Application of polypeptide specific-binding TRB3 in treating or preventing abdominal aortic aneurysm

Abstract

The invention discloses application of polypeptide specific-binding TRB3 in treating or preventing abdominal aortic aneurysm. The amino acid sequence of the polypeptide is showed as SEQ ID NO:1. The polypeptide derivative is a chimeric peptide formed by the polypeptide connecting with a cell penetrating peptide or a dosage form formed by the polypeptide coordinated with a drug excipient. The polypeptide is screened by the surface plasma resonance (Biacore) method, and can specifically bind to TRB3 and blockade the combination of TRB3 and P62 protein. Meanwhile, the polypeptide derivative Pep2-A2 can treat the mice abdominal aortic aneurysm by knocking-out ApoE inducted by angiotensin II. Thus, the polypeptide and the polypeptide derivative can be used as a novel inhibitor for TRB3, and can be used in exploiting vaccine or medicament for inhibiting abdominal aortic aneurysm.

Description

technical field

[0001] The invention relates to the application of a polypeptide and its derivatives in the preparation of medicines for treating or preventing abdominal aortic aneurysms. Background technique

[0002] Abdominal aortic aneurysm refers to an expansive mass in the abdominal aorta that pulses laterally and anteroposteriorly, showing aneurysmal expansion. Usually, an increase of more than 50% in the diameter of the aorta can be defined as an aneurysm. The lesion of this disease has a very high risk of rupture. Once the tumor ruptures, it is often accompanied by shock, and the mortality rate is as high as 78%-92%, which seriously threatens human health. When the tumor is not ruptured, the patient is often accompanied by severe pain around the umbilicus and in the middle and upper abdomen, and concurrently with diseases such as atherosclerosis, diabetes, lower extremity arterial embolism, and hydronephrosis, which seriously affect the quality of life of the patient...

Examples

Embodiment 1

[0058] Example 1: Using surface plasmon resonance to screen peptides that bind to TRB3 protein.

[0059] First, the P62 protein is truncated into different polypeptide fragments, and the peptides are synthesized with a peptide solid-phase synthesizer. This process is carried out by Beijing Saibaisheng Gene Co., Ltd. EXAMPLES The entire screening process was carried out in a surface plasmon resonance instrument BiacoreT200.

[0060] The screening method is as follows:

[0061] 1. The purified protein TRB3 (purchased from R&D Company) was coupled to a CM5 chip (purchased from GE Company) through amino groups, unbound protein was washed away at a flow rate of 10 μL / min, and the surface of the chip was equilibrated for 2 hours.

[0062] 2. Automatically inject 250 μL of different concentrations of polypeptide fragments (200, 50, 12.5, 6.25 nM), and the whole process is carried out at 25°C. The buffer used was HBS-EP buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.005% surfactant). ...

Embodiment 2

[0067] Example 2: ELISA method to verify the binding of peptide A2 to protein TRB3.

[0068] The specific operation steps are as follows:

[0069] 1. Dilute human TRB3 protein and bovine serum albumin (BSA) to 10 μg / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0070] 2. Wash three times with PBS containing 0.1% Tween-20. The plates were coated with 200 μl of blocking solution (10% BSA--PBS), and coated at 37° C. for 2 hours.

[0071] 3. Pour off the coating solution, add 200 μl of 1 μg / ml polypeptide B1, A2 and B3 solutions, and set up positive control wells, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C for 1 hour.

[0072] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl anti-M13 monoclonal antibody diluted with blocking solution 1:4000 to each well, and incubate at room temperature for 1 h.

[0073] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic solution (10...

Embodiment 3

[0076] Example 3: The method of competition ELISA verifies that peptide A2 can compete for the binding of TRB3 and P62 protein.

[0077] The specific operation steps are as follows:

[0078] 1. Dilute human TRB3 protein and BSA to 10 μl / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0079] 2. Wash three times with PBS containing 0.1% Tween-20. The plates were coated with 200 μl of blocking solution (10% BSA--PBS), and coated at 37° C. for 2 hours.

