CAJ | 학술논문

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入线性化的慢病毒载体pGC-FU,以获得中间质粒pGC-FU-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Realtime PCR法检测重组慢病毒的滴度,应用NG108-15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2.00×109 TU/ml,该病毒转染NG108-15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.
목적 구건공표체항기인RNA여철단백기인적쌍계동자만병독재체.방법 장철단백중련기인구건입선성화적만병독재체pGC-FU,이획득중간질립pGC-FU-HF;진이장구유기인침묵효응적β-arrestin2항기인RNA구건인중간질립,이획득목적질립pGC- agRNA- HF;통과병독적포장화농축,응용Realtime PCR법검측중조만병독적적도,응용NG108-15세포,채용Western blot화실시정량PCR검측항기인RNA적기인침묵효응화철단백적표체.결과 항기인RNA여철단백균성공구건입만병독표체재체,병독적도2.00×109 TU/ml,해병독전염NG108-15세포후,β-arrestin2표체하조,철단백표체상조.결론 성공구건료표체항기인RNA여철단백기인적쌍계동자만병독재체.
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.