CN114106141A - Preparation method of beta-amyloid protein polymer, product and application thereof - Google Patents
Preparation method of beta-amyloid protein polymer, product and application thereof Download PDFInfo
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- CN114106141A CN114106141A CN202111520894.3A CN202111520894A CN114106141A CN 114106141 A CN114106141 A CN 114106141A CN 202111520894 A CN202111520894 A CN 202111520894A CN 114106141 A CN114106141 A CN 114106141A
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
Abstract
The invention provides a preparation method of a beta-amyloid polymer, a product and an application thereof, wherein the preparation method of the beta-amyloid polymer comprises the following steps: (1) preparing a peptide membrane from beta-amyloid; (2) dissolving the peptide membrane obtained in the step (1), and then adopting a buffer solution for incubation; (3) separating the incubated solution in the step (2), and collecting the precipitate to obtain insoluble beta-amyloid protein polymer; (4) resuspending the insoluble beta-amyloid polymer obtained in step (3), and performing intermittent ultrasonication to obtain a beta-amyloid collection. The method has simple and easy process, can control the size of the polymer, and the prepared beta-amyloid polymer has better uniformity, and the preparation method can realize the standardized and precise preparation of stable beta-amyloid in different polymerization forms.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a beta-amyloid protein polymer, and a product and application thereof.
Background
Alzheimer Disease (AD) is one of the most common neurodegenerative diseases, mainly manifested by decreased cognitive function, memory impairment and decreased daily living capacity. At present, no radical treatment medicine exists in the Alzheimer disease, and the medicine seriously threatens the health of human beings. In the process of developing the Alzheimer disease medicine, establishing a stable and effective disease model plays a very important role in the promotion of subsequent experiments.
CN101948523A discloses an artificial in vitro preparation method and use of low molecular weight amyloid peptide oligomer, the preparation method comprises: the method comprises the steps of obtaining an A beta polypeptide monomer by using a gene recombination method, dissolving the A beta polypeptide monomer by using anhydrous dimethyl sulfoxide, and then placing the dissolved A beta polypeptide monomer in artificial cerebrospinal fluid for reaction, so that the A beta polypeptide monomer is naturally polymerized to form oligomer. When the amyloid peptide oligomer is assembled in vitro by taking the A beta peptide monomer as a raw material, the composition of artificial cerebrospinal fluid is improved by simulating in vivo physiological conditions, and the obtained product is stable and uniform.
CN112219120A discloses a kit for screening progressive development of alzheimer's disease. The kit comprises reagents for measuring the expression levels of a 56kDa amyloid-beta oligomer and a 72kDa amyloid-beta oligomer in a nasal secretion sample, and the progressive development of Alzheimer's disease is judged by measuring the amount of amyloid-beta oligomer.
The beta-amyloid oligomer is possibly related to Alzheimer disease, an Alzheimer disease experimental model is established through the beta-amyloid oligomer, and the beta-amyloid oligomer plays an important role in developing medicaments for treating the Alzheimer disease. The good experimental model not only plays a crucial role in promoting the experimental process, but also determines the accuracy and repeatability of the experimental result to a great extent.
Currently, chemically synthesized beta-amyloid is mainly used to prepare simple polymers, polymers and insoluble fibers and to establish experimental models of alzheimer's disease. However, the beta-amyloid protein has strong adsorbability, is easy to adsorb to the wall of a tube, has unstable protein property, and is easy to polymerize into a polymer. The existing beta-amyloid oligomer synthesis technology is unstable, the preparation process is easily influenced by various conditions such as temperature, pH value and the like, A beta polymers prepared in different laboratories at different time can generate completely different structures and functions, and the prepared A beta polymers have uncertainty and have obvious differences in the aspects of molecular weight, conformation, neurotoxicity and the like.
Therefore, it is of great interest to develop a method for simultaneously preparing different forms of stable amyloid-beta polymers.
Disclosure of Invention
The invention aims to provide a preparation method of a beta-amyloid protein polymer, and a product and application thereof. The method has simple and easy process, can simultaneously prepare the insoluble beta-amyloid polymer and the beta-amyloid aggregate, can control the size of the polymer, and the prepared beta-amyloid polymer has better uniformity, can be stored in solution for a long time, and has stable property and convenient use. The preparation method can realize the standardized and precise preparation of stable beta-amyloid in different polymerized forms.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a β -amyloid polymer, comprising the steps of:
(1) preparing a peptide membrane from beta-amyloid;
(2) dissolving the peptide membrane obtained in the step (1), and then adopting a buffer solution for incubation;
(3) separating the incubated solution in the step (2), and collecting the precipitate to obtain insoluble beta-amyloid protein polymer;
(4) and (4) resuspending the insoluble beta-amyloid polymer obtained in the step (3), and intermittently carrying out ultrasonic disruption to obtain a beta-amyloid collection.
