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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。
Objective: To clone and express Mycobacterium tuberculosis CFP10 and analyze its immunological activity. Methods: The genomic DNA was extracted from Mycobacterium tuberculosis standard strain H37Rv. The cfp10 gene was amplified by PCR, cloned into T vector pMD18T and transformed into E. coli JM109. The positive clones were identified by colony PCR and sequenced. The cfp10 gene of the correct sequencing pMD18-cfp10 was subcloned into the expression vector PQE30 to construct the recombinant plasmid PQE30-cfp10 and transformed into E. coli JM109 competent cells. Positive recombinant was identified by PCR and double enzyme digestion. IPTG induced CFP10 expression. The affinity layer Analysis of purification, western-blot analysis of its immunocompetence. Results: The recombinant plasmid pQE30-cfp10 was successfully constructed and expressed and purified to obtain CFP10 with a molecular weight of about 10 kDa, which was identified by the serum of tuberculosis patients. Conclusion: The recombinant Mycobacterium tuberculosis CFP10 with immunocompetence was cloned and expressed.