小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装

发布时间：2019-06-20 23:36

【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。
[Abstract]:Objective to construct and identify mouse SMAC specific interference vector and package lentivirus. Methods three pairs of interference sequence siRNA1,siRNA2,siRNA3 and a pair of negative control sequence siRNAn, were designed and synthesized for mouse SMAC gene and the best interference sequence was screened by, Real time PCR method. According to the optimal interference sequence, DNA oligo, ligated GV115 vector was synthesized, and the recombinant interference plasmid was obtained. The positive clone was identified by PCR and the correctness of the construction was identified by sequencing. The recombinant interfering plasmid and auxiliary plasmid pHelper1.0 and pHelper2.0 were co-transfected into 293T cells, and lentivirus packaging was carried out. The cells were collected and concentrated. The virus titer was determined by gradient dilution method. Variance analysis was used for comparison among multiple groups, and SNK-q test was used for further pairwise comparison. Results the three siRNA had different inhibitory effects on SMAC mRNA in hepatocytes, and the inhibitory effect of siRNA1 was the strongest, and the inhibitory efficiency was 70.3%. DNA oligo, was synthesized according to siRNA1 fragment and ligated with lentivirus vector. Sequencing showed that it was constructed correctly. The titer of lentivirus determined by gradient dilution method was 6 脳 108TU / ml. Conclusion the lentivirus specific interference of mouse SMAC gene was successfully constructed, which laid a foundation for the study of the role of SMAC gene in liver failure.
【作者单位】：华中科技大学同济医学院附属同济医院感染科;华中科技大学同济医学院附属协和医院中西医结合科;
【分类号】：R393

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