A kind of chimeric antigen receptor and its application
A chimeric antigen receptor and nanobody technology, applied in the field of tumor cell immunotherapy, can solve problems such as unsatisfactory effect
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Embodiment 1
[0026] Example 1: Determination of CD38Nb-CD8α-CD137-CD3ζ gene sequence and construction of lentiviral expression plasmid
[0027] 1.1 Immunize alpaca with human CD38 protein and screen with phage display technology to obtain anti-human CD38 nanobody sequence. Human CD8α signal peptide (NM_001145873), human CD8α hinge region and transmembrane region (NM_001145873), human CD137 intracellular region (NM_001561) and human CD3ζ intracellular region (NM_198053) gene sequences. The Myc tagged protein is the commonly used 10 amino acid "EQKLISEEDL".
[0028] 1.2 The above gene sequence was sequenced according to human CD8α signal peptide gene, Myc tag sequence, anti-human CD38 nanobody sequence, human CD8α hinge region and transmembrane region gene, human CD137 intracellular region gene, and human CD3ζ intracellular region gene sequence Link, and introduce Kozac sequence at the N-terminus, and introduce different enzyme cutting sites at the junction of each sequence to form the com...
Embodiment 2
[0030] Example 2: Packaging, concentration and titer determination of lentivirus
[0031] A large number of plasmids were extracted by alkaline lysis (Beijing Tiangen Biochemical Technology Co., Ltd. Endotoxin-free plasmid extraction kit), and the lentivirus packaging kit (including lentivirus packaging mixture and HET high-efficiency transfection reagent) of Baienwei Biotechnology Co., Ltd. box) transfected 293T cells packaging virus.
[0032] 2.1 Packaging and concentration of lentivirus:
[0033] 1) On the day before transfection, prepare cells for passage: digest 293T cells with 0.25% trypsin, adjust the cell density with DMEM medium containing 10% serum, and inoculate 6-8×10 cells per 10 cm cell culture dish. 6 Cells into a 10 cm cell culture dish at 37 °C, 5% CO 2 Cultivate in an incubator, and transfect when the cell density grows to 80%-90% after 16h-24h.
[0034] 2) On the next day, 2h-4h before transfection, replace the medium with 5ml P / S-free medium (DMEM+10%FBS)....
Embodiment 3
[0051] Example 3: Preparation and identification of anti-human CD38-CAR-T cells
[0052] 3.1 Isolation, purification and stimulating culture of T cells
[0053] Take peripheral blood from healthy donors, add 50μl of RosetteSep to each 1ml sample TM Human T cell enrichment antibody mixture, mix well; incubate at room temperature for 20min; add an equal volume of PBS+2%FBS to dilute the above sample and carefully add to the upper layer of Ficoll separation medium; centrifuge at 1,200g room temperature for 20min; absorb the middle layer of white membrane on in a centrifuge tube; wash the cells twice with PBS+2% FBS, centrifuge at 1,500rpm for 10min; use X-VIVO containing 5% FBS for the cell pellet TM 15 Resuspended in the culture medium, stained with trypan blue and counted; used X-VIVO magnetic beads for anti-human CD3 / CD28 TM 15 culture medium was washed twice, added to the T cell suspension at a ratio of 1:1, and the cell density was adjusted to 1×10 6 / ml, and 100IU / ml rhI...
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