摘要
采用PCR方法对编码金黄色葡萄球菌纤黏蛋白B的D区和纤黏蛋白原凝集素A的A区基因片段进行了特异性扩增,并通过重叠延伸PCR扩增了FnBPB-ClFA串联基因,构建了克隆质粒pMD19-FnB-PB-ClFA。将该基因片段定向插入到原核表达载体pET-32a(+)中,构建了表达质粒pET-FnBPB-ClFA,并将其转入宿主菌BL21(DE3)。SDS-PAGE分析表明,在1mmol/L IPTG诱导下,在51ku处出现了与目的蛋白一致的外源蛋白带。Western-blot分析表明,所表达的蛋白与目的蛋白一致。上述结果表明,该融合基因在原核细胞中成功表达。
The genes encoding Fn-binding region D of fibronectin-binding proteins B and fibrinogen-binding region A of fibrinogen-binding proteins clumping factor A were amplified by PCR from Staphylococcus aureus chromosomal DNA and the amplified FnBPB-ClFA gene was cloned into pMD19-T vector by overlap extension PCR.The constructed plasmid pET-32a-FnBPB-ClFA was transformed into Escherichia coli BL21（DE3）.The bacterium was induced by 1 mmol/L IPTG and analyzed by SDS-PAGE and Western-blot.The approximately 51 ku exogenous protein was detected by SDS-PAGE.Western-blot indicated that the protein was the interested protein.The above results showed that the fusion protein was successfully expressed in prokaryotic cells.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2011年第2期173-177,共5页
Chinese Veterinary Science
关键词
金黄色葡萄球菌
纤维素结合蛋白B
凝集因子A
重叠延伸PCR
原核表达
Staphylococcus aureus
fibronectin-binding protein B（FnBPB）
clumping factor A（ClFA）
overlap extension PCR
prokaryotic expression