[0080] 3. Pour off the coating solution, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C

[0081] 1h.

[0082] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl of horseradish catalase-labeled polypeptide A2 diluted with blocking solution to each well, and incubate at room temperature for 1 h.

[0083] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic solution (100mmol / L sodium acetate, pH 6.0, add 10μl 30% hydrogen peroxi...

technical field

[0001] The invention relates to the application of a polypeptide and its derivatives in the preparation of medicines for treating or preventing abdominal aortic aneurysms. Background technique

[0002] Abdominal aortic aneurysm refers to an expansive mass in the abdominal aorta that pulses laterally and anteroposteriorly, showing aneurysmal expansion. Usually, an increase of more than 50% in the diameter of the aorta can be defined as an aneurysm. The lesion of this disease has a very high risk of rupture. Once the tumor ruptures, it is often accompanied by shock, and the mortality rate is as high as 78%-92%, which seriously threatens human health. When the tumor is not ruptured, the patient is often accompanied by severe pain around the umbilicus and in the middle and upper abdomen, and concurrently with diseases such as atherosclerosis, diabetes, lower extremity arterial embolism, and hydronephrosis, which seriously affect the quality of life of the patient...

Examples

Embodiment 1

[0058] Example 1: Using surface plasmon resonance to screen peptides that bind to TRB3 protein.

[0059] First, the P62 protein is truncated into different polypeptide fragments, and the peptides are synthesized with a peptide solid-phase synthesizer. This process is carried out by Beijing Saibaisheng Gene Co., Ltd. EXAMPLES The entire screening process was carried out in a surface plasmon resonance instrument BiacoreT200.

[0060] The screening method is as follows:

[0061] 1. The purified protein TRB3 (purchased from R&D Company) was coupled to a CM5 chip (purchased from GE Company) through amino groups, unbound protein was washed away at a flow rate of 10 μL / min, and the surface of the chip was equilibrated for 2 hours.

[0062] 2. Automatically inject 250 μL of different concentrations of polypeptide fragments (200, 50, 12.5, 6.25 nM), and the whole process is carried out at 25°C. The buffer used was HBS-EP buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.005% surfactant). ...

Embodiment 2

[0067] Example 2: ELISA method to verify the binding of peptide A2 to protein TRB3.

[0068] The specific operation steps are as follows:

[0069] 1. Dilute human TRB3 protein and bovine serum albumin (BSA) to 10 μg / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0070] 2. Wash three times with PBS containing 0.1% Tween-20. The plates were coated with 200 μl of blocking solution (10% BSA--PBS), and coated at 37° C. for 2 hours.

[0071] 3. Pour off the coating solution, add 200 μl of 1 μg / ml polypeptide B1, A2 and B3 solutions, and set up positive control wells, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C for 1 hour.

[0072] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl anti-M13 monoclonal antibody diluted with blocking solution 1:4000 to each well, and incubate at room temperature for 1 h.

[0073] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic solution (10...

Embodiment 3

[0076] Example 3: The method of competition ELISA verifies that peptide A2 can compete for the binding of TRB3 and P62 protein.

[0077] The specific operation steps are as follows:

[0078] 1. Dilute human TRB3 protein and BSA to 10 μl / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0079] 2. Wash three times with PBS containing 0.1% Tween-20. The plates were coated with 200 μl of blocking solution (10% BSA--PBS), and coated at 37° C. for 2 hours.

[0080] 3. Pour off the coating solution, add 200 μl of 1 μg / ml P62 protein solution, and incubate at 37°C

[0081] 1h.

[0082] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl of horseradish catalase-labeled polypeptide A2 diluted with blocking solution to each well, and incubate at room temperature for 1 h.

[0083] 5. Wash six times with PBS containing 0.1% Tween-20. Prepare substrate chromogenic solution (100mmol / L sodium acetate, pH 6.0, add 10μl 30% hydrogen peroxi...

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