CN112858685A - Neurodegenerative disease marker beta-spectrin and application thereof - Google Patents

Abstract

The invention provides a neurodegenerative disease marker beta-spectrin and application thereof, wherein the marker comprises phosphorylated beta-spectrin protein. The phosphorylation of the beta-spectrin protein peptide segment is used as a marker of neurodegenerative diseases, and the protein is used for judging and evaluating the symptoms of the neurodegenerative diseases such as AD, PD and the like; the constructed kit sample is simple in pretreatment process, small in sample consumption and high in accuracy, and has important clinical guidance significance for auxiliary diagnosis of AD and PD related indexes.

Description

Neurodegenerative disease marker beta-spectrin and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a neurodegenerative disease marker beta-spectrin and application thereof.

Background

Neurodegenerative diseases refer to diseases caused by neurons of the brain and spinal cord and/or the loss of myelin sheath thereof, and clinically include Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (Huntington), Multiple Sclerosis (MS), and the like.

Alzheimer's Disease (AD), also known as senile dementia, is a progressive degenerative disease of the central nervous system occurring in the elderly, characterized by progressive memory impairment, cognitive decline and loss of daily living ability, accompanied by neuropsychiatric symptoms such as personality changes, seriously affecting social and living abilities, and has become a major public health problem affecting the world. The Alzheimer disease is developed after the age of more than 65 years, however, with the development of society, the living rhythm of people is accelerated, the working pressure is high, and diet and work and rest are irregular, so that the sick people suffering from AD tend to be younger and the development age is reduced to 40-50 years. Because the pathogenesis of the Alzheimer disease is not completely clear and the early symptoms are secret, patients with the Alzheimer disease are easy to miss diagnosis or misdiagnose. Therefore, the method finds the biomarkers with high sensitivity and high accuracy, and has important significance for early diagnosis and drug intervention of the Alzheimer's disease. At present, the diagnosis of AD mainly depends on memory scale, PET, and pathological indicators such as a β, phosphorylated tau in cerebrospinal fluid and blood, but the clinical application of these diagnostic indicators is still controversial, and there is no effective detection basis for early diagnosis of AD.

Parkinson's Disease (PD) is a disease in which motor dysfunction and eventually cognitive dysfunction, most commonly found in the elderly, has an average age of 60 years. The most prominent pathological change in parkinson's disease is degenerative death of mesocerebral nigral Dopamine (DA) neurons, thereby causing a significant reduction in striatal DA content. At present, the exact cause of the pathological changes is not clear, and the diagnosis of PD depends primarily on medical history, clinical symptoms and signs. Since PD is a chronic progressive disease and the disease progression rate varies among patients, early detection of preclinical patients has become one of the hot spots in the study of PD.

CN107449831A discloses the use of protein markers for Alzheimer's disease selected from the group consisting of odour-binding protein 2b, acidic mammalian chitinase, acidic sphingomyelin phosphodiesterase 3a, methyldopa-A-alpha subunit, insulin-like growth factor binding protein 7, procollagen C endonuclease enhancer 1, lactadherin, galactocerebrosidase, alpha-N-acetylglucosaminidase, annexin A11, protein Afamin, lysosomal-ProX-carboxypeptidase, N-acetylgalactosan-6-sulfatase, deoxyribonuclease-1, aminopeptidase N, angiotensin converting enzyme, beta-hexosaminidase beta subunit, sphingolipid activating proprotein, kallikrein-1, pancreatic alpha amylase, heme-binding protein, serum transferrin, Alpha 1 acid glycoprotein 1, fibronectin, leucine-rich alpha 2-glycoprotein, beta 2 glycoprotein 1, immunoglobulin gamma-2 chain C region, poly immunoglobulin receptor, complement protein C3, alpha 2 macroglobulin, alpha-1 antichymotrypsin, alpha 2 antitrypsin, urokinase-type plasminogen activator, plasminogen, complement protein D, prothrombin, ceruloplasmin, aspartic proteinase A, but the protein markers are obtained by using transgenic mouse model, and are used as markers for the diagnosis of human Alzheimer's disease with low accuracy.

