小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装  

Construction of specific interference vector and lentiviral packaging for SMAC gene in mice

作　　者：吴凡[1] 易在凤 田德英[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染科,武汉430030 [2]华中科技大学同济医学院附属协和医院中西医结合科,武汉430022

出　　处：《临床肝胆病杂志》2014年第5期442-445,共4页Journal of Clinical Hepatology

摘　　要：目的构建并鉴定小鼠SMAC特异干扰载体,并进行慢病毒包装。方法针对小鼠SMAC基因设计并合成3对干扰序列siRNA1、siRNA2、siRNA3及一对阴性对照序列siRNAn,脂质体法转染小鼠肝细胞,Real time PCR法筛选出最佳干扰序列。依据该最佳干扰序列合成DNA oligo,连接GV115载体,获得重组干扰质粒,PCR鉴定阳性克隆并测序鉴定构建的正确性。将该重组干扰质粒与辅助质粒pHelper1.0和pHelper2.0共转染293T细胞,进行慢病毒包装,收集细胞上清并浓缩,梯度稀释法测定病毒滴度。多组间比较采用方差分析,进一步两两比较采用SNK-q检验。结果 3条siRNA对肝细胞SMAC mRNA均有不同程度的抑制作用,其中siRNA1的抑制效应最强,抑制效率达到70.3%。依siRNA1片段合成DNA oligo,并与慢病毒载体连接,测序显示其构建正确,梯度稀释法测定慢病毒滴度为6×108TU/ml。结论成功构建了小鼠SMAC基因特异干扰性的慢病毒,为研究SMAC基因在肝衰竭中的作用奠定基础。Objective To construct and identify the specific interference vector for second mitochondria -derived activator of caspase （SMAC）gene in mice and perform lentiviral packaging.Methods According to the SMAC gene sequences in mice,three small interfering RNAs （siRNAs）（siRNA1,siRNA2,and siRNA3）and one negative control sequence （siRNAn）were designed and synthesized,and mouse hepatocytes were transfected with the above siRNAs using Lipofectamine 2000.The inhibitory effects of these siRNAs on SMAC mRNA expression were evaluated by real-time PCR,and the optimal siRNA was screened out accordingly.Oligo DNA was synthesized based on the optimal siRNA and then connected to GV1 15 vector to obtain recombinant interference plasmid.The candidate clones were identified by PCR and sequenced.The recombinant interference plasmid,as well as pHelper 1.0 and pHelper 2.0,was used to transfect 293T cells for lentivirus packaging.Then,cell supernatants were collected and concentrated,and the lentiviral titer was determined by gradient dilution. Comparison between groups was made by analysis of variance,and multiple comparisons were made by SNK-q test.Results The three siRNAs had different inhibitory effects on SMAC mRNA expression in hepatocytes;siRNA1 showed the strongest inhibitory effect,with an inhibition efficiency of 70.3%.Based on siRNA1 ,the oligo DNA was successfully synthesized and correctly connected to the lentiviral vec-tor,as proved by sequencing.The lentiviral titer was 6 ×10^8 TU/ml,as determined by gradient dilution.Conclusion The recombinant lentivirus that inhibits SMAC expression in mice has been constructed successfully,laying the foundation for further studies on the role of SMAC gene in liver failure.

关 键 词：RNA干扰 慢病毒属 

分 类 号：R393[医药卫生—基础医学]