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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 and construct the prokaryotic expression system of tpN37 gene of Treponema pallidum, establish an ELISA based on rTpN37 and evaluate its sensitivity and specificity for serological diagnosis of syphilis. Methods: The tpN37 gene was amplified by PCR to construct the prokaryotic expression system of tpN37 gene. The recombinant protein rTpN37 was detected by SDS-PAGE. The rTpN37 was purified by Ni-NTA affinity chromatography and the immunoreactivity of rTpN37 was identified by Western blot. RTpN37-ELISA was established using rT-pN37 as coating antigen and was used to detect serum of 122 patients with syphilis. The test results were compared with those of RPR and TPPA. Results: The similarity of the cloned tpN37 gene with the related sequences in GenBank was 100%. The expression level of rTpN37 is 20.4% of the total bacterial protein. The purified rTpN37 showed a single protein band after SDS-PAGE. TPPA-positive syphilitic sera can effectively recognize and bind to rTpN37. The positive rate of rTpN37-ELISA was 95.9% (117/122) in serum samples of syphilis patients, which was similar to that of TPPA (96.7%, 118/122) (P> 0.05). The positive rates of rTpN37-ELISA and TPPA were significant Higher than RPR (81.1%, 99/122) (P <0.01). Conclusion: The tpN37 gene of Treponema pallidum and its prokaryotic expression system were successfully cloned and constructed in this study. The rTpN37-rTpN37-ELISA can be used as a rapid, sensitive and specific syphilis serological screening method.