Construction And Identification Of PLVX-CD19-CD28-CD137-TCR? Lentiviral Vector And Its Application In B Cell Leukemia

[Objective]We build the pLVX-CD19-CD28-CD137-TCR lentiviral vector and identified it.To investigate the application in B cell acute lymphoblastic leukemia.Futherly make sure that the important role of targeting CD 19 antigen in B cell lymphocytic leukemia treatment,associating with CD28 and CD13 7 antigen in costim?lating T cell to enhance the proliferation ability of T cells and show a better antitumous effect.[Methods]We biuld the CD19-CD28-CD137-TCR zeta target gene sequences by experimental techniques of genetic engineering and molec?ular biology,then the lentiviral vectors combination with target gene sequences.Through the PCR amplification technique and agarose gel electrophoresis check the expression of CD19-28-137-TCR?,and then verificating the target gene sequences.The constructed vector was transfected into 293FT cells in vitro by CaCL2 method,Immunofluorescence method was used to observe Target vector transfection efficiency and compute lentivirus tite after 48 hours.Weston blot is used to check expression of enzyme digestion of pLVX-CD19-28-137-TCR? in 293FT cells.Search for one willing and suitable for CAR-T cell treatment of B-ALL patients in the Department of Hematology,the first people's Hospital of Yunnan Province,T lymphocytes cell isolated from patients,then transfected T cells in vitro by pLVX-CD19-28-137-TCR zeta lentiviral vector,lead CD19-28-137-TCR zeta antibody gene into T cells.Meanwhile,the CD19-CAR-T cells were constructed by the Beijing Institute of biological immunotherapy,and the cells were transfused by this method:After treatment,the patients' peripheral blood were analyzed by flow cytometry.The bone marrow biopsy sections were stained with HE and the bone marrow cells were observed by electron microscopy to determine the clinical effect.[Results]Basis experiment showed that PCR amplification method detected expression of CD 19-28-137-TCR Stablely.The lentiviral vector was successf?lly transfected into 293FT cells by CaCl2 method.The transfection efficiency was 90%by fluorescence microscopy,The titer of lentivirus titer was 5.5X107TU/ml.The method to detect the expression of Weston blot protein in 293FT cells after digestion and plasmid packaging cell,293FT protein CD 19-CAR gene was digested plasmid the res?lts of beta-actin protein band is very clear,after the plasmid protein was digested to obtain relatively high expression in 293FT cells,but no expression of CD 19 scFv gene after enzyme digestion is not very obvious.CD19-CAR-T in the clinical treatment of 1 cases of acute B lymphocytic leukemia in the treatment of the application of electron microscopy observation of active bone marrow hyperplasia,the patient has a bone marrow fibrosis by CAR-T.After treatment with CAR-T,lymphocytes,gran?locytes,monocytes,primitive myeloid progenitor cells decreased,and the number of nucleated red cells and CD34+ cells increased.The results of immunohistochemistry showed that CD2,CD3,CD5,CD7,CD19,CD20 antibody were negative,but the expression of TdT in the diseased cells,partial expression of Pax-5 antibody and Ki-67 antibody,the results shows that the early lesions of B cells.[Conclusion]The successful construction of pLVX-CD19-CD28-CD137-TCR zeta lentiviral vector,and after agarose gel electrophoresis to identify pLVX-CD19-CD28-CD137-TCR lentiviral vector,WB successf?tlly detected lentivirus and 293FT cells were successf?lly packaged,The virus titer was relatively 5.5X107TU/ml,pLVX-CD19-28-137-TCR successf?lly transfected into T cell,and the treatment of B lymphocytic leukemia plays a certain role,but the side effects associated with bone marrow fibrosis.