The invention discloses preparation of a PD-1 knocked-out CD19 CAR (Chimeric AntigenReceptor)-
T cell. The preparation comprises the following steps of mixing a CD19 CAR
plasmid, a
transposase plasmid thereof, an sgRNA (single guide Ribonucleic Acid)
plasmid for knocking out a PD-1
gene, a CAS9 plasmid and an electrotransfection
reagent, and adding an obtained mixture into a PBMC (
Peripheral Blood Mononuclear
Cell) to carry out electrotransfection; enriching a CD3 positive
T cell by adopting a
magnetic bead which is obtained by
coupling CD3 or CD3 plus CD28 antibodies; screening, culturing and amplifying the
T cell subjected to culture, amplification and
transfection to obtain the PD-1 knocked-out CD19 CAR-T
cell. According to the preparation, a
PiggyBac transposon system is selected and used to improve
transfection efficiency and the expression efficiency and time of
transgene; the preparation procedure of a CAR-T
cell is simplified; the loading capacity of a
system is enhanced. In addition, through the preparation, the use of a
viral vector is avoided when the PD-1
gene is knocked out; the safety is higher.