论文部分内容阅读
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 a recombinant adenovirus vector of GRIM19 for the expression of GRIM19 and to investigate whether GRIM19 has a cytotoxic effect on viral packaging cells and to verify its effect on CHG-5 cells in gliomas effectiveness. Methods The GRIM19 and EGFP genes were subcloned into the shuttle plasmid pAdTrack-CMV by PCR and homologous recombined in the bacterium of BJ5183 (pAdeasy). The positive clones were screened and identified by restriction enzyme digestion. After linearization, the 293T cells were packaged and expanded The virus supernatant was detected by PCR, the virus titer was detected by TCID50 method and the expression of GRIM19 protein was verified by Western immunoblotting after infected with CHG-5 cells. Results After cloning and sequencing, pAd-GRIM19 was cloned by restriction enzyme digestion and sequencing. The green fluorescent protein (GFP) was found to be transfected into 293T cells. The cell growth was good and no obvious cytotoxicity was observed. Recombinant viruses of Ad-GRIM19 and Ad-EGFP were purified by CsCl gradient centrifugation to obtain 5 × 1010U / ml and 8 × 1010U / ml titer of recombinant virus. In vitro infection of CHG-5 cells The infection rate of about 90% could be reached. Western immunoblotting showed that GRIM19 protein expression in Ad-GRIM19 infection group was significantly increased. Conclusion The recombinant adenovirus carrying GRIM19 gene was successfully constructed and laid the foundation for the further study of the effect of GRIM19 on glioma in vivo and in vitro.