TpN17重组抗原的表达及在梅毒血清学诊断中的初步应用

Expression of recombinant TpN17 antigen and preliminary application for syphilis diagnosis

目的用基因工程技术表达梅毒螺旋体TpN17重组抗原,并建立操作简便、特异性好、敏感性高的梅毒血清抗体间接酶联免疫吸附试验(ELISA)检测。方法采用PCR技术扩增梅毒螺旋体TpN17基因,再进行T-A克隆及测序,然后亚克隆到原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白包被于反应板,建立检测血清中梅毒抗体的ELISA间接检测法,利用该法与梅毒螺旋体血凝试验(TPHA)、甲苯胺红不加热血清试验(TRUST)同时检测血清样品,对其结果进行比较研究。结果TpN17重组蛋白在大肠杆菌BL21中得到稳定表达,并成功建立了检测血清中梅毒抗体的ELISA间接检测法及试剂。对135例梅毒可疑患者血清进行检测,ELISA、TPHA和TRUST的检出阳性率分别为82.96%、98.52%和71.11%,而TRUST测出的8例可疑样品及31例阴性样品中,TPHA结果全部阳性、而ELISA只有2例阴性。结论TpN17重组抗原ELISA试剂在特异性和敏感性上明显优于TRUST法。

Objective To produce recombinant TpN17 Treponema pallidum antigen with genetic engineering technique and to establish simple indirect enzyme-linked immunosorbent assay（ELISA） with high sensitivity and specificity for detecting syphilis antibody in serum. Methods The TpN17 gene was amplified from TP genome by PCR. Amplification fragments of the target genes were sequenced after T-A cloning and cloned into prokaryotic expression vector to express recombinant TpN17 protein. The recombinant fusion antigen was purified with affinity chromatography. Based on this recombinant fusion antigen, an indirect enzyme-linked immunosorbent assay （ELISA） was established. Then, the serum samples were tested by this method, and its results were compared with the same sampies detected by treponema pallidum h （TRUST）. Results TpN17 protein was nosorbent assay （ELISA） was established emagglutination assay （TPHA） and toluidine red unheat serum test highly expressed in E. coli BL21, and an indirect enzyme-linked immusuccessfully. The results from 135 showed that the positive rates detected by ELISA.TPHA and TRUST were 82. suspected serum samples of syphilis 96% ,98.52% and 71.11%, However,among 8 suspected sera and 31 negative sera by TRUST,39 were positive according to TPHA, only 2 were negative according to ELISA. Conclusion In terms of sensitivity and specificity, ELISA to the recombinant fusion TpN17 antigen is obviously superior to TRUST.