Construction Of The Expression Vector Of LAG3 Extrancellurar/Transmembrance Region And Preparation Of Its Monoclonal Antibody

Objective:To construct the expression vectors of human lymphocyte activation gene 3(LAG3)which containing its extracellular region and transmembrane region for preparing the monoclonal antibodies(mAb)against LAG3 and laying the foundation for its application in clinical detection of LAG3 and anti-tumor therapy.Methods:1.The cDNA,retrotranscribed by the total RNA extracted from PHA activated peripheral blood mononuclear cells(PBMCs)was used as a template for PCR to amplified the extracellular and transmembrane sequences of LAG3 with the double enzyme digestion primers.The products of PCR were inserted into the pc DNA3.1-m Lumin vector to construct the eukaryotic expression vector of LAG3 which containing its extracellular and transmembrane regions with fluorescence protein m Lumin.The vector was transfected into 3T3 cells,and monoclonal cells expressing LAG3 were selected by geneticin screening and limiting dilution method.After identified by RT-q PCR and observation under fluorescence microscope,the monoclonal cell line with the most predominant expression of LAG3,named as 1-19-2,was selected for mouse immunization.2.The extracellular and transmembrane sequences of LAG3 was amplified from PHA activated PBMCs c DNA.The PCR product was inserted into the p TSB-cop GFP vector to construct five lentiviral expression p TSB-LAG3 TRs-cop GFP.The vector was transfected into HEK293T cells and the virus were replicated and paged.Subsequently,the 3T3 cells were infected with the virus.The monoclonal cells expressing LAG3 were selected by puromycin screening and limiting dilution.After identified by RT-q PCR and observation under fluorescence microscope,the monoclonal cell line with the most predominant expression of LAG3,named as MC-6,was selected for mAb detection.3.The Balb/c mice were injected with 1-19-2 cells and the 3T3 control cells via caudal vein.The anti-LAG3 serum from immunized mice were verified by indirect fluorescence(IF)with flow cytometry(FCM)and fluorescence microscope.The myeloma cells SP2/0 were injected subcutaneously into Balb/c mice to generate solid tumor.The SP2/0 cells were isolated and cells suspension was prepared.The mice splenocytes were mixed with SP2/0 cells at the ratio of 10∶1 and were fused using polyethylene glycol.The monoclonal hybridoma cells were selected by HAT and HT screening and multiple limited dilution subcloning.4.The anti-LAG3-positive monoclonal hybridoma cells were identified by indirect fluorescence with FCM.The antibody function of the mouse anti-human LAG3 antibody also were verified by FCM.5.The ascites,containing high titer LAG3 mAb was prepared by intraperitoneally injection with monoclonal hybridoma cells to Balb/c mice.The LAG3 mAb was purified with Protein A/G from ascites.The protein concentration and effect of the mAb were identified by BCA and FCM.Result:1.The pcDNA3.1-LAG3 TRs-m Lumin eukaryotic expression vector was successfully constructed.The cell line,named as 1-19-2,was the most dominant LAG3~+m Lumin~+3T3 monoclonal cell for LAG3 expression.2.The pTSB-LAG3 TRs-cop GFP lentiviral expression vector was successfully constructed.The cell line,named as MC-6,was the most dominant LAG3~+cop GFP~+3T3 monoclonal cell for LAG3 expression.3.The mice,immunize with 1-19-2 cells,produced specific high-titer antibodies against the human LAG3.4.The mouse anti-human LAG3 mAbs,released by dozens of LAG3 positive monoclonal hybridomas.5.The ascites contained high concentration of mouse anti-human LAG3 mAb and successfully purified by Protein A/G and might be used for flow cytometry detection.Conclusion:The mouse anti-human LAG3 mAbs were successfully prepared.It lays the foundation for the future development of blocking humanized antibodies against LAG3.