转录因子E2F-1基因RNA干扰慢病毒载体的构建与鉴定  

CONSTRUCTION AND IDENTIFICATION OF LENTIVIRAL VECTOR FOR RNA INTERFERENCE OF E2F TRANSCRIPTION FACTOR 1 GENE

作　　者：王晓通[1] 李雷[1] 钱倩[1] 谢玉波[2] 肖强[1] 

机构地区：[1]广西医科大学第一附属医院胃肠腺体外科,南宁530021 [2]广西医科大学第一附属医院麻醉科,南宁530021

出　　处：《广西医科大学学报》2010年第2期165-168,共4页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(30860273)

摘　　要：目的:构建细胞周期相关转录因子E2F-1RNA干扰(RNAi)慢病毒表达载体。方法:选取E2F-1基因的19nt特异性序列,设计针对E2F-1的shRNA序列,应用基因重组技术克隆到pLentiLox3.7(pLL3.7)慢病毒表达载体中,XbaI和NotI进行双酶切和DNA测序鉴定重组克隆,在脂质体的介导下将慢病毒的混合包装载体质粒和包含E2F-1基因RNAi的重组慢病毒载体共转染293FT细胞,包装成病毒48h后,收集病毒颗粒,孔稀释法测定病毒滴度。结果:双酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确;293FT细胞包装E2F-1基因RNAi的重组慢病毒载体成功;收集的细胞培养上清液中,病毒的滴度为5×107TU/mL。结论:成功构建人E2F-1基因RNAi慢病毒载体,为研究E2F-1对于胃癌生长的影响提供了稳定的转染细胞载体。Objective：To construct a lentiviral vector for RNA interference（RNAi）of E2F transcription factor 1（E2F-1）gene.Methods：Two mRNA sequences of E2F-1 were chosen,and the small hairpin RNA sequences（shRNA）were designed.The shRNA sequences were annealed and linked with linearized pLentiLox3.7.The recombinants were identified by double restriction digestion with Xba I/Not I and DNA sequencing.The recombinant lentivirus was harvested from 293FT cells contransfected with the positive recombined plasmid and lentiviral packing materials.Virus titer was determined by hole dilution method.Results：The shRNA sequences were successfully inserted into pLentiLox3.7 vector by double restriction digestion,and the sequences were identified by DNA sequencing.The RNAi of E2F-1 gene of the recombinant lentiviral vector was successfully packed into 293FT cells.The recombinant lentivirus was harvested from 293FT cells with a viral titer of 5×10^7 TU/mL.Conclusion：The lentiviral shRNA expression vector targeting E2F-1 gene has been successfully constructed,which provides a stable vector for further study of the relationship between gastric cancer and E2F-1 gene.

关 键 词：慢病毒载体 E2F-1 RNAI 

分 类 号：R735.2[医药卫生—肿瘤]