逆转录病毒载体介导的SynNotch门控系统构建  

Construction of retroviral vector-mediated SynNotch gating system

作　　者：张野 陈静 王佩佩 林伟 王建勋 ZHANG Ye;CHEN Jing;WANG Peipei;LIN Wei;WANG Jianxun(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学生命科学学院,北京102488 [2]深圳北京中医药大学研究院

出　　处：《山东医药》2022年第15期6-11,共6页Shandong Medical Journal

基　　金：北京中医药大学高层次人才科研启动经费项目(9011451310032)。

摘　　要：目的构建一种逆转录病毒载体介导的SynNotch门控系统。方法对逆转录病毒载体进行设计改造,在其两端插入绝缘子序列(insulator),并基于Gal4/UAS基因表达调控系统进行SynNotch门控系统的构建。首先采用逆转录病毒载体分别构建仅含有Gal4-VP64融合蛋白转录调控元件的门控质粒以及含有UAS激活序列的效应质粒,效应质粒使用绿色荧光蛋白(EGFP)及荧光素酶(luciferase)作为报告基因,共分为6种,其中三种为正向序列设计,将UAS序列以及报告基因正向插入逆转录病毒载体中(5’~3’),分别包括正向无绝缘子、正向单绝缘子、正向双绝缘子;另三种为反向序列设计,将UAS序列以及报告基因反向插入逆转录病毒载体中(3’~5’),分别包括反向无绝缘子、反向单绝缘子、反向双绝缘子。测序成功的质粒进一步通过病毒包装制备病毒载体,包括门控载体和效应载体。PCR法检测逆转录病毒滴度。将病毒载体转导入293T细胞,效应载体单独转导的293T细胞作为未诱导组,门控载体与效应载体共转导的293T细胞作为诱导组,分别通过流式细胞术及荧光素酶化学发光实验检测两组报告基因的表达效率。结果逆病毒载体滴度均在107~108拷贝/mL之间,且不低于阳性对照。流式细胞术的检测结果显示,正向设计的三种效应载体在未诱导的情况下EGFP的表达效率分别为95.5%、69.7%、76.2%,诱导后EGFP的表达效率分别为99.9%、95.3%、97.1%。反向设计的三种效应载体在未诱导的情况下EGFP的表达效率分别为0.085%、0.610%、7.400%,诱导后EGFP的表达效率分别为59.900%、77.900%、89.500%。荧光素酶化学发光实验的检测结果显示,正向设计的三种效应载体在未诱导的情况下luciferase表达的荧光强度均在104以上,诱导后荧光强度均在105以上;反向设计的三种效应载体在未诱导的情况下luciferase表达的荧光强度均在3×103以下,诱导后荧光强�Objective To construct a retroviral vector-mediated SynNotch gating system.Methods The retroviral vector was designed and modified,insulator sequences were inserted at both ends,and the SynNotch gating system was constructed based on the Gal4/UAS gene expression regulation system.First,a retroviral vector was used to construct a gated plasmid containing only Gal4-VP64 fusion protein transcriptional regulatory elements and an effector plasmid contain⁃ing the UAS activation sequence.The effector plasmids used green fluorescent protein(EGFP)and luciferase as reporter genes,and were divided into 6 types,three of which were designed for the forward sequence,and the UAS sequence and the reporter gene were inserted into the retroviral vector(5'-3')in the forward direction,including the forward non-insula⁃tor,forward single insulator,and forward double insulator,respectively;the other three were designed for reverse se⁃quence,and the UAS sequence and the reporter gene were reversely inserted into the retroviral vector(3'-5'),including reverse non-insulator,reverse single insulator,and reverse double insulator,respectively.After the plasmid was construct⁃ed,it was verified by sequencing,and the successfully sequenced plasmid was further prepared by virus packaging to pre⁃pare viral vectors,including gated vectors and effector vectors.PCR method was used to detect retrovirus titers.The viral vector was transduced into 293T cells,the 293T cells transduced by the effector vector alone were used as the uninduced group,and the 293T cells co-transduced by the gated vector and the effector vector were used as the induced group.Flow cytometry and luciferase chemiluminescence experiment were used to detect the expression efficiency of reporter genes in the two groups.Results The titers of retroviral vectors were 107-108 copies/mL,and were not lower than those of the pos⁃itive control.The results of flow cytometry showed that the expression efficiencies of EGFP were 95.5%,69.7%,and 76.2%in the three forwardly designed e

关 键 词：逆转录病毒载体 SynNotch受体 嵌合抗原受体T细胞 绝缘子 

分 类 号：R733[医药卫生—肿瘤]