Chimeric T cell growth factor and application thereof
A growth factor and cell technology, applied in the field of genetic engineering, can solve the problems of reducing the biological effect of T cells, short survival time, weakening the therapeutic effect of T cells, etc., and achieve the effect of enhancing the therapeutic effect and broad application prospects
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Embodiment 1
[0053] Example 1 Construction of chimeric T cell growth factor
[0054] The inventors have overcome the first disadvantage of native IL-2 by mutating the amino acid residues in the native IL-2 sequence that bind to CD25.
[0055] The amino acid sequence of native IL-2 is:
[0056] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMTFKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWI TFCQSIISTLT (SEQ ID NO: 1).
[0057] attached figure 1 Shown is a schematic diagram of the binding of native IL-2 to CD25. figure 1 The black mark in A is IL-2 and CD25 binding region 1 (L40-Y45); figure 1 The black mark in B is IL-2 and CD25 binding domain 2 (L63-E68).
[0058] The two CD25 binding regions separate the natural IL-2 amino acid sequence into three segments, wherein the amino acid sequence of IL-2 segment one is: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM (SEQ ID NO: 2);
[0059] The amino acid sequence of IL-2 fragment two is: MPKKATELKHLQCLEEE (SEQ ID NO: 3); ...
Embodiment 2
[0091] Example 2 Combination of chimeric T cell growth factor and natural IL-2 with CD25
[0092] Main experimental materials: purified chimeric T cell growth factor, natural human IL-2 (Peprotech, #200-02), CD25-Fc (Acrobiosystems, #ILA-H5251), HRP-anti-human IgG (abcam, #ab81202 ), TMB (Thermo Scientific, #N301).
[0093] experimental method:
[0094] 1) Dilute natural IL-2 (Peprotech, #200-02) and the chimeric T cell growth factor prepared in Example 1 with PBS to a final concentration of 5 μg / ml; was overnight;
[0095] 2) 5% skimmed milk powder, sealed at 37°C for 2 hours;
[0096] 3) Add double-diluted CD25-Fc (Acrobiosystems, #ILA-H5251), and incubate in a water bath at 37°C for 1 hour;
[0097] 4) After washing five times, add HRP-anti-human IgG (abcam, #ab81202), and incubate in a 37°C water bath for half an hour;
[0098] 5) After washing five times, add TMB to develop color for 10 minutes, and terminate with 2M sulfuric acid;
[0099] 6) Microplate reader dete...
Embodiment 3
[0101] Example 3 Effect of Chimeric T Cell Growth Factor and Natural IL-2 on Treg Proliferation
[0102] Main experimental materials: purified chimeric T cell growth factor, natural human IL-2 (Peprotech, #200-02), anti-CD3 / anti-CD28 antibody (STEMCELL, #10971), fluorescently labeled antibody: APC Mouse Anti-Human CD3 (BD, #555335); PE-Cy TM 7Mouse Anti-Human CD4 (BD, #560909); PE anti-human CD25 Antibody (Biolegend, #302605); FITC anti-human CD127 (IL-7Rα) Antibody (Biolegend, #351311).
[0103] experimental method:
[0104] 1) Use Ficoll-Paque (GE Healthcare, #17144002) density gradient centrifugation to separate and purify peripheral blood mononuclear cells (PBMC) from healthy adults;
[0105] 2) Sorting total T cells by magnetic bead sorting (STEMCELL, #17961);
[0106] 3) Dilute T cells with RPMI 1640 complete medium to a final concentration of 1×10 6 Cells / ml, 96-well culture plate, 100ul / well;
[0107] 4) Co-culture the isolated and purified T cells with anti-CD3 / a...
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