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目的对结核分枝杆菌Mtb81抗原基因进行克隆、表达及纯化,并评价其抗原性。方法应用聚合酶链反应技术扩增结核分枝杆菌H37Rv Mtb81 DNA序列;将目的基因构建大肠杆菌高效表达株;亲和层析柱纯化重组蛋白;经SDS-PAGE电泳鉴定;通用ELISA法进行重组蛋白的抗原性检测分析。结果获得了结核分枝杆菌抗原Mtb81基因,在大肠杆菌BL21中高效表达,Western印迹结果证实该重组蛋白能与抗结核分支杆菌多克隆抗体发生特异免疫结合反应。ELISA分析中,该纯化的重组抗原敏感性、特异性和准确性分别为:35.8%、98.3%和56.7%,具有良好的抗原性和较高的特异性。结论结核分枝杆菌Mtb81重组蛋白抗原具备良好的抗原性与特异性,为结核病诊断、重组疫苗应用和免疫效应检测以及抗原、抗体的大规模制备打下基础。
Objective To clone, express and purify Mtb81 antigen of Mycobacterium tuberculosis and evaluate its antigenicity. Methods The DNA sequence of Mycobacterium tuberculosis H37Rv Mtb81 was amplified by polymerase chain reaction (PCR). The target gene was constructed and expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography and identified by SDS-PAGE electrophoresis. The recombinant protein Antigenicity test analysis. Results Mtb81 gene of Mycobacterium tuberculosis was obtained and highly expressed in E. coli BL21. The results of Western blotting confirmed that the recombinant protein could specifically bind to the anti-Mycobacterium tuberculosis polyclonal antibody. The sensitivity, specificity and accuracy of the purified recombinant antigen were 35.8%, 98.3% and 56.7%, respectively, with good antigenicity and high specificity. Conclusion Mycobacterium tuberculosis Mtb81 recombinant protein antigen has good antigenicity and specificity, which lays the foundation for the diagnosis of tuberculosis, the application of recombinant vaccine, the detection of immune effect and the large-scale preparation of antigens and antibodies.