The method comprises the steps of preparing beta-amyloid into a peptide membrane, dissolving the obtained peptide membrane, incubating the peptide membrane in a buffer solution, polymerizing the beta-amyloid in a sample into beta-amyloid fibrils, centrifuging at a high speed, separating supernatant in the solution by using an SEC chromatographic column, wherein beta-amyloid monomers are positioned in a small molecular weight component, soluble beta-amyloid oligomers are positioned in a high molecular weight component, breaking the beta-amyloid fibrils by ultrasonic waves to obtain a beta-amyloid aggregate, and separating the obtained beta-amyloid aggregate by using a chromatographic method to obtain the beta-amyloid monomers and oligomers. Two different forms of beta-amyloid polymer can be obtained simultaneously by means of polymerization and redispersion, and the beta-amyloid polymer has better uniformity. The amyloid beta polymers described in the present invention include insoluble amyloid beta polymers, amyloid beta aggregates and soluble amyloid beta oligomers.
Preferably, in the step (1), the preparation method of the peptide membrane comprises the following steps: the beta-amyloid protein was mixed with HFIP and incubated, and after evaporation of HFIP, dried to give a peptide membrane.
In the present invention, β -amyloid is dissolved by HFIP (hexafluoroisopropanol), and HFIP is evaporated to give a peptide membrane.
Preferably, the beta-amyloid protein comprises A beta 1-42 or A beta 40.
Preferably, the beta-amyloid protein is subjected to equilibrium before mixing, the equilibrium temperature is 25-37 ℃, for example, 25 ℃, 27 ℃, 29 ℃, 30 ℃, 31 ℃, 33 ℃, 35 ℃ or 37 ℃, and the equilibration time is 15-20 min, for example, 15min, 16min, 17min, 18min, 19min or 20 min.
The beta-amyloid protein is placed in a refrigerator at-20 ℃ before being mixed, and needs to be balanced for a period of time at room temperature before being mixed with HFIP.
Preferably, the HFIP is subjected to an ice bath before mixing, the temperature of the ice bath is 0 to 4 ℃, for example, 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃, and the time of the ice bath is more than 1h, for example, 1h, 1.5h, 2h or 2.5 h.
Preferably, the concentration of β -amyloid in the mixed solution obtained by the mixing is 0.5 to 2mM, and may be, for example, 0.5mM, 1mM, 1.5mM, or 2 mM.
Preferably, the incubation temperature is 35-37 ℃, for example, 35 ℃, 36 ℃ or 37 ℃, and the incubation time is 1-2 h, for example, 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h or 2 h.
In the invention, the self-polymerized A beta monomer can be released by incubating beta-amyloid at 35-37 ℃ by using HFIP.
Preferably, the temperature of the evaporation is 35-37 ℃, for example, 35 ℃, 36 ℃ or 37 ℃, and the time of the evaporation is 4-12 h, for example, 4h, 6h, 8h, 10h or 12 h.
Preferably, the drying is carried out in the presence of a drying agent comprising any one of silica gel, activated alumina or calcium chloride or a combination of at least two thereof.
Preferably, the drying temperature is-20 to-10 ℃, for example, -20 ℃, -18 ℃, -16 ℃, -14 ℃, -12 ℃ or-10 ℃ and the like, and the drying time is 8 to 12 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours and the like.
Preferably, in step (2), the solvent used for the dissolution is DMSO.
Preferably, in the step (2), the concentration of the peptide film in the solution obtained by the dissolution is 50 to 100. mu.g/. mu.L, for example, 50. mu.g/. mu.L, 60. mu.g/. mu.L, 70. mu.g/. mu.L, 80. mu.g/. mu.L, 90. mu.g/. mu.L, or 50. mu.g/. mu.L.
In the invention, the peptide membrane is a beta-amyloid protein membrane obtained by incubating beta-amyloid protein through HFIP and drying.
