cFLIP_L和cFLIP_S腺病毒载体的构建及其在心肌细胞中的表达  

Construction of cFLIP_L and cFLIP_S adenoviral vectors and their expression in cardiomyocytes

作　　者：刘滴 吴辉 李云曌[1,2] 杨俊 杨简 丁家望 范致星 张静 Liu Di;Wu Hui;Li Yunzhao;Yang Jun;Yang Jian;Ding Jiawang;Fan Zhixing;Zhang Jing(Institute of Cardiovascular Diseases,First Clinical Medical College of Three Gorges University,Yichang 443003,China;Department of Cardiology,First Clinical Medical College of Three Gorges University,Yichang 443003,China;Central Laboratory,First Clinical Medical College of Three Gorges University,Yichang 443003,China)

机构地区：[1]三峡大学第一临床医学院三峡大学心血管病研究所,宜昌443003 [2]三峡大学第一临床医学院心内科,宜昌443003 [3]三峡大学第一临床医学院中心实验室,宜昌443003

出　　处：《解剖学杂志》2019年第6期557-560,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金青年项目（81600234）

摘　　要：目的:构建并鉴定cFLIPL和cFLIPS重组质粒,并观察其在乳鼠心肌细胞中表达并检测转染效率.方法:利用基因合成技术获取cFLIPL和cFLIPS基因,分别连接pAd-CMV-GFPa1-IRES载体质粒,并转移至穿梭质粒pAd-cFLIPL和pAd-cFLIPS,经挑选、扩培后进行酶切验证及测序鉴定、包装、扩增、纯化获得一定滴度的pAd-cFLIPL和pAd-cFLIPS,之后感染原代乳鼠心肌细胞,观察两者在心肌细胞中的荧光表达并用流式细胞仪检测转染效率.结果:酶切验证与理论值相符,测序鉴定结果与目的基因序列一致;pAd-cFLIPL和pAd-cFLIPS感染人胚肾293A细胞后可见大量绿色荧光蛋白表达和聚集,10 d后出现典型的细胞病变,腺病毒包装成功,并测得滴度均为1×10^9 pfu/ml;同时,在荧光显微镜下可见重组腺病毒感染可在乳鼠心肌细胞中稳定表达并发出绿色荧光,流式细胞仪检测结果显示其转染效率为90.30%±3.62%.结论:成功构建cFLIPL和cFLIPS重组腺病毒表达载体且证实其能安全、有效地感染乳鼠心肌细胞.Objective:To construct and identify cFLIPL and cFLIPS recombinant plasmids,and observe their fluorescence expression in neonatal rat cardiomyocytes,and detect the transfection efficiency.Methods:The cFLIPL and cFLIPS genes were obtained by gene synthesis and the pAd-CMV-GFPa1-IRES vector plasmid was ligated to construct the shuttle plasmids pAd-cFLIPL and pAd-cFLIPS.After selection and expansion,the enzyme digestion verification and sequencing identification were performed.After packaging,amplification and purification,a certain titer of pAd-cFLIPL and pAdcFLIPS was obtained,which was infected into primary neonatal rat cardiomyocytes,and the fluorescence expression of cardiomyocytes was observed and the efficacy of transfection was detected by flow cytometry.Results:The enzyme digestion was consistent with the theoretical value.The sequencing results were consistent with the target gene sequence.After the infection of human embryonic kidney 293A cells by pAd-cFLIPL and pAd-cFLIPS,a large amount of green fluorescent protein was expressed and aggregated.After 10 days the typical results appeared.The cytopathic effect of adenovirus packaging was successful and the titer was 1×10^9 pfu/ml.Meanwhile,under the fluorescence microscope,the neonatal rat cardiomyocytes expressed green fluorescent protein.The results of flow cytometry showed that the transfection efficiency was 90.30%±3.62%.Conclusion:The recombinant adenoviral expression vector of cFLIPL and cFLIPS is successfully constructed and proved to be ef fective in infecting neonatal rat cardiomyocytes.

关 键 词：白细胞介素1β转换酶抑制蛋白 腺病毒载体 质粒 转染 心肌细胞 

分 类 号：R73[医药卫生—肿瘤]