梅毒螺旋体重组蛋白rTpN17或rTpN47为抗原的ELISA与TRUST和TPHA血清学检测效果的比较

Comparison of serological detection effects of ELISA using rTpN17 or rTpN47 of Treponema pallidum as antigen with that of TPHA and TRUST

目的:克隆并构建梅毒螺旋体tpn17、tpn47基因原核表达系统,建立基于rTpN17、rTpN47的ELISAs,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tpn17和tpn47基因并构建tpn17和tpn47基因原核表达系统。采用SDS-PAGE检测目的重组蛋白rTpN17和rTpN47表达情况,Ni-NTA亲和层析法提纯rTpN17和rTpN47,Western blot检测rTpN17和rTpN47免疫反应性。分别以rTpN17和rTpN47为包被抗原,建立检测血清标本中梅毒抗体的ELISAs(rTpN17-ELISA和rTpN47-ELISA),检测200例健康人、25例类风湿关节炎(RA)和17例系统性红斑狼疮(SLE)患者血清样本,并与甲苯胺红不加热血清试验(TRUST)和梅毒螺旋体间接血凝试验(TPHA)检测效果进行比较。结果:克隆的tpn17和tpn47基因与GenBank中相关序列相似性为100%。rTpN17和rTpN47表达量分别为细菌总蛋白的37.2%和26.8%。提纯后的rTpN17和rTpN47能与梅毒抗体阳性血清发生明显的结合反应。TPHA检测阳性率最高(99.1%,P<0.001),rTpN17-ELISA和rTpN47-ELISA检测阳性率相近(85.3%和84.3%,P=0.427),TRUST检测阳性率低于rTpN17-ELISA(P=0.001),但与rTpN47-ELISA相近(P=0.014)。所有健康人血清、RA和SLE患者血清标本的rTpN17-ELISA、rTpN47-ELISA和TPHA检测结果均为阴性,但有7.1%(3/42)RA和SLE患者血清标本TRUST检测结果阳性。结论:以rTpN17和rTpN47为抗原的ELISAs可作为快速、简便、敏感性和特异性较高的梅毒血清学筛查方法,而且rTpN17和rTpN47仍保持原有免疫反应特异性。

Objective： To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems, to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis. Methods： The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni -NTA affinity chromatography was applied to extract rTpN17 and rTpN47,while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen ,respectively ,ELISAs （rTpN17-ELISA and rTpN47- ELISA） were established to detect serum samples from 200 healthy individuals,25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA. Results： The sequence similarity of the cloned tpn17 and tpn47 genes was 100% compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2% and 26.8% of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum. The positive detection rate of TPHA （99. 1%） was the highest （P〈0. 001）. The positive detection rates of rTpN17- ELISA （85.3%） and rTpN47-ELISA （84. 3%） were similar （P〉0. 05）. The positive detection rates of TRUST （72.5%） was lower than that of rTpN17-ELISA （P=0. 001） but similar to that of rTpN47-ELISA （P = 0. 014）. The detection results of all the serum samples from healthy individuals,RA patients and SLE patients were negative,whereas 7. 1% （3/42） of the samples from RA or SLE patients were positive. Conclusions： rTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.