Preferably, the buffered solution comprises any one of HEPES, PBS, DMEM/F12 or Ham's F12 or a combination of at least two thereof, preferably Ham's F12.
In the present invention, the Ham's F12 does not contain phenol red, and the beta-amyloid forms fibers in the buffer solution.
Preferably, the peptide membrane is contained in the buffer solution in an amount of 0.1 to 0.2% by mass, for example, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, or the like.
Preferably, the incubation temperature is 0-4 ℃, for example, 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃, and the incubation time is 12-24 h, for example, 12h, 14h, 16h, 18h, 20h, 22h or 24 h.
Preferably, in step (3), the separation mode is centrifugal separation.
Preferably, the rotation speed of the centrifugation is 10000rpm or more, such as 10000rpm, 11000rpm, 12000rpm or 13000rpm, the time of the centrifugation is 10-15 min, such as 10min, 11min, 12min, 13min, 14min or 15min, and the temperature of the centrifugation is 0-4 ℃, such as 0 ℃, 1 ℃, 2 ℃, 3 ℃ or 4 ℃.
Preferably, in step (3), the isolated precipitate comprises insoluble beta-amyloid polymer and the isolated supernatant comprises soluble beta-amyloid oligomers.
In the present invention, the supernatant obtained by centrifugation contains the beta-amyloid monomer and some oligomers of soluble beta-amyloid.
Preferably, in the step (3), the soluble β -amyloid oligomer is separated from the supernatant obtained by the separation by using a chromatography column.
Preferably, in step (4), the resuspended medium comprises water and/or PBS buffer.
Preferably, in step (4), the frequency of the intermittent ultrasonication is 20000 to 60000Hz, and for example, 20000Hz, 30000Hz, 40000Hz, 50000Hz, 60000Hz, etc.
Preferably, in the step (4), the total time of the intermittent ultrasonic disruption is 2-5 min, such as 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5 min.
Preferably, in the step (4), the ultrasonic time of the intermittent ultrasonic crushing is 5-10 s, such as 5s, 6s, 7s, 8s, 9s or 10s, and the intermittent time of the intermittent ultrasonic crushing is 5-10 s, such as 5s, 6s, 7s, 8s, 9s or 10 s.
In the present invention, the desired size of beta-amyloid can be controlled according to the electron microscope results by adjusting the incubation time and the ultrasonic conditions.
As a preferred embodiment of the present invention, the method for preparing the amyloid-beta polymer comprises the steps of:
(1) preparation of beta-amyloid into peptide membranes:
the method comprises the steps of balancing beta-amyloid at 25-37 ℃ for 15-20 min, carrying out ice bath on HFIP at 0-4 ℃ for more than 1h, mixing the beta-amyloid with the HFIP, incubating at 35-37 ℃ for 1-2 h, evaporating the HFIP, drying, wherein the evaporation temperature is 35-37 ℃ and the evaporation time is 4-12 h, and obtaining a peptide membrane, wherein the drying treatment temperature is-20-10 ℃ and the drying treatment time is 8-12 h.
(2) Dissolving the peptide membrane obtained in the step (1), and then adopting a buffer solution for incubation:
dissolving a peptide membrane by using a solvent, wherein the concentration of the peptide membrane in DMSO is 50-100 mug/muL, incubating for 12-24 h at 0-4 ℃ by using a buffer solution, and the mass percentage of the peptide membrane in the buffer solution is 0.1-0.2%.
(3) Separating the solution incubated in the step (2), and collecting the precipitate to obtain insoluble beta-amyloid protein polymer:
centrifuging the incubated solution for 10-15 min under the conditions that the rotating speed is more than 10000rpm and the temperature is 0-4 ℃, wherein the precipitate obtained by separation comprises insoluble beta-amyloid protein polymers, the supernatant obtained by separation comprises soluble beta-amyloid protein oligomers, and separating the supernatant obtained by separation by adopting a chromatographic column to obtain the soluble beta-amyloid protein oligomers.
(4) Resuspending the insoluble beta-amyloid polymer obtained in step (3), and performing intermittent ultrasonication to obtain a beta-amyloid collection:
and (3) resuspending the insoluble beta-amyloid polymer obtained in the step (3) by using a medium, and intermittently ultrasonically crushing for 2-5 min under the condition that the frequency is 20000-60000 Hz, wherein the ultrasonic time of the intermittent ultrasonic crushing is 5-10 s, and the intermittent time is 5-10 s, so as to obtain a beta-amyloid collection.
