| 孙爱华,孙百莉,严杰.rTpNl7和rTpN47及其融合蛋白酶联免疫吸附试验检测梅毒血清标本的效果[J].Chinese journal of Epidemiology,2007,28(10):1016-1020 |
| rTpNl7和rTpN47及其融合蛋白酶联免疫吸附试验检测梅毒血清标本的效果 |
| Study on ELISAs with rTpN17,rTpN47 and its fusion protein as antigens in the detection of serum samples from syphilis patients |
| Received:February 01, 2007 |
| DOI： |
| KeyWord: 梅毒螺旋体 基因 重组表达 酶联免疫吸附试验 |
| English Key Word: Trepnnema pallidum Gene Recombinant expression Enzyme linked immunoaorbent aslay |
| FundProject:浙江省医药卫生科研基金资助项日(2004A018) |
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| Abstract: |
| 目的 构建梅毒螺旋体tpnl7、tpn47基因及tpnl7-tpn47融合基因原核表达系统，建立基于rTpNl7、rTpN47和rTpNl7-TpN47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 采用常规分子生物学方法扩增并克隆梅毒螺旋体tpnl7和tpn47基因，以连接引物PCR构建tpnl7-Lpn47人工融合基因，建立上述目的基因原棱表达系统。采用SDS-PAGE检测目的重组蛋白rTpNl7、rTpN47和rTpNl7-TpN47表达情况，Ni-NTA亲和层析法提纯上述目的系组蛋白，WesLern blot检测其免疫性。分别以rTpNl7、rTpN47和rTpNl7-TpN47为包被抗原，建立相应ELISAs检测健康人群、类风湿关节炎(RA)和系统性红斑狼疮(SLE)患者及梅毒患者血清标本中梅毒抗体并与现行TRusT和TPHA进行比较。结果 克隆的tpnl7、tpn47摹围及构建的tpnl7-tpn47融合基用与GenBank中相关序列相似性为100%。rTpNl7、rTpN47和rTpNl7-TpN47表达量分别为细菌总蛋白的37.2%、23.3%和29.8%，其提纯物电泳后均娃示单一条带，并均能与梅毒抗体阳性血清发生明显的结合反应。rTpNl7-ELISA、rTpN47-ELISA和rTpNl7-TpN47．ELIsA对所有健康人血清、RA和sI．E患者血清标本的检测结果均为阴性，对梅毒患者血清标本检测阳性率分别为84.4%、82.3%和98.1%，其中rTpNl7-ELIsA和rTpN47-ELIsA检测阳性率低于TPHA(P<0.01)，rTpNl7-TpN47．ELIsA检测阳性率相似(P>0.05)，但均高于TRUST(71.4%)。结论研究中建立的rTpNl7-ELISA、rTpN47-ELIsA，尤其是rTpNl7-TpN47-ELIsA，有望成为快速、简便、安全的梅毒患者血清学筛查方法。 |
| English Abstract: |
| Objective To construct the prokaryotic expression systems of tpnl7 and tpn47 genes and tpn17-tpn47 fusion of Treponemu pallidum,and to establish ELISAs based on rTpN17,rTpN47 and rTpNl7-TpN47 as antigens to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosed syphilis.Methods tpn17 and tpn47 genes were amplified and cloned by routine molecular biological methods.PCR with linking primers was used to ponstruct artificial fusion gene tpn17-tpn47.The prokaryotic expression systems of the genes were then constructed.SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17,rTpN47 and rTpN17-TpN47.Ni-NTA affinity chromatography was applied to extract the three recombinant proteins,while Western blot was performed to determine their immunity.Using rTgN17，rTpN47 and rTpNi7-TpN47 as the coated antigens,ELISAs(rTpN17-ELISA,rTpN47-ELISA and rTpN17-TpN47-ELISA) were established to detect serum samples from 200 healthy individuals,25 RA patients,17 SLE patients and 419 syphilis patients.Results of the ELISAs were compared to those with TRUST and TPHA.Results The sequence similarities of the cloned tpn17 and tpn47 genes and the constructed tpn17-tpn47 fusion gene were 100%,compared with the corresponding sequences in GenBank.The expression outputs of rTpN17,rTpN47 and rTpN17-TpN47 were 37.2%，23.3% and 29.8% of the total bacterial proteins,respectively.Each of the three purified recombinant proteins showed a single fragment in gel after electrophoresis,and could take place remarkable conjugation reactions to the positive sera from syphilis patients.The detection results of rTpN17-ELISA,rTpN47-ELISA and rTpN17-TpN47-ELISA were negative far the scrum samples from healthy individuals,RA and SLE patients,while presented 84.4%,82.3% and 98.1% positive detection rates for the serum samples from syphilis patients.The positive detection rates of rTpN17-ELISA and rTpN47-ELISA were lower than that of TPHA (P<0.O1)，while the positive detection rate of rTpNl7-TpN47-ELISA was similar to that of TPHA (P>0.05),All the positive detection rates from ELISA tests were higher than that of TRUST (71.4%),Conclusion rTpN17-ELISA,rTpN47-ELISA and especially rTpN17-TpN47-ELISA established in this study were of great hope as it was rapid,simple,convenient,safe,with high sensitivity and specificity for serological screening and detection of syphilis. |
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