梅毒螺旋体TP0772蛋白的原核表达和免疫反应性鉴定

Prokaryotic Expression of TP0772 Protein of Treponema Pallidum and Identification of Its Immunoreactivity

目的:利用原核基因工程技术克隆表达梅毒螺旋体TP0772基因,探讨其在梅毒血清学诊断中的应用。方法:PCR扩增获得TP0772基因,构建pET-28b-TP0772重组质粒,转化到大肠杆菌BL21中,以IPTG诱导蛋白质表达,经镍柱纯化后通过质谱技术鉴定。采用免疫印迹法测定其与梅毒患者血清的免疫反应性。建立基于重组TP0772抗原的ELISA间接法并对30份TPPA阳性血清和25份TPP阴性血清进行方法学评价。结果:PCR扩增获得约850 bp的基因片段,成功构建原核表达载体pET-28b-TP0772。目的蛋白分子量约为32kDa,以包涵体的表达形式存在,约占菌体总蛋白的30%。经质谱技术和免疫印迹分析证实重组蛋白为TP0772蛋白,并能够与梅毒患者血清发生特异性结合反应。ELISA测定TPPA阳性血清和阴性血清的符合率分别为93%(28/30)和96%(24/25)。结论:通过DNA重组技术成功获得了重组TP0772蛋白,其与梅毒阳性血清具有良好的免疫反应性,为优化梅毒的血清学诊断方法奠定基础。

Objective To express TP0772 protein of Treponema pallidum in E. coli by genetic engineering technology and identi- fy its immunoreactivity with syphilitic sera. Methods TP0772 gene was amplified by PCR from treponemal genomie DNA and cloned into vector pET-28b. The recombinant plasmid pET-28b-TP0772 was transformed into E. eoli BL21 （ DE3 ） for protein expression on IPTG induction. The expressed protein was purified by Ni2 ~ affinity chromatography and then identified by mass spectrometry. The im- munoreactivity of TP0772 with syphilitic sera was investigated by Western blotteehnie. An indirect ELISA method with recombinant TP0772 antigen was developed and applied to test 30 TPPA positive and 25 TPPA negative sera. Results TP0772 gene fragrnent a- bout 850bp in length was amplified by PCR. The recombinant plasmid pET-28-TP0772 was constructed correctly. Protein with molecu- lar mass of 32kDa was highly expressed and accumulated in inclusion body. The target protein was expressed in approximately 30% of total bacterial proteins. The result of mass spectrometric analysis demonstrated that the purified protein were TP0772 protein which could react with syphilitic sera. The coincidence rate was 93% （28/30）TPPA positive sera and 96% （24/25）in TPPA negative sera with the result of TP0772-ELISA. Conclusion Recombinant TP0772 antigen was successfully obtained by recombinant DNA technology and had strong and specific immunoreactivity with syphilitic sera. TP0772 protein could potentially be useful for the development of improved immunodiagnostic test of syphilis.