CN106755563A discloses an application of a biomarker in the diagnosis of Parkinson, wherein the biomarker is UROD, the invention discovers that the expression of the UROD is down-regulated in a Parkinson patient for the first time, and the detection of the expression level of the UROD can be one of the indexes of early diagnosis of Parkinson, but the biomarker cannot be used as a marker of other neurodegenerative diseases.

For the reasons, the current neurodegenerative diseases do not have typical diagnosis markers, and the analysis of the change of the markers on the molecular level is beneficial to providing more accurate prognosis and diagnosis information for the neurodegenerative disease patients.

Disclosure of Invention

Aiming at the defects and actual needs of the prior art, the invention provides a neurodegenerative disease marker beta-spectrin and application thereof, wherein the marker beta-spectrin protein is co-localized with a key protein beta-amyloid plaque of AD morbidity and co-localized with a key protein alpha-Synuclein of PD morbidity in neurons, and can be used as a marker of neurodegenerative diseases such as AD, PD and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a neurodegenerative disease marker comprising phosphorylated β -spectrin protein.

In the present invention, Spectrin is a multifunctional neuronal membrane-skeleton protein in the brain, involved in the dynamic regulation of the actin cytoskeleton, and plays an important role in maintaining the cell morphology and the integrity of the membrane structure, and Spectrin protein is composed of α -and β -subunits, wherein Sptbn1(Spectrin beta nonerythrocytic1) is a member of the β -Spectrin gene family, and the co-localization of β -Spectrin and β -amyloid plaques is observed in the brain of sporadic AD patients, suggesting that the protein is a component of β -amyloid plaques, and structural or functional abnormalities thereof may be related to the formation or deposition of β -amyloid plaques, and thus, changes in the expression level thereof may serve as a diagnostic indicator of AD; in addition, studies have shown that beta-Synuclein is co-localized in neurons with a key protein for the onset of PD, alpha-Synuclein has direct interaction, suggesting that alpha-Synuclein may regulate neurite outgrowth by interacting with cytoskeletal proteins such as beta-spectrin, and may cause pathological changes in the actin cytoskeleton and downstream neurotoxicity. Thus, altered levels of β -spectrin expression may also be diagnostic of PD.

Preferably, the phosphorylation site of the beta-spectrin protein is shown as SEQ ID NO. 1;

SEQ ID NO:1：tsskESSPVPSPTLDR。

in the present invention, the first amino acid of the peptide fragment as shown in SEQ ID NO. 1 is indicated by lower case letters, the latter lower case letters indicate amino acids which are phosphorylated, and the upper case letters indicate amino acids which are not phosphorylated.

In a second aspect, the invention provides a beta-spectrin protein detection kit, which comprises a first antibody.

Preferably, the first antibody is a monoclonal antibody to the β -spectrin protein.

Preferably, the concentration of the first antibody is 0.01 to 5. mu.g/mL, and may be, for example, 0.01. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 2.5. mu.g/mL, 3. mu.g/mL, 3.5. mu.g/mL, 4. mu.g/mL, 4.5. mu.g/mL, or 5. mu.g/mL.

Preferably, the kit further comprises an enzyme-labeled secondary antibody.

Preferably, the enzyme-labeled secondary antibody comprises HRP-labeled goat anti-mouse IgG and/or HRP-labeled goat anti-rabbit IgG.

Preferably, the concentration of the enzyme-labeled secondary antibody is 0.01-5. mu.g/mL, for example, 0.01. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL, 2.5. mu.g/mL, 3. mu.g/mL, 3.5. mu.g/mL, 4. mu.g/mL, 4.5. mu.g/mL, or 5. mu.g/mL.

In the invention, an enzyme-labeled secondary antibody is diluted by adopting HRP protective solution according to the volume ratio of the enzyme-labeled secondary antibody to the protective solution of 1 (2000-10000), and the specification of the enzyme-labeled secondary antibody is 12mL per bottle; the preparation method of the HRP protective solution comprises the following steps: to 1g BSA and 2.5g EDTA was added 800mL 1 XPBS to 1000 mL.

