Improved tumor inhibitory peptide capable of pecifically binding to PD-1 and application of tumor inhibitory peptide
A tumor suppressor, PD-1 technology, applied in the field of disease treatment to achieve good biological activity
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Embodiment 1
[0028] Embodiment one: the preparation of inhibitory peptide mutant
[0029] According to the previous research of the applicant, the preparation method of the inhibitory peptide is disclosed in CN107383174A, and the specific polypeptide sequence is:
[0030] KWQEE GQAIHS TNLTQYPHRSGLRVGCHDWRTWPHNYPC GALPRK NYSLFPQDHTPCYIWYYSPDLQHMPNNVKHNSSRSPDLLASKPPVTESPWW HIDTQ MSGYMA. The three places underlined are the extracellular region of the protein receptor that specifically binds to PD-1, and a disulfide bond is formed between the 11th amino acid and the 77th amino acid.
[0031] According to the binding characteristics of the inhibitory peptide and the extracellular region, the applicant designed 400 simulated structures at three binding sites, and finally obtained 20 optimized mutation combinations based on the protein binding isoelectric point and three-dimensional structure simulation data. Forms, after verification in the later stage, only 6 have significantly improved a...
Embodiment 2
[0032] Example 2 Determination of the affinity of inhibitory peptides to PD-1
[0033] (1) Prepare the polypeptide with 1×PBS to a solution with a concentration of 1 mg / mL, and prepare the anti-PD-1 polypeptide to a solution with a concentration of 100 μg / mL, take 2.5 μL and drop it on a carboxylated SPR chip (purchased from Plexera company, Kx5 type SPR standard substrate), and the polypeptide was immobilized on the SPR chip through the specific reaction of streptavidin and biotin. Then, the PD-1 solution (diluted in 1×PBS) with concentrations of 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 50 μg / mL was passed through the chip surface as the mobile phase. The binding time was 150 s, the dissociation time was 130 s, and the regeneration time 200s. 1×PBS was used as the dissociation solution, and 0.5% phosphoric acid was used as the reconstitution solution. The binding-dissociation curves of polypeptides and TNF-α were recorded on a K×5 SPR instrument (Plexera).
[0034] (2) Fitting th...
Embodiment 3
[0038] Example 3 Biological Activity Detection of Mutant Polypeptide Inhibitors
[0039](1) Select JurKat / PD-1 / NFAT cells preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee with the registration number 10298 as target cells, and use DMEM / F12+10% FBS medium to illuminate 2.5×10 4 cells / well, spread evenly in a 96-well plate at a density of 100 μl / well (corning), 37°C, 5% CO 2 Incubate overnight in the incubator;
[0040] (2) Use 1640+10% fetal bovine serum medium to prepare different concentrations of mutant polypeptide solutions and dilute to 1 μg / mL, then dilute 10 times down to 7 concentration points, and suck off the DMEM / F12+ in the above 96-well plate 10% FBS medium, add the prepared peptide inhibitor solution to 96-well plate according to 50 μl / well;
[0041] (3) Use 1640+10% fetal bovine serum medium to prepare target cell suspension, and follow the 5×10 4 per well, 50 μl / well density evenly spread to the ab...
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