Construction of recombined adeno-associated virus vector expressing Dll4 short RNA interference and preparation of high-titer virus

Objective: To construct the recombinant adeno-associated virus vector expressing tumor angiogenesis-related gene Dll4 short RNA interference（RNAi） and to use it for the preparation of high-titer virus. Methods: pDC316-Dll4-shRNA plasmid,constructed with Dll4 siRNA,was digested with EcoRI and SalI.A 1 500bp fragment,containing U6 promoter and Dll4 siRNA,was obtained and inserted into the adeno-associated virus vector plasmid pSNAV2.0 digested with EcoRI+SalI.Positive vectors were analyzed through enzyme digestion and DNA sequencing.The recombinant plasmid was transfected into BHK-21 cells using LipofectamineTM2000.G418-resistant cells were obtained and infected with HSV1-rc/△UL2,which have the function of packaging recombinant adeno-associated virus（rAAV）.After purification,target vector and virus were collected. Results: The target vector,pSNAV2.0-shRNA-Dll4,was successfully constructed,and the rAAV with a titer of 2.4×1011vg/L（vg:vectorgenome） was obtained. Conclusion: rAAV vector may be used in further investigation of Dll4 function in tumor angiogenesis and gene therapy.