Construction and Expression of Tp0453 Recombinant Protein of Treponema pallidum and Development of Indirect ELISA for Diagnosinq Syphilis  

梅毒螺旋体Tp0453重组蛋白的表达及在梅毒血清学诊断中的应用(英文)

作　　者：刘双全[1] 吴移谋[1] 赵飞骏[1] 曾铁兵[1] 

机构地区：[1]南华大学医学院病原生物学研究所,衡阳421001

出　　处：《Chinese Journal of Sexually Transmitted Infections》2005年第1期30-34,共5页中华性传播感染杂志（英文版）

基　　金：Financially supported by Key grant from the Education Committee of Hunan Province (No. 02A046)

摘　　要：Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.目的克隆、表达梅毒螺旋体(Treponema Pallidum,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ELISA法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Mr约为32×103的特异蛋白带,免疫印迹检测其只与Tp IgG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%(30/30);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。

关 键 词：Treponema pallidum recombinant protein SERODIAGNOSIS enzyme link immunosorbent assay 

分 类 号：R759.1[医药卫生—皮肤病学与性病学]