB was amplified from Staphylococcus aureus chromosomal DNA by PCR technique,and the PCR product was about 3 458bp DNA segment.Using T-A cloning technique,the PCR product was cloned into pGEM T easy vector successfully and was named plasmid pGEM-fnbB.pGEM-fnbB and pET28a( )were digested by BamHI and XhoI double enzymes,then the purified fnbB gene was subcloned into the expression Vector pET28a( ),and the prokaryotic expression vector pET28a-fnbB was thus constructed successfully.The reconstructed plasmid pET28a-fnbB was transformed into E.coli BL21(DE3)competent cell.The bacterium was induced by IPTG(1 mmol/L)and analyzed by SDS-PAGE,approximately 165 kDa exogenous protein was observed on the SDS-PAGE.Western blot analysis indicated the protein had antigenic activity of FnBP-B.These results were expected to lay foundation for further studies on and development of diagnosis reagent and the new DNA vaccine of FnBP-B in the prevention of Staphylococcus aureus.