CN109490527B

CN109490527B - Application of DBN1 protein in preparation of neurodegenerative disease diagnosis drug

Info

Publication number: CN109490527B
Application number: CN201811325523.8A
Authority: CN
Inventors: 池在龙; 甘宜静
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2020-01-21
Anticipated expiration: 2038-11-08
Also published as: CN109490527A

Abstract

The invention provides an application of DBN1 protein in preparing a neurodegenerative disease diagnosis medicament, wherein the application in diagnosis comprises early diagnosis of neurodegenerative diseases and evaluation of nerve injury severity; the diagnosis and evaluation method is carried out by measuring the content of DBN1 protein in peripheral blood plasma, and DBN1 protein of patients with neurodegenerative diseases has obvious increased expression; DBN1 protein is used as a neurodegenerative disease marker protein, and the DBN1 protein content in the blood of a patient is detected by an enzyme-linked immunosorbent assay quantitative kit to evaluate clinical symptoms and judge whether neurodegenerative diseases exist or not before clinical examination changes occur and monitor the nerve injury degree; the detection means is convenient on the whole, and secondary damage to patients can not be caused, so that the method has important clinical significance and social and economic benefits on prediction, prevention and evaluation of related diseases.

Description

Application of DBN1 protein in preparation of neurodegenerative disease diagnosis drug

Technical Field

The invention relates to the technical field of medical biology, in particular to application of DBN1 protein in preparation of a neurodegenerative disease diagnosis medicine.

Background

Neurodegenerative Diseases (Neurodegenerative Diseases) refer to degenerative Diseases of the nervous system caused by degeneration and apoptosis of neurons. The clinical symptoms and pathological anatomy are divided into the following groups:

1. dyspostural and motor syndromes, e.g. Parkinson's disease, multiple system atrophy

2. Progressive dementia syndromes, e.g. Alzheimer's disease, frontotemporal dementia

3. Progressive dementia with other neurological abnormalities, e.g. Huntington's chorea, degeneration of the basal cortex

4. Progressive ataxia syndrome, e.g. spinocerebellar ataxia

5. Slowly progressing muscle weakness and muscular atrophy syndrome, e.g. motor neuron disease

6. Sensory and motor disorders, e.g. hereditary motor sensory neuropathy (CMT)

7. Progressive blindness or external ophthalmoplegia, e.g. glaucoma, hereditary optic neuropathy

8. Syndromes characterised by sensorineural deafness, e.g. hereditary hearing loss with atrophy of the nervous system

It has been found that Tau protein plays an important role in axonal conduction by regulating the dynamic stability of neuronal microtubules, and tauopathies have been studied extensively in neurodegenerative diseases such as retinal diseases and Alzheimer's Disease (AD), but have been limited to studies in retinal tissues and cerebrospinal fluid. Its expression level in brain or retinal tissue of patients with neurodegenerative diseases and in cerebrospinal fluid (CSF) is significantly increased. The detection of expression in brain or retinal tissues requires pathological specimens, and cerebrospinal fluid also requires lumbar puncture for acquisition, which causes burden to patients and has difficulty in clinical application.

On the other hand, glaucoma, as a retinal neurodegenerative disease, can only be clinically examined to find and evaluate the degree of optic nerve damage, and no effective biomarker is available for diagnosis and evaluation at present. However, some patients cannot accurately observe the fundus and accurately evaluate the degree of nerve damage due to turbid refractive medium and the like. More seriously, because there are no symptoms at an early stage, many patients have lost 30-50% of retinal ganglion cells when they are diagnosed as glaucoma by the hospital. Can develop a biochemical marker which can be applied to early diagnosis and evaluation of nerve injury, and has great clinical significance.

