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Sirpa protein affinity cyclic peptide and application thereof

A cyclic peptide and amino acid technology, applied in the direction of cyclic peptide components, peptide/protein components, peptides, etc., can solve the problems of inability to quickly terminate immune adverse events, poor tissue permeability, high production costs, etc., and achieve good medical application prospects, in vitro Strong affinity and good effect

Active Publication Date: 2020-05-08
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, monoclonal antibodies targeting the CD47 / SIRP-α pathway have been marketed and are used in immunotherapy of tumors. However, antibody drugs have high production costs, poor tissue penetration, and long half-life, and cannot quickly terminate immune adverse events. Small molecules such as peptides Convenient drug synthesis, good tissue permeability, and low immunogenicity

Method used

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  • Sirpa protein affinity cyclic peptide and application thereof
  • Sirpa protein affinity cyclic peptide and application thereof
  • Sirpa protein affinity cyclic peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Determination of phage titer

[0045]Inoculate a single colony of ER2738 in 10 mL LB medium, and culture it on a shaker until logarithmic phase (OD 600nm value around 0.5). Phage were serially diluted 10-fold with LB medium. Dilution range: Amplified phage culture supernatant: 10 8 ~10 12 ; Unamplified panning eluates: 10 1 ~10 5 . Put 200 μL of bacteria that have reached the logarithmic phase into a microcentrifuge tube, and then add 10 μL of different dilutions of phage to each tube, shake and mix quickly, and incubate at room temperature for 5 minutes. Add the infected bacteria to the upper agar culture tube pre-warmed at 45°C, mix quickly, and immediately pour it on the LB / IPTG / Xgal plate pre-warmed at 37°C to spread evenly. After the plate was cooled for 15 minutes, it was inverted and incubated overnight at 37°C. Checked the tablet the next day, counted ~10 2 The number of spots on the plate of phage. This number was then multiplied by the dilution f...

Embodiment 2

[0081] (1) Human Sirpɑ protein labeling: use PBST to adjust the protein concentration to 200nM, take 100μL for later use; add 50μL BST to the solid fluorescent dye, and vortex to mix the dye; use PBST to adjust the concentration of the dye to 100nM; the volume ratio is 1:1 The ratio of protein and dye was mixed and incubated at room temperature for 30 min in the dark.

[0082] (2) Centrifuge the sample at 4°C and 15 000g for 10 minutes. Keep the supernatant, discard the pellet, and complete the protein labeling.

[0083] (3) MST detection: prepare 16 200 μL EP tubes, labeled 1-16, add 20 μL 400 μM affinity loop peptide solution to the first EP tube, and then double the affinity loop peptide solution in tube 1 with PBST Ratio dilute to tubes 2-16; add 10 μL labeled human Sirpɑ protein to tubes 1-16 and mix well; after incubating at room temperature for 5 minutes, use a capillary to absorb the mixed solution in tubes 1-16, and follow the steps from high concentration to low con...

Embodiment 3

[0086] (1) Use 1640 medium (containing 10% FBS) for CHO-K1-hSirpɑ cells at 37°C, 5% CO2 Cultivate in the incubator to a good state. Collect cells, adjust cell density, press 3×10 5 / tube into 1.5mL EP tubes. Centrifuge at 3000rpm / min at 4°C for 5min, discard the supernatant to obtain the cell pellet for later use.

[0087] (2) SP4 and SP5 cyclic peptide solutions with different concentrations (0, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM) were serially diluted in the reaction system of 50 μL PBS (pH7.2), and SP1- A solution of SP6 cyclic peptide at a concentration of 200 μM.

[0088] (3) Add the cyclic peptide solution prepared in the previous step to the cell pellet in (1), vortex gently to mix, incubate on ice for 30 minutes, and take a tube of cell pellet to reconstitute with 50 μL PBS (pH7.2) buffer suspended as a negative control.

[0089] (4) After the incubation, add 15ng of hCD47 protein to each tube (except the negative control tube), gently vortex to mix...

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Abstract

The invention relates to a Sirpa protein affinity cyclic peptide and application thereof. The amino acid sequence of the affinity cyclic peptide is shown in SEQ ID NO.1-6. The invention further provides a medicine composition or a kit with the cyclic peptide. The affinity cyclic peptide provided by the invention is affine with a Sirpa extracellular domain, is capable of blocking interactions of Sirpa and CD47, and further has an antitumor effect.

Description

Technical field: [0001] The invention belongs to the field of biotechnology and pharmacy, and specifically relates to Sirpɑ protein affinity cyclic peptide and its application. Background technique: [0002] For a long time, tumor has become one of the major and serious diseases that constantly threaten human life and health with its increasing mortality and morbidity, and people are constantly looking for ways to treat tumors. Traditional tumor treatment measures include chemotherapy, radiotherapy and surgical treatment. Although these methods have been used clinically for several years, they are limited and have relatively large side effects on the human body. At present, immunotherapy is attracting the most attention in the field of tumor treatment. Tumor immunotherapy is to stimulate the body's own immune mechanism, and to kill tumor cells by improving autoimmunity to achieve the purpose of anti-tumor. In 2013, "Science" magazine listed tumor immunotherapy as the top te...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/64G01N33/68A61K38/08A61K38/12A61P35/00
CPCC07K7/06C07K7/64G01N33/68A61P35/00A61K38/00Y02A50/30
Inventor 高艳锋李艳营翟文杰祁元明王鸿飞
Owner ZHENGZHOU UNIV
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