牛GM-CSF与金黄色葡萄球菌黏附素FnBPB共表达质粒的构建与原核表达

Construction and Prokaryotic Expression of the Co-expression Plasmid with FnBPB and GM-CSF Gene Encoding Staphylococcus aureus Adhesion

采用PCR方法对牛粒细胞-巨噬细胞集落刺激因子(GM-CSF)和金黄色葡萄球菌FnBPB的D区进行特异性的扩增,并通过重叠延伸PCR扩增GM-CSF-FnBPB串联基因,构建了克隆质粒pMD19-GM-CSF-FnBPB。利用表达载体pET-32a(+)对该融合基因片段进行原核表达,SDS-PAGE分析表明,在1mmol/L IPTG诱导浓度下,在39ku处出现了与目的蛋白一致的外源蛋白带,Western blot分析表明,该蛋白具有反应原性,进而证明该融合基因成功在原核细胞中表达。

Granulocyte-macrophage colony stimulating factor（GM-CSF） and fibronectin-binding proteins B（FnBPB） and fibrinogen-binding（GM-CSF） were amplified by PCR from Staphylococcus aureus chromosomal DNA and the amplified sequence GM-CSF-FnBPB was cloned into pMD19-T vector by overlap extension PCR.The fusion gene was successfully coloned into expression vector pET-32a.The constructed plasmid pET-32a-GM-CSF-FnBPB was transformed into E.coli BL21（DE3） competent cell.The bactera were induced by 1 mmol/L IPTG and analyzed by SDS-PAGE and Western blot.Approximately 39 ku exogenous protein was observed by SDS-PAGE.Western blot analysis indicated the protein had antigenicity of Staphylococcus aureus.The results showed that the fusion gene was successfully expressed in prokaryotic cells.