梅毒螺旋体Tp0319重组蛋白的表达、纯化及其在临床诊断中的初步应用  

Expression and purification of Tp0319 recombinant protein of Treponema pallidum and its application in diagnosis of syphilis

作　　者：肖勇健[1] 伍宁[2] 刘双全[3] 赵飞俊[2] 吴移谋[2] 

机构地区：[1]南华大学附属第二医院检验科,湖南衡阳421001 [2]南华大学病原生物学研究所,湖南衡阳421001 [3]南华大学附属第一医院检验科,湖南衡阳421001

出　　处：《临床检验杂志》2009年第4期252-255,共4页Chinese Journal of Clinical Laboratory Science

基　　金：湖南省教育厅重点基金项目(06A061)

摘　　要：目的克隆、表达及纯化梅毒螺旋体(Tp)外膜蛋白Tp 0319,并建立间接ELISA方法,探讨其在梅毒血清学诊断中的价值。方法构建重组质粒PQE32/Tp 0319,IPTG诱导表达,SDS-PAGE和western blot鉴定表达结果;Ni-NTA亲和层析法纯化重组蛋白,间接ELISA检测梅毒患者血清中特异性IgG抗体。结果成功构建了PQE32/Tp 0319重组质粒;经SDS-PAGE检测,诱导产物显示有一条相对分子质量约为30 000的特异蛋白质条带,western blot分析证明其只与人抗Tp IgG发生特异性反应;将Tp 0139用于间接ELISA检测抗Tp抗体阴、阳性参比血清,特异性和敏感性均为100%;检测梅毒患者和健康献血者血清各150份,结果与TPPA法符合率为95.3%。结论制备的Tp 0319重组蛋白有较好的免疫反应性,可望用于临床梅毒血清学诊断。Objective To clone, express, and purify Tp 0319 outer membrane protein of Treponema pallidum and to develop an indirect ELISA for diagnosing syphilis. Methods The expression plasmid PQE32/Tp 0319 was conventionally constructed. The recombinant Tp 0319 protein was produced in E. coli M15 after induction by IPTG. The Tp 0319 protein was analyzed by SDS-PAGE and Westem blotting, and then purified with Ni-NTA affinity chromatography. Indirect ELISA was developed to detect the syphilis antibody in human sera. Results The recombinant plasmid PQE32/Tp 0319 was constructed successfully and the fusion protein with relative molecular weight near 30 000 Dalton was revealed by SDS-PAGE. Western blotting proved that the recombinant protein specifically reacted with anti -Tp antibodies in sera from syphilis patients. The results of the indirect ELISA indicated the sensitivity and the specificity were both 100%. The concordance of 300 sera （150 from blood donors and 150 from syphilis patients）detected in parallel by the ELISA and the TPPA was 95.3%. Conclusions The data suggest that the prepared recombinant protein Tp 0319 of Treponema pallidum has high immunoreactivity. The recombinant protein can be used to develop ELISA kit for diagnosing syphilis.

关 键 词：梅毒螺旋体 重组蛋白 血清学试验 间接ELISA 

分 类 号：Q786[生物学—分子生物学]