Abstract: Objective To construct pEGFP-N1-AMPKα2 expression vector and observe its up-regulation effect on human AMPKα2 gene in the SH-SY5Y cell line. Methods Human AMPKα2 gene fragment were amplified and cloned into the pEGFP-N1- AMPKα2 vector. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The recombinant vector was transfected by lipofectamine into the SH-SY5Y cells. After the screening by G418, the expression levels of AMPKα2 mRNA and protein were detected by RT-PCR and Western blot. ROS was measured by flow cytometry in cells transfected with recombinant vector. Results The expression vector pEGFP-N1-AMPKα2 was successfully constructed, which was confirmed by the DNA sequencing and the enzyme digestion analysis. The vector pEGFP-N1-AMPKα2 can up-regulate protein expression of AMPKα2 effectively after transfection in the SH-SY5Y cells. ROS increased in cells transfected with pEGFP-N1-AMPKα2. Conclusion pEGFP-N1-AMPKα2 expression vector was successfully constructed. The protein expression of AMPKα2 gene was up-regulated effectively in SH-SY5Y cells transfacted with pEGFP-N1-AMPKα2, which laid a basis for its application in the research of cell injury induced by local anesthetic.