结核分枝杆菌phoS2基因在大肠埃希菌中的高效表达及其产物抗原性分析

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。

High-level expression of Mycobacterium tuberculosis phoS2 gene in E. coli and antigenicity analysis of the recombinant protein

Objective To express phoS2 in Escherichia coli,purify the recombinant protein,preliminary evaluate its antigenicity and specificity by Western blot. Methods Expression plasmid of phoS2 was constructed with DNA recombinant technique. Positive clones were chosen using emzyme digestion and polymerase chain reaction. Recombinant plasmid was transformed into E. coli. Then phoS2 was induced to express. The expression of phoS2 was identified by SDS-polyacrylamid gel electrophoresis （SDS-PAGE） ; the recombinant protein was purified; the antigenicity and specificity of phoS2 protein was analyzed by Western blot. Results phoS2 was highly expressed in E. coli ,the amount of phoS2 protein in E. coli BL21 occupied more than 40% of total bacterial proteins. Purified protein could react strongly with serum samples from the patients with tuberculosis, but could not react with serum samples from healthy control. Conclusion The recombinant phoS2 protein was expressed mostly in inclusion body form in E. coli. The results indicated that the recombinant phoS2 antigen showed higher antigenic specificity and immunoreactivity, had potential application value in diagnosis of tuberculosis.