Construction And Identification Of PHBLV-BCL2/IgH-CAR Plasmid Of Follicular Lymphoma

Objective:Constructe pHBLV-BCL2/Ig H-CAR plasmid to lay the foundations for the preparation of chimeric antigen receptor BCL2/Ig H-CD28-CD3 zeta lentivirus,to explore a new method for immunotherapy of FL and other malignant lymphoma.Methods:（1）The design of chimeric antigen receptor Hing-TM-CD28-CD3 zeta expression box,The hinge region（hinge）and transmembrane region（TM）from Ig G4 Fc（Genbank:AAB59394.1）,costimulatory molecules from CD28（Genbank:AF222341.1）,intracellular signal peptide from CD3zeta（Genbank:AK313946.1）,The corresponding functional area of the gene by biological synthesis,and each segment is directly connected.（2）Use Eco RI and Bam HI cut up the Hinge-Tm-CD28-CD3 zeta fragments,and it was cloned into Eco Rl and Bam Hl sites of vector pHBLV-CMV-MCS-EF1-Zs Green get plasmid pHBLV-CD28-CD3 zeta,then verify it with agarose gel electrophoresis,double enzyme digestion.（3）BCL2/IgH was selected as the extracellular target antigen,Trizol method was used to extract total m RNA from DOHH2 cells,RT-PCR amplification and sequencing to obtain BCL2/Ig H fusion gene sequence,comparing with the BCL2 gene and Ig H gene sequences of Genbank.According to the sequencing results,BCL2 and Ig H gene CDS（Coding sequence）region were synthesized by chemical method.（4）BCL2/IgH target gene was obtained by EcoRI single enzyme,it was cloned into EcoRl sites of vector pHBLV-CD28-CD3 zeta get plasmid pHBLV-CD28-CD3 zeta.Verify it with colony PCR and DNA sequencing identification.Results:（1）In this study,the chimeric antigen receptor Hing-TM-CD28-CD3 zeta expression box was successfully designed and synthetised.（2）Constructed the pHBLV-CD28-CD3 zeta plasmid,agarose gel electrophoresis and enzyme digestion showed that the target gene was cloned into Hing-TM-CD28-CD3 zeta lentiviral vector.（3）In this study,BCL2/Ig H gene was extracted and synthesized from human follicular lymphoma DOHH-2 cell line,sequencing results were consistent with the sequence of BCL2/Ig H Genebank（NM₀00633.2,NC₀00014.9）（4）pHBLV-BCL2/IgH-CAR plasmid was successfully constructed,the results of colony PCR were in line with expectations,and the DNA sequence was identical to the designed base sequence.Conclusion:（1）The construction of the lentivirus plasmid pHBLV-CD28-CD3 zeta provide experimental conditions for the next step to clone BCL2/Ig H gene into lentiviral vector.（2）The construction of the plasmid pHBLV-BCL2/Ig H-CAR laid the foundation for the packaging and transfection of lentiviral vector expressing BCL2/Ig H-CAR gene.（3）This experiment selects the BCL2/Ig H fusion gene as a target antigen,to expand the scope of application of CAR-T in tumor cell immunotherapy,for other types of malignant lymphoma specific immunotherapy,CAR-T precise treatment provides a valuable reference.