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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.coliDH5α,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p ET-28ampt64,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。
Objective: To clone and express Mycobacterium tuberculosis antigen MPT64 and analyze its immunological activity. Methods: The mpt64 gene was amplified by PCR using Mycobacterium tuberculosis H37Rv genomic DNA as a template. The mpt64 gene was cloned into the T vector pMD18-T and transformed into E. coli DH5α. The positive clones were identified by colony PCR and sequenced. The mpt64 gene of pMD18-T-mpt64 was subcloned into the expression vector pET-28a to construct the recombinant plasmid p ET-28ampt64, which was then transformed into E. coli BL21. Positive recombinant plasmids were identified by PCR and restriction enzyme digestion. IPTG induced MPT64 expression , Affinity chromatography purification, western-blot analysis of its immunocompetence. Results: The recombinant plasmid pET28a-mpt64 was constructed successfully and expressed and purified to obtain MPT64 with a molecular weight of about 26 kDa, which can be recognized by the serum of tuberculosis patients. Conclusion: The recombinant Mycobacterium tuberculosis MPT64 protein with immunocompetence was cloned and expressed.