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金黄色葡萄球菌纤连蛋白结合蛋白A基因的
克隆及其原核表达
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贺宽军1，郭闯2，张涌1
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(1. 西北农林科技大学 生物工程研究所,陕西 杨凌712100；
2.内蒙古民族大学 动物科技学院，内蒙古 通辽028042)
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摘要：
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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3600bp 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.coli BL21(DE3)感受态细胞中，经1mmol/L IPTG诱导和SDS-PAGE分析，在约165ku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。 |
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关键词：
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金黄色葡萄球菌； fnbA； 克隆； 原核表达 |
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中图分类号：S 852.611：Q 786 文献标识码：
A 文章编号：1673-4696(2006)04-0266-04 |
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Cloning and expression of fibronectin-binding protein A
gene of Staphylococcus aureus in E.coli |
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HE Kuan-jun1, GUO Chuang2，ZHANG
Yong1 |
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(1.Research Institute of Bio-Engineering, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100,
China；2.College of Animal Science and Technology, Nei Monggol University For Nationalities, Tongliao 028042, China) |
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Abstract: he 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 3600 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 HindⅢ 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（1mmol/L）. SDS-PAGE analysis revealed a band of approximately 165ku in molecular weight from the induced E. coli BL21 competent cells. Western-blotting analysis indicated that the protein had antigenic activity of FnBP-A. |
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Key words: Staphylococcus aureus； fnbA； cloning； prokaryotic expression |
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