摘要
目的:分别构建以甲胎蛋白(alpha fetoprotein,AFP)300bp启动子和800bp增强子-300bp启动子调控复制的条件复制型腺病毒(CRAd),对两种病毒的条件复制性以及溶瘤作用进行比较,为肝癌靶向性治疗提供更优良的载体。方法:以HepG2基因组DNA为模板,PCR扩增AFP基因启动子(AFPp)和增强子(AFPe),构建表达质粒pAFPp-EGFPluc和pAFPep-EGFPluc,通过检测报告基因EGFP和luciferase的表达鉴定启动子和增强子的活性后,构建穿梭质粒pDC311-AFPp-E1A,pDC311-AFPep-E1A并包装出腺病毒Ad.AFPp-E1A和Ad.AFPep-E1A。利用Western blot、病毒增殖、细胞病变、细胞活力等鉴定并比较两种病毒的复制能力和溶瘤作用。结果:Ad.AFPp-E1A和Ad.AFPep-E1A均可在AFP阳性细胞中选择性复制并且具有一定的溶瘤作用,以后者的选择复制性和溶瘤性更为明显。感染病毒Ad.AFPp-E1A后的HepG2、Hep3B细胞的存活率分别为(54.23±7.13)%、(61.18±12.63)%;感染病毒Ad.AFPep-E1A后的HepG2、Hep3B细胞存活率分别为(26.65±5.43)%、(24.49±3.31)%。结论:被截短的800bp增强子片断,在对AFP启动子起到增强作用的同时,可以为腺病毒包装进更多的治疗基因提供空间,从而为肝癌靶向治疗提供更为良好的条件复制型病毒载体。
Objective： To construct two conditional replication-competent adenoviral vectors （CRAs） driven by alpha fetoprotein （AFP）300bp promoter and 300bp promoter±AFP 800bp enhancer respectively,and to find a better CRA vector for liver cancer targeting therapy. Methods： The minimal essential DNA fragment of the AFP gene promoter and enhancer was amplified through PCR from the genome of HepG2 cells. Construct the plasmid pAFPp-EGFPIuc and pAFPep-EGFPluc to evaluate the activity of the AFP promoter and enhancer by the way of flow cytometry and detect fluorescence. Then construct the shuttle plasmid pDC311-AFPp-E1A and pDC311-AFPep-E1A and construct the conditional replicative adnovirus （CRAs）Ad.AFPp-E1A and Ad.AFPep-E1A. The ability of conditional replication was detected and compared by western blot, virus multiplication test and CPE. At last measure the oncolytie efficacy of these two viruses in AFP positive cells like HepG2 and Hep3B. Results： Both AFPp-E1A and Ad.AFPep-E1A could be selectively replicated in AFP positive cells and could be selectively inhibited AFP positive cells and the latter adenovirus was better. After using Ad. AFPp-E1A, the survival rates of HepG2 and Hep3B were （54.23± 7.13）% and （61.18± 12.63）% respectively. After using Ad. AF- Pep-E1A, the survival rates of HepG2 and Hep3B were （26.65± 5.43）% and （24.49± 3.31）% respectively. Conclusions： We use the short AFP 800bp enhancer for the first time.It can enhance the activity of AFP promoter, at the same time, it provides more room to add more therapy gene for adenovirus package. Thereby,Ad.AFPep-E1A is a promising vector candidate for hepatocarcinoma targeting therapy.
出处
《现代生物医学进展》
CAS
2009年第10期1801-1804,1831,共5页
Progress in Modern Biomedicine
基金
国家重点基础研究计划(No.2004CB518804)
国家自然科学基金杰出青年项目(No.30325043)