In a second aspect, the present invention provides an insoluble amyloid-beta polymer prepared by the method for preparing an amyloid-beta polymer according to the first aspect.
In a third aspect, the present invention provides a β -amyloid collection prepared by the method for preparing a β -amyloid polymer according to the first aspect.
In the present invention, the amyloid beta set is a mixture of oligomers of soluble amyloid beta and oligomers of insoluble amyloid beta, and the oligomers of soluble amyloid beta are separated by centrifugation, and the precipitate obtained by centrifugation is insoluble oligomers of amyloid beta, and the supernatant obtained by centrifugation is soluble oligomers of amyloid beta.
In a fourth aspect, the present invention provides a soluble amyloid beta oligomer prepared by the method for preparing a amyloid beta polymer according to the first aspect.
In a fifth aspect, the present invention provides the use of an insoluble amyloid beta polymer according to the second aspect, a collection of amyloid beta proteins according to the third aspect or a soluble amyloid beta oligomer according to the fourth aspect for the modeling of a disease.
Preferably, the disease model comprises an alzheimer's disease model.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the preparation method disclosed by the invention, the beta-amyloid is polymerized into an insoluble beta-amyloid polymer, and then the beta-amyloid polymer is prepared in an ultrasonic smashing mode, so that the obtained beta-amyloid polymer has better uniformity.
(2) The preparation method can ensure that the beta-amyloid polymers in different forms are prepared from the same batch of beta-amyloid monomers, and the initial states of the materials are the same.
(3) The beta-amyloid protein polymer prepared by the method has stable property, better guarantees unique variables for subsequent experiments, and the prepared samples are more diverse and can be used for establishing a subsequent Alzheimer disease model, quantifying the samples and the like.
Drawings
FIG. 1 is a graph showing the results of transmission electron microscopy examination of insoluble A β 1-42 polymers in test example 1.
FIG. 2 is a graph showing the results of transmission electron microscopy examination of the A β 1-42 pool in test example 1.
FIG. 3 is a graph showing the result of Western blotting in test example 2.
FIG. 4 is a graph showing the result of Western blotting in test example 3.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the field or according to the product specification. The reagents or the manufacturer without labels are all conventional products which can be purchased through regular channels.
In the following examples and comparative examples, the sources of the materials, reagents and equipment are as follows:
the A beta 1-42 used in the invention is synthesized by Hubei Qiangyao Biotechnology limited, and the amino acid sequence of the A beta 1-42 is as follows: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA are provided.
Example 1
This example provides a method for preparing a polymer of β -amyloid protein, a β 1-42, which can prepare insoluble a β 1-42 polymer, a β 1-42 monomer, soluble a β 1-42 oligomer, and a β 1-42 pool. The preparation method of the beta-amyloid protein polymer comprises the following steps:
(1) preparing A beta 1-42 into a peptide membrane:
equilibrating Abeta 1-42 at 25 deg.C for 15min, ice-cooling HFIP at 4 deg.C for 1h, mixing Abeta 1-42 with HFIP to obtain a mixture, incubating at 35 deg.C for 1h with Abeta 1-42 concentration of 1mM, evaporating HFIP, drying at-20 deg.C for 6h to obtain peptide membrane, and drying at-20 deg.C for 12 h.
(2) After the peptide membrane was dissolved, incubation was performed with buffer:
peptide membranes were dissolved in DMSO at a concentration of 50 μ M and incubated with Ham 'sF12 (phenol red free) at 4 ℃ for 12h, at a mass percentage of 0.1% in Ham's F12.
(3) Separating the solution incubated in the step (2), and collecting the precipitate to obtain insoluble beta-amyloid protein polymer:
centrifuging the incubated solution for 10min at the rotation speed of 13000rpm and the temperature of 4 ℃, wherein the precipitate obtained by separation comprises insoluble Abeta 1-42 polymer, the supernatant obtained by separation comprises Abeta 1-42 monomer and soluble Abeta 1-42 oligomer, and separating the Abeta 1-42 monomer and the soluble Abeta 1-42 oligomer from the supernatant obtained by separation by adopting SEC chromatographic columns.
