金黄色葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

Cloning and Expression of fnbB Gene of Staphylococcus aureus in E.coli

金黄色葡萄球菌(Staphylococcus aureus)是引起奶牛乳房炎主要致病菌之一,主要通过其菌体表面的黏附素侵入寄主细胞引起疾病,为奶牛业造成巨大损失。金黄色葡萄球菌表面蛋白纤连蛋白结合蛋白(fibronectin-binding protein,FnBP)是其关键的黏附因子,在研制抗金黄色葡萄球菌的新型疫苗中占有重要地位.本文根据GenBank中纤连蛋白结合蛋白B基因(fnbB)序列设计特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增,获得3458bp的DNA片段。使用T-A克隆技术,将PCR产物克隆至pGEM Teasy Vector中,成功构建出了克隆质粒pGEM-fnbB。以BamHI和XhoI双酶切pGEM-fnbB和pET28a(+),并将纯化的基因fnbB亚克隆至pET28a(+)中,构建出原核表达质粒pET28a-fnbB,并将其转化至E.coliBL21(DE3)感受态细胞中,经1mmol/L的IPTG诱导和SDS-PAGE分析,在约165ku处出现了与预期目的蛋白相一致的外源蛋白带,Western blot分析结果表明该蛋白具有金黄色葡萄球菌的抗原性。金黄色葡萄球菌pET28a-fnbB成功表达为金黄色葡萄球菌引起的奶牛乳房炎的诊断和研究新型疫苗奠定基础。

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 fthronectin-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.