梅毒螺旋体特异性抗原TP17、TP0453的表达及以其作为诊断抗原的间接ELISA方法的建立

目的表达、纯化梅毒螺旋体特异性外膜蛋白TP17和TP0453,并以其作为包被抗原,建立新型梅毒酶联免疫诊断方法。方法 PCR扩增TP17和TP0453基因,分别克隆入pET-22b(+)和pET-28a(+)载体,构建重组表达质粒pET-22b(+)-TP17和pET-28a(+)-TP0453,转化E.coliBL21(DE3),IPTG诱导表达,表达产物纯化后进行Western blot鉴定。以纯化的重组TP17和TP0453蛋白作为诊断抗原,建立间接ELISA方法,并对该方法进行验证。结果 PCR分别扩增出500和800bp的TP17和TP0453基因片段,测序结果与GenBank中登录的CDS序列完全一致。重组表达质粒pET-22b(+)-TP17和pET-28a(+)-TP0453经双酶切鉴定正确;重组TP17和TP0453蛋白的相对分子质量分别约为18000和29000。表达量分别约占菌体总蛋白的18%和24%,纯化的重组TP17和TP0453蛋白的纯度分别>95%和>90%。两种重组蛋白均能与梅毒标准血清发生特异性反应。检测70份梅毒参考血清和定值质控血清的批内变异系数(CV)≤4.35%,批间CV≤6.69%,表明精密性良好;对国家标准血清盘的检测灵敏度为94.4%,特异性为97.1%,符合率为95.7%,最低检出限为0.25NCU/ml。结论表达和纯化的重组TP17和TP0453蛋白具有良好的抗原性和特异性,可用于梅毒血清学诊断。

Expression of Specific Antigens TP17 and TP0453 of Treponema pallidum and Development of An Indirect ELISA Method Using The Expressed Products as Diagnostic Antigens

Objective To express and purify the outer membrane proteins TP17 and TP0453 of Treponema pallidum and use them as coating antigens to develop a novel ELISA method for diagnosis of syphilis.Methods TP17 and TP0453 genes were amplified by PCR and cloned into vectors pET-22b(+)and pET-28a(+)respectively.The constructed recombinant plasmids pET-22b(+)TP17 and pET-28a(+)-TP0453 were transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed products were purified then identified by Western blot.An indirect ELISA method was developed using the purified TP17 and TP0453 proteins as diagnostic antigens and verified.Results TP17 and TP0453 gene fragments,at lengths of 500 and 800 bp respectively,were amplified by PCR,of which the sequencing results were completely consistent with the CDS sequence reported in GenBank.Restriction analysis proved that recombinant plasmids pET-22b(+)-TP17 and pET-28a(+)-TP0453 were constructed correctly.Recombinant TP17 and TP0453 proteins,with relative molecular masses of 18 000 and 29 000 respectively,contained about 18% and about 24% of total somatic proteins respectively.The purified TP17 and TP0453 proteins reached purities of more than 95% and more than 90% respectively,both of which showed specific reactions with standard syphilis serum.The intra-and inter-coefficients of variation of determination results of 70 reference serum and quantitative quality control serum samples were not more than 4.35% and not more than 6.69% respectively,indicating high precision of the developed method.The sensitivity,specificity,coincidence rate and minimum detection limit of the developed method for national standard serum plate were 94.4%,97.1%,95.7% and 0.25 NCU/ml respectively.Conclusions The expressed and purified recombinant TP17 and TP0453 proteins showed high antigenicities and specificities,which might be used for the serological diagnosis of syphilis.