Preparation and application of a novel HCV diagnostic antigen fused to streptavidin / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection.</p><p><b>METHODS</b>A recombinant plasmid pET-11d-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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>