家蚕核多角体病毒双穿梭载体的构建及GFP和HBeAg融合蛋白的表达

用ＡｃＮＰＶ穿梭载体Ａｃ－Ｂａｃｍｉｄ与野生型ＢｍＮＰＶＤＮＡ共转染家蚕ＢｍＮ细胞，通过同源重组得到整合了ｌａｃＺ／Ｔｎ７ａｔ／ｍｉｎｉＦ表达盒的ＢｍＮＰＶＢａｃｍｉｄ，除能在大肠杆菌／ＢｍＮ细胞中穿梭复制外，还能感染ＢｍＮ－ＰＶ的非允许细胞Ｓｆ９，成功地构建了ＢｍＮＰＶ的双穿梭载体．经与重组转座质粒ｐＦＧＨｅ转座，获得了插入ｇｆｐ／ＨＢＶｅ融合基因的重组ｒＢｍ－Ｂａｃｍｉｄ（ｒＢｍＧＨｅ），在家蚕细胞中表达了既能发射绿色荧光又具有ＨＢｅＡｇ抗原性的双功能融合蛋白．Ｂｍ－Ｂａｃｍｉｄ为杆状病毒ＢａｃｔｏＢａｃ策略增添了一个新成员

Construction of bi shuttle Vector for Bombyx mori Nuclear Polyhedrois Virus and Expression of a GFP/HBeAg Fusion Protein

A bi shuttle vector Bm Bacmid was constructed by co infecting into BmN cells with wild type genomic DNA from BmNPV and Ac Bacmid DNA.It not only could replicate in E,coli cells as a large plasmid but also remain infections when introduced into BmN or Sf9 cells.The Bm Bacmid comprises of BmNPV DNA and a transposon cassette which includes a mini freplicon,a kanamycin resistance gene (ka),lac Z and an attachment site for Tn7 (att Tn7).Recombinant virus rBmGHe was obtained after fransposition of a donor plasmid carrying green fluorescent protein gene (gfp) and Hapatitis B e antigen gene (HBVe) into att Tn7.SDS PAGE analysis shows that both gfp and HBeAg gene were highly expressed in BmN cells.The fusion protein was determined by ELISA kit meanwhile the intensity of luminescence was observed under the fluorescent microscope.The results have demonstrated that the expressed GFP HBeAg fusion protein not only can generate striking green fluorescence but also have antigenic activity.