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目的建立重组结核分枝杆菌CFP10蛋白的基因克隆表达、纯化和复性技术,研究其免疫学特性,并评价该重组蛋白在结核病血清学诊断方面的价值。方法用PCR法从结核分枝杆菌H37Rv基因组中扩增出1hp基因片段,连接到表达载体PET30a上,在大肠杆菌中表达;组氨酸标签(His-Tag)镍柱层析纯化重组蛋白;用ELISA方法进行抗原性初步评价。结果构建了含CFP10重组质粒的大肠杆菌工程菌,发现目的蛋白主要以可溶形式存在;经镍柱层析获得了纯的重组蛋白,纯度达90%以上;用纯化的CFP10蛋白做抗原,ELISA方法检测105份临床诊断结核病人血清,阳性率达42%。结论目的基因克隆入宿主菌中并表达成功,具有天然结构和高纯度的重组融合蛋白有可能成为结核病血清学诊断的组合抗原之一。
Objective To establish the cloning, expression and purification of recombinant Mycobacterium tuberculosis CFP10 protein and to study the immunological characteristics of the recombinant protein and to evaluate its value in serological diagnosis of tuberculosis. Methods The 1hp gene fragment was amplified from the Mycobacterium tuberculosis H37Rv genome by PCR and ligated into the expression vector PET30a and expressed in E. coli. His-Tag was used to purify the recombinant protein. ELISA method for antigenicity preliminary evaluation. Results The E. coli strain containing CFP10 recombinant plasmid was constructed and found to be soluble in soluble form. The pure recombinant protein was obtained by nickel column chromatography with the purity of over 90%. The purified CFP10 protein was used as the antigen and ELISA Methods The serum of 105 clinically diagnosed TB patients was tested, the positive rate was 42%. Conclusion The target gene was cloned into host bacteria and expressed successfully. Recombinant fusion protein with natural structure and high purity may be one of the combinatorial antigens for serodiagnosis of tuberculosis.