Ⅰ型HEV ORF3-pEGFPN1真核表达质粒的构建与鉴定

Cloning idetification and eukaryotic expression of genetype 1 HEV ORF3 in LO2 cell line

目的:构建表达Ⅰ型HEV ORF3-pEGFPN1融合蛋白的真核表达载体;获得重组质粒稳定转染的LO2细胞系。方法:从猕猴胆汁提取RNA,利用RT-PCR及PCR技术从HEV基因组中扩增出ORF3基因片断,EcoRⅠ/BamHⅠ双酶切后连接到经同样酶切的pEGFPN1真核表达载体,转化DH5a菌株感受细胞,获得阳性重组质粒ORF3-pEGFPN1。将阳性克隆用脂质体法转染LO2细胞系,G418筛选抗性克隆,SDS-PAGE、Western blot分析鉴定ORF3-pEGFPN1融合蛋白的表达。结果:真核表达质粒转染LO2细胞,SDS-PAGE显示在39.8kD左右蛋白表达量明显高于对照组,Western blot在39.8kD左右有一条强的棕色条带。结论:成功构建ORF3-pEGFPN1真核表达质粒,能在LO2细胞表达融合蛋白,为进一步研究该蛋白生物学功能奠定基础。

Objective： In order to construct an eukaryotic expression vector for expressing genetype 1 HEV recombinant ORF3-pEGFPN1 fusion protein and to obtain a stable transfected LO2 cell line. Methods： The coding region of ORF3 gene of HEV was amplified by PCR and was digested by EcoR I/BamH I. This fragment was inserted into eukaryotic expression vector pEGFPN1 with T4 ligase and transformed E. Coli DH5α. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into LO2 cell by Lipofect MAINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. Results： The expression of fusion gene was analyzed by SDS-PAGE and Western blotting. Recombinant vector ORF3-pEGFPN1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected LO2 cell selected by G418 was confirmed by SDS-PAGE and Western blotting. Conclustion： It is concluded that the stable transfected LO2 cell line can express ORF3-pEGFP fusion protein. This lays a foundation for further studies on genetype 1 HEV ORF3.