结核分枝杆菌38kD-16kD融合蛋白克隆表达及血清学诊断价值

Expression and immunological characteristics of recombinant fusion protein 38kD-16kD of Mycobacterium tuberculosis

目的构建重组结核分枝杆菌38 kD-16 kD融合蛋白,评价其在结核病血清学诊断方面的价值。方法通过聚合酶链反应扩增获得16 kD和38 kD基因片段,分别连接到表达载体pET21a中,同时将2片段2次连接到表达载体pET21a中,形成38 kD-16 kD(3 816),16 kD,38 kD重组载体;经表达、纯化和复性后,通过Western blot分析,以酶联免疫吸附试验检测184例血清抗体,肺结核患者96例、非结核肺部疾病患者50例,正常对照38例。结果成功构建重组质粒pET21a-3816,表达蛋白经SDS-PAGE后显示相对分子质量约为65 kD,重组蛋白经镍柱纯化后,纯度均达90%以上;经Western blotting证实重组蛋白能与结核病患者血清反应;重组蛋白3816血清检测,其敏感性为80.2%(77/96),特异性为90.9%(80/88)。结论成功表达和纯化了3816融合蛋白,其对血清学检测证明重组融合蛋白有希望成为结核病血清学诊断的有效候选者之一。

Objective To clone, express and purify the recombinant fusion protein 38 kD-16 kD of Mycobacterium tuberculosis, and to evaluate its potential value in TB serodiagnosis. Methods The genes coding 16 kD and 38 kD proteins were amplified by polymerase chain reaction （PCR） from the genome of Mycobacterium tuberculosis H37Rv,and then cloned into expression vector pET21a,respectively. The fusion proteins 38 kD-16 kD were expressed in E. coli BL21 （DE3）. It was purified by his-tag of the fusion protein and renaturated by semipermeable membrane. Recombinant protein was analyzed by Western-blot. The purified recombinant protein was used as antigens to screen the sera from the patients with pulmonary TB （n=96）,as well as other pulmonary disease （n=50）, and clinically healthy controls （n = 38） by ELISA. Results The recombinant plasmid pET21a-38 kD-16 kD was obtained. The relative molecular mass of the protein was about 65 kD by SDS-PAGE analysis. Specific immunogenicity of the recombinant protein was confirmed by western blot analysis. The results of ELISA showed that the sensitivity and specificity of 38 kD- 16 kD antigen were 80. 2%（77/96） and 90. 9% （80/88）, respectively. Conclusion The recombi-nant fusion protein 38 kD-16 kD was successfully expressed and purified, and may be a potential candidate of serodiagnostic reagent.