大鼠CRH基因RNA干扰慢病毒载体的构建与鉴定

摘要

目的：构建针对大鼠促肾上腺皮质激素释放激素（CRH）基因的RNA干扰（RNAi）慢病毒表达载体并进行鉴定。方法针对CRH基因设计4个RNAi靶点并分别构建入慢病毒骨架载体，测序鉴定。过表达质粒和RNAi质粒共转染293T工具细胞后，用Western blot进行外源筛靶以确定有效靶序列。有效RNAi病毒质粒与辅助质粒通过Lipofectamine 2000共转染293T细胞，培养48 h后，收集细胞培养上清液，将其浓缩后用孔稀释法测定病毒滴度。结果 Western blot外源筛靶显示3个靶点能有效敲减目的基因的表达。测定浓缩病毒悬液的滴度为1．5×109 T U／ml。结论成功构建了CRH基因的RNAi慢病毒载体，可用于CRH在应激反应中作用的研究。%Objective To construct and identify the RNA interference(RNAi) lentivirus vector targeting rat corticotrophin‐releasing hormone (CRH ) gene .Methods Four RNAi vector plasmids were constructed by different interference target points of CRH gene and confirmed by sequencing . The 293T cells were co‐transfected with over‐expression plasmids and RNAi plasmids and the effective sequence of siRNA targeting CRH gene was identified by Western blot .The effective RNAi plasmid and the subsidiary plasmid were transfected into 293T cells to obtain packed lentivirus particles .Forty‐eight hours later ,the supernatant was obtained to condense for viral titer determination by 96‐well dilution .Results Three effective target points were shown by Western blot . The concentrated titer of virus suspension was 1.5 × 109 TU/ml .Conclusion RNAi lentivirus vector of CRH gene has been successfully constructed and could be used for studying the role of CRH gene in stress response .