Construction And Qualification Of A Lentivirus Vector For Stable Transduction Of HLA-A*0201 Molecule Into Pig Cells

BackgroundThe humanized mouse model with the reconstruction of the human immune system can simulate the process of human immunity in mice,and has great value in basic and translational research related to human immunity.The humanized mouse model of human immune system(called Thy/HSC model or BLT model)established by joint transplantation of human fetal thymus and CD34+ human fetal liver cells has sound human immune function and is widely used.However,the construction of this model relies on the use of human fetal tissue;and this limits the wide application of this model.Previous studies have shown that human T cells can develop efficiently in the fetal pig thymus transplanted in immunodeficient mice;and this shows that the fetal pig thymus is expected to be used to replace human fetal thymus for the construction of humanized mouse models.However,the human T cells developed in the pig thymus have not undergone the HLA molecule-mediated thymus positive selection process and cannot efficiently produce HLA-restricted T cells,which limits the application value of the humanized mouse model based on the pig fetal thymus.Therefore,the study of this master's thesis intends to construct a lentiviral vector capable of stably transducing human HLA-A*0201 molecules into pig cells,which provides the foundation for the establishment of a humanized mouse model based on the transplantation of HLA transgenic pig thymus and artificial blood hepatocytes.ObjectiveMHC molecules participate in positive selection and negative selection in the maturation process of T cells,which give T cells the function of MHC restriction and self-tolerance.Human T cells developed in fetal pig thymus can develop into mature human T cells by cross-reaction with pig MHC(SLA)molecules on the surface of pig thymus epithelial cells,and their HLA-restricted reactivity is defective.The transduction of human HLA molecules into porcine thymus epithelial cells is expected to improve the antigen-specific immune response function of human T cells developed from porcine thymus.Therefore,the main goal of this subject is to construct a lentiviral vector that can stably transduce human HLA-A*0201 molecules into pig cells.Methods1 By the method of homologous recombination,a lentiviral core plasmid expressing the HLA-A2 chimeric molecule gene was constructed.2.We packaged the lentiviral vector by a four-plasmid system,and infected Mel888 cells and determined the titer of the lentiviral vector.by using the flow cytometer.3.The lentivirus containing the HLA-A2 chimeric molecule was used to infect the pig PK-15 cell line,and we identified the ability of the lentiviral vector to transduce human HLA molecules into pig cells.4.The melanoma cells were incubated with PK-15 cells expressing HLA-A2 chimeric molecules to detect the antigen presentation function of HLA-A2 chimeric molecules.Results1.1.We constructed a plasmid expressing HLA-A2 chimeric molecule gene2.We successfully packaged the lentivirus containing the HLA-A2 chimeric molecule.3.We determined the effect of the lentiviral vector in infecting pig cells4..We determined the antigen presentation function of HLA-A2 chimeric molecules Conclusions1.We successfully constructed a lentiviral vector that can stably transduce HLA A2 molecules into pig cells..2.?3 chimeric HLA-A2 molecules can be highly expressed on the surface of pig cells.3..The ?3 chimeric HLA-A2 molecules can present antigens on the target cell surface to effector cells.