血清学诊断中结核杆菌磷酸烯醇型丙酮酸羧激酶的应用

目的]在原核系统中表达结核杆菌磷酸烯醇型丙酮酸羧激酶(phosphoenolpyruvate car-boxykinase PEPCK),并研究该蛋白在诊断结核病人血清抗体中的应用价值.方法]应用基因重组技术表达重组蛋白结核杆菌磷酸烯醇型丙酮酸羧激酶,经亲和层析法纯化表达产物.用表达的重组蛋白免疫小鼠,研究其免疫学特性.间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测结核病人血清中特异性IgG抗体,并与结核杆菌抗体胶体金法诊断试剂盒检测结果对比.结果]试验表明转化入大肠杆菌中的重组质粒能够表达并纯化出相对分子量为72 kDa的重组蛋白;Western blot证实重组蛋白能够与小鼠抗BCG血清发生特异性反应;重组蛋白免疫小鼠后,小鼠血清中的抗体滴度可达1∶1280以上;重组蛋白用作ELISA包被抗原检测病人血清阳性率为17.3%(30/173),其中排菌病人的阳性率为32.5%(13/42),不排菌病人的阳性率为12.9%.该方法结果与结核杆菌抗体胶体金法诊断试剂盒的检测结果相比,敏感性为51.0%,特异性为96.7%.结论]结核杆菌PEPCK具有较好的免疫原性和抗原性,有可能作为结核病血清学诊断的一组抗原之一.

Recombinant phosphoenolpyruvate carboxykinase from Mycobacteriumtu-berculosis for sero diagnosis

Objective] To express phosphoenolpyruvate carboxykinase (PEPCK) of tuberculosis and evaluate its immune diagnostic ability. Methods] PEPCK was cloned and expressed in prokaryotic expression system. We analyzed immune response of recombinant PEPCK with Western blot and studied the immunity of it through mice immunization. We also investigated the sensitivity and specificity of immunodiagnosis of PEPCK via detecting the sera from tubercular patients by using ELISA. The results were evaluated by comparing with a Mycobacterium tuberculosis antibody Colloidal Gold Diagnostic Kit. Results] The recombinant protein was 72 kDa and specifically reacted with anti-BCG antibody. Specific humoral immune response was elicited after mouse immunization with PEPCK protein and the high titer specific antibody was generated (1:1280). Antibodies were detected against M. tuberculosis 17.3% in all tuberculosis patients, 32.5% in patients harboring Mycobacterium currently and 12.9% in patients without M. tuberculosis. The sensitivity and specificity of ELISA were 51.0% and 96.7%. Conclusion] These results indicated that the recombinant PEPCK might be one of the suitable antigens for serodiagnosis of tuberculosis.