Screening And Functional Research On Jurkat Cell Strain Of Stable Chimeric Antigen Receptor Expression

Adoptive cellular immunotherapy (ACI), including adoptive NK cell therapy and adoptive T cell therapy, plays its anti-tumor role by in vitro activating and amplifying tumor-specific and/or non-specific killer cells. ACI,which took action faster and had little side effects, has been growing dramatically owing to its advantages The design of Chimeric antigen receptor CAR combines the scFv that has a high affinity to tumor-associated antigen (TAA) with an activation motif of T lymphocytes which has a potency of killing.T lymphocytes are expected to express CAR by gene transfer and then can specifically recognize and kill tumor cells. This strategy may introduce fresh vitality into adoptive cellular immunotherapy.Objective:It’s fact that VEGF is overexpreesed in almost all kinds of human solid tumors. This research was to design a new gene structure FLT1CAR of chimeric antigen receptor special to VEGF and to screen the Jurkat cells expressing FLT1CAR and finally to detect the chemotactic effect of the stable transfected cell trend to VEGF.Method:The constructed chimeric antigen receptor gene was inserted into the lentiviral vector named pLV120gn-FLT1CAR Then Jurkat cells were infected with the virus collected from the293T cells which were co-transfected with recombinant vector of plasimd pLV120gn-FLT1CAR, packaging plasimd pCMVdelta8.91and helper plasimd pMD.G.Finally,we selected the stable transfected cell strains by meams of G418screening and cloning selection.To detect the integration and epression of interest gene in the stable transfected cell strains selected,PCR and FCAS was used respectively.The chemotactic effect of the stable transfected cell trend to VEGF was tested by Transwell assay.Results:The agarose gel electrophoresis showed that the constructed lentiviral vector of plasimd pLV120gn-FLT1CAR is correct.PCR and FCAS showed that FLT1CAR has integrated into Jurkat cells and stably expressed. Transwell assay showed that the cell strains screened Clonel and Clone2have obvious chemotactic effect to VEGF. Conclusion:Successfully,we have gained the cell clones that expressing FLT1CAR stably.They have a distinct chemotactic trend to VEGF.Our results provides a theoretical basis for the subsequent clinical cell therapy.