牛Nanog基因真核表达载体构建及有效干扰序列筛选

Construction of Eukaryotic Expression Vector of Bovine Nanog Gene and Screening for Effective siRNA

【目的】构建牛Nanog基因的真核表达载体pVenus-Nanog,筛选对其有效的干扰序列。【方法】以含有牛Nanog基因的PMD-18T-Nanog重组质粒为模板,经PCR扩增目的片段,将其克隆到真核表达载体pVenus上,酶切及测序鉴定正确后命名为pVenus-Nanog。将pVenus-Nanog用脂质体瞬时转染牛皮肤成纤维细胞,用荧光显微镜观察、RT-PCR和Western blotting分别检测Nanog的表达情况。针对牛Nanog基因的CDS区设计并合成3对干扰序列,分别命名为S1、S2、S3及阴性对照N.C.,用脂质体共转染pVenus-Nanog和siRNA于成纤维细胞,用半定量RT-PCR分析干扰效率。【结果】酶切分析与测序结果表明重组载体pVenus-Nanog构建成功,荧光显微镜观察、RT-PCR和Western blotting结果显示其能够在牛成纤维细胞中高效表达。脂质体共转染pVenus-Nanog和siRNA后,半定量RT-PCR结果显示S1效果最好,干扰效率达75%,S2和S3分别为20%和70%。【结论】成功构建了牛Nanog真核表达载体pVenus-Nanog,且获高效表达。通过脂质体共转染法筛选出了牛Nanog基因的有效干扰序列,为研究该基因在胚胎干细胞及早期胚胎中维持多能性调控网络中的作用机制奠定了基础。

【Objective】This study was performed for the construction of an eukaryotic expression vector of bovine Nanog gene,namely the pVenus-Nanog,and then to screen a valid siRNA sequence targeting the CDS of the Nanog gene.【Method】A DNA fragment of 900 bp was amplified from recombinant plasmid PMD-18T-Nanog using PCR and then cloned into an expression vector pVenus.After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pVenus-Nanog that was subsequently transfected into bovine skin fibroblasts using Liposome 2000.Fluorescent microscopy observation,RT-PCR and Western blotting were used to detect the expression of the bovine Nanog gene.Afterwards,three candidate siRNAs（S1,S2 and S3） targeting the CDS of bovine Nanog gene and a negative control（N.C.） were designed and synthesized.They were cotransfected together with the pVenus-Nanog into the bovine skin fibroblasts,respectively.The interference efficiency was assessed using the semi-quantitative RT-PCR method.【Result】Both restriction enzyme digestion and sequencing assays showed that the recombinant vector pVenus-Nanog was successfully constructed.Fluorescent microscopy observation,RT-PCR and Western blotting indicated that the bovine Nanog gene expressed efficiently in the fibroblasts.Semi-quantitative RT-PCR showed that the RNAi efficiencies of S1,S2 and S3 were 75%,20% and 70%,respectively.【Conclusion】A bovine Nanog gene eukaryotic expression vector was successfully constructed and the Nanog gene expressed efficiently in the bovine fibroblasts.An effective siRNA targeting the CDS of bovine Nanog gene was also identified.This study has paved a way for future research on biological functions of bovine Nanog gene,in particular its role in early embryo development and molecular mechanisms for the pluripotential maintenance of ES cells.