Preparation method and application of CAR-T cell targeting B7H3
A B7H3-CAR, LV-B7H3 technology, applied in the field of immunology, can solve problems such as protein expression limitations, and achieve the effect of high killing activity
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[0032] A method for preparing CAR-T cells targeting B7H3, comprising the steps of:
[0033] (1) prepare PBMC cells;
[0034] Take 20ml of peripheral blood from healthy volunteers, centrifuge (1300g, 10 minutes) and save the autologous plasma for later use. The remaining blood cells are diluted with an equal volume of normal saline, added to the upper layer of the lymphocyte separation medium, centrifuged (600g, 25 minutes), and the intermediate white blood The membrane layer cells were washed with physiological saline, centrifuged (400 g, 10 minutes), and the supernatant was discarded to obtain PBMC cells.
[0035] (2) Mix the shuttle plasmid LV-B7H3 3.75 μg containing the CAR structure, the helper plasmid psPAX2 3.75 μg, and the envelope plasmid VSV-G 2.5 μg in 300 μl opti-MEM medium, and in another 300 μl opti-MEM medium Add 20 μl PEI reagent dropwise, shake and mix, and let stand at room temperature for 5 minutes, add the mixture containing PEI reagent to the plasmid mixture...
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