梅毒螺旋体Tp0453重组蛋白的表达及在梅毒血清学诊断中的初步应用

Expression of Tp0453 recombinant protein of Treponema pallidum and development of the indirected ELISA for diagnosing syphilis

目的克隆、表达梅毒螺旋体(Tp)外膜蛋白Tp0453,建立间接ELISA法,探讨其在梅毒血清学诊断中的应用。方法构建重组质粒pQE32/Tp0453,IPTG诱导表达,SDS-PAGE和免疫印迹鉴定表达结果;Ni-NTA亲和层析法纯化重组蛋白,间接酶联免疫吸附试验(ELISA)检测人血清中特异性IgG抗体。结果成功构建了重组质粒pQE32/Tp0453;SDS-PAGE检测诱导产物显示有一分子量约为32000的特异蛋白带,免疫印迹检测其只与TpIgG抗体具有阳性反应;重组蛋白用作ELISA包被抗原检测Tp阴阳性参比血清,特异度和灵敏度均为100%;检测患者血清中特异性IgG抗体结果与TPPA法符合率为98·2%。结论表达的Tp0453重组蛋白具有较好的免疫反应活性,可望用于梅毒的血清学诊断。

Objective To clone, and express Tp0453 outer membrane protein of Treponema pallidum and develop an indirect ELISA for sero diagnosis of syphilis. Methods The immuno-dominant epitope of Tp0453 was amplified by PCR and subcloned into the expression vector pQE32. The recombinant protein was expressed in E. coli M15 and purified with Ni-NTA affinity chromatography columns. Indirect ELISA was developed to detect the antibody to Tp in human sera. Results 60 control sera was tested by ELISA. The sensitivities was 100% （30/30）, and the specificities was 100%. While detecting uninfected and infected T. pallidum human sera, the sensitivities of ELISA was 96. 8% compared with the results of the TPPA tests, and the specificities was 100% when the results of ELISA was compared with those of the TPPA test. The concordance of results between the ELISA test and the TPPA test was 98. 2%. Conclusion The recombinant Tp0453 outer membrane protein showed excellent immuno-reactive activity, and were suitable for development of ELISA for sero-diagnosis of syphilis.