Preferably, the kit further comprises Phos-tag^TMAny one or the combination of at least two of prefabricated gel, positive control, negative control, antibody diluent, developing solution, stopping solution, confining solution or washing solution.

In the present invention, Phos-tag^TMThe preformed gel is a metal chelate, Phos-tag, with a specific affinity for compounds bearing phosphate groups^TMThe prefabricated gel contains zinc ions and is added with 50 mu mol/L of Phostag in advance^TMAcrylamide can be directly used after being unpacked, and Phos-tag in SDS-PAGE gel during electrophoresis^TMThe phosphorylated protein is captured, and the phosphorylated protein is separated from the non-phosphorylated protein by slowing the migration rate of the phosphorylated protein.

Preferably, the positive control is a beta-spectrin protein standard.

Preferably, the concentration of the positive control is 50-100 ng/mL, for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

Preferably, the negative control is bovine serum albumin.

Preferably, the concentration of the negative control is 50-100 ng/mL, for example, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.

In the invention, the positive control adopts an antibody diluent to dilute the beta-spectrin standard substance according to the volume ratio of 1 (20-100), and the specification is 1mL per tube; the negative control substance adopts an antibody diluent to dilute the negative protein according to the volume ratio of 1 (20-100), and the specification is 1mL per tube.

The kit has high sensitivity and good accuracy, and can be used for auxiliary diagnosis of AD, PD and other neurodegenerative diseases.

In a third aspect, the present invention provides a method of using the kit of the second aspect, the method comprising the steps of:

(1) pre-treating a sample to be detected, and loading the sample to Phos-tag^TMPreparing glue, simultaneously arranging a positive control group and a negative control group, carrying out polypropylene gel electrophoresis, and transferring the protein on the electrophoresis gel to a pvdf membrane or an NC membrane;

(2) adding a first antibody, incubating and washing;

(3) adding enzyme-labeled secondary antibody, incubating and washing;

(4) and (4) measuring a protein band of the sample, and analyzing the phosphorylation level of the protein.

Preferably, the sample to be tested is derived from any one of body fluid, blood or tissue or a combination of at least two of them.

In a fourth aspect, the present invention provides a marker according to the first aspect and/or a kit according to the second aspect, for use in the preparation of a neurodegenerative disease detection reagent and/or a detection medicament.

Preferably, the neurodegenerative disease includes any one of alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, or multiple sclerosis, or a combination of at least two thereof.

Compared with the prior art, the invention has the following beneficial effects:

(1) the phosphorylation of the beta-spectrin protein peptide segment SEQ ID NO:1 is used as a marker of neurodegenerative diseases, and the marker is used for judging and evaluating the symptoms of the neurodegenerative diseases such as AD, PD and the like;

(2) the invention is based on Phos-tag^TMThe SDS-PAGE technology, the kit sample constructed by taking the beta-spectrin protein as the marker has simple pre-treatment process, small sample consumption, high precision and auxiliary treatmentThe diagnosis assistance of the related indexes of AD and PD has important clinical guidance significance.

Drawings

FIG. 1 is a graph showing the expression level of phosphorylated β -selectin protein in AD model mice and wild-type mice;

FIG. 2 shows the results of quantitative analysis of the protein bands in FIG. 1.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

EXAMPLE 1 sample Collection and processing

(1) Collecting

The experimental mice (AD model mice and wild type mice) are anesthetized with isoflurane, the neck is cut off and killed, the head is cut off, the hippocampal tissue of the brain of the mice is stripped by a small forceps, the hippocampal tissue is put into an EP tube and then quickly frozen on dry ice or in liquid nitrogen, and the hippocampal tissue is stored in a refrigerator at the temperature of minus 80 ℃ for standby.

