梅毒螺旋体Tp0453和TpN47抗原的制备及应用

    朱玉兰，黄宗炎，刘胜牙，王佃鹏，叶健忠，张登峰

    深圳国际旅行卫生保健中心，广东 深圳 518033

    摘要：目的表达纯化梅毒螺旋体Tp0453和TpN47蛋白，评价2种蛋白的检测效果，并建立一种新的梅毒抗体检测方法。方法以梅毒螺旋体Nichols菌株基因组DNA为模板，通过PCR扩增Tp0453和TpN47蛋白基因，构建原核表达载体；转化表达菌株后经IPTG诱导表达，并优化表达条件，大规模培养后采用Ni2+亲和柱纯化目的蛋白；纯化蛋白用过碘酸钠法进行HRP标记，采用双抗原夹心ELISA法分别评价两种蛋白的检测效果，在此基础上，按比例混合两种抗原，并建立双抗原夹心ELISA检测法。结果构建了Tp0453和TpN47蛋白的原核表达载体，在低温（27℃）和低剂量IPTG（0.1mmol/L）诱导条件下，有效的实现了Tp0453和TpN47蛋白的可溶性表达。采用Ni2+亲和纯化方法成功纯化出了高纯度的Tp0453和TpN47融合蛋白，ELISA检测结果显示Tp0453比TpN47具有更强的反应性，两种蛋白混合，建立的双抗原夹心ELISA检测法通过血清学试验表明具有很好的敏感度和特异性。结论实现了梅毒螺旋体Tp0453和TpN47蛋白快速高效的表达，并结合2种抗原优势，建立了一种高效的梅毒双抗原夹心ELISA检测方法。关键词：梅毒螺旋体；表达；Ni2+亲和纯化；双抗原夹心；纯化；应用

    中图分类号：R337.1　文献标识码：B

    Expression and purification of membraneimmunogens Tp0453 and TpN47

    of Treponema pallidum and their applicationin test of syphilis

    ZHU Yu-lan*, HUANG Zong-yan, LIU Sheng-ya,WANG Dian-peng, YE Jian-zhong, ZHANG Deng-feng

    Shenzhen International Travel Health CareCenter, Shenzhen， Guangdong 518033,China

    Abstract:   Objective  To express and purify Tp0453 and TpN47 protein of Tp (Treponemapallidum), to evaluate the effects of detection of these twoproteins, and develop a new test in syphilis serodiagnosis. Methods   Tp0453 and TpN47 genes of Tp were amplified byPCR with the template of the genomic DNA of Nichols strain andcloned into the prokaryotic express vector. The recombinantsexpressed the proteins by the IPTG induction, fusion proteins withhis tag were purified using Ni2+ affinity chromatography, then werelabeled using HRP by NaIO4 method. The reactivity of these twoproteins was detected, and then we combined these two proteins totest the reactivity.   Results   We constructedthe recombinant vectors, then in the conditions of low temperature(27℃) and low IPTG (0.1mmol/L), both Tp0453 and TpN47 wereexpressed mainly in form of soluble protein, high purity fusionproteins of Tp0453 and TpN47 with his tag were got bychromatography purification, ELISA tests show that Tp0453 was morereactive than TpN47 with sera from syphilis patients. On thesebases, a new double-antigen-sandwich ELISA (DAgS-ELISA) wasdeveloped and showed high sensitivities and specificities.Conclusion   Soluble proteins of Tp0453 and TpN47 wereexpressed and purified, combined with the advantages of twoproteins, we developed an effective DAgS-ELISA test for syphilisdiagonsis.

    Key words:   Treponema pallidum;Expression; Ni2+ affinity chromatography; Double-antigen-sandwich;Purify; Application

    《中国国境卫生检疫杂志》