重组抗基因RNA与铁蛋白基因双启动子慢病毒表达载体的构建

Construction of lentivirns vector expressing antigene RNA and ferritin gene

目的构建共表达抗基因RNA与铁蛋白基因的双启动子慢病毒载体。方法将铁蛋白重链基因构建人线性化的慢病毒载体pGC-FU，以获得中间质粒DGC-U-HF；进而将具有基因沉默效应的β-arrestin2抗基因RNA构建人中间质粒，以获得目的质粒pGC-agRNA-HF；通过病毒的包装和浓缩，应用RealtimePCR法检测重组慢病毒的滴度，应用NGl08-15细胞，采用Western blot和实时定量PCR检测抗基因RNA的基因沉默效应和铁蛋白的表达。结果抗基因RNA与铁蛋白均成功构建入慢病毒表达载体，病毒滴度2．00×109TU／ml，该病毒转染NGl08．15细胞后，β-arrestin2表达下调，铁蛋白表达上调。结论成功构建了表达抗基因RNA与铁蛋白基因的双启动子慢病毒载体。

Objective To construct lentivirus vector expressing antigene RNA and ferritin gene.Methods Intermediate plasmid pGC-FU-HF was constructed by transfecting lentivirus vector pGC-FU with heavy chain ferritin subunit gene. The target plasmid pGC-agRNA-HF was subsequently constructed by transfecting the intermediate plasmid with β-arrestin 2 antigene RNA. The NG108-15 cells were transfected with the target plasmid. The titre of lentivirus vector was measured by RT-PCR. The expression of antigene RNA and ferritin gene was determined by Western blot and RT-PCR. Results Lentivirus vector was successfully transfected with antigene RNA and ferritin gene. The titre of lentivirus vector was 2.00 × 109 TU/ml. The expression of β-arrestin2 protein was down-regulated and the expression of ferritin protein up-regulated in the NG108-15 cells after being transfected with the lentivirus vector. Conclusion Lentivirus vector expressing antigene RNA and ferritin gene has been successfully constructed.