Application of a polypeptide specifically binding to trb3 in the treatment of abdominal aortic aneurysm
An abdominal aortic aneurysm and species-specific technology, applied in the direction of medical preparations containing active ingredients, drug combinations, anti-tumor drugs, etc., can solve the problem of lack of TRB3 protein inhibitors and achieve significant curative effect
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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 a 96-well ELISA plate at 4°C overnight.
[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 (...
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 at 4°C overnight.
[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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