HEV ORF3真核表达载体的构建、鉴定和真核表达

Cloning idetification and eukaryotic expression of HEV ORF3 in L02 cell line

目的构建表达戊型肝炎病毒(HEV)ORF3-pXF2RH融合蛋白的真核表达载体;获得重组质粒稳定转染的L02细胞系。方法利用PCR技术从HEV基因组中扩增出ORF3基因片断,HindⅢ/EcolⅠ双酶切后连接到经同样酶切的pXF2RH真核表达载体,转化DH5α菌株感受态细胞,获得阳性重组质粒ORF3-pXF2RH。将阳性克隆用脂质体法转染L02细胞系,G418筛选抗性克隆,SDS-PAGE、Western blot分析鉴定ORF3-pXF2RH融合蛋白的表达。结果真核表达质粒转染L02细胞,SDS-PAGE显示在28.5kD左右蛋白表达量明显高于对照组,Western blot在28.5kD左右有一条强的棕色条带。结论成功构建ORF3-pXF2RH真核表达质粒,在L02细胞表达融合蛋白,为进一步研究该蛋白生物学功能奠定了基础。

In order to construct a eukaryotic expression vector for expressing hepatitis E virus recombinant ORF3-pXF2RH fusion protein and to obtain a stable transfected L02 cell line. The coding region of ORF3 gene of HEV was amplified by PCR and was digested by HindⅢ/EcolⅠ . This fragment was inserted into eukaryotic expression vector pXF2RH with T4 ligase and transformed E. coli DH5α. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into L02 cell by Lipofect AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The expression of fusion gene was analyzed by SDS-PAGE and Western blotting. Recombinant vector ORF3-pXF2RH was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected L02 cell membrane selected by G418 was confirmed by SDS-PAGE and Western blotting. It is concluded that the stable transfected HepG2 cell line can express ORF3-pEGFP and ORF3-pXF2RH fusion protein. This lays a foundation for further studies on HEV ORF3.