庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达

Expression of E2 Glycoprotein Gene of Hepatitis G Virus in Insect Cells

用ＰＣＲ扩增出ＨＧＶＥ２全基因，克隆进杆状病毒表达载体ｐＦＡＳＴＢＡＣＨＴａ中，构建成重组转座载体ｐＦＡＳＴＢＡＣＥ２，转化ＤＨ１０ＢＡＣ大肠杆菌感受态细胞，筛选阳性菌落，抽提大分子质粒ＤＮＡ，获得含ＨＧＶＥ２基因的重组杆状病毒穿梭载体，转染昆虫草地夜蛾Ｓｆ９细胞，出现细胞病变后，收集含有重组病毒颗粒的培养上清，重新感染草地夜蛾Ｓｆ９单层细胞及甜菜夜蛾幼虫，分别收集Ｓｆ９细胞和甜菜夜蛾幼虫体内的血淋巴细胞，进行１２％ＳＤＳ聚丙烯酰胺凝胶电泳，可见表达的融合蛋白带，经亲和层析进行蛋白纯化，用ＥＬＩＳＡ方法检测各类血清标本。

Hepatitis G virus E2 glycoprotein gene was amplified by polymerase chain reaction, and was inserted into baculovirus expression vector pF AST B AC HTa, constructing a recombinant transposing vector pF AST B AC E2. The plasmid pF AST B AC E2 was transformed into DH10B AC competent E. coli cells. High molecular weight DNA was prepared from the overnight cultures from the selected E. coli colonies, which was recombinant baculovirus shuttle vector containing HGV E2 gene, named Bacmid E2. The Bacmid E2 was transfected into Spodoptera fragiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells and larvae of Spodoptera exigua were infected with the recombinant virus to express the target protein. The E2 glycoprotein recovering from the Sf9 cells and hemolymph cells of larvae of Spodoptera exigua exhibited a molecular mass of approximataly 54000D. Purified HGV E2 glycoprotein using affinity chromatography was used to develop an ELISA for detection of HGV E2 antibodies in human sera.