重组结核分枝杆菌CFP10的克隆与表达

目的:克隆和表达结核分枝杆菌CFP10,并分析其免疫学活性。方法:自结核分枝杆菌标准株H37Rv提取基因组DNA,PCR扩增cfp10基因,克隆至T载体pMD18T,转化入大肠杆菌JM109,菌落PCR鉴定阳性克隆并测序分析。将测序正确的pMD18-cfp10的cfp10基因亚克隆至表达载体PQE30,构建重组质粒PQE30-cfp10,转化大肠杆菌JM109感受态细胞,PCR和双酶切鉴定阳性重组体,IPTG诱导CFP10表达,亲和层析纯化,western-blot分析其免疫活性。结果:成功构建PQE30-cfp10重组表达质粒,表达、纯化获得分子量约10kDa的CFP10,并能被结核病人血清识别。结论:克隆表达获得具有免疫活性的重组结核分枝杆菌CFP10。

Cloning and Expression of Mycobacterium tuberculosis CFP10

Objective To clone and express CFP10 of Mycobacterium tuberculosis and analyze its immunological activity.Methods The gene cfp10 was amplifified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and was cloned into T vector pMD18-T.The positive cloning was identified by clony PCR and sequencing.The recombinant plasmid PQE30-cfp10 was constructed through subcloning gene cfp10 into prokaryotic expresson vector PQE30 and then transformed into competent cell of E.coli JM109.The protein CFP10 was induced by IPTG and purified with affinity chromatography,the immunological activity of CFP10 was analyzed by western-blot.Results The recombinant expression plasmid PQE30-cfp10 was constructed.The recombinant protein CFP10 was expressed in E.coli JM109 containing recombinant plasmid PQE30-cfp10 and was purified from E.coli JM109 containing recombinant plasmid PQE30-cfp10 lysate with affinity chromatography.The purified CFP10 reacted with positive sera of TB.Conclusion Recombinant protein CFP10 recognized specifically by sera of TB patients was obtained through cloning and expression.