The research finds that the brain development regulatory protein Drebrin (also called DBN1) plays an important role in the central nervous system as a nerve skeleton protein actin regulatory protein and is related to the occurrence and development of neurodegenerative diseases. Thus, there is a need in the art for a diagnosis and risk of neurodegenerative disease in a population, i.e. for an assessment and guidance of the risk of suffering from said disease for a precise treatment regimen. The need for assessment of the severity of the disease and/or the risk of prediction of the disease provides a need for a detection method that is convenient to detect on a detection means without causing secondary harm to the patient. The method has important clinical significance and social and economic benefits for prediction and prevention of related diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a new application of DBN1 protein so as to provide a convenient and rapid method and a medicament for diagnosing neurodegeneration.

To achieve the above objects, the present invention provides use of the DBN1 protein in the preparation of a medicament for the diagnosis of neurodegenerative diseases, including early diagnosis of neurodegenerative diseases and assessment of the severity of nerve damage, by determining the content of DBN1 protein in peripheral blood plasma.

In embodiments of the invention, wherein the neurodegenerative disease includes dyspostural and motor syndromes, including parkinson's disease, multiple system atrophy; progressive dementia syndromes, including alzheimer's disease, frontotemporal dementia; progressive dementia with other neurological abnormalities including huntington's disease, corticobasal degeneration; progressive ataxia syndrome, including spinocerebellar ataxia; slowly progressing muscle weakness and muscle wasting syndrome, including motor neuron disease; sensory and motor disorders, including hereditary motor sensory neuropathy (CMT); progressive blindness or external ophthalmoplegia, including glaucoma, hereditary optic neuropathy; syndromes characterized by neurosensory deafness, including hereditary hearing loss with atrophy of the nervous system; and other neurodegenerative diseases.

In an embodiment of the invention, wherein progressive blindness includes especially primary angle-closure glaucoma and primary angle-opening glaucoma in glaucoma disease.

In an embodiment of the invention, wherein said diagnosis is detected by an enzyme linked immunosorbent assay kit.

In an embodiment of the invention, wherein said medicament comprises a kit.

In an embodiment of the invention, wherein the kit is an enzyme linked immunosorbent assay kit.

In an embodiment of the invention, wherein said kit comprises an antibody to said DBN1 protein.

In an embodiment of the invention, wherein the antibody comprises capture antibody a and/or enzyme-labeled detection antibody B.

In an embodiment of the invention, the kit further comprises a pre-coated plate, a standard product, a coating buffer solution, a sample diluent, a confining liquid, horseradish peroxidase, an eluent and a color-developing agent.

On the basis of research, the DBN1 protein plays an important role in the central nervous system, the DBN1 protein in blood is denatured in the nervous system, and the expression of the DBN1 protein in the blood is increased, so that experiments prove that the DBN1 protein of patients with neurodegenerative diseases has obvious expression increasing conditions, the DBN1 protein is used as a neurodegenerative disease marker protein, the DBN1 protein content in the blood of the patients is detected by an enzyme-linked immunosorbent assay quantitative detection kit to diagnose whether the patients have the neurodegenerative diseases, the detection means is convenient to detect on the whole, secondary damage to the patients is avoided, and the important clinical significance and the social and economic benefits on the prediction and prevention of related diseases are achieved.

Drawings

FIG. 1 shows the Western Blot experiment results of the retinas of the animal models with optic nerve clamping injuries of 3d, 7d and 14d and the control group;

FIG. 2 is the result of Western Blot assay quantitative analysis of optic

nerve clamp injuries

3d, 7d, 14d and control retinas of animal models;

FIG. 3 is a graph showing the change of the expression level of DBN1 protein in serum of an animal model of optic nerve clamping injury;

FIG. 4 is a graph showing the results of quantitative analysis of immunofluorescent staining of Neuro-2A cells treated with the cytotoxic agent NMDA and DBN1 protein of the control group;

FIG. 5 is a graph of the thickness of an optic nerve fiber layer in each quadrant for a patient with open, closed, or ciliary beating after dividing the thickness into eight quadrants;

FIG. 6 shows the expression level of DBN1 protein in the plasma of glaucoma patients and controls;

FIG. 7 is a graph showing the plasma Tau expression levels in glaucoma patients and in control groups;

FIG. 8 is a graph of the percentage of normal range, cut-off and significant thinning in the eight quadrants of the thickness of the optic nerve fiber layer in patients with positive and negative DBN1 protein;

FIG. 9 is a Logistic regression analysis chart of the DBN1 protein expression level and the optic nerve fiber layer thickness.

