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目的从H37Rv标准株克隆出结核菌Rv2653c基因并进行表达与初步分析。方法根据H37Rv基因组序列合成引物,以PCR方法钓取Rv2653c基因,克隆到pGEM-T上,挑取阳性克隆进行测序,将正确编码基因克隆到pET24b载体上,在BL21大肠埃希菌菌株中以IPTG诱导表达重组蛋白。以金属螯合层析初步分离纯化重组蛋白,采用western blot方法对重组抗原进行抗原性初步分析。结果首先获得了重组的Rv2653c编码的蛋白,表达产率不低于20%,初步纯化纯度约90%,以人血清进行的western blot分析发现,Rv2653c重组蛋白有较好的抗原性。结论获得了Rv2653c重组蛋白为进一步寻探讨其功能及应用价值奠定基础。
Objective To clone and express Mycobacterium tuberculosis Rv2653c gene from H37Rv strain. Methods According to the H37Rv genome sequence, the Rv2653c gene was amplified by PCR and cloned into pGEM-T. The positive clones were selected and sequenced. The correct coding gene was cloned into pET24b vector. Induced the expression of recombinant protein. The recombinant protein was separated and purified by metal chelate chromatography. The antigenicity of the recombinant antigen was analyzed by western blot. Results The recombinant protein of Rv2653c was obtained firstly. The expression yield was not less than 20% and the initial purity was about 90%. Western blot analysis using human serum showed that Rv2653c recombinant protein had good antigenicity. Conclusion The recombinant protein Rv2653c obtained further lay the foundation for exploring its function and application value.