A kind of antigen-specific T-lymphocyte hybridoma and its preparation method and application
A technology of lymphocytes and hybridomas, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-cytokine/lymphokine/interferon immunoglobulin, etc., can solve the problems of difficult proliferation, difficult T cell culture, instability And other issues
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Embodiment 1
[0058] Acquisition of antigen-specific lymphoid cells
[0059] First, OVA transgenic mice can be vaccinated with OVA albumin at a dose of 25 μg to make OVA 323-339 Specific T cell proliferation. Mice are sacrificed after 7 days and spleen and lymph node cells are collected. Join NH 4 OH to final concentration 1% lyse red blood cells in the cell suspension, wash the cells 2-3 times by centrifugation with DMEM basal medium; obtain a lymphocyte sample containing a single antigen-reactive T cell; then in the lymphocyte sample, combined with the characteristics of T cell expression protein, using a mouse CD4 isolation kit, according to its instructions, using immunomagnetic bead technology and combined antibodies, negative screening CD4 + T cells, isolate antigen-specific CD4 + T lymphocytes.
Embodiment 2
[0061] Preparation of antigen-specific T lymphocytes for hybridoma cells
[0062] Antigen-specific CD4 obtained in Example 1 + T cells were mixed with mouse thymoma cell line BW5147.G.1.4 in a 5:1 ratio, and cell fusion was performed by polyethylene glycol (PEG) cell fusion method. After mixing the two cells, centrifuge, discard the medium, add 1 ml of 50% PEG to the cell pellet within 1 min, shake the centrifuge tube while adding, mix the PEG with the cells, let stand for 1 min, add incomplete medium to the centrifuge tube, terminate the PEG action, centrifuge and discard the supernatant, dilute the fused cells to DMEM selective medium containing 20% fetal bovine serum, 1× HAT containing hypoxanthine, aminotetradiene and thymidine, at 1 ×10 per well 5 Individual cells are added to a 96-well cell culture plate at 200 μl per well. Place the culture plate into 5% CO 2 Continue incubating at 37°C incubator. Hybridoma cells of T lymphocytes are prepared after 10-14 days, see preparati...
Embodiment 3
[0064] Screening of hybridoma cells of T lymphocytes
[0065] The monoclonal obtained in Example 2 was amplified to a 24-well plate to expand the culture, and the monoclonals were screened after 2-3 days. The biological activity of monoclonal cells was detected by in vitro antigen presentation experiments, and the specific antigen OVA was mixed with splenocytes of ordinary C57BL / 6 mice of the same strain 323-339 The mixture is added to the medium of monoclonal cells and incubated the monoclonal cells overnight (see David H et al., Methods Mol Biol, 2013, 960: 297–307 for specific methods). After 24 h, the content of mouse interleukin 2 (mIL2) in the cell culture solution supernatant was detected using ELISA. Positive clones secreting mIL-2 were amplified into 6-well plates for amplification culture, and hybridoma cells in 6-well plates were rechecked 2-3 days later with in vitro antigen presentation experiments. For retesting, use OVA of different concentrations 323-339 Stimulatio...
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