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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48h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 TU/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 express RNA interference (RNAi) lentiviral vector for rat corticotropin-releasing hormone (CRH) gene. Methods Four RNAi targeting CRH genes were designed and constructed into lentiviral vector respectively and sequenced. 293T kit cells were co-transfected with the overexpression plasmid and the RNAi plasmid and then screened by Western blot to determine the effective target sequence. The effective RNAi virus plasmid and helper plasmids were cotransfected into 293T cells by Lipofectamine 2000. After cultured for 48h, the cell culture supernatants were collected, and the virus titers were determined by pore dilution after concentration. Results Western blotting showed that three targets could effectively knockdown the target gene expression. The titer of the concentrated virus suspension was determined to be 1.5 × 10 9 TU / ml. Conclusion The RNAi lentiviral vector of CRH gene was successfully constructed and could be used in the study of the role of CRH in stress response.