大鼠瘦素受体RNAi慢病毒载体的构建及鉴定

Construction and identification of lentivial RNA interference vector of rat leptin receptor gene

关键词:  瘦素受体  RNA干扰  慢病毒载体

Key words:  OBRb  RNAi  lentivirus vector

摘要:

［目的］构建大鼠OBRb基因的RNAi慢病毒载体，并评估其对OBRb基因表达的沉寂作用。［方法］ 设计大鼠OBRb的siRNA靶序列，合成包含各正反义靶序列的互补单链寡核苷酸，退火后插入到慢病毒载体质粒pRNA-lentivector-VGFP上，构建pRNA-Lenti-OBRb-VGFP表达重组体；为了评估RNAi基因沉寂作用，将构建的慢病毒载体转染表达OBRb的C6大鼠神经胶质瘤细胞，利用Realtime PCR方法检测OBRb mRNA表达水平。［结果］ 将目的序列连接到慢病毒载体上，成功构建pRNA-Lenti-OBRb-VGFP表达重组体；通过PCR、电泳初步鉴定该重组体有OBRb表达，并进一步进行基因测序证实插入序列正确；成功将慢病毒载体转染到C6大鼠神经胶质瘤细胞，转染效率可达40%；荧光实时定量PCR检测OBRb基因表达量，结果干扰组的OBRb mRNA表达水平明显低于非干扰序列的对照组，可使OBRb mRNA表达量下调近80%。［结论］成功构建大鼠OBRb 的RNAi慢病毒载体，为进一步的体内研究奠定了基础。

Abstract:

［Objective］To construct the lentiviral RNA interference ( RNAi) vectors of rat OBRb gene and evaluate the effects of silencing OBRb gene expression by siRNA.［Methods］The target sequence of siRNA-OBRb was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter, target sequence and PolyⅢ terminator. Then, the effects of RNAi to reduce gene expression were further confirmed by realtime PCR in transfected rat glioma cells expressing OBRb.［Results］The rat lentivirus vectors expressing OBRb-specific shRNA were synthesized, and the specificity of designed target sequence was primarily identified by PCR and electrophoresis, and further confirmed by gene sequence analysis. Rat glioma cells were successfully transfected with pRNA-Lenti-OBRb-VGFP, and the transfection rates were 40%. The real-time RT-PCR analysis confirmed that the levels of OBRb mRNA were reduced significantly as compared with the control group, and suppressed by approximately 80% in cells transfected with pRNA-Lenti-OBRb-VGFP.［Conclusions］The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.