端粒酶基因的腺病毒包装条件优化及其活性功能验证  

Packaging condition optimization and activity functional validation of adenovirus telomerase gene

作　　者：贺小英 荆乾鸽 姜欣颖 袁婷 吴宪 佟瑞莹 王晶妍 马利兵 HE Xiao-ying;JING Qian-ge;JIANG Xin-ying;YUAN Ting;WU Xian;TONG Rui-ying;WANG Jing-yan;MALi-bing(School of Life Science and Technology,Inner Mongolia University of Science & Technology,Baotou,Inner Mongolia 014010,China)

机构地区：[1]内蒙古科技大学生命科学与技术学院

出　　处：《南方农业学报》2019年第6期1356-1361,共6页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31560331,31660340);内蒙古自然科学基金项目[2017MS(LH)0213,2017MS0337]

摘　　要：【目的】构建携带人类端粒酶催化亚基基因(hTERT)的腺病毒载体并优化其包装条件,为进一步探究端粒酶与细胞重编程调控间的作用机理提供参考依据。【方法】利用腺病毒载体pAdEasy-1构建携带hTERT基因的重组腺病毒载体,分别采用脂质体法和电转法及不同浓度胎牛血清在HKE293A细胞中进行包装,筛选出最佳病毒包装条件,收集重组腺病毒rAd-hTERT并体外感染小鼠胎儿成纤维细胞,验证重组腺病毒rAd-hTERT的活性功能。【结果】以腺病毒载体pAdEasy-1为骨架质粒,成功构建获得携带hTERT基因的重组腺病毒载体(pAd-hTERT),其最佳病毒包装条件是采用脂质体法结合15%胎牛血清,此条件下收集获得的病毒滴度最高(1×1011 IFU/mL)。以携带hTERT基因的腺病毒rAd-hTERT感染小鼠胎儿成纤维细胞72 h后,可扩增出片段大小约3450 bp的目的基因条带,即hTERT基因在小鼠胎儿成纤维细胞中成功表达。【结论】采用脂质体法结合15%胎牛血清包装获得的携带hTERT基因重组腺病毒具有较高病毒滴度,感染小鼠胎儿成纤维细胞能成功表达hTERT基因,进一步证实端粒酶具有蛋白功能的生物学活性。【Objective】The aim of the study was to construct an adenoviral vector containing the telomerase catalytic subunit(hTERT)gene,optimize its packaging conditions and lay a foundation for further study of the interaction mechanism between telomerase and cell reprogramming.【Method】The adenoviral vector containing the hTERT gene was constructed by using the AdEasy-1 system of recombinant adenovirus,and then it was packaged in HKE293A cells by liposome, electroporation and different serum concentrations of fetal bovine. The optimal packaging condition was selected. Recombinant adenovirus rAd-hTERT was collected and infected mouse fetal fibroblasts in vitro,and activity function of recombinant adenovirus rAd-hTERT was verified.【Result】The recombinant adenoviral vector(pAd-hTERT)carrying hTERT gene was successfully constructed with recombinant adenovirus rAd-hTERT as skeleton plasmid. The optimal packaging condition was liposome method combining with 15% serum of fetal bovine. The titer of virus obtained under such condition was the highest(1×1011 IFU/mL). After 72 h of adenovirus(rAd-hTERT)carrying hTERT gene infected mouse fetal fibroblasts,target gene bands of 3450 bp was amplified,which meaned that hTERT gene was successfully expressed in mouse fetal fibroblasts.【Conclusion】The recombinant adenovirus carrying hTERT gene has been successfully prepared by liposome method combining with 15% serum of fetal bovine. It has high virus titer. Infecting mouse fetal fibroblasts can successfully express hTERT gene,which further verifies that telomerase has biological activity of functional protein.

关 键 词：端粒酶 腺病毒 细胞重编程 载体构建 病毒包装 功能验证 

分 类 号：S814.8[农业科学—畜牧学]