大鼠瘦素受体RNAi慢病毒载体的构建及鉴定

[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。

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

[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.[Conclusion] The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.