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目的克隆结核分枝杆菌Mtb81蛋白的编码基因Rv1837c,并在大肠杆菌中表达、纯化,获得重组蛋白Mtb81。方法以结核分枝杆菌H37Rv基因组为模板,PCR扩增Rv1837c基因序列,克隆入原核表达载体pET-28a,构建重组表达质粒,转化大肠杆菌后用IPTG诱导表达,纯化表达产物,获得重组Mtb81。ELISA检测重组Mtb81蛋白的敏感性和特异性。结果重组质粒pETRv1837c测序表明具有正确的编码序列,质粒构建成功,重组蛋白Mtb81在大肠杆菌中以包涵体和可溶性形式稳定表达。以Mtb81为抗原ELISA检测结核病患者血清抗体总敏感性为20.83%,特异性为95.83%。结论结核分枝杆菌Mtb81重组蛋白能在大肠杆菌工程菌种成功表达,为进一步的应用研究奠定基础。
Objective To clone the gene Rv1837c encoding Mycobacterium tuberculosis Mtb81 and express in E. coli and purify it to obtain the recombinant protein Mtb81. Methods The Mycobacterium tuberculosis H37Rv genome was used as a template to amplify the Rv1837c gene sequence and cloned into the prokaryotic expression vector pET-28a. The recombinant plasmid was constructed and transformed into E.coli. After induced by IPTG, the recombinant protein was purified and the recombinant Mtb81 was obtained. Sensitivity and specificity of ELISA for the detection of recombinant Mtb81 protein. Results The recombinant plasmid pETRv1837c showed correct coding sequence and the plasmid was successfully constructed. The recombinant protein Mtb81 was stably expressed in E. coli with inclusion bodies and soluble forms. The overall sensitivity and the specificity of the serum antibody against Mtb81 antigen were 20.83% and 95.83%, respectively. Conclusion The recombinant Mtb81 protein of Mycobacterium tuberculosis can be successfully expressed in Escherichia coli engineering strains, which lays the foundation for further application research.