结核分枝杆菌Rv0057-Rv1352融合蛋白的制备及其初步应用

Preparation and preliminary application of fusion protein Rv0057-Rv1352 of Mycobacterium tuberculosis

目的制备结核分枝杆菌(Mycobacterium tuberculosis,M.tb)Rv0057-Rv1352融合蛋白,并通过化学发光酶免疫法分析其抗原性,以评价其应用价值。方法根据Rv0057和Rv1352蛋白序列,合成Rv0057和Rv1352基因片段并进行扩增,分别插入质粒pET30aSETB中,构建重组质粒Rv0057/pET30aSETB和Rv1352/pET30aSETB。用NheⅠ和XhoⅠ双酶切重组质粒Rv1352/pET30aSETB,SpeⅠ和XhoⅠ双酶切重组质粒Rv0057/pET30aSETB,将回收的Rv1352基因片段插入Rv0057/pET30aSETB质粒中,构建重组质粒Rv0057-Rv1352/pET30aSETB,转化感受态大肠埃希菌BL21(DE3),IPTG诱导表达。表达的包涵体蛋白经His.Bind蛋白纯化试剂盒纯化后,以其作为抗原建立化学发光酶免疫分析法,检测患者血清中抗结核抗体,并与市售M.tb抗体诊断试剂盒和痰涂片法进行比较。结果构建的重组质粒Rv0057-Rv1352/pET30aSETB的核苷酸序列与设计序列完全一致;表达的重组融合蛋白Rv0057-Rv1352相对分子质量约为30 500,主要以包涵体形式存在,表达量约占菌体总蛋白的30%;纯化的重组融合蛋白的浓度为1.6 mg/ml,纯度为95%;以其为抗原建立的化学发光酶免疫分析法检测患者血清中抗结核抗体的灵敏度和特异性分别为66.70%和90.00%,而市售M.tb抗体诊断试剂盒的灵敏度和特异性分别为80.00%和63.33%;30例活动性肺结核患者经痰涂片检测,灵敏度为20.0%,化学发光酶免疫分析法检测灵敏度为66.7%,二者差异有统计学意义(P<0.01)。结论成功制备了M.tb Rv0057-Rv1352融合蛋白,以其为抗原建立的化学发光酶免疫分析法检测抗结核抗体具有较高的灵敏度和特异性,在结核病血清学快速诊断方面具有广阔的应用前景。

Objective To prepare the fusion protein Rv0057-Rv1352 of Mycobacterium tuberculosis（M.tb）,analyze its antigenicity by chemiluminescent EIA and evaluate its application.Methods Rv0057 and Rv1352 genes were synthesized,amplified and inserted into vector pET30aSETB to construct recombinant plasmid Rv0057/pET30aSETB and Rv1352/pET30aSETB respectively.Recombinant plasmid Rv1352/pET30aSETB was digested with NheⅠ and XhoⅠ,while Rv0057/pET30aSETB with SpeⅠ and XhoⅠ,and the recovered Rv1352 gene fragment was inserted into plasmid Rv0057/pET30aSETB.The constructed recombinant plasmid Rv0057-Rv1352/pET30aSETB was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed protein,in a form of inclusion body,was purified by His.Bind protein purification kit and used as an antigen for development of chemiluminescent EIA.The antibodies against M.tb in sera of patients were determined by the developed method,and the results were compared with those by commercial M.tb antibody diagnostic kit and by sputum smearing examination.Results The nucleotide sequence of constructed recombinant plasmid Rv0057-Rv1352/pET30aSETB was completely consistent with that designed.The expressed recombinant fusion protein Rv0057-Rv1352,with a relative molecular mass of about 30 500,mainly existed in a form of inclusion body and contained about 30% of total somatic protein.The concentration and purity of purified recombinant fusion protein were 1.6 mg / ml and 95% respectively.The sensitivity and specificity of developed chemiluminescent EIA using the fusion protein as an antigen were 66.70% and 90.00% for the antibody against M.tb in sera,while those of commercial M.tb antibody diagnostic kit were 80.00% and 63.33 % respectively.The sensitivity of sputum smearing examination for 30 cases of active pulmonary tuberculosis was 20.0%,which showed significant difference with that of chemiluminescent EIA（66.7%）（P ＆lt; 0.01）.Conclusion M.tb Rv0057-Rv1352 fusion protein was successfully prepared.Chemiluminescent EIA developed using the fusion protein as an antigen showed high sensitivity and specificity for antibody against M.tb and was of a broad prospect in application to rapid serological diagnosis of tuberculosis.