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目的： 研制特异的抗ＧＢＶ－Ｃ／ＨＧＶ抗体诊断试剂。方法： ＰＣＲ扩增ＧＢＶ－Ｃ／ＨＧＶ ＮＳ５ 基因片段并定向克隆至转座载体ｐＦａｓｔＢａｃＨＴａ，转化ＤＨ１０Ｂａｃ感受态细胞，３７℃振荡培养４ ｈ 使发生转座，于三抗选择平皿筛选重组ｂａｃｍ ｉｄ。脂质体介导转染ｓｆ９ 细胞，将转染上清再次感染ｓｆ９ 细胞，以批量表达ＮＳ５重组蛋白。利用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ方法分析、检测重组蛋白。结果： 序列测定证实克隆的基因片段是ＧＢＶ－Ｃ／ＨＧＶ 的ＮＳ５ 基因片段，且阅读框架正确。ＳＤＳ－ＰＡＧＥ分析显示在相对分子质量４．１５×１０４ 处有重组蛋白的表达条带，薄层扫描占总蛋白量的１１．７％ 。Ｗｅｓｔｅｒｎ ｂｌｏｔ 发现重组蛋白可与ＧＢＶ－Ｃ／ＨＧＶ ＲＮＡ阳性患者混合血清发生较强的免疫反应。结论： ＧＢＶ－Ｃ／ＨＧＶ ＮＳ５ 重组蛋白可以用于ＧＢＶ－Ｃ／ＨＧＶ感染的检测。
Objective: To develop a specific anti-GBV-C / HGV antibody diagnostic reagent. Methods: The GBV-C / HGV NS5 gene fragment was amplified by PCR and cloned into the transposase vector pFastBacHTa. The recombinant plasmid was transformed into DH10Bac competent cells. After shaking for 4 h at 37 ℃, transfection was performed and the recombinant bacm id was screened on the three selective antigens. Lipofectamine was transfected into sf9 cells, which were transfected into sf9 cells again to express recombinant NS5 in bulk. The recombinant protein was detected by SDS-PAGE and Western blot analysis. Results: Sequence analysis confirmed that the cloned gene fragment was the NS5 gene fragment of GBV-C / HGV, and the reading frame was correct. SDS-PAGE analysis showed that the recombinant protein was expressed at a molecular weight of 4.15 × 104, and the thin layer scan accounted for 11.7% of the total protein. Western blot showed that the recombinant protein with GBV-C / HGV RNA-positive serum mixed with strong immune response. Conclusion: The recombinant protein of GBV-C / HGV NS5 can be used for the detection of GBV-C / HGV infection.