〔Abstract〕 Objective To obtain recombinant adeno-associated virus capable of over-expressing OPTN and TAU-P301-L genes, to detect its infection of 293 T cellsin vitro, and to provide new ideas for the study of Alzheimer's disease(AD). Methods The plasmid containing the gene of interest was extracted from Escherichia coli containing the gene of interest, and the target gene was obtained by PCR amplification. The target gene OPTN was constructed intopAAV-IRES-ZsGreen1vector, and the target gene Tau-P301 L-V5 was constructed intopAAV-CMV-mkate-IRESvector. Plasmid positive clones were identified and sequenced. The recombinant plasmidspAAV-IRES-ZsGreen1-AAV9-OPTNandpAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 were co-transfected into 293 T cells with the packaging plasmidpAAV-RC9 and the helper plasmidpHelperby LipofiterTM2000, respectively, to obtainAAV9 virus. Using the same method, the corresponding green and redAAV9 control virus were constructed without using the target gene.The titer of recombinant adeno-associated virus was determined by qPCR method. The virulence and safety of 293 T cells were detected by viral vector, and the protein expression was identified by Western blot and RT-PCR. MTT assay was used to detect the effect of OPTN protein and TAU-P301-L protein on the activity of 293 T cells. Results The sequencing results showed that theAAV-IRES-ZsGreen1-AAV9-OPTNandpAAV-CMV-mkate-IRES-AAV9-Tau-P301L-V5 adeno-associated virus vectors were successfully constructed. The virus titer determined by qPCR was 1.0×1012vg/ml. The transfection efficiency of virus vector-infected 293 T cells was 33.6% and 20.5%. Western blot and RT-PCR showed protein overexpression. MTT results showed that the OPTN group rescued the apoptosis of the TAU-P301-L group. Conclusion Adeno-associated virus over-expressing OPTN and TAU-P301-L genes was successfully constructed. OPTN protein can reduce the cytotoxicity of TAU-P301-L protein.