结核分枝杆菌重组蛋白Rv0222的表达及血清学鉴定

Expression and serodiagnosis of recombinant protein Rv0222 from Mycobacterium tuberculosis

目的构建Mtb重组蛋白Rv0222的重组质粒,评价其用于结核病血清学诊断的价值。方法通过PCR从Mtb染色体基因组中扩增出Rv0222基因片段,插入pMD18-T载体后,再亚克隆入表达载体pET30a中,诱导表达重组菌,组氨酸标签(His-tag)亲和层析纯化蛋白,通过免疫印迹法分析重组蛋白的免疫反应性,通过间接ELISA方法检测142例血清样本,来自82例结核病患者(痰涂片或痰培养阳性,抗结核治疗症状减轻者),28例非结核病患者(8例肺癌、15例肺炎和5例肺气肿)和32例健康体检者,检测数据通过专业医学软件MedCalc 11.5.0分析ROC曲线下面积和最佳阳性判定值。结果成功构建重组载体,重组蛋白Rv0222经电泳后显示相对分子质量约为27 000,镍柱纯化后蛋白纯度达95%以上,通过蛋白免疫印迹法证实重组蛋白Rv0222与结核病患者血清能特异性结合。酶联免疫检测ROC曲线下面积为0.893,最佳阳性判定值为0.12时,结核病患者组抗体检测敏感度为69.5%(57/82),非结核病患者和健康体检者抗体检测特异度为90.0%(54/60),结核病患者与非结核病患者及健康体检者相比,差异有统计学意义(t=25.78,P<0.0001)。结论成功构建pET30a-Rv0222表达载体,获得高纯度的重组蛋白Rv0222,ELISA结果显示Rv0222蛋白有望成为结核病血清学诊断的有效抗原之一。

Objective To construct the recombinant plasmid of Mycobacterium tuberculosis Rv0222 in E.coli,and to study its potential value for serodiagnosis of pulmonary tuberculosis（TB）.Methods The gene coding Rv0222 protein was amplified by polymerase chain reaction（PCR） from genome of Mycobacterium tuberculosis H37Rv,and then cloned into vector pMD18-T and subcloned into expression vector pET30a.Recombinant Rv0222 was expressed and purified.The antigenicity of the recombinant protein was analyzed by using Western blot.The purified recombinant Rv0222 protein was evaluated with the sera from pulmonary TB patients（n=82）,the other pulmonary disease patients（n=28）,and the healthy controls（n=32） by ELISA.The data were analyzed using professional medical software MedCalc 11.5.0.Results Recombinant plasmid of Rv0222 was established,and the relative molecular mass was about 27 000 by SDS-PAGE analysis.The protein purified by Ni-NTA was in a purity over 95%,which was confirmed by western blot.The results from ELISA showed that its sensitivity and specificity were 69.5%（57/82） and 90.0%（54/60）,respectively.There was significant difference between TB patients and the controls（t=25.78,P0.0001）.Conclusion The recombinant protein Rv0222 was successfully obtained and might be used as a potential candidate antigen for TB serodiagnosis.