(4) Resuspending the insoluble Abeta 1-42 polymer obtained in step (3), and performing intermittent ultrasonication to obtain an Abeta 1-42 aggregate:
and (3) resuspending the insoluble Abeta 1-42 polymer obtained in the step (3) with a PBS buffer solution, and carrying out intermittent ultrasonic crushing for 2min under the condition that the frequency is 60000Hz, wherein the ultrasonic time of the intermittent ultrasonic crushing is 10s, and the intermittent time is 10s, so as to obtain a soluble Abeta 1-42 set.
Example 2
This example provides a method for preparing a polymer of β -amyloid protein, a β 1-42, which can prepare insoluble a β 1-42 polymer, a β 1-42 monomer, soluble a β 1-42 oligomer, and a β 1-42 pool. The preparation method of the beta-amyloid protein polymer comprises the following steps:
(1) preparation of beta-amyloid into peptide membranes:
equilibrating Abeta 1-42 at 37 deg.C for 20min, ice-cooling HFIP at 4 deg.C for more than 2h, mixing Abeta 1-42 and HFIP to obtain a mixture, incubating at 37 deg.C for 2h with Abeta 1-42 concentration of 200 μ M, evaporating HFIP, drying at 35 deg.C for 8h to obtain peptide membrane, and drying at-20 deg.C for 8 h.
(2) After the peptide membrane was dissolved, incubation was performed with buffer:
peptide membranes were dissolved in DMSO at a concentration of 80 μ M and incubated with Ham 'sF12 (phenol red free) at 4 ℃ for 24h, at a mass percent of 0.2% in Ham's F12.
(3) Separating the solution after the incubation in the step (2), and collecting precipitates to obtain insoluble Abeta 1-42 polymers:
centrifuging the incubated solution for 15min at the rotation speed of 12000rpm and the temperature of 4 ℃, wherein the separated precipitate comprises insoluble Abeta 1-42 polymer, the separated supernatant comprises Abeta 1-42 monomer and soluble Abeta 1-42 oligomer, and separating the Abeta 1-42 monomer and the soluble Abeta 1-42 oligomer from the separated supernatant by using SEC chromatographic columns.
(4) Resuspending the insoluble Abeta 1-42 polymer obtained in step (3), and performing intermittent ultrasonication to obtain an Abeta 1-42 aggregate:
and (3) re-suspending the insoluble Abeta 1-42 polymer obtained in the step (3) with water, and carrying out intermittent ultrasonic crushing for 5min under the condition that the frequency is 40000Hz, wherein the ultrasonic time of the intermittent ultrasonic crushing is 5s, and the intermittent time is 5s, so as to obtain an Abeta 1-42 set.
Example 3
This example provides a method for preparing a polymer of β -amyloid protein, a β 1-42, which can prepare insoluble a β 1-42 polymer, a β 1-42 monomer, soluble a β 1-42 oligomer, and a β 1-42 pool. The preparation method of the beta-amyloid protein polymer comprises the following steps:
(1) preparing A beta 1-42 into a peptide membrane:
equilibrating Abeta 1-42 at 30 deg.C for 18min, ice-cooling HFIP at 4 deg.C for 1.5 hr or more, mixing Abeta 1-42 with HFIP to obtain a mixture, incubating at 36 deg.C for 1.5 hr with Abeta 1-42 concentration of 150 μ M, evaporating HFIP, drying at 36 deg.C for 10 hr to obtain peptide membrane, and drying at-20 deg.C for 10 hr.
(2) After the peptide membrane was dissolved, incubation was performed with buffer:
peptide membranes were dissolved in DMSO at a concentration of 100. mu.M and incubated with Ham 'sF12 (without phenol red) at 0 ℃ for 18h, at a mass percentage of 0.15% in Ham's F12 (without phenol red).
(3) Separating the solution after the incubation in the step (2), and collecting precipitates to obtain insoluble Abeta 1-42 polymers:
centrifuging the incubated solution for 12min at the rotation speed of 12500rpm and the temperature of 4 ℃, separating the obtained precipitate to obtain insoluble Abeta 1-42 polymer, separating the obtained supernatant to obtain Abeta 1-42 monomer and soluble Abeta 1-42 oligomer, and separating the separated supernatant to obtain the Abeta 1-42 monomer and the soluble Abeta 1-42 oligomer by using SEC chromatographic columns.
