Universal chimeric antigen receptor T cell targeting claudin 18.2, construction method and application of universal chimeric antigen receptor T cell
A chimeric antigen receptor and targeting technology, applied in the field of molecular biomedicine, can solve problems such as unknown functions, and achieve the effect of simple structure and high tumor killing activity
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Embodiment 1
[0033] Example 1 Construction of lentiviral expression vector
[0034] A nucleic acid molecule encoding the signal transduction region was synthesized, the nucleotide sequence of which was shown in SEQ ID NO.5, and named pGSI.
[0035] 3ug pGSI and recombinant lentiviral vector plasmid pCDH-EF1 were double-digested with BamHI and SalI restriction endonucleases respectively, and the digested products were recovered from the gel and ligated with T4 DNA ligase, ligated overnight at 4°C, transformed into DH5α competent cells, and taken Spread 100 μL of the bacterial solution onto an LB plate containing ampicillin resistance, and incubate overnight at 37°C. Single clones were picked for colony PCR, positive clones were sent for sequencing, and the clones with correct sequencing results were saved and plasmids were extracted, named pCDH-EF1-emCAR.
[0036] A nucleic acid molecule encoding the antigen-binding region was synthesized, the nucleotide sequence of which was shown in SEQ ...
Embodiment 2
[0038] Example 2 Lentiviral packaging
[0039] Take out a tube of frozen 293T cells from liquid nitrogen and quickly place them in a 37°C water bath until the ice cubes disappear, add dropwise to a 15ml centrifuge tube containing 5ml preheated medium, centrifuge at 1200rpm for 3min, discard the supernatant, and culture with 293T Medium (10% FBS+DMEM) resuspended cells were inoculated into 150mm culture dishes, 37°C, 5% CO 2 Cultivate in saturated humidity. When the confluence of the cells reaches more than 90%, discard the old medium, add 5ml sterilized PBS solution, shake gently, discard the PBS solution after washing the cells, add 2ml 0.25% Trypsin-EDTA digestion solution, and digest for 1-2min until Cells are fully digested. Add serum-containing medium to stop the digestion, centrifuge the cell suspension at 1200rpm for 3min, resuspend the centrifuged cells with medium, inoculate 1.2×107 cells in each 150mm culture dish for packaging lentivirus, 37°C, 5% CO 2 Culture in...
Embodiment 3
[0042] Example 3 Lentivirus purification
[0043] At 24h and 48h after the medium exchange, the supernatant was collected and temporarily stored at 4°C, and 20ml of fresh medium was replaced. Centrifuge the collected liquid at 4°C and 3500rpm for 15min, discard the precipitate, concentrate the supernatant with a Millipore protein ultrafiltration column (10KD) and measure the virus titer at the same time, dilute the lentivirus claudin CAR) to 1*10 8 TU / ml, the aliquoted lentivirus (claudin CAR) was stored at -80°C.
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