Polypeptide for inducing CD4<+>CD25<+> regulatory T cells and application
A regulatory and cellular technology, applied in the field of immunology, to achieve broad application prospects and the effect of inhibiting inflammatory pathological reactions
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Embodiment 1
[0047] Example 1: After in vivo immunization with peptides or pretreatment of mouse spleen and lymph node cells in vitro, CD4 + CD25 + Foxp3 + Changes in the number of T cells;
[0048] 1) Synthesize Schistosoma japonicum-T cell epitope peptide SJMHE1, its amino acid sequence (SEQ.ID.NO.1) is VPGGGTALLRCIPVLDTLSTKNED(Val Pro Gly Gly Gly Thr Ala Leu Leu Arg Cys Ile ProVal Leu Asp Thr Leu Ser Thr Lys Asn Glu Asp). And synthesize the control peptide OVA 323-339 , its sequence is (SEQ.ID.NO.2): ISQAVHAAEINEAGRY (Ile Ser Gln Ala Val His Ala Ala Glu Ile Asn GluAla Gly Arg Tyr); the synthetic purity of the peptide and the control peptide are both ≥99%. The above-mentioned peptides were all produced by Shanghai Shenggong Company, and the purity of the peptides was greater than 99%, which was identified by HPLC.
[0049] 2) The peptide (SJMHE1), control peptide (OVA 323-339 ), PBS and IFA to make emulsifier, IFA was purchased from Sigma company, every 100 μl emulsifier contains ...
Embodiment 2
[0053] Example 2: Peptide immunization in vivo or pretreatment of mouse CD4 in vitro + CD25 + After Tregs cells, the effect on the suppressive function of Tregs;
[0054] 1) Immunize the mice according to the method in the above-mentioned Example 1, separate the spleen and lymph nodes of the immunized mice in each group, make a single cell suspension, lyse the erythrocytes with the erythrocyte lysate, and use Mouse CD4 + CD25 + Regulatory T cell IsolationKit (purchased from Miltenyi Biotec Company, item number: 130-091-041) to isolate mouse CD4 + CD25 + , CD4 + CD25 - T cells; non-CD4 + Cells were treated with 50 μg / ml mitomycin at 37°C, 5% CO 2 After 30 minutes of treatment in the incubator, it is APC. Take 5×10 4 / well purified CD4 + CD25 + T cells, 1×10 5 / well CD4+ CD25 - T cells, 1×10 5 / well APC, add 0.1 μg / ml SJMHE1 or OVA 323-339 , under anti-CD3 stimulation, 37 °C, 5% CO 2 After culturing in the incubator for 3 days, the 3 H incorporation assay for de...
Embodiment 3
[0057] Example 3: Peptide immunization in vivo or pretreatment of mouse CD4 in vitro + CD25 + After Tregs cells, the peptide on CD4 + CD25 + The mechanism of action of Tregs inhibitory effect;
[0058] 1) Immunize the mice according to the method in the above-mentioned Example 1, separate the spleen and lymph nodes of the immunized mice in each group, make a single cell suspension, and after lysing the erythrocytes with the erythrocyte lysate, separate the small cells according to the method in the above-mentioned Example 2. mouse CD4 + CD25 + , CD4 + CD25 - T cells; non-CD4 + Cells were treated with 50 μg / ml mitomycin at 37°C, 5% CO 2 After 30 minutes of treatment in the incubator, it is APC. Add 0.4 μm Millicell (Millipore Company, semi-permeable membrane separator, which can separate cells and only allow soluble molecules to pass through) in the 24-well culture plate, and take 2.5×10 4 / well purified CD4 + CD25 + Add T cells to the upper part of Millicell, 5×10 ...
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