重组结核分枝杆菌 MPT64的克隆与表达

目的：克隆和表达结核分枝杆菌抗原 MPT64，并分析其免疫活性。方法：以结核分枝杆菌 H37Rv 基因组 DNA 为模板，PCR 扩增 mpt64基因，克隆至 T 载体 pMD18-T，转化入 E.coli DH5α，菌落 PCR 鉴定阳性克隆并测序分析。将测序正确的 pMD18-T-mpt64的 mpt64基因亚克隆至表达载体 pET-28a，构建重组质粒 pET-28a-mpt64，转化 E.coli BL21中，PCR 和双酶切鉴定阳性重组子，IPTG 诱导 MPT64表达，亲和层析纯化，western-blot 分析其免疫活性。结果：成功构建 pET28a-mpt64重组表达质粒，表达、纯化获得分子量约为26kDa 的 MPT64，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 MPT64蛋白。

Cloning and Expression of Mycobacterium tuberculosis MPT64

Objective To clone and express antigen MPT64 of Mycobacterium tuberculosis and analyze its immunological activity. Methods The gene mpt64 was amplified by PCR from bacterium tuberculosis H37Rv genomic DNA was cloned into T vector pMD18-T.The positive clone was identified by clony PCR and sequencing. The recombinant plasmid pET28a-mpt64 was constructed through subcloning gene mpt64 into prokaryotic expression vector pET28a and then transformed into compen-tent cell of E.coli BL21 .The protein MPT64 was induced by IPTG and purified with affinity chromatography, the immunological activity of MPT64 was analyzed by western-blot. Results The recombinant expression plasmid pET28a-mpt64 was constructed. The recombinant protein MPT64 was expressed in E.coli BL21 and purified by affinity chromatography. The purified MPT64 reacted with positive sera of TB. Conclusion Recombinant protein MPT64 recognized specifically by sera of TB patients was obtained through cloning and expression.