Construction of recombinant adeno-associated virus vector rAAV2-PD-L1 and its biological efficiency in transfecting mouse vascular endothelial cells / 第二军医大学学报

ABSTRACT

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 4×1012 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 co-stimulation.