(4) Resuspending the insoluble Abeta 1-42 polymer obtained in step (3), and performing intermittent ultrasonication to obtain an Abeta 1-42 aggregate:
and (3) resuspending the insoluble Abeta 1-42 polymer obtained in the step (3) with water, and carrying out intermittent ultrasonic crushing for 4min under the condition that the frequency is 50000Hz, wherein the ultrasonic time of the intermittent ultrasonic crushing is 6s, and the intermittent time is 6s, so as to obtain an Abeta 1-42 set.
Example 4
This example provides a method for preparing a β -amyloid polymer, which differs from example 1 only in that a β 1-42 is replaced with a β 40, and the other preparation steps are the same as example 1. The beta-amyloid protein polymer similar to that in example 1 can be obtained by using Abeta 40 as a starting raw material, and comprises insoluble Abeta 40 polymer, Abeta 40 aggregate, Abeta 40 monomer and Abeta 40 oligomer, and the Abeta 40 polymer obtained in the example has stable properties and is convenient to use.
Example 5
This example provides a method for preparing a β -amyloid polymer, which differs from example 1 only in that in step (2), the incubation buffer Ham's F12 is replaced with PBS buffer, and the other preparation steps are the same as example 1. Similar beta-amyloid polymer as in example 1 was obtained using PBS buffer as incubation matrix, and the obtained beta-amyloid polymer also had good homogeneity.
Comparative example 1
This comparative example provides a method for preparing a beta-amyloid monomer, the beta-amyloid being a β 1-42, the method comprising the steps of:
(1) adding 1mg of A beta 1-42 into 220 mu L of HFIP, and incubating for 30min at 25 ℃ to completely dissolve the A beta 1-42;
(2) transferring the solution obtained in the step (1) into a silicified centrifugal tube, and thoroughly drying HFIP by using a nitrogen blowing instrument to obtain a peptide membrane;
(3) dissolving the obtained peptide membrane in 40 mu L of DMSO, and adding HEPES buffer solution with the pH value of 7.4 and the concentration of L0 mM to make the final volume of the solution be 1mL, wherein the mass percentage content of the peptide membrane in HEPES is 0.1%;
(4) and (4) oscillating the solution obtained in the step (3) for 24 hours at the temperature of 37 ℃ and rotating at the speed of 600rpm to obtain the beta-amyloid protein monomer.
Comparative example 2
This comparative example provides a method of preparing a polymer of amyloid beta, which is a β 1-42, comprising the steps of:
(1) adding 1mg of A beta 1-42 into 220 mu L of HFIP, and incubating for 30min at 25 ℃ to completely dissolve the A beta 1-42;
(2) transferring the solution obtained in the step (1) into a silicified centrifugal tube, and thoroughly drying HFIP by using a nitrogen blowing instrument to obtain a peptide membrane;
(3) dissolving a peptide membrane by using 40 mu L DMSO, and adding the dissolved peptide membrane into a buffer solution to ensure that the final volume of the solution is 1mL, wherein the mass percentage content of the peptide membrane in HEPES is 0.1%, and the composition of the buffer solution is as follows: NaCl 125mmol/L, KCl 3.3.3 mmol/L, KH2PO4 1.2mmol/L，NaHCO3 26mmol/L、CaCl2 2.5mmol/L、MgSO42.4mmol/L, glucose 5mmol/L and amino acid 0.3 g/L.
(4) And (4) oscillating the solution obtained in the step (3) for 24 hours at the temperature of 37 ℃ and rotating at the speed of 600rpm to obtain the beta-amyloid protein polymer.
Comparative example 1A beta 1-42 and HEPES buffer solution are mixed, shake incubation is carried out to obtain A beta 1-42 monomers, oligomers including dimers or trimers and the like exist in the obtained monomer solution; comparative example 2 A.beta.1-42 was incubated with a specific buffer to give a beta-amyloid polymer, but the homogeneity and stability thereof could not be ensured. Both methods can only prepare one form of polymer at a time, while the preparation method described in example 1 can prepare insoluble a β 1-42 polymer, a β 1-42 aggregate and a β 1-42 monomer and soluble a β 1-42 oligomer at the same time, and the purity of the obtained a β 1-42 monomer is higher by separating and purifying the supernatant using SEC chromatography column in example 1. And the obtained A beta collections are scattered from insoluble A beta 1-42 polymers, so that the obtained samples are more stable and uniform in source.
Test example 1
Test samples: insoluble A β 1-42 polymers and A β 1-42 pools from example 1.
The test method comprises the following steps: and observing and recording the appearance of each sample by adopting a transmission electron microscope.
