牛结核分枝杆菌CFP10-ESAT6融合蛋白的原核表达及其在牛结核病检测中的应用 |
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引用本文: | 徐贤坤,熊毅,龙剑明,刘棋,曾宪垠. 牛结核分枝杆菌CFP10-ESAT6融合蛋白的原核表达及其在牛结核病检测中的应用[J]. 中国兽医科学, 2009, 39(9) |
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作者姓名: | 徐贤坤 熊毅 龙剑明 刘棋 曾宪垠 |
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作者单位: | 徐贤坤(广西动物疫病预防控制中心,广西,南宁,530001;四川农业大学生命理学院,四川,雅安,625014);熊毅,刘棋(广西动物疫病预防控制中心,广西,南宁,530001);龙剑明(广西大学动物科学技术学院,广西,南宁,530005);曾宪垠(四川农业大学生命理学院,四川,雅安,625014) |
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基金项目: | 广西科技厅科技攻关项目,国家农业公益性行业科研专项,广西科技创新能力建设项目 |
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摘 要: | 采用重组PCR技术从牛结核分枝杆菌AN5基因组DNA中扩增CFP10-ESAT6融合基因,并将其定向克隆至原核表达载体pET-32a,构建了原核表达质粒pET-CFP10/ESAT6.重组子经酶切及测序鉴定后转化至大肠杆菌BL21(DE3),IPTG诱导后经SDS-PAGE电泳鉴定,获得约42 ku带有6×His蛋白标签的rHIS-CFP10/ESAT6融合蛋白,表达量约占菌体总蛋白的40%.用HIS蛋白纯化柱纯化该蛋白,Western-blot分析显示,该融合蛋白能与抗牛结核分枝杆菌阳性血清发生特异性反应.将重组的该融合蛋白用于刺激单次皮内变态反应阳性牛全血,可以产生高水平的IFN-γ,其特异性优于结核菌素.结果表明,获得的重组融合蛋白rHIS-CFP10/ESAT6为牛结核病的诊断奠定了基础.
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关 键 词: | 牛结核病 融合蛋白 |
Prokaryotic expression of recombinant CFP10-ESAT6 fusion protein from Mycobacterium tuberculosis and its application to detection of bovine tuberculosis |
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Abstract: | The gene encoding protein CFP10-ESAT6 was amplified from Mycobacterium tuberculosis strain AN5 chromosomal DNA by recombinant PCR and cloned into the expression vector pET-32a to gen-erate the recombinant plasmid pET-CFP10/ESAT6. After being identified by BamH 1 +HindⅢ digestion and sequencing,the recombinant expression plasmid was transformed into Escherichia coli BL21(DE3). The fused protein rHIS-CFP10/ESAT6 was expressed with induction by IPTG. SDS-PAGE analysis showed that the fusion protein rHIS-CFP10/ESAT6 was 42 ku in molecular weight,and made up 40% of whole bacterial proteins. The soluble protein was purified with HIS affinity chromatography column. West-ern-blotting analysis indicated that the purified recombinant protein possessed good immunological activity, and could induce to generate high level of IFN-γ in whole blood,and the IFN-γ response to rHIS-CFP10/ ESAT-6 was better than that to PPDa and PPDb. The results showed that the recombinant fusion protein rHIS-CFP10/ESAT6 provided a basis for the diagnosis of tuberculosis. |
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Keywords: | CFP-10 ESAT-6 |
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