细胞死亡调控基因GRIM19重组腺病毒的构建及在恶性胶质瘤CHG-5细胞中的表达  

Construction of recombinant GRIM19 gene adenovirus and its expression in glioma cell CHG-5

作　　者：姚声涛[1] 黄轶[2,3] 曾昭淳[2] 唐文渊[1] 

机构地区：[1]重庆医科大学附属第一医院神经外科,重庆400016 [2]重庆医科大学基础医学院生物化学与分子生物学教研室,重庆400016 [3]附属儿童医院临床分子医学中心,重庆400014

出　　处：《第三军医大学学报》2008年第23期2194-2197,共4页Journal of Third Military Medical University

摘　　要：目的构建细胞死亡调控基因GRIM19(genes associated with retinoid-IFN-induced mortality 19)重组腺病毒载体,观察GRIM19对病毒包装细胞是否具有细胞毒作用,并验证其对脑胶质瘤CHG-5细胞的感染效率。方法采用PCR方法分别将GRIM19以及EGFP基因亚克隆至穿梭质粒pAdTrack-CMV,在BJ5183(pAdeasy)菌内同源重组,筛选阳性克隆,酶切测序鉴定,线性化后转染293T细胞进行包装、扩增、纯化,PCR检测病毒上清,TCID50法测定病毒滴度并感染CHG-5细胞后Western免疫印迹法验证GRIM19蛋白表达。结果连接重组后经酶切和测序法筛选出pAd-GRIM19;转染293T包装细胞,观察到绿色荧光蛋白(GFP)明显表达,细胞生长状态良好,未见明显细胞毒作用。Ad-GRIM19及Ad-EGFP初纯病毒经氯化铯梯度离心纯化最终获得约5×1010U/ml与8×1010U/ml滴度的重组病毒;将其体外感染胶质瘤CHG-5细胞,均能达到90%左右的感染率,Western免疫印迹法表明Ad-GRIM19感染组GRIM19蛋白表达明显升高。结论成功构建了携带GRIM19基因的重组腺病毒,为体内外进一步研究GRIM19对脑胶质瘤的作用奠定了基础。Objective To construct the adenoviral recombinant plasmid encoding the genes associated with retinoid-IFN-induced mortality 19 （GRIM19）, and observe its cytotoxic effect on the viral packaging cells and evaluate the expression of GRIM19 in human glioma cell line CHG-5. Methods The GRIM19 gene and IRES2EGFP gene were respectively subcloned into pAdTrack-CMV plasmid. After confirmed by restriction enzyme digestion and sequencing, the recombinant plasmid of pAdTrack-CMV-GRIM19 was linearized with Pine I and transformed into competent BJ5183 cells containing pAdeasy-1 vector. Then the positive clones with homologous recombination （pAd-GRIM19） was confirmed by Pac I digestion and transfected into the packaging 293 T cells to generate virus and the viral supernatant containing the GRIM19 gene was confirmed by PCR. After the virus was tittered by using TCIDs0 method, CHG-5 cells were infected with the recombinant adenovirus and the expression of GRIMI9 was detected by Western blot analysis. Results The recombinant adenoviral plasmid pAd-GRIM19 was successfully constructed. The adenoviral liters for GRIM19 and EGFP control recombinant were 5×10^10 U/ml and 8×10^10U/ml respectively. The recombinant virus infected CHG-5 cells with great efficiency. Conclusion The recombinant adenoviral plasmid of pAd-GRIM19 and pAd-EGFP control are constructed successfully. GRIM19 is overexpressed in CHG-5 cells, providing a basis for further study of GRIM19 in vitro and in vivo.

关 键 词：GRIM19 腺病毒 胶质瘤细胞 CHG-5 

分 类 号：R394-33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]