Construction And Identification Of PHBLV-CCND1/IgH-CAR Lentivirus Expression Vector Of Mantel Cell Lymphoma

Objective:To construct the lentivirus pHBLV-CCND1/IgH-CAR,the purpose of this study is to lay a experimental foundation for a new immunotherapy method of MCL and other malignant lymphoma or will maximize the accuracy of CAR-T treatment.Methods:Total RNA was extracted from jeko-1 cell line by TRIZOL method for amplification by RT-PCR.The sequencing results were compared with the sequences of CCND1/IgH gene in NCBI Genbank.The fusion gene of CCND1/IgH was synthesized according to the coding sequence region of CCND1 and IgH gene.The CCND1/IgH fusion gene was directly linked to the Hinge-TM-CD28-CD3?fragment by overlapping extension PCR,and the CCND1/IgH-CAR fragment was obtained.Cut the lentivirus vector with EcoR I and BamH I double enzyme,and then CCND1/IgH-CAR fragment was cloned into the lentivirus vector.Colony PCR and DNA sequencing was used to verify whether the bases were correct.Three plasmids were co-transfected into 293T cells to package the lentivirus and the titer of lentivirus was detected successfully.Results:In this study,the 2~ndd generation chimeric antigen receptor CCND1/IgH-CAR was obtained by overlapping extension PCR method.CCND1/IgH-CAR and lentivirus vector were linked by double enzyme to obtain pHBLV-CCND1/IgH-CAR plasmid.The pHBLV-CCND1/IgH-CAR plasmid was constructed correctly by DNA sequencing and colonyPCR.Threeplasmidswerecotransfectedtopackagelentivirus pHBLV-CCND1/IgH-CAR and the titer of lentivirus was 2×10~8TU/ml.Conclusion:ThechimericantigenreceptorpHBLV-CCND1/IgH-CARlentivirus expression vector is constructed successfully,which might provide valuable reference about adoptive immunotherapy for MCL and other types of malignant lymphoma.