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目的在大肠埃希菌中高效表达结核杆菌phoS2,通过免疫印迹反应初步鉴定重组蛋白的抗原性和特异性。方法采用DNA重组技术构建结核分枝杆菌phoS2抗原表达载体,用双酶切和PCR等方法鉴定转化子,重组质粒转化大肠埃希菌,诱导表达phoS2;用SDS-PAGE初步鉴定其表达量;将表达产物进行纯化;重组蛋白用Western blot分析其抗原性和特异性。结果phoS2基因在大肠埃希菌中得到高效表达,表达量占全菌蛋白的40%以上;重组蛋白与结核病患者血清标本呈强阳性反应,与健康人血清标本呈阴性反应。结论重组phoS2蛋白在大肠埃希菌中主要以包涵体形式表达,有很好的抗原特异性和免疫原性,对结核病诊断有潜在的应用价值。
Objective To express mycobacterium tuberculosis phoS2 efficiently in Escherichia coli and identify the antigenicity and specificity of the recombinant protein by immunoblotting. Methods The recombinant plasmid was constructed by DNA recombinant technique. The transformant was identified by double enzyme digestion and PCR. The recombinant plasmid was transformed into Escherichia coli to induce the expression of phoS2. The expression of phoS2 was identified by SDS-PAGE. The expressed product was purified. The recombinant protein was analyzed by Western blot for its antigenicity and specificity. Results The phoS2 gene was highly expressed in Escherichia coli and its expression level accounted for more than 40% of the total bacterial proteins. The recombinant protein showed strong positive reaction with serum samples of patients with tuberculosis and negative reaction with serum samples of healthy people. Conclusion The recombinant phoS2 protein is mainly expressed in the form of inclusion bodies in Escherichia coli. It has good antigen specificity and immunogenicity, and has potential value in the diagnosis of tuberculosis.