Detailed Description

The present invention will be described in further detail with reference to examples.

The invention relates to a new discovery that DBN1 protein is highly expressed in neurodegenerative diseases, is secreted by cells and enters blood circulation, so that the protein level in the serum of patients with neurodegenerative diseases is increased. Therefore, the DBN1 protein can be used as a novel neurodegenerative disease serum marker and can be used for screening, tracking, early diagnosis, treatment guidance and prognosis judgment of neurodegenerative diseases.

The amino acid sequence of the DBN1 protein is shown in a sequence table SEQ ID NO: 1 is shown.

The kit for detecting neurodegenerative diseases comprises a capture antibody A which specifically recognizes DBN1 protein, wherein the capture antibody A is coated on a pre-coated plate.

The kit for detecting the serum DBN1 protein antigen by using the ELISA detection technology is developed based on the DBN1 protein-resistant specific antibody, and the kit is suitable for screening, tracking and early diagnosis of neurodegenerative diseases and guiding treatment and judging prognosis.

The ELISA kit for detecting DBN1 protein antigen provided by the invention is characterized by mainly comprising: the kit comprises a pre-coated plate coated with a DBN1 protein capture specific antibody, an enzyme-labeled detection antibody, a standard product, a coating buffer solution, a sample diluent, a confining liquid, horseradish peroxidase, an eluent and a color developing agent. Both the capture antibody and the detection antibody can be combined with DBN1 protein antigen.

Example 1 discloses the purification of antigens and the preparation of antibodies;

1. expression, purification and identification of DBN1 protein:

the DBN1 protein is obtained from saccharomyces cerevisiae which is genetically engineered in the previous research, and is subjected to galactose-induced overexpression, agarose affinity medium (glutathione) separation and purification, and Western-Blotting identification.

2. Preparation of serum samples:

placing the whole blood sample at room temperature for 2 hours or overnight at 4 ℃, centrifuging at 1000g for about 20 minutes, and taking the supernatant to immediately detect; or subpackaging, and storing at-20 deg.C or-80 deg.C, but avoiding repeated freeze thawing. The thawed sample should be centrifuged again and then examined. The samples tested did not contain NaN3, as NaN3 inhibited the activity of horseradish peroxidase (HRP).

Preparation methods of various buffers and reagents of ELISA method:

(1) coating buffer solution: 0.05M Na2CO3-NaHCO3 pH 9.6

(2) Sample diluent: pH 7.4PBS solution

(3) Eluent: PBST solution pH 7.4

(4) Sealing liquid: 0.5% BSA in PBS pH 7.4

(5) Color developing agent: developer A and developer B (for on-site use)

(6) Stopping liquid: 2mol/L H2SO4 solution (concentrated sulfuric acid is slowly dropped into distilled water in timing and mixed with the distilled water at the same time)

ELISA method for determination of anti-DBN 1 protein IgG antibody concentration in serum to aid early diagnosis of gastric cancer: the specific operation steps are as follows:

(1) coating: diluting the purified human DBN1 protein solution to 1 mu g/mL by using a coating buffer solution, adding the diluted solution into a 96-well enzyme label plate, wherein each well is 100 mu L, and coating the solution at 37 ℃ for 2 hours or overnight at 4 ℃; washing the plate for 3 times by using a washing liquid, and spin-drying;

(2) and (3) sealing: adding 200 mu L of confining liquid, and keeping the temperature at room temperature for 2 hours; washing the plate for 3 times by using a washing liquid, and spin-drying;

(3) diluting and loading the standard substance and the sample: the standard and the serum sample to be tested are diluted to 100 mu L with sample buffer solution at a ratio of 1: 100, and added into the respective antigen determination well plate. And (3) paying attention to no air bubbles, adding the sample to the bottom of the hole of the quincunx standard plate in a sample adding mode, keeping the sample from touching the hole wall as far as possible, slightly shaking and uniformly mixing, and covering or laminating a film on the ELISA plate. If the serum samples to be detected are more, a multi-tube micro liquid adding device is recommended to be used for sample adding. The standard substance and the sample to be detected are prepared within 15 minutes before use, are discarded after use, and the freshly prepared standard substance is used for next detection.

