摘要
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3 600bp的DNA片段。将PCR产物克隆至pGEM T easy载体中,成功地构建了克隆质粒pGEM-fnbA。以HindⅢ和XhoⅠ双酶切pGEM-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(DE3)感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在约165 ku处出现了与预期目的蛋白一致的外源蛋白带。Western-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性。
The fnbA gene encoding fibronectin-binding protein A （FnBP-A） was amplified from Staphylococcus aureus chromosomal DNA by PCR. Using the T-A cloning technique, the PCR product about 3 600 bp in length was cloned into a pGEM T easy vector and the cloned product was designated plasmid pGEM-fnbA, pGEM-fnbA and pET28a（＋） were digested by Hind Ⅲ and XhoⅠ , then the purified gene fnbA was sub-cloned into expression vector pET28a （＋） , and the prokaryotic expression vector pET28a-fnbA was constructed. The constructed pET28a-fnbA was transformed into E. coli BL21 （DE3） competent cells and then induced by IPTG（1 mmol/L）. SDS-PAGE analysis revealed a band of approximately 165 ku in molecular weight from the induced E. coli BL21 competent cells. Western-blotting analysis indicated that the protein had antigenic activity of FnBP-A.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第4期266-269,共4页
Chinese Veterinary Science
基金
国家奶业重大科技专项基金(2002BA518A01-2)
