结核分枝杆菌 rPstS1-HspX 融合蛋白的制备及其血清学诊断价值

目的 表达、纯化结核分枝杆菌rPstS1 - HspX( rPH)融合蛋白,评价其在结核病血清学诊断方面的价值.方法 异丙基βD-硫代吡喃半乳糖苷(IPTG)诱导rPH融合蛋白表达,并用镍离子螯合亲和层析柱纯化融合蛋白.Western印迹分析纯化后融合蛋白的免疫反应性.最后用ELISA法检测194份血清抗体样本,其中来源于肺结核患者94份,非结核肺部疾病患者52份,健康对照者48份.结果 rPH融合蛋白在大肠埃希菌中主要以可溶性非包涵体形式表达,相对分子质量为51 000.经亲和层析后得到了浓度为0.62ng/mL,纯度达92％的融合蛋白.Western印迹实验证实纯化后融合蛋白能与结核病阳性血清发生特异性免疫应答.重组蛋白rPH血清检测的灵敏度为77.6％ (73/94),特异性为92.0％(92/100).结论 成功表达和纯化了rPH融合蛋白,其用于结核病血清学诊断具有较好的灵敏度和较高的特异性,是ELISA的可靠抗原.

Production of recombinant fusion protein rPstS1-HspX and application in serodiagnosis of tuberculosis

Objective To express and purify rPstS1-HspX （rPH）fusion protein of Mycobacterium tuberculosis H37Rv and evaluate its application in serodiagnosis of tuberculosis. Methods The expression of rPH fusion protein was induced with isopropyl-β-thiogalactopyranoside （IPTG）. Then the fusion protein was purified by nickel affinity chromatography, and the immunoreactivity of purified rPH was evaluated by Western blot. Finally, the purified fusion protein was used as antigens to screen the sera from the patients with pulmonary TB （ n = 94）, as well as other pulmonary disease （ n = 52）, and clinically healthy controls （ n = 48） by ELISA. Results The expressed 51 000 rPH fusion protein mainly existed in soluble form. After nickel aginity chromatography, the purity of rPH reached 92% （0.62 ng/mL）. Specific immunoreactivity of purified rPH to positive serum from tuberculosis patients was confirmed by Western blot. The results of ELISA showed that the sensitivity and specificity of rPH were 77.6% （73/94） and 92.0% （92/100）, respectively. Conclusions The rPH fusion protein is successfully expressed and purified. Purified rPH fusion protein, which can provide a satisfactory sensitivity and specificity in serodiagnosis of tuberculosis, may become effective antigen in ELISA.