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Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application

A low-molecular-weight amyloid peptide technology, which is applied in the field of artificial in vitro preparation of low-molecular-weight amyloid peptide oligomers, can solve the problems of uneven Aβ oligomer composition, unstable conditions, low yield and effect, and achieve Highly reproducible effect

Inactive Publication Date: 2011-01-19
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Many scholars have used a variety of methods to prepare Aβ oligomers, but the yield and effect are relatively low, and the components of the obtained Aβ oligomers are not uniform, containing more high molecular weight aggregates and fibrous components, which are difficult to further purify
For example, oligomers prepared by chemical cross-linking have differences in composition, which cannot truly reflect the action characteristics of low-molecular-weight Aβ oligomers
Moreover, the conditions of each preparation method are unstable, and the obtained results are difficult to compare
Some methods and materials have relatively large adverse reactions, affecting the health of experimenters and users

Method used

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  • Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application
  • Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application
  • Artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The acquisition of embodiment 1 amyloid peptide monomer

[0042] 1. Gene recombination method

[0043] The Aβ42 gene (Genebank accession number BC065529) was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and installed on the prokaryotic expression vector with 6×His tag to form the NAbeB-pET30a expression plasmid. Then Escherichia coli BL21 was transformed to induce expression. Take the NAbeB-pET30a-BL21 frozen bacterial solution, streak it on a Kana-resistant LB culture plate, and incubate at 37°C for 12-16 hours; pick a single colony that grows well and put it in 7ml of Kana-resistant LB culture medium, 260rpm , cultivated overnight at 37°C; after about 4 hours of cultivation, the OD value of the bacterial solution reached 0.6, and at this time, IPTG with a final concentration of 1 mmol / l was added to induce it; after 6 hours, 12,000 rpm, 4°C for 15 minutes to collect the bacterial cells; washed the bacterial cells twice with PBS , 12000...

Embodiment 2

[0049] The assembly of embodiment 2 amyloid peptide oligomers

[0050] Dissolve the peptide film obtained in Example 1 with 20 μl of anhydrous dimethyl sulfoxide (DMSO) (Sigma), and finally place it in a modified artificial cerebrospinal fluid buffer system (replenish the volume to 1 ml), place at 4°C for 24 hours, Allow it to polymerize naturally, and detect the preparation of oligomers by Western blot.

[0051] The composition of the improved artificial cerebrospinal fluid buffer system: NaCl 125mmol / L, KCl 3.3mmol / L, KH 2 PO 4 1.2mmol / L, NaHCO 3 26mmol / L, CaCl 2 2.5mmol / L, MgSO 4 2.4mmol / L, glucose 5mmol / L, amino acid 0.3g / L.

Embodiment 3

[0053] Example 3 Western blot (Western blot) experiment

[0054] 1. Purpose of the experiment

[0055] Validation of Monomer and Oligomer Obtainment by Molecular Weight and Immunoreactivity

[0056] 2. Experimental method

[0057] Take the 5 μg sample collected at 0s and 24h during the assembly process, add 1 / 4 volume of 5×sample buffer, mix well and load the sample, and first pass the protein through the stacking gel with a voltage of 100V. When the sample enters the separating gel, adjust the voltage to keep it constant at 120V. When the bromophenol blue swims to the bottom of the gel, end the electrophoresis, remove the gel, and stain it with Coomassie Brilliant Blue R-250 routinely; put the gel and nitrocellulose membrane into containers containing blotting buffer Equilibrate in the chamber for 10 minutes, put filter paper, gel, NC membrane, and filter paper in order to form a "sandwich" shape, pour the transfer buffer, with the gel side facing the negative electrode an...

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Abstract

The invention discloses an artificial in-vitro preparation method of low molecular weight amyloid peptide oligomer and application, which is characterized in that the monomer of A beta polypeptide obtained with a gene recombination method is placed in a buffer system to react after dissolved by anhydrous dimethyl sulfoxide, thus naturally polymerizing to obtain oligomer. The method which obtains the monomer by gene recombination is economic and environmentally-friendly; when A beta serves as raw material to assemble amyloid peptide oligomer in vitro, a stable and even product is obtained by simulating in vivo physiological conditions and improving artificial cerebrospinal fluid components. The oligomer prepared with the method can serve as the standard reference for the detection and the therapy researches of various Alzheimer diseases, and can be applied in the diagnostic reagent and the therapy drug of the Alzheimer disease.

Description

Technical field: [0001] The invention relates to an artificial in vitro preparation method of a low molecular weight amyloid peptide oligomer. The invention also relates to the application of the oligomer in the preparation of diagnostic reagents and therapeutic drugs for Alzheimer's disease. Background technique: [0002] Amyloid peptide oligomers (also known as Aβ oligomers) are considered to be the initiating factors of early pathological changes in Alzheimer's disease (AD). The latest research shows that only low-molecular-weight Aβ oligomers are toxic substances that cause AD neuron damage, and high-molecular-weight aggregates and fiber components do not have this effect. The artificial preparation of the amyloid peptide oligomer outside the human body is helpful for studying the pathogenic mechanism and treatment method of Alzheimer's disease. [0003] Amyloid peptides readily form mixtures including fibrils and high or low molecular weight aggregates without fibrous...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/70A61K38/17A61P25/28G01N33/53G01N30/00G01N23/22
Inventor 张莹洪涛彭向雷
Owner BEIJING JIAOTONG UNIV
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