(4) And (3) incubation: the ELISA plate is placed at 37 ℃ for reaction for 120 minutes, and liquid in the hole is completely thrown away without washing.

(5) Adding an enzyme: 100 μ L of DBN1 protein IgG antibody labeled with horseradish peroxidase was added to each well at 37 ℃ for 60 minutes. And (5) throwing off liquid in the holes, and patting the holes dry for 5 times with the upper washing plate.

(6) Color development: after the mixture is patted dry, 50 mu L of color developing agent A is firstly dropped into each hole, 50 mu L of color developing agent B is added, the mixture is gently shaken and uniformly mixed, and the mixture is shaded at 37 ℃ for developing for 15 minutes.

(7) And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well in sequence. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. The stop solution should be added as soon as the substrate reaction time is reached.

(8) And (4) judging a result:

i. the optical density (OD value) of each well was measured sequentially at a wavelength of 450nm using an enzyme-linked analyzer.

A450 is an abbreviation for absorbance at 450 nm.

At present, no international reference standard exists for DBN1 protein antibodies, so that relative units are adopted for calibrating the detection result.

Determination of the value of anti-DBN 1 protein in serum

Quality control

Each test result must meet the following criteria:

a450 of standard serum 1: less than or equal to 0.100

A450 of standard serum 2: not less than 0.700

If the above criteria are not met, the result is deemed invalid and must be retested.

Example 2 preparation of experimental model and validation of results

Rat optic nerve injury animal model: the temporal side of the eyeball of the rat is cut off the bulbar conjunctiva, the tissues around the eyeball are separated bluntly, and the optic nerve is closed by using a No. 5 surgical forceps at a position 2mm right behind the eyeball for 5 seconds to manufacture an optic nerve injury animal model.

After the optic nerve of the rat is clamped, retinal tissues are taken and subjected to immunoblotting and immunofluorescence staining, the expression condition of the retinal DBN1 protein is detected, and the expression condition is quantified by Image J software.

Neuro-2A cells were treated with 100uM NMDA for 24 hours and their Drebrin levels were measured by immunofluorescence assay.

The results of Western Blot experiments on the retinas of the control group and the

optic nerve block

3d, 7d and 14d, which show that the DBN1 protein is mostly distributed in the retinal ganglion cell layer and the inner plexiform layer, are shown in FIGS. 1-2: the expression level of DBN1 protein in retinal tissues gradually decreases. Heart blood is taken through a rat model, placed in a refrigerator for 2 hours, centrifuged at 2000g and 4 ℃ for 10 minutes, transferred to a 1.5ml EP tube, stored at-80 ℃, and tested by an ELISA technology for the change of the DBN1 protein expression level in serum of an optic nerve clamping animal model, as shown in figure 3, the DBN1 protein expression level is obviously increased until the day 7 of clamping, and the expression level falls back after the day 14 of clamping. The animal model research confirms that the expression level of DBN1 protein in blood is obviously increased when optic nerve is damaged, and correlation exists between the expression level of the DBN1 protein in the blood and the nerve damage.

Immunofluorescence staining results of Neuro-2A cells treated with the cytotoxic agent NMDA and the control DBN1 protein showed that as shown in fig. 4: in normal Neuro-2A cells, DBN1 protein is mainly distributed at the edge of a cell membrane, and after NMDA treatment, the expression level of DBN1 protein is obviously reduced (P is less than 0.01) and the distribution is changed. As a result of this experiment, it was also found that, as in the in vivo study, the expression level of DBN1 protein decreased when nerve cells were injured, and when it was released outside the cells, the expression level of DBN1 protein in blood increased.

