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目的表达、纯化梅毒螺旋体(Tp)重组蛋白Tp0259(rTp0259),鉴定其免疫原性,并探讨其在梅毒血清诊断中的价值。方法 PCR扩增tp0259基因,构建pET-28a(+)/Tp0259原核表达载体,转化至E.coli BL21中诱导表达。采用Ni-NTA亲和层析法纯化重组蛋白,并进行Western blot鉴定。以重组蛋白Tp0259为抗原,采用Western blot方法检测梅毒患者血清(一期梅毒患者血清21份,二期梅毒患者血清13份,潜伏梅毒患者血清6份,并与梅毒诊断金标准比较)。结果 PCR扩增获得600bp左右大小的目的基因片段并成功构建原核表达载体pET28a-Tp0259,经诱导高效表达分子质量单位为28ku的重组蛋白。Western blot证实该重组蛋白为Tp0259假定蛋白,能被梅毒患者血清识别。以Tp0259为抗原采用Western blot检测梅毒患者血清特异性抗体结果与梅毒诊断金标准(临床症状、病史结合血清学诊断方法)间的κ值为0.944,表明这两种方法检测结果的一致性较好。结论成功表达、纯化了可溶性rTp0259,该蛋白具有良好的免疫反应性并具有较高的血清学诊断价值。
Objective To express and purify Tp recombinant protein Tp0259 (rTp0259) and identify its immunogenicity, and to explore its value in the diagnosis of syphilis. Methods The tp0259 gene was amplified by PCR. The prokaryotic expression vector pET-28a (+) / Tp0259 was constructed and transformed into E. coli BL21 to induce expression. The recombinant protein was purified by Ni-NTA affinity chromatography and identified by Western blot. The recombinant protein Tp0259 was used as antigen to detect the serum of syphilis patients (21 cases of syphilis, 13 cases of secondary syphilis, 6 cases of latent syphilis, and syphilis diagnostic gold standard) by Western blot. Results The target gene fragment of 600bp was obtained by PCR amplification and the prokaryotic expression vector pET28a-Tp0259 was successfully constructed. The recombinant protein with high molecular mass of 28ku was induced by high performance liquid chromatography. Western blot confirmed that the recombinant protein Tp0259 hypothetical protein, syphilis serum can be identified. Using Tp0259 as antigen to detect syphilis serum-specific antibody results with syphilis diagnostic criteria (clinical symptoms, history combined with serological diagnostic method) κ value was 0.944, indicating that the consistency of the results of these two methods is better . Conclusion The soluble rTp0259 was successfully expressed and purified. The protein has good immunoreactivity and high serological diagnostic value.