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通过分析丙肝病毒（ＨＣＶ） 的非结构４ 区（ＮＳ４） 和５ 区（ＮＳ５）蛋白的氨基酸序列，推测确定抗原决定簇位点。用ＲＴ－ ＰＣＲ 技术从中国丙肝病人血清中扩增克隆含抗原决定簇基因的ＮＳ４ 及ＮＳ５ 基因，将该两段基因分别克隆至质粒表达载体ｐＥＴ２８ａ（ ＋ ） 内，转化大肠杆菌ＢＬ２１（ＤＥ３） ，构建成功了高效表达ＮＳ４ 和ＮＳ５ 蛋白的工程菌，ＩＰＴＧ 诱导后两蛋白在工程菌内的表达量分别约占菌体蛋白的３３ ％ 和２８ ％ 。经Ｓｅｐｈａｒｃｒｙｌ Ｓ－ ２００ 分子筛及Ｎｉ－ ＮＴＡＳｅｐｈａｒｏｓｅ 螯合层析纯化，获得了较纯的重组ＮＳ４ 和ＮＳ５ 蛋白。用纯化的ＮＳ４ 和ＮＳ５ 蛋白作抗原ＥＬＩＳＡ 法检测ＨＣＶ抗体阴、阳性血清，证实它们有较好的抗原性和特异性。
The antigenic determinant site was presumed to be determined by analyzing the amino acid sequences of the nonstructural 4 (NS4) and 5 (NS5) proteins of hepatitis C virus (HCV). The NS4 and NS5 genes containing the epitope genes were amplified by RT-PCR from the serum of hepatitis C patients in China. The two genes were cloned into plasmid pET28a (+) and transformed into E. coli BL21 (DE3) The constructed engineering bacteria were successfully constructed to express NS4 and NS5 protein efficiently. The expression of the two proteins in the engineered bacteria induced by IPTG accounted for about 33% and 28% of the bacterial proteins, respectively. Purified by Sephacryl S-200 molecular sieve and Ni-NT Sepharose chelation chromatography, the pure recombinant NS4 and NS5 proteins were obtained. The purified NS4 and NS5 proteins were used as antigen ELISA to detect the positive and negative HCV sera, confirming their good antigenicity and specificity.