梅毒螺旋体重组蛋白rTpN37为抗原的ELISA的建立与RPR和TPPA血清学检测效果的比较

Comparison of serological detection effects of ELISA using rTpN37 of Treponema pallidum as antigen with that of TPPA and RPR

目的:克隆并构建梅毒螺旋体tpN37基因原核表达系统,建立基于rTpN37的ELISA并对其用于梅毒血清学诊断的敏感性和特异性进行评价。方法:采用PCR扩增tpN37基因,构建tpN37基因原核表达系统。采用SDS-PAGE检测目的重组蛋白rTpN37表达情况,Ni-NTA亲和层析法提纯rTpN37,Western blot鉴定rTpN37的免疫反应性。以rT-pN37为包被抗原,建立rTpN37-ELISA并用于检测122例梅毒病人血清,其检测结果与RPR和TPPA进行比较。结果:克隆的tpN37基因与GenBank中相关序列相似性为100%。rTpN37表达量为细菌总蛋白的20.4%。提纯的rTpN37在SDS-PAGE后显示为单一的蛋白条带。TPPA阳性的梅毒病人血清能有效识别rTpN37并与之结合。rTpN37-ELISA对梅毒病人血清标本检测阳性率为95.9%(117/122),与TPPA检测阳性率(96.7%,118/122)相近(P>0.05),rTpN37-ELISA和TPPA检测阳性率均显著高于RPR(81.1%,99/122)(P<0.01)。结论:本研究中成功地克隆并构建了梅毒螺旋体tpN37基因及其原核表达系统。以rTpN37为包被抗原的rTpN37-ELISA可作为快速、敏感和特异的梅毒血清学筛查方法。

Objective：To clone tpN37 gene of Treponema pallidum and then construct its prokaryotic expression system,and to establish ELISA based on rTpN37 and to evaluate the sensitivity and specificity of the ELISA in detection of serological diagnosis of syphilis.Methods： tpN37 gene was amplified by PCR and its prokaryotic expression system was then constructed.SDS-PAGE was used to measure the expression of target recombinant protein rTpN37,Ni-NTA affinity chromatography was applied to extract rTpN37,and Western blot was performed to determine the immunoreactivity of rTpN37.By using rTpN37 as the coated antigen,a novel ELISA（rTpN37-ELISA） was established to detect serum specimens of 122 syphilis patients,and the detection result was compared to those by RPR and TPPA.Results： Sequence similarity of the cloned tpN37 gene was 100% compared to the corresponding sequence in GenBank.The expression output of rTpN37 was approximately 20.4% of the total bacterial proteins.The extracted rTpN37 showed a single protein band in gel after SDS-PAGE.TPPA positive syphilis patients′ sera could recognize and combine rTpN37 as well.The positive detection rate of rTpN37-ELISA was 95.9%（117/122）,similar to that of TPPA（96.7%,118/122）（P0.05）.Both the positive rates of rTpN37-ELISA and TPPA were significantly higher than that of RPR（81.1%,99/122）（P0.01）.Conclusion： Treponema pallidum tpN37 gene and its prokaryotic expression system are successfully cloned and constructed,respectively.rTpN37-ELISA by using rTpN37 as the coated antigen is a rapid,simple and convenient,sensitive and specific methods for serological screening and diagnosis of syphilis.