RpN47重组抗原的表达及其产物反应原性的初步鉴定

目的探讨梅毒螺旋体外膜蛋白（TpN47）重组抗原及其临床意义。方法采用PCR技术扩增TpN47重组抗原,再进行T-A克隆及测序,然后亚克隆至原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白作为抗原制备酶联免疫吸附试验（ELISA）检测试剂盒,对正常人血清200份、类风湿性关节炎（RA）阳性血清55份、系统性红斑狼疮（SLE）阳性血清34份及梅毒可疑患者阳性血清135份进行检测,其结果与梅毒血凝试验（TPHA）、梅毒甲苯胺红不加热血清试验（TRUST）的检测结果进行比较。结果成功地构建了pET32-TpN47重组表达载体,重组的TpN47蛋白在大肠杆菌BL21中得到稳定表达。对梅毒可疑阳性血清采用ELISA、TPHA、TRUST检出阳性率分别为81.48%、98.52%、71.11%;TRUST测出的8份可疑阳性样品及31份阴性样品TPHA检测结果均为阳性,而ELISA检测结果阳性19份。ELISA检测阳性率与TRUST和TPHA比较,差异均有统计学意义（均P〈0.01）。结论TpN47重组抗原具有较好的免疫反应活性,ELISA试剂特异性、敏感性次于TPHA,但明显高于TRUST。

Expression of recombinant Treponema pallidum TpN47 antigen and preliminary analysis on the reactivity of the expressed products

Objective To investigate recombinant antigen(TpN47)for outer membrane protein of Treponema pallidum and the clinical application of this fusion antigen.Methods The TpN47 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 TpN47 protein.The recombinant fusion antigen was purified with affinity chromatography and an Enzyme-linked immunosorbent assay(ELISA)kit was prepared.Then,the sera of 200 normal persons,the sera positive of 55 rheumatoid arthritis(RA)and 34 systemic lupus erythematosus(SLE)patients,135 suspected serum samples of syphilis antibodies were tested by this method,and its results were compared with the same samples detected by treponema pallidum hemagglutination assay(TPHA)and toluidine red unheat serum test(TRUST).Results The expression vector pET32-TpN47 was constructed successfully,and TpN47 protein was highly expressed in E.coli BL21.The results from suspected serum sample of syphilis showed that the positive rates detected by ELISA,TPHA,TRUST were 81.48%,98.52%,71.11%,among 8 suspected positive sera and 31 negative sera by TRUST,The results of TPHA were all positive,19 were positive according to ELISA.The positive rate of ELISA to TRUST and TPHA have statistical significant difference(P<0.01).Conclusion The recombinant fusion TpN47 antigen possessed highly immuno-reactive activity,according to the sensitivity and specificity,ELISA is less than TPHA,but is obviously superior to TRUST.