HCV免疫学诊断抗原与链亲和素融合蛋白的制备及应用研究

目的 制备带有链亲和素（SA）标签的丙型肝炎病毒（HCV）融合蛋白,并初步探讨其在抗-HCV ELISA检测中的应用.方法 构建了HCV诊断抗原与链亲和素融合蛋白重组表达质粒pET-11d-C44P-SA,在大肠埃希菌BL21（DE3）中表达,并用Western Blot进行鉴定,表达的融合蛋白经镍金属螯合（Ni-NTA）层析柱进行纯化,透析复性后分别包被生物素化以及普通酶联板,进行人血清抗-HCV检测.结果 成功构建了带有SA标签的重组表达质粒,其在大肠埃希菌BL21（DE3）中实现高效表达,制备的融合蛋白纯度可达90%以上.建立ELISA法进行人血清抗-HCV检测显示：融合蛋白包被生物素化酶联板检测灵敏度与特异性明显高于普通酶联板.结论 构建的融合蛋白作为包被抗原,结合利用SA标签与生物素化酶联板,明显提高了抗-HCV检出的灵敏度与特异性,该策略为进一步提高抗-HCV检测试剂的质量打下了基础.

Preparation and application of a novel HCV diagnostic antigen fused to streptavidin

Objective To prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection. Methods A recombinant plasmid pET-11 d-C44P-SA was constructed, which coding a novel HCV diagnostic antigens( C44P) and streptavidin (SA) fusion protein, and the fusion protein was generated with BL21(DE3) E Coli and identified by Western Blot analysis. Then the fusion protein was purified through the Ni-NTA affinity chromatography and over 90% purity has been achieved. Anti-HCV ELISAs were developed when the fusion protein was used in the biotin-pre-coated microplate or ordinary microplate, and then the sensitivity and specificity of the ELISA were evaluated with confirmed human sera panels. Results The fusion protein was expressed in high yields and purified successfully, the ELISA detection of anti-HCV with human sera panel indicated that its sensitivity and specificity is higher when SA-tagged HCV antigen ( C44P-SA) coated in biotin-pre-coated microplate, compared to C44P or C44P-SA coated in ordinary microplate. Conclusion The sensitivity and specificity of anti-HCV ELISA can be improved when a novel HCV diagnostic antigen fused to SA combined with the biotin- pre-coated microplate. This study laid a foundation for improving the performance of HCV diagnostics.