小鼠FOXP3-siRNA慢病毒载体的构建及其对小鼠动脉粥样硬化的影响

Construction of a recombinant lentivirus vctor siRNA targeting mouse FOXP3 gene and effects on the progression of the atherosclerosis plaque in mouse

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 recombinant lentivirus vector RNAi targeting mouse FOXP3 gene and its effect on the progression of aortic atherosclerosis plaque in hypercholesterolemic apoliprotein（apo）E-/- mice. Method：siRNA of FOXP3 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairp in RNAs （shRNA） of siRNA, in total four siRNA sequence, and prepare double DNA oligo, to conjugate lentivirus vector enzymied by Hpa Ⅰ and Xho Ⅰ, and transfer into competent cell DH-5α, identified by PCR and sequence analysis. To construct FOXP3-siRNA lentivirus vector, we identify its function by Western-blot and inject through the tail vein to apoE-/- mice to observe its effect on atherosclerosis. Result：The recombinant lentivirus vector for RNAi targeting mouse FOXP3 gene is concord with our protocol by PCR and sequencing confirm. It can knock down mouse Foxp3^＋ CD4^＋ CD25^＋ Treg cells effectively and in vivo It can promote significant the progression of the atherosclerosis plaque in apoE-/- mice. Conclusion：Our studies indicate that lentiviral siRNA can provide highly efficient and specific FOXP3 knockdown.