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

Prokaryotic expression of recombinant CFP10-ESAT6 fusion protein from Mycobacterium tuberculosis and its application to detection of bovine tuberculosis

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。

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 generate the recombinant plasmid pET-CFP10/ESAT6. After being identified by BamHⅠ＋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. Western-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.