大鼠细胞因子信号转导抑制因子3基因RNA干扰慢病毒载体的构建与鉴定

Construction and Identification of Lentiviral-Mediated RNA Interference Vector of Rat Suppressors of Cytokine Signaling 3 Gene

目的研究构建大鼠细胞因子信号转导抑制因子3(SOCS3)基因RNA干扰(RNAi)慢病毒载体的方法。方法根据大鼠SOCS3基因(NM053565),用Ambion在线软件选择3个靶序列,设计并合成包含各正反义靶序列的互补单链寡核苷酸,与经BamHⅠ和XhoⅠ酶切后的慢病毒载体质粒pRNA-Lenti-绿色荧光蛋白(GFP)(含U6启动子和GFP)连接产生pRNA-Lenti-SOCS3-GFP慢病毒重组质粒,然后分别转染C6大鼠神经胶质瘤细胞,通过荧光显微镜观察细胞转染情况,利用荧光实时定量PCR方法检测RNAi组(siRNA1、siRNA2、siRNA3),空白细胞组和阴性序列组(siRNA-Negative)SOCS3的表达情况,筛选出干扰效果最佳的重组质粒。此慢病毒重组质粒与慢病毒包装混合物共转染293T细胞,包装产生慢病毒,收集病毒上清,采取系列稀释法测定慢病毒滴度。结果将目的序列成功连接到载体上,并经测序分析证实载体构建成功。荧光实时定量PCR检测显示慢病毒重组质粒感染C6大鼠神经胶质瘤细胞后,与空白细胞组比较,3个RNAi组均不同程度地抑制SOCS3表达,其中siRNA1组抑制效果最佳,可使SOCS3 mRNA表达量下调达80%。系列稀释法检测慢病毒悬液的滴度为1.0×1010TU.L-1。结论运用pRNA-Lenti-GFP载体构建的pRNA-Lenti-SOCS3-GFP慢病毒载体可有效地抑制大鼠SOCS3的表达。大鼠SOCS3基因RNAi慢病毒载体的构建成功,为SOCS3相关疾病的深入研究和治疗奠定基础。

Objective To study the construction of the lentiviral-mediated RNA interference（RNAi） vector targering rat suppressors of cytokine signaling 3（SOCS3） gene.Methods Three target sequences were selected by on-line designer software on Ambion according to rat SOCS3 mRNA sequence（NM053565）,the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized.After annealing,these double strands DNA were cloned to pRNA-Lenti-green fluorescent protein（GFP）,which contained U6 promoter and GFP.The resulting Lentiviral vector containing SOCS3 shRNA was named pRNA-Lenti-SOCS3-GFP.After the rat glioma cells（C6）were transduced with the constructed 1entiviral vectors,real-time polymerase chain reaction was used to evaluate the level of SOCS3 expression（including siRNA1 group,siRNA2 group,siRNA3 group,vacuity group and siRNA-Negative group）.The pRNA-Lenti-SOCS3-GFP and Lentivector Pakaging plasmid mix were cotransfected into 293T to package Lentivirus particles.Culture supematant was harvested,then the virus titer was determined by serial dilution assay.Results The SOCS3 mRNA sequence was successfully cloned to pRNA-Lenti-GFP,which was proved by PCR and DNA sequence.Compared with control group,the SOCS3 mRNA expressions were obviously suppressed in all 3 experimental groups,especially the expression rate in siRNA1 group was reduced by 80%.The Lentiviral particle titer was determined by serial dilution assay with 1.0×10^10 TU·L^-1.Conclusion The lentiviral-mediated RNAi vector of rat SOCS3 gene has been constructed successfully,this may provide a potential tool for studying and treating SOCS3-related diseases.