丙型肝炎病毒NS3-NS4基因的表达及鉴定

目的　构建丙型肝炎病毒 (HCV)非结构蛋白NS3 NS4全长基因重组质粒并诱导表达 ,鉴定NS3 NS4蛋白的抗原性。方法　应用PCR技术从pUC19/HCV中扩增目的基因 ,构建重组表达质粒 ,进行原核表达 ,用SDS PAGE、ELISA技术对表达产物的抗原性进行鉴定。结果　成功构建、表达了重组质粒pBV2 2 0 /NS3 NS4 ,用 5 0份质控标准血清对表达蛋白质进行抗原性检测 ,与第二代诊断试剂相比 ,总符合率为 96 %。结论　全长NS3 NS4蛋白是一个优势免疫原性区 ,应该是HCVEIA诊断试剂的有效成分。

The expression and identification of NS3-NS4 gene of hepatitis C virus

Objective To construct and expr es s a recombinant plasmid of nonstructural protein NS3-NS4 of hepatitis C virus ( HCV), and to identify the antigenicity of the expressed protein. Methods A gene region encompassing the nonstructural protei n NS3-NS4 of HCV was amplified by polymerase chain reaction (PCR) from the pUC1 9/HCV template. The recombinant expression plasmid containing the pBV220/NS3-NS 4 sequence was constructed, and the nonfused NS3-NS4 recombinant protein was ex pressed in E.coli DH5α efficiently. The recombinant protein was det ected by SDS-PAGE and ELISA. Results We successfully constructed and expressed the recom binant plasmid in prokaryote. Its antigenicity was detected with 50 standard ser a. Compared with the second-generation diagnostic Kit, the total detection rate was 96%. Conclusion The whole NS3-NS4 protein, a region of dominant immunogenicity, should be the effective component of the HCV diagnostic Kit and provide the clue for developing HCV DNA vaccine.