IDO1 related vaccine and application thereof
A technology for vaccines and uses, applied in the field of biomedicine
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Embodiment 1
[0050] Example 1 Immunogenicity prediction of polypeptides
[0051] According to the selected allele typing of HLA-A11:01 and HLA-A24:02, the invention uses the self-developed biological information analysis process to cut the full length of the IDO1 protein into a polypeptide sequence of 8-11 mer amino acids, and the IDO1 gene encodes The length of the protein is 403 amino acids, the molecular weight is 45326 Daltons, and the cleaved polypeptide sequence is 1578. The presentation ability prediction and affinity prediction were performed on these 1578 polypeptides, respectively. The presentation ability score is represented by a numerical value between 0 and 1. The higher the score value, the stronger the presentation ability, and the score greater than 0.1 indicates that the polypeptide has the presentation ability. Affinity score is expressed by IC50 score, IC50 less than 500 indicates that the polypeptide has affinity, and IC50 less than 50 indicates that the polypeptide h...
Embodiment 2
[0056] Example 2 Affinity verification of polypeptide T2
[0057] Take 2×10 5 T2 cells were plated with 500 μL containing human beta 2 Microglobulin (final concentration, 3 μg / ml) in IMDM serum-free medium was resuspended in a 24-well plate, and the peptides listed in Table 1 (final concentration 100 μM) were added. 2 ), cultured overnight. Two replicate wells per group; T2 cells without peptide added were used as background controls. Cells were harvested by centrifugation at 200g for 5 minutes. After the cells were washed twice with PBS, the cells were directly incubated with anti-HLA-A11:01 / HLA-A24:02 FITC monoclonal antibody at 4°C for 30 minutes. Then analyzed by flow cytometer. Fluorescence Index (FI) was calculated with the following formula: FI = [Mean Fluorescence Intensity (MFI) sample - MFI background ] / MFI background , where MFI background Represents values without peptides. FI > 1.5 indicates that the peptide has high affinity for HLA-A11:01 or HLA-A24:0...
Embodiment 3
[0061] Example 3 Mass spectrometry experiments verify that peptides are presented by HLA molecules on the surface of tumor cells
[0062] The present invention enriches the polypeptide-MHC complex on the cell surface by means of co-immunoprecipitation-mass spectrometry combination, and identifies whether the MHC molecule on the tumor cell surface presents the polypeptide. The specific method is as follows:
[0063] 1) Separation and purification of MHC-I-restricted T-cell epitope peptides: use pan-MHC-I A / B / C antibody (clone number: w6 / 32) and sepharose CL-4B beads coupled with protein A molecules on the surface Bind at 4°C for 1 hour, and use NanoDrop to detect the residual antibody content in the supernatant. The antibody binding rate > 90% is considered qualified, and pan-MHC-I A / B / C-binding sepharose is prepared and used at 4°C. Add 40ml of RIPA lysate to SKMEL5 and HCT8 cell samples respectively, incubate at 4°C for 1 hour, centrifuge at 12,000 rpm for 30min, add sepharo...
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