梅毒螺旋体Tp0453重组蛋白的表达纯化及免疫活性研究

目的表达梅毒螺旋体膜蛋白Tp0453(28-288 aa),纯化表达产物并进行免疫活性分析,为探索Tp0453重组蛋白在梅毒血清学诊断中的应用提供实验依据。方法通过生物信息学分析,挑选Tp0453抗原的优势表位,进行诱导表达;以W estern-b lot和间接ELISA法检测重组蛋白的免疫反应性;用重组蛋白免疫新西兰家兔,评价其免疫原性。结果纯化后获得了相对分子量约为32×103的融合蛋白。W estern-b lot检测其能与梅毒患者阳性血清发生特异性反应;同时以重组蛋白为包被抗原建立间接ELISA法,检测梅毒诊断试剂参考血清,其阴阳性符合率均为100%。用纯化的Tp0453重组蛋白免疫新西兰家兔,能诱导其产生特异性免疫应答,ELISA法测定免疫血清中特异性抗体滴度在1∶1280以上。结论基因重组表达的Tp0453重组蛋白具有良好的免疫原性和免疫反应性,为进一步研究其在梅毒血清学诊断中的应用和其生物学功能奠定了基础。

Expression and Purification of Tp0453 Recombinant Protein of Treponema Pallidum and Its Immunocompetence Analysis

Objective To express Tp0453 outer membrane protein of Treponema pallidum(T.pallidum),and assess the immunocompetence.Methods The immuno-dominant epitope of Tp0453 was ligased into the expression vector pQE32,and expressed in E.coli M15.its immuno-competence was analyzed by Western-blot and ELISA.Results A fusion protein with molecular weight about 32KDa was attained after expression and purification.Western blot proved that the recombinant protein can specifically react with T.pallidum IgG positive sera.The sensitivities and the specificities of the diagnostic reagent detected by 60 control sera were 100%(30/30),100%(30/30) repectively.Specific humoral response was elicited after introducing recombinant protein in zealand rabbit and the specific antibody titer was above 1:1280 detected by indirect ELISA.Conclusion The expressed recombinant protein showed excellent immuno-competence,and the results lay the foundation for the research on its function to T.pallidum infection and development of quick diagnostic kit applying to detect T.pallidum infection.