The transmission electron microscope results of the insoluble A β 1-42 polymer obtained in example 1 are shown in FIG. 1, and it can be seen from the figure that a fibrous β -amyloid polymer, i.e., an insoluble A β 1-42 polymer, is formed in the precipitate. The transmission electron microscope results of the A.beta.1-42 pools obtained in example 1 are shown in FIG. 2, from which it can be seen that the A.beta.1-42 pools were obtained after batch sonication of the precipitates.
Test example 2
The supernatant obtained in the centrifugation in the step (3) in example 1 was passed through a SEC column, and the fractions obtained were analyzed by western blotting.
The western blotting method comprises the following steps:
(1) preparing glue and loading: samples were loaded with Sample Buffer at 1:1 using 16% Tris-Tricine gel.
(2) Electrophoresis: constant pressure was used first 75V, 15min, then 125V, 120 min.
(3) Film transfer: and (3) opening the electric transfer printing clamp after taking out the gel, filling a special sponge pad on each side, putting a piece of filter paper respectively, flatly putting the gel on the filter paper on the cathode side, finally flatly putting the NC film on the gel, removing bubbles, clamping the electric transfer printing clamp, and performing transfer printing for 2 hours by using a constant current of 120 mA.
(4) Blocking was performed with 1% bovine serum albumin for one hour at room temperature.
(5) The A.beta.antibodies m266 and 4G8 were incubated together overnight at 4 ℃.
(6) Wash with PBST for 1h, with PBST changed every 1 h.
(7) And (4) incubating for 1h at normal temperature by using a secondary antibody of anti-Mouse-HRP.
(8) Wash with PBST for 1h, with PBST changed every 1 h.
(9) Color development was performed using TMB.
The western blot results are shown in FIG. 3. from FIG. 3, it can be seen that different groups of western blots were obtained after passing the supernatant through the SEC column, wherein lanes 7 and 8 are oligomers, and lanes 17-19 are monomers, the molecular weight of which is 4 kDa.
Test example 3
The insoluble a β 1-42 polymer and a β 1-42 pool obtained in example 1 were analyzed by western blotting, the existing a β 1-42 monomer was used as a standard, the experimental procedure was referred to test example 2, and the western blotting results are shown in fig. 4, wherein lane 1, lane 2: insoluble a β 1-42 polymer, lane 3, lane 4: a β 1-42 pool, lane 5, lane 6, lane 7: a beta 1-42 monomer.
In conclusion, the preparation method of the beta-amyloid polymer provided by the invention is simple and easy to use, can simultaneously prepare the insoluble beta-amyloid polymer and the beta-amyloid aggregate, can control the size of the polymer, and has better uniformity. The beta-amyloid protein polymer prepared by the method has stable property, better guarantees unique variables for subsequent experiments, and the prepared samples are more diverse and can be used for establishing a subsequent Alzheimer's disease model, quantifying the samples and the like.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. A method for preparing a β -amyloid polymer, comprising the steps of:
(1) preparing a peptide membrane from beta-amyloid;
(2) dissolving the peptide membrane obtained in the step (1), and then adopting a buffer solution for incubation;
(3) separating the incubated solution in the step (2), and collecting the precipitate to obtain insoluble beta-amyloid protein polymer;
(4) resuspending the insoluble beta-amyloid polymer obtained in step (3), and performing intermittent ultrasonication to obtain a beta-amyloid collection.
2. The method for preparing a β -amyloid polymer according to claim 1, wherein the method for preparing the peptide membrane in step (1) comprises the steps of:
mixing beta-amyloid protein with HFIP, incubating, evaporating HFIP, and drying to obtain a peptide membrane;
preferably, the beta-amyloid protein comprises a β 1-42 or a β 40;
preferably, the beta-amyloid needs to be balanced before mixing, the balancing temperature is 25-37 ℃, and the balancing time is 15-20 min;
preferably, the HFIP needs to be subjected to ice bath before mixing, the temperature of the ice bath is 0-4 ℃, and the time of the ice bath is more than 1 h;
preferably, in the mixed solution obtained by mixing, the concentration of the beta-amyloid is 0.5-2 mM;
preferably, the incubation temperature is 35-37 ℃, and the incubation time is 1-2 h;
preferably, the evaporation temperature is 35-37 ℃, and the evaporation time is 4-12 h;
preferably, the drying is carried out in the presence of a drying agent, wherein the drying agent comprises any one or a combination of at least two of silica gel, activated alumina or calcium chloride;
preferably, the drying temperature is-20 to-10 ℃, and the drying time is 8 to 12 hours.
