结核分枝杆菌Rv3872基因的克隆、表达和纯化

Cloning,expression and purification of Rv3872 of Mycobacterium tuberculosis

目的原核表达并纯化结核分枝杆菌Rv3872蛋白。方法聚合酶链反应(PCR)技术从结核分枝杆菌标准株H37Rv全基因组中扩增出的表达Rv3872基因序列,将其克隆到pMD18-T载体后,测序分析。亚克隆该目的基因到pET28a表达载体,最终转化至大肠杆菌BL21(DE3),获得正确的阳性克隆子后大量表达重组Rv3872蛋白,再经纯化、定量后,通过Westernblot分析其抗原性。结果扩增Rv3872基因经序列测定与GenBank公布的序列完全一致,表达蛋白经SDS-PAGE分析,在约15000kD处有表达条带,纯化后的重组蛋白占总蛋白的90%以上。免疫印迹分析显示重组的Rv3872蛋白具有抗原性。结论成功表达结核分枝杆菌的Rv3872蛋白,为结核病血清学诊断候选抗原筛选和开发应用打下基础。

Objective To study the expression and purification of Rv3872 protein of Mycobacterium tuberculosis in E. coll. Methods Rv3872 gene were amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis,and first cloned into vector pMD18-T. After sequencing was confirmed,the gene was subeloned to expression vector pET28a. Recombinant Rv3872 protein was got by expression and purification. The antigenicity of the fused protein was analyzed by Western-blot. Results The DNA sequences of Rv3872 were identical with that published by GenBank. The relative molecular mass of the protein was about 150 000 by SDS-PAGE analysis. The protein purified by Ni-NTA was above 90% , and confirmed by Western-blot analysis. Conclusions Successfully expression Rv3872 protein establishes a foundation of further function research and diagnostic value.