摘要目的 探讨梅毒螺旋体重组蛋白TP0993在梅毒血清学诊断中的应用价值.方法 通过生物信息学分析,获取TP0993基因序列,构建原核载体进行诱导表达;Ni-NTA亲合层析柱纯化重组蛋白,Western印迹检测其免疫抗原性,用重组蛋白直接注射免疫新西兰家兔,评价其免疫原性.用纯化的TP0993重组蛋白包被微孔板,建立间接酶联免疫吸附试验(ELISA)方法,检测临床梅毒患者血清和健康体检者正常血清,同时与梅毒螺旋体明胶凝集试验(TPPA)进行比较,根据重组蛋白与梅毒阴阳性血清的反应情况,评价重组抗原在梅毒血清学诊断中的应用价值.结果 成功构建PET-28a(+)-0993原核表达载体,经表达、纯化后获得相对分子质量约为34 000的重组蛋白;Western印迹检测显示该重组蛋白能与梅毒患者阳性血清发生特异性反应;利用纯化的TP0993重组蛋白免疫新西兰兔,能诱导新西兰兔产生特异性免疫应答.以重组蛋白为包被抗原建立间接ELISA法,对TPPA法检测的480份临床血清进行检测,与TPPA法比较ELISA法的灵敏度为88.3％,特异度为85.8％,ELISA法与TPPA法符合率为86.5％.结论 重组表达的TP0993蛋白具有较好的免疫活性,可作为梅毒血清学诊断的候选抗原之一.

abstractsObjective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.