Construction of recombinant adeno-associated virus vector rAAV2-PD-L1 and its biological efficiency in transfecting mouse vascular endothelial cells

Objective:To construct a recombinant adeno-associated virus vector rAAV2-PD-L1 and to investigate the biological efficiency of rAAV2-PD-L1-transfected vascular endothelial cells in co-stimulating secretion of cytokines by T cells.Methods: Mouse PD-L1 cDNA was amplified by RT-PCR from total RNA of mouse liver tissues and was cloned into shuttle vector pSNAV1;the products were then transferred into BHK21 cells by lipofectamine and rAAV2-PD-L1 was screened out.Mouse vascular endothelial cell line 2F-2B was infected with rAAV2-PD-L1 and were co-cultured with activated mouse T cells,and the IFNγ content was identified by ELISA in the supernatant.Results: The sequences of PD-L1 cDNA and pSNAV-PD-L1 were confirmed to be correct.The recombinant rAAV2-PD-L1 was verified by PCR and SDS-PAGE analysis.The virus physical titer was 4×10~(12) virus genome/ml and the protein concentration was 0.355 mg/ml.There was a high expression of PD-L1 in mouse vascular endothelial cells infected with rAAV2-PD-L1.The content of IFNγ in the culture supernatant was significantly decreased 48 hours after co-culture.Conclusion: The recombinant rAAV2-PD-L1 can infect vascular endothelial cells and inhibit secretion of IFNγ by activated T cells through costimulation.