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目的:构建小鼠FOXP3基因特异性si RNA慢病毒载体,并对其进行功能性研究。方法:根据Gene-Bank提供的小鼠FOXP3cDNA序列,设计4条RNA干扰靶点序列,制备双链DNA oligo,与制备好的双酶切慢病毒载体连接,再转入细菌感受态细胞DH-5a,行PCR鉴定出阳性克隆并测序,制备成FOXP3-si RNA慢病毒载体,利用Western-blot方法对构建的载体进行功能性研究;将载体通过尾静脉注入小鼠体内,观察其对小鼠动脉粥样硬化形成的影响。结果:构建的小鼠FOXP3基因si RNA慢病毒载体,经PCR和DNA测序证实与设计完全一致,并对Foxp3+CD4+CD25+调节性T细胞有显著敲减效应;其能显著促进动脉粥样硬化形成。结论:体内外实验表明,成功构建了小鼠FOXP3基因的特异性si RNA慢病毒载体。
Objective: To construct a mouse FOXP3 gene-specific si RNA lentivirus vector and study its function. Methods: According to the mouse FOXP3 cDNA sequence provided by Gene-Bank, four RNA interference target sequences were designed to prepare a double-stranded DNA oligo, which was ligated with the prepared double-digested lentiviral vector and then transferred into bacterial competent cells DH-5a The positive clones were identified by PCR and sequenced. The FOXP3-si RNA lentiviral vector was prepared, and the function of the constructed vector was analyzed by Western-blot. The vector was injected into mice via the tail vein to observe its effect on mouse arteries The impact of atherosclerosis formation. Results: The mouse FOXP3 si RNA lentiviral vector was confirmed by PCR and DNA sequencing to be completely consistent with the design and had a significant knockdown effect on Foxp3 + CD4 + CD25 + regulatory T cells; it could significantly promote atherosclerosis form. Conclusion: In vitro and in vivo experiments showed that the siRNA lentiviral vector of mouse FOXP3 gene was successfully constructed.