HBV前C/C基因与绿色荧光蛋白基因融合表达载体的构建及在HEK293细胞中的表达

目的 :构建含HBV前C C基因与绿色荧光蛋白基因的融合表达载体 ,并在真核细胞中表达。方法 :用PCR法扩增含 1.3倍HBVayw型全基因组真核表达载体pHBV1.3的前C C基因片断 ,应用含绿色荧光蛋白基因的真核表达质粒构建融合表达载体 ,采用脂质体介导方法将其转染到HEK2 93细胞 ,并用荧光显微镜及ELISA法检测融合蛋白的表达。结果 :经PCR及酶切鉴定 ,证实成功构建了含HBV前C C基因的真核表达重组体pEGFP -HB VC ,显微镜下观察及ELISA法证实在HEK2 93细胞中表达了相应融合蛋白。结论 :构建的重组表达载体能在真核细胞中表达目的蛋白与GFP的双功能融合蛋白 ,适合于针对HBV前C C区的相关研究。

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR CONTAINING HEPATITIS B VIRUS PRE C/C GENE AND GREEN FLUORESCENT PROTEIN GENE AND ITS EXPRESSION IN HEK293 CELLS

Objective:To construct eukaryotic expression vector containing HBV preC/C gene and green fluorescent protein gene, then transfect it into HEK293 cells, so that it can be used to study the biological significance of HBVpreC/Cgene mutation in vitro. Methods: The HBVpreC/Cgene fragment was amplified by PCR from pHBV1.3 which contains 1.3-fold-whole length genome of HBV subtype ayw and cloned into pEGFP-C1 to construct pEGFP-HBVC,then transfected into HEK293 cells by means of lipoinfection. ELISA was used to detect the expression of fusion protein. Results: The insertion of HBVpreC/C gene fragment in the recombinant plasmid is confirmed by PCR as well as enzyme digestion method, and the plasmid can express the fusion protein in HEK293 cells. Conclusion: The recombinant expression vector can express double function fusion protein, and it can be used by any research targeting HBV preC/C gene.