Abstract: ob＜x＞jective To construct Eya1 S298A overexpressed lentiviral ex＜x＞pression vector and investigate the effect of site 298 mutation of EYA1 on apoptosis of MES23.5 dopaminergic cells. Methods The recombinant ex＜x＞pression plasmid of lentivirus carrying Eya1S298A gene was constructed by PCR amplification, Golden gate and LR reaction. The MES23.5 cell lines with stable ex＜x＞pression of Eya1S298A were screened by purinomycin. The cells were divided into two groups: the mutant group stably expressing Eya1S298A and the wild-type group stably expressing Eya1. Cell injury model was established after 2h treatment with 6-OHDA, and the effects of site 298 mutation on cell viability and Bcl-2 protein ex＜x＞pression were detected by CCK8 and Western blot. Results Sequencing and PCR results showed that the 298th S of the Eya1 gene was mutated into A, and the Eya1S298A gene was successfully li＜x＞nked to the lentiviral vector. CCK8 results showed that the cel l viability level of the Eya1S298A mutant group was significantly lower than that of the Eya1 wild-type group, and Western blot results showed that the Bcl-2 protein ex＜x＞pression level of the mutant group was significantly higher than that of the wild-type g roup. Conclusion The lentiviral ex＜x＞pression plasmid of Eya1S298A gene was successfully constructed and the stable ex＜x＞pression cell line of Eya1S298A was established. Moreover, the 298 mutation of Eya1 reduced the antiapoptotic effect of Eya1 on MES23.5 cells . This study lay a foundation for further study of the role of Eya1 in the antiapoptosis of dopaminergic neurons.