梅毒螺旋体Tp0453和TpN47抗原的制备及应用

Expression and purification of membrane immunogens Tp0453 and TpN47 of Treponema pallidum and their application in test of syphilis

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。

Objective To express and purify Tp0453 and TpN47 protein of Tp （Treponema pallidum）, to evaluate the effects of detection of these two proteins, and develop a new test in syphilis serodiagnosis. Methods Tp0453 and TpN47 genes of Tp were amplified by PCR with the template of the genomic DNA of Nichols strain and cloned into the prokaryotic express vector. The recombinants expressed the proteins by the IPTG induction, fusion proteins with his tag were purified using Ni2＋ affinity chromatography, then were labeled using HRP by NaIO4 method. The reactivity of these two proteins was detected, and then we combined these two proteins to test the reactivity. Results We constructed the recombinant vectors, then in the conditions of low temperature （27℃ ） and low IPTG （0.1mmol/L）, both Tp0453 and TpN47 were expressed mainly in form of soluble protein, high purity fusion proteins of Tp0453 and TpN47 with his tag were got by chromatography purification, ELISA tests show that Tp0453 was more reactive than TpN47 with sera from syphilis patients. On these bases, a new double-antigen-sandwich ELISA （DAgS-ELISA） was developed and showed high sensitivities and specificities. Conclusion Soluble proteins of Tp0453 and TpN47 were expressed and purified, combined with the advantages of two proteins, we developed an effective DAgS-ELISA test for syphilis diagonsis.