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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 Mtb recombinant protein Rv0222 and evaluate its value in the serological diagnosis of tuberculosis. Methods The Rv0222 gene fragment was amplified by PCR from the genome of Mtb and inserted into the pMD18-T vector. The recombinant plasmid was subcloned into the expression vector pET30a to induce the expression of recombinant strain. His tag was purified by affinity chromatography Immunoblotting was used to analyze the immunoreactivity of the recombinant protein. 142 serum samples were detected by indirect ELISA from 82 patients with tuberculosis (positive for sputum smear or sputum culture and the alleviation of symptoms of anti-tuberculosis treatment), 28 non-tuberculosis Patients (8 with lung cancer, 15 with pneumonia and 5 with emphysema) and 32 with health examination were analyzed by MedCalc 11.5.0 using the medical software MedCalc 11.5.0 to analyze the area under the ROC curve and the best positive predictive value. Results The recombinant vector was successfully constructed. The recombinant protein Rv0222 showed a relative molecular mass of about 27 000 after electrophoresis and a protein purity of more than 95% after purification by nickel column. The recombinant protein Rv0222 could specifically bind to the serum of patients with tuberculosis by Western blotting. The area under the ROC curve of ELISA was 0.893, the best positive determination was 0.12, the detection sensitivity was 69.5% (57/82) in tuberculosis patients, the specificity of antibody detection in non-tuberculosis patients and healthy subjects was 90.0% (54/60). There was a significant difference between TB patients and non-TB patients and healthy subjects (t = 25.78, P <0.0001). Conclusion The pET30a-Rv0222 expression vector was successfully constructed and the recombinant protein Rv0222 with high purity was obtained. ELISA showed that Rv0222 protein could be one of the effective antigens in serodiagnosis of tuberculosis.