携带白蛋白启动子和IDO基因重组腺病毒的构建

目的 构建携带小鼠白蛋白启动子和IDO基因的重组腺病毒载体,研究肝脏Hepa l石细胞的IDO基因mRNA及蛋白表达情况.方法 酶切含有小鼠全长IDO cDNA的IDO质粒,亚克隆至穿梭载体pAdTrack.ALB上,在BJ5183细菌中和AdEasy-1进行同源重组,生成并筛选阳性克隆,测序、鉴定正确后,转染AD-293细胞进行包装、扩增,检测病毒滴度,RT-PCR和荧光显微镜鉴定重组腺病毒转染AD-293细胞后IDO的表达.重组腺病毒进一步感染Hepa 1-6细胞,RT-PCR和WesternBlot法分别检测IDO基因在细胞内表达情况.结果 经酶切及测序证实携带白蛋白启动子和IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,病毒感染滴度为2.9×10~6pfu/ml.感染Hepa 1-6细胞后,RT-PCR和Western Blot可以检测到IDO mRNA水平和蛋白水平表达.结论 构建了携带白蛋白启动子和IDO基因的重组腺病毒载体.

Construction and identification of a recombinant adenovirus vector with indoleamine 2,3-deoxygenase and chimeric albumin promoter

Objective To construct a recombinant adenovirus vector encoding for indoleamine 2,3-dioxygenase(IDO)and chimetric albumin promoter,evaluate the mRNA and protein expression levels in Hepa 1-6cell.Methods Full-length mouse derived IDO cDNA was subeloned into pAdTraek-ALB shuttle Plasmid.The product was linearized to homologous recombination with AdEasy-l vector in BJ5183 bacteria.The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing.The recombined adenoviruses DNA were transfected into AD-293 cells for packaging and amplification of Ad-ALB/IDO.The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells.The recombinant viruses with Hepa 1-6 cells were cultured and the mRNA and protein expression levels monitored bv RT-PCR and Western blot, respectively. Results Construction of recombinant andenoviruses containing IDO and albumin promoter was confirmed by restriction endonuclease digestion and sequencing.The expression of IDO was identified by RT-PCR in transfected AD-293 cell.The virus titer was 2.9×10~6 pfu/ml.The IDO mRNA and protein expression levels were detectable after transfected Hepa 1-6 cells by RT-PCR and Western blot. Conclusion A recombinant adenovirus Ad-ALB/IDO was susceessfullyconstructed.