Polypeptide for inhibiting enzyme digestion of beta secretase and application thereof

A technology of secretase and secretase inhibitor, which is applied to polypeptides and their application fields in the preparation of drugs for treating Alzheimer's disease, and can solve the problem of easily causing allergic reactions, large specific antibody molecules, and difficult to pass through the blood-brain barrier, etc. problem, to achieve the effect of small molecular weight of polypeptide, small side effects and poor antigenicity

Inactive Publication Date: 2010-10-20
TSINGHUA UNIV
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of polypeptide for the specific antibody molecule of the β-secretase cleavage site on APP, which is difficult to pass through the blood-brain barrier, and the antibody has antigenicity and is easy to cause allergic reactions. The sequence is: His-Asp-Pro-Ala-Pro-Rrg-Thr, or the above-mentioned amino acid sequence is substituted, deleted, or one or several amino acids are added, and it has the effect of inhibiting β-secretase digestion, inhibiting Aβ aggregation and Polypeptides Inhibiting the Cytotoxicity of Aβ

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polypeptide for inhibiting enzyme digestion of beta secretase and application thereof
  • Polypeptide for inhibiting enzyme digestion of beta secretase and application thereof
  • Polypeptide for inhibiting enzyme digestion of beta secretase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1. The specific binding of Pbss1 to the β-secretase cleavage site on APP and Aβ42

[0034] The β-secretase cleavage site polypeptide on APP prepared in PBS buffer (Shanghai Jier Polypeptide Co.), Aβ42 (American Peptide Co.) and the negative control polypeptide were coated with 1 μg / well on a 96-well high-affinity microtiter plate, overnight at 4°C. Block the blank site on the microplate plate that is not bound to the polypeptide with BSA, then add 11 μg / well of Pbss linked with 6 histidines as tags, and let it react for 2 hours at room temperature. After washing, the HRP-conjugated antibody capable of binding to the histidine tag was added and reacted at room temperature for 1 h. After washing, the substrate TMB was added, and after 15 minutes of color development, the absorbance at 450 nm was measured with a multifunctional microplate reader.

[0035] Repeated three times under the same conditions, the results are shown in figure 1 , the data in the figure is...

Embodiment 2

[0036] Example 2. Amino acid substitution, deletion or insertion of Pbss1 still binds specifically to the cleavage site of β-secretase of APP and Aβ42

[0037] The N-terminal acetylation of the polypeptide Pbss1 of the present invention, the deletion of alanine (sequence: His-Asp-Pro-Pro-Rrg-Thr), and the replacement of alanine with glycine (sequence: His-Asp-Pro-Pro- Gly-Pro-Rrg-Thr) or insert histidine and connect leucine at the end (sequence: His-Asp-Pro-Ala-His-Pro-Rrg-Thr-Leu), then according to Example 1 The test method detects the specific binding to the β-secretase cleavage site of APP and Aβ42, and uses the ELISA method to measure the OD value. The results are shown in figure 2 . It shows that Pbss1 still retains the ability to simultaneously bind to the β-secretase cleavage site on APP and Aβ42 after certain modifications, certain amino acid substitutions, deletions, or addition of amino acids in the polypeptide (compared with the negative control, P<0.01 ).

Embodiment 3

[0038] Example 3. Pbss1 inhibits the aggregation of Aβ42 in vitro

[0039] 1) Dissolve Aβ42 (American Peptide Company, USA) hexafluoroisopropanol (HFIP) purchased abroad to 1 mg / ml, ultrasonically treat at room temperature for 10 min, dispense into epidorf tubes, volatilize HFIP in vacuum, and then place at -20 Store at ℃. Before use, the HFIP-treated Aβ42 was left at room temperature for 20 min, then dimethyl sulfoxide (DMSO) was added to make the concentration of Aβ42 1 mg / ml, and then diluted to the required concentration with 0.02M PBS buffer at pH 7.4.

[0040] 2) Pbss1 was dissolved in 0.02M PBS buffer at pH 7.4, and then added to the Aβ42 solution so that the final concentration of Aβ42 was 10 μM and the final concentration of Pbss1 was 10 μM and 100 μM. And the Aβ42 solution without Pbss1 was used as the control. All samples were placed at 37°C for 24 hours.

[0041] 3) Thioflavin (ThT) was dissolved in pH 6.5, 50 mM phosphate buffer to a concentration of 5 μM. Tak...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the field of biotechnology and in particular discloses polypeptide, which has the amino acid sequence of His-Asp-Pro-Ala-Pro-Rrg-Thr. The invention also provides application of the polypeptide as a beta secretase inhibitor and an A beta aggregation inhibitor and in preparing medicaments for treating Alzheimer disease. Experiments show that: the polypeptide can specifically combine restriction enzyme cutting site of beta secretase on APP to inhibit the enzyme digestion of the beta secretase, the generation of A beta40 and A beta42 and the aggregation and cytotoxicity of the A beta42. After the polypeptide is injected into an AD transgenic mouse for animal memory experiments, results show that the polypeptide can obviously improve the spacial memory capacity of the mouse and decrease the number of cerebral amyloid plaques. The polypeptide has small molecular weight, is easy to pass through a blood brain barrier, has poor antigenicity and small side effect than an antibody, and has wide clinical application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polypeptide and its application in the preparation of drugs for treating Alzheimer's disease. Background technique [0002] Alzheimer's Disease, hereinafter referred to as AD, commonly known as senile dementia, is formed by the aggregation of non-toxic β-amyloid monomer molecules (β-Amyloid (Aβ40 / 42 hereinafter referred to as Aβ)) to form toxic oligosaccharides. Aggregate-induced neurodegenerative diseases in the elderly are mainly characterized by memory loss and the formation of senile plaques in the brain. Medical statistics show that 5-6% of the elderly over the age of 60 in my country and European and American countries suffer from Alzheimer's disease. The disease has been ranked as the fourth leading cause of death after heart disease, cancer and stroke. my country's existing population over 60 years old is about 100 million. According to the estimation of 5% prevalence rat...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K7/06A61K38/08A61P25/28
Inventor 刘瑞田杨世高
Owner TSINGHUA UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap