Chimeric antigen receptor comprising Fcgamma RI and application thereof
A chimeric antigen receptor and chimeric receptor technology, applied in the field of biomedicine, can solve problems such as low affinity
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Problems solved by technology
 Example 1 Construction of pCAR-Fcγ receptor I lentiviral overexpression vector
 NCBI obtained the signal peptide region and extracellular region gene sequence and corresponding amino acid sequence of Fcγ receptor Ⅰ, and obtained the gene sequence and corresponding amino acid sequence of transmembrane region CD28, intracellular signal region 4-1BB and CD3ζ by the same method to form Fcγ receptor Ⅰ chimeric gene, figure 1 It is a schematic diagram of the chimeric gene structure of Fcγ receptor Ⅰ. BstBI and XbaI restriction sites were added to both ends of the gene, the total length was 1692bp, and it was sent to Shanghai Jierui Bioengineering Co., Ltd. for synthesis, and the vector PGH-Z1528G for cloning the target gene was obtained. The lentiviral overexpression vector pCAR was purchased from Aikangde Biomedical Technology (Suzhou) Co., Ltd. and Hanbio, and the specific map refers to figure 2 .
 The PGH-Z1528G and pCAR plasmid vectors were respectivel...
 Example 2 Preparation of pCAR-Fcγ receptor I lentivirus
 The lentivirus preparation, purification and concentration of the chimeric gene Fcγ receptor I adopt the following methods:
 1. Cryopreservation of HEK293T cells
 When the cells are frozen, the cell density is preferably 60% to 80%. At this time, the cells are in the logarithmic growth phase, the proliferation is vigorous, and the cells are in good condition.
 1) Take out the cells, add trypsin to digest, stop the digestion, transfer the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min;
 2) Prepare the cell cryopreservation solution according to the ratio of DMSO to FBS of 1:9 (V / V), and the prepared cell cryopreservation solution needs to be pre-cooled in a 4°C refrigerator;
 3) After centrifugation, discard the supernatant of the culture medium, add pre-cooled cryopreservation solution, resuspend the cell pellet with a pipette gun, dispense...
 Example 3 Preliminary verification of overexpression of Fcγ receptor Ⅰ in Jurkat cells
 Jurkat cells are human peripheral blood leukemia cells, which are widely used in the research of blood tumors and immunology. Because their physiological characteristics are the closest to those of T lymphocytes, this disclosure uses Jurkat cells to initially verify the overexpression of Fcγ receptor Ⅰ. On the one hand, it can be judged whether the packaged virus is correct and infectious; on the other hand, it can be judged whether the Fcγ receptor I protein can be correctly transcribed and translated.
 Recovery of Jurkat cells
 For the specific steps of recovery of Jurkat cells, refer to the recovery of HEK293T cells above, but it should be noted that the medium used for Jurkat cells is RPMI 1640, and 10% FBS can be added before use.
 Passaging of Jurkat cells
 Jurkat cells are suspension cells. When the cell density is 60%-80% under a micr...
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