大鼠PEDF基因RNA干扰慢病毒载体的构建与鉴定

目的 构建针对大鼠PEDF 基因的RNA干扰（RNAi）慢病毒表达载体并进行鉴定,探讨PEDF基因对缺血心肌血管新生的影响.方法 构建PEDF基因过表达质粒,测序鉴定.针对PEDF基因不同干扰靶点构建4种RNAi 病毒载体质粒pGCsi-U6/Neo/GFP/shRNA,测序鉴定.过表达质粒和RNAi质粒共转染293T细胞后应用Western blot方法进行外源筛靶确定PEDF基因RNAi 有效靶序列.有效RNAi 病毒质粒和其他3种辅助包装原件载体质粒通过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 过表达质粒及4种RNAi质粒构建成功.Western blot外源筛靶显示2个靶点能有效敲减目的基因的表达.PEDF shRNA慢病毒表达载体Serpinf1-RNAi-LV经293T细胞包装成功,收集293T细胞分泌的病毒上清测定浓缩病毒悬液的滴度为2×1012Tu/L.结论 成功构建了PEDF基因的RNAi慢病毒载体,为研究PEDF基因对缺血心肌血管新生的影响打下基础.

Construction and identification of RNA interference lentivirus vector of rat PEDF gene

Objective To construct and identify the RNA interference （RNAi） lentivirus vector targeting rat PEDF gene, and to investigate the effect of PEDF gene on the neogenesis of ischemic myocardial vessels. Methods Over - expression plasmid of PEDF gene was constructed and confirmed by sequencing. Four RNAi vector plasmids, pGCsi - U6/ Neo/GFP/ shRNA, were constructed by different interference target points of PEDF gene and confirmed by sequencing. 293T cells were co - transfected with over - expression plasmids and RNAi plasmids, and the effective sequence of siRNA targeting PEDF gene was identified by Western blot. The effective RNAi plasmid and a lentivirus packaging mix were co - transfected into 293T cells to obtain packaged lentivirus particles. At the end of 48 hours of culture, the supernatant was obtained to condense for viral titer determination by 96 - well dilution. Results Over - expression plasmids and four RNAi plasmids were successfully constructed. Two effective target points were shown by Western blot. The RNAi vector Serpinfl - RNAi - LV of PEDF gene producing PEDF shRNA was successfully packaged by 293T cells. The concentrated titer of virus suspension was 2 x 1012 Tu/L. Conclusion RNAi lentivirus vector of PEDF gene has been successfully constructed and can provide basis for the study of the role of the PEDF gene on the neogenesis of ischemic myocardial vessels.