3. The method for preparing a β -amyloid polymer according to claim 1 or 2, wherein in the step (2), the solvent used for the dissolution is DMSO;
preferably, in the step (2), the concentration of the peptide membrane in the solution obtained by dissolving is 50-100 mug/muL;
preferably, the buffered solution comprises any one of HEPES, PBS, DMEM/F12 or Ham's F12 or a combination of at least two thereof, preferably Ham's F12;
preferably, the mass percentage content of the peptide membrane in the buffer solution is 0.1-0.2%;
preferably, the incubation temperature is 0-4 ℃, and the incubation time is 12-24 h.
4. The method for preparing a β -amyloid polymer according to any one of claims 1 to 3, wherein in the step (3), the separation is performed by centrifugation;
preferably, the rotating speed of the centrifugation is more than 10000rpm, the time of the centrifugation is 10-15 min, and the temperature of the centrifugation is 0-4 ℃;
preferably, in step (3), the isolated precipitate comprises insoluble beta-amyloid polymer and the isolated supernatant comprises soluble beta-amyloid oligomer;
preferably, in the step (3), the soluble β -amyloid oligomer is separated from the supernatant obtained by the separation by using a chromatography column.
5. The method for preparing a β -amyloid polymer according to any one of claims 1 to 4, wherein in step (4), the resuspended medium comprises water and/or PBS buffer.
6. The method for preparing a β -amyloid polymer according to any one of claims 1 to 5, wherein in step (4), the frequency of the intermittent ultrasonication is 20000 to 60000 Hz;
preferably, in the step (4), the total time of the intermittent ultrasonic crushing is 2-5 min;
preferably, in the step (4), the ultrasonic time of the intermittent ultrasonic crushing is 5-10 s, and the intermittent time of the intermittent ultrasonic crushing is 5-10 s.
7. An insoluble amyloid-beta polymer, wherein the insoluble amyloid-beta polymer is produced by the method for producing an amyloid-beta polymer according to any one of claims 1 to 6.
8. A amyloid beta-assembly prepared by the method for preparing the amyloid beta polymer according to any one of claims 1 to 6.
9. A soluble amyloid-beta oligomer prepared by the method for preparing the amyloid-beta polymer according to any one of claims 1 to 6.
10. Use of the insoluble amyloid beta polymer of claim 7, the amyloid beta collection of claim 8, or the soluble amyloid beta oligomer of claim 9 in the establishment of a disease model;
preferably, the disease model comprises an alzheimer's disease model.
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|---|---|---|---|---|
| US5854215A (en) * | 1995-03-14 | 1998-12-29 | Praecis Pharmaceuticals Incorporated | Modulators of β-amyloid peptide aggregation |
| JP2008032554A (en) * | 2006-07-28 | 2008-02-14 | Japan Science & Technology Agency | BIOCHIP AND AMYLOID SENSOR USING beta-AMYLOID OLIGOMER, AND DETECTION METHOD OF beta-AMYLOID EXISTING IN BIOSAMPLE |
| US20090087916A1 (en) * | 2004-03-31 | 2009-04-02 | Angesmg, Inc. | Assay method for identifying drug candidate |
| US20100240868A1 (en) * | 2005-06-30 | 2010-09-23 | Henryk Mach | Composition and Method for a Producing Stable Amyloid Beta Oligomers of High Molecular Weight |
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|---|---|---|---|---|
| US5854215A (en) * | 1995-03-14 | 1998-12-29 | Praecis Pharmaceuticals Incorporated | Modulators of β-amyloid peptide aggregation |
| US20090087916A1 (en) * | 2004-03-31 | 2009-04-02 | Angesmg, Inc. | Assay method for identifying drug candidate |
| US20100240868A1 (en) * | 2005-06-30 | 2010-09-23 | Henryk Mach | Composition and Method for a Producing Stable Amyloid Beta Oligomers of High Molecular Weight |
| JP2008032554A (en) * | 2006-07-28 | 2008-02-14 | Japan Science & Technology Agency | BIOCHIP AND AMYLOID SENSOR USING beta-AMYLOID OLIGOMER, AND DETECTION METHOD OF beta-AMYLOID EXISTING IN BIOSAMPLE |
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