酶切与PCR联用合成梅毒抗原TpN17全长DNA及其表达活化和ELISA检测

Expression and detection of full length gene TpN17 of Treponema pallidum by enzyme companiced with PCR

目的在大肠埃希菌中重组表达梅毒密螺旋体TpN17抗原,用其建立梅毒诊断血清学方法。方法酶切与PCR联用,合成全长TpN17基因,亚克隆后克隆到pET-HIS质粒并转化到大肠杆菌BL21株中表达。亲和层析纯化。纯化rTpN17包被微孔板,ELISA方法检测临床8份梅毒阳性血清和4份阴性血清。结果获得了重组表达并纯化的TpN17,ELISA检测结果表明重组蛋白能被梅毒患者阳性血清所识别,特异性100%,灵敏度100%。结论证明了该重组蛋白具有良好的免疫反应性,可用于梅毒感染的血清学检测。

Objective To express the recombinant TpN17 protein of Treponema pallidum （TP） in E.goli, and to develop ELISA for serological diagnosis of syphilis. Methods Synthesis of full length Gene TpN17 by enzyme companied with PCR, subclone and then cloned into the expression vector pET-HIS, and expressed in E.coli BL21. Purified with Ni- NTA affinity chromatography columns. Micro well plate was coated with purified rTpN17, and then 4 negative and 8 positive sera was tested by ELISA. Results Gained purified rTpN17 protein, and the results of ELISA showed 100% of the sensitivity and 100% of specificity . Conclusion The rTpN17 protein can be used for development of ELISA for serological diagnosis of syphilis.