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A kind of PD-1 targeting blocking peptide and its application

A PD-1, targeting technology, applied in the biological field to achieve anti-tumor effect, significant effect, and the effect of restoring lymphocyte activity

Active Publication Date: 2022-07-12
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the characteristics of the PD-1 / PD-L1 interaction, that is, the surface-surface interaction interface of the protein is relatively flat, the range of action sites is large and highly variable, so it is becoming a challenge to find small molecule inhibitors with blocking effects. a challenging job

Method used

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  • A kind of PD-1 targeting blocking peptide and its application
  • A kind of PD-1 targeting blocking peptide and its application
  • A kind of PD-1 targeting blocking peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation and activation of host bacteria:

[0032] The following operations need to be sterile in the ultra-clean workbench:

[0033] 1. Melt 100 mL of LB solid medium, add 100 μL of tetracycline stock solution when the temperature drops to 50-60 °C, and pour about 20 mL into a sterile petri dish (Φ=0.9 cm) after mixing to prepare a final concentration of 20 μg / mL Tetracycline resistance-LB plate (Tet-LB plate);

[0034] 2. Streak the strain E.coli ER2738 stored at -70°C on a Tet-LB plate, invert at 37°C overnight (about 16-18h), and store it in a 4°C refrigerator for two weeks;

[0035] 3. Prepare a sterile test tube containing 2 mL of LB liquid medium, add 2 μL of tetracycline stock solution with a concentration of 20 mg / mL, and the final concentration is 20 μg / mL;

[0036] 4. Pick the ER2738 monoclonal colony on the Tet-LB plate into 2 mL of Tet-LB medium, and shake and culture at 37°C overnight for use.

Embodiment 2

[0038] Amplification of phage libraries:

[0039] 1. Inoculate 200 μL of overnight cultured ER2738 in 200 mL of sterilized LB liquid medium, and incubate with vigorous shaking at 37°C for about 2 to 2.5 hours to reach the logarithmic growth phase (OD600 nm absorbance value is 0.4 to 0.6);

[0040] 2. Dilute 1.5 μL of the original phage library in 150 μL of TBS buffer solution; infect 200 mL of ER2738 bacterial broth in log phase with 100 μL of the diluted phage library;

[0041] 3. Mix well and let stand for 15 minutes, then vigorously shake at 220-250 rpm at 37°C for 4.5-5 hours;

[0042]4. Collect the culture in a sterilized centrifuge cup, pre-cool at 4°C, and centrifuge at 10,000 rpm for 10 min;

[0043] 5. Transfer the supernatant phage solution obtained after centrifugation to a new centrifuge cup, add 1 / 5 volume of PEG / NaCl (about 40 mL), mix well and place at 4°C overnight (about 16-18h) to allow the phage Precipitation is complete;

[0044] 6. Centrifuge the sample...

Embodiment 3

[0050] Phage titer determination:

[0051] 1. Preparation of the lower IPTG / X-gal / LB plate: Heat and melt 100 mL of solid LB medium and cool it to about 55°C, add 100 μL IPTG / X-gal stock solution evenly, mix quickly and pour the sterile plate (Φ = 0.9 cm). After cooling, cover with plastic wrap and pre-warm at 37°C for later use;

[0052] 2. Preparation of the upper layer of LB semi-solid agar: the semi-solid LB medium was heated and melted and cooled to 55°C, and each 3 mL of it was dispensed into sterile EP tubes, and the water bath was kept at 45°C for later use;

[0053] 3. Transfer the ER2738 cultured overnight in step 3 of 1.2.1 into LB liquid medium at 1:100 (a total of 2-3 mL of culture can be prepared), and shake vigorously at 37°C for about 2 hours to make it logarithmic growth period;

[0054] 4. Pack ER2738 in logarithmic growth phase into 1.5mL sterilized Eppendorf (EP) at 200μL / branch;

[0055] 5. Prepare serial dilutions of phage solution:

[0056] (1) Dist...

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Abstract

The invention discloses a PD-1 targeting blocking peptide and an application thereof, belonging to the field of anti-tumor immunotherapy. The present invention utilizes phage display technology to obtain a blocking peptide that can specifically target PD-1, and the blocking peptide sequence is shown in SEQ ID NO. The phage monoclonal with specific affinity to PD-1 protein is obtained by panning, which can target tumor cells that naturally express PD-1; the polypeptide sequence displayed on the phage surface is obtained by sequencing and chemically synthesized, which can regulate human PBMC The cytokines IFN-γ and IL-2 are secreted; the PD-1 blocking peptide is encapsulated to make nanoparticles, and the in vivo efficacy experiment has proved that it has a significant anti-tumor effect; the PD-1 targeting blocking peptide of the present invention can be combined with PD-1 ‑1 specifically binds, blocks the combination of PD1 and PD‑L1, restores the function of T cells, and can be widely used in tumor targeting, immune regulation and anti-tumor fields.

Description

technical field [0001] The invention relates to a targeting blocking peptide that can specifically bind to PD-1, and belongs to the field of biotechnology. Specifically, it relates to the biopanning method of the blocking peptide, the targeting effect at the cell level and the immunostimulatory effect and its application in anti-tumor research. Background technique [0002] Phage display technology was created by George P. Smith in 1985. By engineering phage, exogenous polypeptide or protein is fused and expressed with a certain capsid protein of phage, under the condition of ensuring its ability to infect and reproduce. , so that the exogenous amino acid sequence is displayed at the end of the capsid protein. The random dodecapeptide phage display library is a combinatorial library by fusing random dodecapeptide to the M13 phage minor capsid protein (pIII). The displayed dodecapeptide is expressed at the N-terminus of pill. Through at least three rounds of adsorption, el...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K19/00A61K38/10A61P35/00A61P37/02
CPCC07K7/08A61P35/00A61P37/02C07K2319/00A61K38/00Y02P20/55
Inventor 邱郑王旻陶慧敏
Owner CHINA PHARM UNIV
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