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Polypeptide combined with various amyloid protein monomers simultaneously and application thereof

A technology of amyloid and monomers, applied in the direction of immunoglobulin, medical preparations containing active ingredients, peptides, etc., can solve the problems of patient death and achieve the effect of eliminating side effects

Active Publication Date: 2011-05-18
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unfortunately in the second phase of clinical trials, although most patients still showed good results, 6% of patients showed symptoms and pathological changes of meningitis, and some patients even died

Method used

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  • Polypeptide combined with various amyloid protein monomers simultaneously and application thereof
  • Polypeptide combined with various amyloid protein monomers simultaneously and application thereof
  • Polypeptide combined with various amyloid protein monomers simultaneously and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A polypeptide (PEMX for short) that binds amyloid monomers at the same time, its amino acid sequence is: Cys-Trp-His-Thr-Asp-Thr-Arg-Ser-Cys, or the above amino acid sequence has been substituted, deleted, Or add one or several amino acids and have the function of inhibiting the formation of amyloid oligomers. The invention can effectively stimulate the body to produce specific antibodies combined with multiple amyloid amyloid transition state monomers, and the antibodies can inhibit amyloid aggregation and cytotoxicity.

[0027]In a specific embodiment of the present invention, the N-terminal of the polypeptide of the present invention is modified with PEG2000 (referred to as PEG-PEMX) (sequence: PEG-Cys-Trp-His-Thr-Asp-Thr-Arg-Ser- Cys), replacement of aspartic acid with alanine (referred to as PEMX-D / A) (sequence: Cys-Trp-His-Thr-Ala-Thr-Arg-Ser-Cys), deletion of tryptophan (referred to as for: PEMX-?W) (sequence: Cys-His-Thr-Asp-Thr-Arg-Ser-Cys) or inserted glycine...

Embodiment 2

[0038] Example 2: PEMX can bind to various amyloid monomers

[0039] 100 μl Ab42, PrP, amylin, a-synuclein (American Peptide Company, USA), insulin, lysozyme (Sigma-Aldrich, USA) monomers (1 μg / well) were respectively coated on 96-well ELISA microplates , BSA as negative control, placed at 37°C for 2 h, blocked with 3% BSA at 37°C for 2 h, 200 μL / well. The plate was washed 3 times with PBS, 100 μl of PEMX solution with histidine tag was added, incubated at room temperature for 1 h, and washed three times with PBS containing 0.1% Tween-20. Add 100 μl of HRP-linked histidine tag-recognizing antibody (9E10) (1:3000) to each well, incubate at room temperature for 1 h, and wash 3 times with PBS containing 0.1% Tween-20. Add 100 μL of substrate solution TMB to each well, place at 37°C for 20 min, then add 50 μl of 1 mmol / L sulfuric acid to each well to terminate the reaction, and measure the OD value (wavelength: 450 nm) on an enzyme-linked immunosorbent detector ( figure 1 ). fi...

Embodiment 3

[0040] Example 3: PEMX with substituted, deleted or inserted amino acids still binds to Aβ42

[0041] The N-terminus of the polypeptide PEMX was modified with PEG2000 (referred to as PEG-PEMX) (sequence: PEG-Cys-Trp-His-Thr-Asp-Thr-Arg-Ser-Cys), and aspartic acid was replaced with alanine amino acid (abbreviated as PEMX-D / A) (sequence: Cys-Trp-His-Thr-Ala-Thr-Arg-Ser-Cys), delete tryptophan (abbreviated as: PEMX-?W) (sequence: : Cys-His-Thr-Asp-Thr-Arg-Ser-Cys) or inserted glycine (referred to as: PEMX-G) (sequence: Cys-Trp-His-Thr-Asp-Thr-Arg-Gly-Ser- Cys). In order to be able to detect the binding of the modified polypeptides to Aβ42, these polypeptides compete with unmodified PEMX with a tag to bind to Aβ42, and then ELISA is used to detect the binding of the latter to Aβ42. Add 100 ml of a mixture of 1 mg unmodified tagged PEMX and 10 mg modified PEMX to a 96-well ELISA plate coated with Aβ42 monomer, and then use ELISA to detect the binding of tagged PEMX to Aβ42 , mea...

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Abstract

The invention belongs to the field of peptide-containing medicinal preparations, and in particular relates to polypeptide combined with various amyloid protein monomers simultaneously and application thereof, which can be applied to preparation of an antibody for identifying various amyloid protein transitional monomers, and diagnosis, treatment and test research of amyloid protein diseases. In the polypeptide combined with various amyloid protein monomers simultaneously (PEMX) provided by the invention, the amino acid sequence is shown as Cys-Trp-His-Thr-Asp-Thr-Arg-Ser-Cys; or the amino acid sequence is subjected to substitution, deficiency or addition of one or more amino acids. By the polypeptide combined with various amyloid protein monomers simultaneously and the application thereof, an organism can be effectively stimulated to generate a specific antibody combined with various amyloid protein transitional monomers, and the antibody can inhibit aggregation and cytotoxicity of amyloid protein.

Description

technical field [0001] The invention belongs to the field of peptide-containing pharmaceutical preparations, and specifically relates to a polypeptide that simultaneously binds to amyloid monomers and its application; the invention can be applied to the preparation of antibodies that recognize a variety of amyloid transition state monomers, amyloid Disease diagnosis, treatment and experimental research. Background technique [0002] Over the years, various diseases caused by the aggregation of amyloid or polypeptide itself have caused more and more harm to human health. Some protein monomers are non-toxic or have little toxicity, but they can aggregate into oligomers or fibrils with toxic effects and cause a series of diseases, such as Beta-amyloid (A-Beta ) caused by Alzheimer's disease (commonly known as senile dementia) (Alzheimer' disease, AD), caused by alpha-synuclein (Parkinson' disease, PD), by prion protein (Prion protein (PrP)) Caused at least ten kinds of enceph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K16/00A61K39/00A61P3/10A61P25/00A61P25/16A61P25/28A61P43/00
Inventor 刘瑞田薛頔
Owner TSINGHUA UNIV
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