Example 3: exemplary diagnostic applications of the diagnostic kit according to the invention

We collected 232 glaucoma patients including 164 primary angle-closure glaucoma (closed glaucoma, PACG), 46 primary angle-opening glaucoma (open glaucoma, POAG) and 22 glaucoma ciliaris syndrome (celiac, PS) patients, as well as 50 control groups at the eye-vision hospital, western medicine university, wenzhou, from 2016 to 2017, 12 months.

The diagnosis kit provided by the invention detects the expression level of plasma Drebrin by using an enzyme-linked immunosorbent assay (ELISA) kit, and collects and analyzes clinical examination results of intraocular pressure, OCT (optical coherence tomography), visual field and the like of a patient, and the results are shown in table 1:

the visual field index in table 1 is close to 100, which is a normal value, and a smaller value indicates severe visual field damage; the average variation of the visual field index is close to 0 and is normal, and the larger the negative value is, the larger the visual field damage is; the standard variation of the model is close to 0 and is a normal value, and the larger the deviation is, the larger the visual field damage is. Compared with a control group, the visual field index of the patient with trichiasis is not obviously changed, the patient with trichiasis and trichiasis is obviously reduced, the deviation from the normal value is larger, and the visual field damage is more serious; compared with the control group, the thickness of optic nerve fiber layer of the trichiasis patient is not obviously changed, and the thickness of the optic nerve fiber layer of the trichiasis patient is obviously reduced by the trichiasis patient and the trichiasis patient (P is less than 0.0001); the normal value of the cup/disc ratio is about 0.3-0.5, generally more than 0.6, the patient is considered as glaucoma, compared with the normal value, the cup/disc ratio of the patient with trichiasis is higher than 0.5, the cup/disc ratio of the patient with trichiasis is higher than 0.7, and the optic nerve injuries of trichiasis, trichiasis and opening are gradually increased by being proved. FIG. 5 shows the optic nerve fiber layer thickness further divided into eight quadrants, each quadrant exhibiting substantially the thinnest open, second-closed, and third-ciliary. The thinner the optic nerve fiber layer thickness, the more severe the optic nerve damage.

By comparing the ratios of normal range (upper segment), critical value (middle segment) and obvious thinning (lower segment) in eight quadrants of the thicknesses of the optic nerve fiber layers of the patients with glaucoma and the patients with negative DBN1 protein, as shown in fig. 8, the percentage of thinning of the thickness of the patients with positive DBN1 protein is higher than that of the patients with negative DBN1 protein in the IT quadrant, and the significant difference is achieved. The percentage of thickness reduction IN the ST quadrant was also higher than that IN the DBN1 protein negative group, and Logistic regression analysis of DBN1 protein expression and optic nerve fiber layer thickness IN each quadrant showed that P value < 0.05 IN the IN, IT and ST quadrants represents that plasma DBN1 protein expression is increased and the optic nerve fiber layer thickness is reduced, i.e. correlated with nerve injury.

The results of the expression levels of DBN1 in serum of 232 glaucoma patients and 50 control groups are shown in FIG. 6, and compared with the control group, the expression levels of DBN1 in the trichiasis patients are not significantly different, the expression levels of the trichiasis patients are significantly increased (P < 0.05), and the expression levels of the green-switched patients are most significantly increased (P < 0.01);

the 232 glaucoma patients and the control group were tested at the same time, and the expression level of Tau in serum is shown in FIG. 7, and the content of Tau is significantly increased in plasma of all of the patients with trichiasis, anuria and olemia (P < 0.001, P < 0.0001) relative to the expression level of DBN1 protein;

the results show that the glaucoma patient can be identified by checking the expression level of the DBN1 protein in the serum, and the damage degree of the optic nerve of the glaucoma can be identified. In contrast, the detection of serum Tau expression levels did not correctly reflect the severity of optic nerve injury.

