Synthesis And Validation Of A Novel Second-generation Anti-CEA CAR Lentivirus Expression Vector

Objective:This study is to construct a novel second-generation chimeric antigen receptor CEAscFv-CD137-CD3? lentivirus expression vector.Pack lentivirus and infect 293T cells.Test whether 293T cells can efficiently express the chimeric antigen receptor contained.Provide the experimental basis for CEA positive tumors of adoptive immunotherapy.Materials and methods:In this study,the novel second-generation anti-CEA chimeric antigen receptor was composed of CEAscFv(C2-45,750bp),hinge domain,transmembrane domain,CD 137 costimulatory domain and CD3? chain.The primers were designed to amplify the Hinge-TM-CD137-CD3? gene fragment located in the plasmid pCLK1,and the restriction enzymes Ecorl was added to it's 3'end.Also,the primers were designed to amplify the C2-45 gene fragment located in the plasmid pC2-45,and the restriction enzymes Notl and a signal peptide gene fragment were added to the 5' end of the C2-45 gene fragment.Finally,the gene fragment C2-45 and the gene fragment Hinge-TM-CD 137-CD3? were spliced together by overlap extension PCR to form a chimeric antigen receptor C2-45/CAR.Chimeric antigen receptor C2-45/CAR and linearized vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro was stitched by ClonExpressTM MultiS One Step Cloning Kit to form the plasmid pC2-45/CAR.After the plasmid pC2-45/CAR was transformed and identified,lentivirus was prepared using a lentivirus packaging kit,and the virus titer was measured.The lentivirus infected 293T cells,and the expression of the target gene was detected by Western blot.Results:CEA monoclonal antibody C2-45 and gene fragments Hinge-TM-CD137-CD3? were spliced into chimeric antigen receptor C2-45-Hinge-TM-CD137-CD3?(C2-45/CAR)by overlap extension PCR.C2-45/CAR and linearized vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro were successfully spliced by ClonExpressTM MultiS One Step Cloning Kit.Gene sequencing verified that the target gene C2-45/CAR was correct and cloned into the lentiviral expression vector.The lentivirus was successfully packaged with a virus titer of 1×106TU/mL.After lentivirus infected 293T cells,protein was extracted from the cells,and the expression of C2-45/CAR was confirmed by CD3?antibody western blot(molecular weight about 23KDa).Conclusions:The second-generation chimeric antigen receptor C2-45/CAR lentiviral expression vector was successfully constructed.After lentivirus LV-C2-45/CAR infected 293T cells,Western blot analysis showed that 293T cells lentivirus infected by lentivirus LV-C2-45/CAR showed C2-45/CAR expression with a molecular weight of approximately 23KDa.