梅毒螺旋体Tp0453特异性抗原优势表位区段的克隆表达及其在血清学诊断中的初步评价

Gene cloning and expression of the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum and its potential use in serodiagnosis of syphilis

目的应用基因工程技术在大肠埃希菌中表达梅毒螺旋体（Tp）Tp0453（62～224位氨基酸）重组蛋白，为提高梅毒血清学诊断试剂的特异性提供实验基础。方法采用PCR法从Tp全基因组中扩增目的片段Tp0453（第184～672位核苷酸），TA克隆后构建原核表达质粒pQE30-Tp0453，酶切鉴定后转化E．coliM15，异丙基-β-D-硫代吡喃半乳糖苷诱导表达，Ni2＋亲和层析柱纯化目的蛋白，表达产物经Western印迹鉴定其免疫反应性。以纯化的重组抗原建立双抗原夹心法ELSA，检测经Tp抗体明胶颗粒凝集试验（TPPA）法确证的梅毒阳性血清48份、阴性血清40份。结果PCR法扩增出约490bp目的片段，成功构建原核表达质粒pQE30-Tp0453，并在E．coliM15中表达，蛋白表达率为18％，十二烷基硫酸钠聚丙烯酰胺凝胶电泳初步测定目的蛋白的相对分子质量为2l000，破菌后电泳证实目的蛋白以包涵体形式表达，纯化后蛋白纯度〉95％，Western印迹证实该蛋白能与梅毒患者阳性血清发生特异性反应。双抗原夹心法ELISA检测88份临床标本，灵敏度为97．9％（47／48），特异度为100．0％（40／40），与TPPA的总符合率为98．9％（87／88）。结论所表达的Tp0453重组蛋白具有良好的免疫反应性，为开发临床检测效果更好的梅毒诊断试剂盒提供了实验基础。

Objective To clone and express the Tp0453 antigen immuno-dominant epitope fragment of Treponerna pallidum （Tp） in Escherichia coli, in an effort to develop serological tests with increased specificity for the diagnosis of syphilis. Methods The gene encoding Tp0453 recombinant outer membrane protein fragment was amplified by polymerase chain reaction （PCR）, and inserted into expression vector pQE30 after T A cloning, then confirmed by restriction map. The constructed recombinant plasmid pQE30-Tp0453 was transformed to E. coli M15 for expression induced by isopropyl ^-D-l-thiogalactopyranoside. The expressed product was identified by Western blot, and purified by Ni2＋-NTA agarose column chromatography. A double antigen sandwich enzyme- linked immunosorbent assays （ELISA） was established by using the recombinant Tp0453 protein to test sera from 48 patients with positive Treponema pallidum particle agglutination test （TPPA）, and 40 negative sera as control. Results The PCR amplicon of the target gene was about 490 bp. The recombinant plasmid pQE30-Tp0453 was correctly constructed and successfully expressed in E. coli M15. The expressed product, with a relative molecular of about 21 000, existed in a form of inclusion body, accounting for about 18% of total somatic protein, and reached a purity of more than 95% after purification. Western blot showed specific reaction of the expressed protein with Tp positive serum. The ELlSA tests with the 88 clinical samples yielded a sensitivity of 97.9% （47/48）, and specificity of 100.0% （40/40）. The consistency of results between the ELISA test and the TPPA test was 98.9% （87/88）. Conclusion The expressed Tp0453 fragment has showed good immunoreactivity with serum from patients with syphilis, providing the foundation of further development of serological diagnostic kit with increased specificity for the diagnosis of TP infection.