庚型肝炎病毒(HGV)E2区cDNA的克隆与表达

Cloning and expression for cDNA of E2 region of hepatitis G virus isolated from Chinese blood donor

目的 克隆ＨＧＶ Ｅ２ 基因并在原核细胞系统中表达ＨＧＶ Ｅ２ 蛋白。方法 从ＨＧＶＲＮＡ 阳性血浆中抽提病毒ＲＮＡ，利用半巢式ＲＴＰＣＲ 法扩增ＨＧＶ Ｅ２ 基因片段并进行序列分析。然后将该片段克隆到ｐＧＥＸ５ｘ１ 表达载体上，进行诱导表达。表达产物用抗ＨＧＶ 阳性血浆作Ｗｅｓｔｅｒｎ ｂｌｏｔ 活性鉴定。结果 获得ＨＧＶ Ｅ２ Ｎ 端长度为７７９ 个核苷酸的基因片段。该片段与美国株ＨＧＶ（Ｕ４４４０２） 、西非株ＧＢＶＣ（ Ｕ３６３８０） 、中国株ＨＧＶ（ Ｕ７５３５６） 的核苷酸序列同源性分别为８４ ％ 、８５ ％ 、９１ ％ ，推测的氨基酸序列同源性分别为８８ ％ 、９３ ％ 、９４ ％ 。表达产物为相对分子质量约５０ ×１０３的ＧＳＴＥ２ 融合蛋白，在细胞内形成包涵体。Ｗｅｓｔｅｒｎ ｂｌｏｔ 反应中在约５０ ×１０３ 处有显色条带。结论本研究成功地在原核细胞系统中表达了具有抗原性的ＨＧＶ Ｅ２ 蛋白，为进一步研究ＨＧＶ Ｅ２ 蛋白及Ｅ２ 抗体的生物学功能打下了基础。

Objective To clone and express HGV E2 gene. Methods HGV RNA was extracted from a HGV positive serum and amplified with semi nested RT PCR. The PCR product was inserted into pGEM T vector and sequenced. Then it was inserted into an expression vector pGEX 5x 1 and expressed in E.coli BL21. Results We got a 779bp fragment. The nucleotide homology of HGV E2 with other three GBV C/HGV E2 sequences from GenBank was 84% to 91%, the amino acid sequence homology was 88% to 94%. The recombinant plasmid pGEX 5x 1 E2 was transformed into E.coli BL21 and inducted by 1mM IPTG. A 50kD expected protein of HGV E2 fusion protein was synthesized. The expressed product formed inclusion bodies in cells. The E2 protein could be recognized by HGV positive sera in Western blot. Conclusion We succeeded in expressing HGV E2 protein in E.coli and the expressed product has antigenicity.