From the above, it is obvious to those skilled in the art that the DBN1 protein has obvious differential expression in patients with primary angle-closure glaucoma and patients with primary open-angle glaucoma, and is clearly closely related to the occurrence and development of glaucoma, and accordingly, antibodies specific to the DBN1 protein, including various monoclonal antibodies and polyclonal antibodies, can be used for detecting patients with primary angle-closure glaucoma and primary open-angle glaucoma, or for preparing reagents or kits for detecting patients with primary angle-closure glaucoma and primary open-angle glaucoma, because they can be used for detecting the expression level of the DBN1 protein.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

SEQUENCE LISTING

<110> Wenzhou university of medical science

Application of <120> DBN1 protein in preparation of neurodegenerative disease diagnosis drug

<141>2018-10-24

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<170>SIPOSequenceListing 1.0

<210>1

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Met Ala Gly Val Ser Phe Ser Gly His Arg Leu Glu Leu Leu Ala Ala

15 10 15

Tyr Glu Glu Val Ile Arg Glu Glu Ser Ala Ala Asp Trp Ala Leu Tyr

20 25 30

Thr Tyr Glu Asp Gly Ser Asp Asp Leu Lys Leu Ala Ala Ser Gly Glu

35 40 45

Gly Gly Leu Gln Glu Leu Ser Gly His Phe Glu Asn Gln Lys Val Met

50 55 60

Tyr Gly Phe Cys Ser Val Lys Asp Ser Gln Ala Ala Leu Pro Lys Tyr

65 70 75 80

Val Leu Ile Asn Trp Val Gly Glu Asp Val Pro Asp Ala Arg Lys Cys

85 90 95

Ala Cys Ala Ser His Val Ala Lys Val Ala Glu Phe Phe Gln Gly Val

100 105 110

Asp Val Ile Val Asn Ala Ser Ser Val Glu Asp Ile Asp Ala Gly Ala

115 120 125

Ile Gly Gln Arg Leu Ser Asn Gly Leu Ala Arg Leu Ser Ser Pro Val

130 135 140

Leu His Arg Leu Arg Leu Arg Glu Asp Glu Asn Ala Glu Pro Val Gly

145 150 155 160

Thr Thr Tyr Gln Lys Thr Asp Ala Ala Val Glu Met Lys Arg Ile Asn

165170 175

Arg Glu Gln Phe Trp Glu Gln Ala Lys Lys Glu Glu Glu Leu Arg Lys

180 185 190

Glu Glu Glu Arg Lys Lys Ala Leu Asp Glu Arg Leu Arg Phe Glu Gln

195 200 205

Glu Arg Met Glu Gln Glu Arg Gln Glu Gln Glu Glu Arg Glu Arg Arg

210 215 220

Tyr Arg Glu Arg Glu Gln Gln Ile Glu Glu His Arg Arg Lys Gln Gln

225 230 235 240

Thr Leu Glu Ala Glu Glu Ala Lys Arg Arg Leu Lys Glu Gln Ser Ile

245 250 255

Phe Gly Asp His Arg Asp Glu Glu Glu Glu Thr His Met Lys Lys Ser

260 265 270

Glu Ser Glu Val Glu Glu Ala Ala Ala Ile Ile Ala Gln Arg Pro Asp

275 280 285

Asn Pro Arg Glu Phe Phe Lys Gln Gln Glu Arg Val Ala Ser Ala Ser

290 295 300

Ala Gly Ser Cys Asp Val Pro Ser Pro Phe Asn His Arg Pro Gly Ser

305 310 315 320

His Leu Asp Ser His Arg Arg Met Ala Pro Thr Pro Ile Pro Thr Arg

325330 335

Ser Pro Ser Asp Ser Ser Thr Ala Ser Thr Pro Val Ala Glu Gln Ile

340 345 350

Glu Arg Ala Leu Asp Glu Val Thr Ser Ser Gln Pro Pro Pro Leu Pro

355 360 365

Pro Pro Pro Pro Pro Ala Gln Glu Thr Gln Glu Pro Ser Pro Ile Leu

370 375 380

Asp Ser Glu Glu Thr Arg Ala Ala Ala Pro Gln Ala Trp Ala Gly Pro

385 390 395 400

Met Glu Glu Pro Pro Gln Ala Gln Ala Pro Pro Arg Gly Pro Gly Ser

405 410 415