(2) Protein extraction and quantification

Taking out the sample, thawing the sample on ice, adding a proper amount of lysate (7M urea, 2M thiourea and 0.1% CHAPS), and uniformly mixing the lysate and the CHAPS in a vortex manner; ultrasonic 60s, 0.2s on, 2s off, 22% amplitude; extracting at room temperature for 30 min;

centrifuging at 4 deg.C for 20min at 15,000g, carefully taking out supernatant, packaging, and freezing at-80 deg.C;

the extracted proteins were quantified using the Bradford method, and then all samples of the AD model group and the healthy control group were mixed in-group, respectively.

(3) Proteolytic cleavage

Putting 200 μ g of protein solution into a centrifuge tube, adding 4 μ L of reducing agent, reacting at 60 deg.C for 1h, adding 2 μ L of cysteine blocking solution, and incubating at room temperature for 10 min; adding the protein solution after reductive alkylation into a 10K ultrafiltration tube, centrifuging for 20min at 12,000 revolutions, and discarding the solution at the bottom of the collection tube; adding 100 μ L of dissolving Buffer (Dissolution Buffer) in iTRAQ kit, centrifuging for 20min at 12,000 rpm, discarding the solution at the bottom of the collection tube, and repeating for 3 times; replacing a new collecting pipe, adding 4 mu g of trypsin into the ultrafiltration pipe, wherein the mass ratio of the trypsin to the protein is 1:50, the volume is 50 mu L in total, and reacting at 37 ℃ overnight; centrifuging for 20min at 12,000 rpm the next day, and centrifuging the peptide fragment solution after enzymolysis and digestion at the bottom of a collecting pipe; add 50. mu.L lysis Buffer (lysis Buffer5) into the ultrafiltration tube, centrifuge for 20min at 12,000 rpm, combine with the above steps, collect the bottom of the tube to obtain 100. mu.L protein sample after enzymolysis.

(4) iTRAQ sample labeling

Taking out of the refrigerator

The reagent is balanced to room temperature and then centrifuged to ensure

Centrifuging the reagent to the bottom of the tube; to each tube

Adding 150 mu L of isopropanol into the reagent, carrying out vortex oscillation, and centrifuging to the bottom of the tube; transfer 50. mu.L of sample (100. mu.g of enzymatic hydrolysate) to a new centrifuge tube and add

The reagent is vortexed and oscillated, centrifuged to the bottom of the tube, and reacted for 2 hours at room temperature; adding 100 mu L of water to stop the reaction; and (4) carrying out vortex oscillation on the mixed and marked sample, centrifuging to the bottom of the tube, carrying out vacuum freezing and centrifugal drying, and storing for later use.

Example 2LC-MS/MS Mass Spectrometry analysis and data processing

Separating by using 400 mu g of BSA subjected to enzymolysis (the column temperature is 45 ℃, and the detection wavelength is 214nm), and detecting the condition of the system; the mixed labeled sample was dissolved with 100. mu.L of mobile phase A (0.1% formic acid) and separated with a linear gradient of mobile phase B (80% acetonitrile and 0.1% formic acid), centrifuged at 14,000g for 20min, and the supernatant was taken and separated with a flow rate of 0.7 mL/min; redissolving the components obtained by high pH reverse phase separation with 20 μ L of 2% methanol and 0.1% formic acid, centrifuging for 10min at 12,000 rpm, sucking the supernatant to obtain a sample with a volume of 10 μ L, and loading by a sandwich method. The flow rate of the Loading Pump (Loading Pump) was 350nL/min, the holding time was 15min, and the separation flow rate was 350 nL/min.

Mass spectrometry of iTRAQ was performed by an ABI-5600 mass spectrometer, and the resulting mass spectra raw file was processed using the commercial software ProteinPilot available from ABI corporation.

As a result, compared with normal mice, the peptide fragment of the beta-spectrin protein SEQ ID NO 1 of the beta-spectrin protein in the hippocampal region of the early-stage AD model mice shows that the phosphorylation level of phosphorylation sites is reduced, and the peptide fragment of the beta-spectrin protein is reduced by 0.621 times.

