金黄色葡萄球菌FnBA活性基因的克隆及其原核表达

Cloning and expression of fibronectin-binding protein a reactive gene of Staphylococcus aureus in E.coli

根据GenBank中纤连蛋白结合蛋白A基因(FnBA)序列设计了1对特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增;结果获得了1 735 bp的DNA片段。将PCR产物克隆至pMD18-T载体中,成功地构建了克隆质粒pMD18-T-FnBA。以HindⅢ和BamHⅠ双酶切pMD18-T-FnBA和pET28a(+),将纯化的基因FnBA亚克隆至pET28a(+)中,构建了原核表达质粒pET28a-FnBA,并将其转化至E.coliBL21感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在约85 000处出现了与预期目的蛋白一致的外源蛋白带。又经West-ern-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性。

The gene encoding fibronectin-binding protein A （FnBA） was amplified from Staphylococcus aureus chromosomal DNA by PCR. Using the T-A cloning technique,the PCR product about 1 735 bp in length was cloned into a pMD18-T vector and was designated plasmid pMD18-FnBA, pMD18-FnBA and pET28a（＋） were digested by BamⅢ and Hind Ⅲ , then the purified FnBA gene was sub-cloned into expression vector pET28a（＋） ,and the pro- karyotie expression vector pET28a-FnBA was constructed. The constructed pET28a-FnBA was transformed into E. coli BL21 competent cells and then induced by IPTG（1 mmol/L）. SDS-PAGE analysis revealed a band of approximately 85 000 in molecular weight from the induced E. coli BL21 competent cells. Western-blotting analysis indicated that the protein had antigenic activity of FnBA.