靶向TNF-α基因shRNA干扰片段的筛选和重组体的构建

Screening of Effective shRNA Targeting TNF-α and Constructing of Recombinant Plasmid

本研究利用RNA干扰技术,筛选出可以高效、特异性抑制肿瘤坏死因子-α(TNF-α)表达的小发夹状RNA(shRNA)片段,设计并构建重组质粒,进行序列分析,为探索TNF-α相关疾病的基因治疗提供新途径。取原代培养小鼠腹腔巨噬细胞,置于15%DMEM中,调细胞浓度为2×107/L,接种于6孔板,3ml/well;细胞用脂多糖(LPS)激活,ELISA法测不同时间培养上清中TNF-α的浓度;将针对TNF-α基因的5段shRNA干扰片段的表达框经PCR扩增、纯化后,与转染试剂混合转染入巨噬细胞,细胞用LPS激活24小时后用ELISA法测培养上清中TNF-α的浓度,RT-PCR法测细胞TNF-αmRNA的表达;将最有效抑制TNF-α表达的干扰片段插入质粒载体,转化E.coliDH5α感受态菌株,提取质粒,进行测序分析。结果表明:LPS刺激巨噬细胞24小时后,细胞分泌TNF-α达高峰;转染了干扰序列1的细胞培养上清中TNF-α浓度与对照组相比下降最明显(p<0.05),达59.46%,TNF-αmRNA的表达被抑制了61.2%;将干扰序列1DNA序列克隆到载体上,经测序鉴定确实为所需序列。结论:成功筛选出靶向TNF-α基因的shRNA干扰片段并构建重组质粒,可进一步利用克隆所得到的shRNA干扰TNF-αmRNA的表达,为TNF-α相关疾病的基因治疗提供新手段。

The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha （TNF-α）, to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for seaching gene therapy of TNF-α related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2×10^7 cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37℃ in a fully humidified atmosphere with 5% CO2. Cells were stimulated with lipopolysaccharide （LPS） and the concentration of TNF-α in the supernatant at different time points was determined by enzyme-linked immunosorbent assay （ELISA）. The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the ceils were stimulated with LPS for 24 hours. The concentration of TNF-α in the supematant and the expression of TNF-α mRNA were determined by ELISA and reverse transcription polymerase chain reaction （RT-PCR） respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-α in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-α by 59.46% and the expression of TNF-α mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-α was successfuly screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-α related disease.