Example 3 protein level validation on AD model

In the embodiment, the phosphorylation level of the beta-spectrin protein is verified by adopting western blot, and the expression level of the phosphorylated beta-spectrin protein is detected by using Phos-tagSDS-PAGE (Phos-tagSDS-PAGE), so that the phosphorylation beta-spectrin protein of a 3-month-old AD model mouse (APP/PS1) is obviously reduced relative to a Wild type mouse (Wild-type) and the level of the total beta-spectrin protein is kept unchanged as shown in figure 1; the results of quantitative analysis of protein bands in FIG. 1 are shown in FIG. 2, in which the relative expression levels (Fold change) of p- β -selectin and total β -selectin proteins were obtained from p- β -selectin/β -selectin and β -selectin/GAPDH, respectively. Since 3-month-old AD model mice do not have cognitive impairment symptoms, but have abnormal synaptic function, the change of phosphorylation level of beta-spectrin protein is suggested to precede the change of protein level, so that the phosphorylated beta-spectrin can be used as a biological index related to the abnormal synaptic function of AD cognitive impairment.

In conclusion, the phosphorylation of the peptide segment SEQ ID NO. 1 of the beta-spectrin protein is used as a marker of neurodegenerative diseases, and the protein is used for judging and evaluating the symptoms of the neurodegenerative diseases such as AD, PD and the like; the invention is based on Phos-tag^TMSDS-PAGE technology, kit sample prophase constructed by taking beta-spectrin protein as markerSimple process, less sample consumption and high accuracy, and has important clinical guidance significance for auxiliary diagnosis of AD and PD related indexes.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> neurodegenerative disease marker beta-spectrin and application thereof

<130> 20191128

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 16

<212> PRT

<213> Artificial Synthesis

<400> 1

Thr Ser Ser Lys Glu Ser Ser Pro Val Pro Ser Pro Thr Leu Asp Arg

1 5 10 15

Claims (10)

1. A neurodegenerative disease marker comprising phosphorylated β -selectin protein.

2. The marker according to claim 1, wherein the phosphorylation site of phosphorylated β -spectrin protein is shown as SEQ ID NO 1.

3. A beta-spectrin protein detection kit, which is characterized by comprising a first antibody;

preferably, the first antibody is a monoclonal antibody for the β -spectrin protein;

preferably, the concentration of the first antibody is 0.01-5 mug/mL.

4. The kit of claim 3, further comprising an enzyme-labeled secondary antibody;

preferably, the enzyme-labeled secondary antibody comprises HRP-labeled goat anti-mouse IgG and/or HRP-labeled goat anti-rabbit IgG;

preferably, the concentration of the enzyme-labeled secondary antibody is 0.01-5 mu g/mL.

5. The kit according to claim 3 or 4, characterized in that it further comprises Phos-tag^TMAny one or the combination of at least two of prefabricated gel, positive control, negative control, antibody diluent, developing solution, stopping solution, confining solution or washing solution.

6. The kit according to any one of claims 3 to 5, wherein the positive control is a β -spectrin protein standard;

preferably, the concentration of the positive control is 50-100 ng/mL.

7. The kit of any one of claims 3-6, wherein the negative control is bovine serum albumin;

preferably, the concentration of the negative control substance is 50-100 ng/mL.

8. A method of using the kit of any one of claims 3 to 7, wherein the method comprises the steps of:

(1) pre-treating a sample to be detected, and loading the sample to Phos-tag^TMPreparing glue, simultaneously arranging a positive control group and a negative control group, carrying out polypropylene gel electrophoresis, and transferring the protein on the electrophoresis gel to a pvdf membrane or an NC membrane;

(2) adding a first antibody, incubating and washing;

(3) adding enzyme-labeled secondary antibody, incubating and washing;

(4) and (4) measuring a protein band of the sample, and analyzing the phosphorylation level of the protein.

9. The method of claim 8, wherein the sample to be tested is derived from any one of a body fluid, blood or tissue or a combination of at least two thereof.

10. Use of a marker according to claim 1 or 2 and/or a kit according to any one of claims 3 to 7 for the preparation of a neurodegenerative disease detection reagent and/or a detection medicament;

preferably, the neurodegenerative disease includes any one of alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, or multiple sclerosis, or a combination of at least two thereof.

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