Pro Ala Glu Asp Leu Met Phe Met Glu Ser Ala Glu Gln Ala Val Leu

420 425 430

Ala Ala Pro Val Glu Pro Ala Thr Ala Asp Ala Thr Glu Ile His Asp

435 440 445

Ala Ala Asp Thr Ile Glu Thr Asp Thr Ala Thr Ala Asp Thr Thr Val

450 455 460

Ala Asn Asn Val Pro Pro Ala Ala Thr Ser Leu Ile Asp Leu Trp Pro

465 470 475 480

Gly Asn Gly Glu Gly Ala Ser Thr Leu Gln Gly Glu Pro Arg Ala Pro

485 490495

Thr Pro Pro Ser Gly Thr Glu Val Thr Leu Ala Glu Val Pro Leu Leu

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Asp Glu Val Ala Pro Glu Pro Leu Leu Pro Ala Gly Glu Gly Cys Ala

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Thr Leu Leu Asn Phe Asp Glu Leu Pro Glu Pro Pro Ala Thr Phe Cys

530 535 540

Asp Pro Glu Glu Val Glu Gly Glu Ser Leu Ala Ala Pro Gln Thr Pro

545 550 555 560

Thr Leu Pro Ser Ala Leu Glu Glu Leu Glu Gln Glu Gln Glu Pro Glu

565 570 575

Pro His Leu Leu Thr Asn Gly Glu Thr Thr Gln Lys Glu Gly Thr Gln

580 585 590

Ala Ser Glu Gly Tyr Phe Ser Gln Ser Gln Glu Glu Glu Phe Ala Gln

595 600 605

Ser Glu Glu Leu Cys Ala Lys Ala Pro Pro Pro Val Phe Tyr Asn Lys

610 615 620

Pro Pro Glu Ile Asp Ile Thr Cys Trp Asp Ala Asp Pro Val Pro Glu

625 630 635 640

Glu Glu Glu Gly Phe Glu Gly Gly Asp

645

Claims

Use of the DBN1 protein for the preparation of a medicament for the diagnosis of neurodegenerative diseases, which is diagnosed as primary angle-closure glaucoma and primary angle-opening glaucoma among glaucoma diseases, early diagnosis and evaluation of nerve damage severity, the diagnosis being performed by measuring the content of the DBN1 protein in peripheral blood plasma, the DBN1 protein expression level in the peripheral blood plasma being increased relative to the DBN1 protein expression level in a normal group, and the diagnosis being determined as primary angle-closure glaucoma or primary angle-opening glaucoma disease, the DBN1 protein expression level being positively correlated with the nerve damage severity of primary angle-closure glaucoma and primary angle-opening glaucoma.
2. The use of the DBN1 protein of claim 1 for the preparation of a medicament for the diagnosis of neurodegenerative disease, wherein the medicament comprises a kit.
3. The use of the DBN1 protein of claim 2 in the preparation of a medicament for the diagnosis of neurodegenerative disease, wherein the kit is an enzyme linked immunosorbent kit.
4. The use of the DBN1 protein of claim 3 for the preparation of a medicament for the diagnosis of neurodegenerative diseases, wherein the kit comprises an antibody to the DBN1 protein.
5. The use of the DBN1 protein of claim 4 in the preparation of a medicament for the diagnosis of neurodegenerative diseases, wherein the antibody comprises a capture antibody a and/or an enzyme-labeled detection antibody B.
6. The use of the DBN1 protein in the preparation of a medicament for the diagnosis of neurodegenerative disease as claimed in claim 5, wherein the kit further comprises a pre-coated plate, a standard, a coating buffer, a sample diluent, a blocking solution, horseradish peroxidase, an eluent, and a color-developing agent.