Short-peptide inhibitor and use thereof
A technology of inhibitors and short peptides, applied in the field of biomedicine, can solve the problems of poor solubility, high cytotoxicity, and limitations, and achieve the effects of reducing toxicity, obvious aggregation inhibition, and strong binding ability
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[0039] The preparation method of described assembly comprises the steps:
[0040] a1. making a solution of the six polypeptides Aβ1-20, Aβ1-25, Aβ1-28, Aβ20-1, Aβ25-1 and Aβ28-1 and marker molecules;
[0041] b1. Mix the marker molecule with the six polypeptides one by one to form a co-assembly;
[0042] c1. Add the solution obtained in step b1 dropwise onto a conductive substrate, such as graphite or metal substrates such as gold, silver, copper, platinum, and semiconductor substrates such as silicon, to form an assembly.
[0043] Scanning tunneling microscope images of short peptide inhibitor assemblies were obtained at the solid / air interface after solvent removal.
[0044] d1. The stable assembly core fragments of Aβ1-20, Aβ1-25, Aβ1-28, Aβ20-1, Aβ25-1 and Aβ28-1 obtained by STM (respectively GYEVHHQKLVFF, GYEVHHQKLVFFAEDVG, GYEVHHQKLVFFAEDVGSNK, DAEFRHDSGYEVHH, DAEFRHDSGYEVHH and DAEFRHDSG ) take the same fragment as GYEVHH, which is the selected short peptide inhibitor...
Embodiment 1
[0085] Example 1 Using Scanning Probe Microscopy to Obtain the Assembly Core Fragment of the Nitrogen Terminal of the β-Amyloid Peptide
[0086] 1. The chemical structure of the substance used
[0087] Short peptide inhibitor (GYEVHH (SEQ ID NO:1), Shanghai Keyept Biotechnology Co., Ltd., with a purity of 98%), the labeled molecule is 4,4'-bipyridyl (4,4'-bipyridyl, 4Bpy, Sigma- Aldrich) sequence and structure are shown below: Short peptide inhibitor: N 2 H-GlyTyrGluValHisHis-COOH; marker molecule 4Bpy:
[0088]
[0089] 2. Specific method
[0090] 1) Using scanning tunneling microscopy to obtain nitrogen-terminal stable assembly fragments.
[0091] Dissolve β-amyloid peptides (Aβ1-20, Aβ1-25, Aβ1-28 and anti-sequence Aβ20-1, Aβ25-1, and Aβ28-1) and marker molecules in aqueous solution, sonicate for 30 seconds, and mix thoroughly. Take out 15 microliters of the solution, drop it on the surface of newly cleaved highly oriented pyrolytic graphite (ZYB grade, Veeco, USA), ...
Embodiment 2
[0110] Example 2 Detecting the neurotoxicity of β-amyloid polypeptide Aβ1-42 and the inhibition of short peptide inhibitors to β-amyloid polypeptide neurotoxicity by using the neurocytotoxicity test method
[0111] SH-SY5Y cells (ECACC, UK) were inoculated in 96-well plates (purchased from Corning Incorporated, USA), 150 μL of cell culture medium per well, and cultured in an incubator at 37°C. When about 60% of the cells adhere to the wall and are in good condition, the mixed system of β-amyloid peptide and short peptide inhibitor and β-amyloid peptide can be added to the cell culture medium for cytotoxicity test. The cell concentration is 15000 pcs / hole.
[0112] Aβ1-42, Aβ1-42 / GYEVHH and Aβ1-42 / KLVFF were added to the cell culture medium respectively, and the final concentrations were Aβ1-42: 10 μM, 20 μM, 40 μM; GYEVHH: 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 120 μM, 160 μM, 200 μM; Aβ1-42 / GYEVHH: 10 μM:—, 10 μM: 1 μM, 10 μM: 5 μM, 10 μM: 10 μM, 10 μM: 20 μM, 10 μM: 40 μM; when...
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