梅毒血清学诊断用抗原Gpd蛋白的重组表达及鉴定

摘要

目的 利用基因工程技术重组表达梅毒螺旋体Gpd蛋白,探讨其在梅毒血清学诊断中的应用.方法 PCR扩增Gpd基因序列,构建表达载体pET-28b-Gpd,将表达载体转化感受态细胞E.coli BL21(DE3)并以IPTG诱导蛋白表达,经镍柱亲和层析纯化后,利用质谱和免疫印迹法鉴定重组蛋白.以重组蛋白建立间接ELISA法,并检测20份TPPA阳性和20份TPPA阴性血清去评价其在梅毒血清学诊断中的应用.结果 PCR扩增出约1.1 kb的Gpd片段,成功构建重组质粒pET-28b-Gpd.表达的重组蛋白的相对分子质量约41×103,主要以包涵体形式存在,占菌体总蛋白的40%.质谱和免疫印迹分析证实重组蛋白为Gpd蛋白,并能够与梅毒患者血清发生免疫学反应.间接ELISA法测定TPPA阳性血清和TPPA阴性血清的符合率分别为95%(19/20)和100%(20/20).结论 重组Gpd蛋白能够与梅毒患者血清发生特异性的免疫学反应,是一种可潜在应用于梅毒血清学诊断的新抗原.%Objective To express recombinant Gpd protein of Treponema pallidum by genetic engineering technology and investigate its application in serodiagnosis of syphilis. Methods Gpd gene was amplified by polymerase chain reaction(PCR) and cloned into plasmid vector pET-28b. The recombinant plasmid was transformed into competent cell Escherichia Coli(E. Coli) BL2KDE3) for protein expression under isopropy-β-D-thiogalactoside(IPTG) induction. The expressed protein was purified by Ni2+ affinity chromatography and then identified by mass spectrometry. The immunoreactivity was identified by Western blotting. An indirect enzyme linked immunosorbent assay(ELISA) method with Gpd protein was developed and applied to test 20 Treponema pallidun particle agglutination(TPPA) positive and 20 negative cases. Results Gpd gene fragment with a length of 1. 1 kb was amplified. The prokarotic expression plasmid pET-28b-Gpd was constructed correctly. Recombinant protein with a relative molecular mass of 41× 103 was highly expressed about 40% of total bacterial proteins in inclusion body. The result of mass spectrometry and Western blotting analysis demonstrated that the recombinant protein was Gpd protein which could react with syphilitic sera. The coincidence rate was 95% (19/20) in TPPA positive sera and 100%(20/20) in TPPA negative sera with the result of Gpd-ELISA. Conclusion Recombinant Gpd protein could react with syphilitic sera and could be an alternative antigen for serodiagnosis of syphilis.