PI3K-AKT信号通路抑制剂AKT-DN重组质粒的构建及鉴定

Objective] To construct the phosphatidylinositol 3-kinase(PI3K)/ protein kinase B(PKB/Akt) signaling pathway inhibitor AKT-DN lentivirus plasmid which is also known as a dominant-negative construct of protein kinase B(AKT). [Methods] The AKT-DN gene cloned from expression vector pSRα-AKT-DN was inserted into the lentivirus vector pCDH-CMV-MCS-EF1-copGFP. The obtained recombinant plasmid pCDH-AKT-DN, packaging vector psPAX2 and envelope vector pMD2.G were co-transfected into the 293T cells. The expression of green fluorescent protein (GFP) that reflecting transfection efficiency was observed by fluorescent microscope after 48h， with the empty vector Mock as negative control. The human umbilical vein endothelial cells (HUVECs) were infected by Lentivirus-Mock(Lv-Mock) or Lentivirus-pCDH-AKT-DN(Lv-AKT-DN) respectively. Phosphorylated AKT(p-AKT) protein in PI3K-AKT signaling pathway was detected by western blotting. [Results] The successful construction of recombinant plasmid pCDH-AKT-DN was confirmed by restriction endonuclease and nucleic acid sequencing. The empty vector and pCDH-IκBαDN plasmid were used to package lentivirus respectively. Expression of p-AKT protein in Lv-Mock and Lv-AKT-DN infected HUVECs were detected by western blotting. The result showed that expression of p-AKT protein in PI3K - AKT signaling pathway showed a significant decrease after the AKT-DN overexpression in HUVECs. [Conclusion] A lentivirus vector containing AKT-DN gene was successfully constructed. The recombinant lentivirus can infect HUVECs and effectively inhibit the activation of PI3K-AKT signaling pathway.