CN113354724A

CN113354724A - Sclerostin and application thereof in preparation of related products for treating or preventing Alzheimer's disease

Info

Publication number: CN113354724A
Application number: CN202110467570.1A
Authority: CN
Inventors: 蒋青; 郭保生; 石天舒
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-09-07
Also published as: WO2022227449A1

Abstract

The invention belongs to the field of research on related medicaments for Alzheimer's disease, and particularly discloses sclerostin and application thereof in preparation of related products for treating or preventing Alzheimer's disease. The sclerostin is a protein secreted by bone cells and used for regulating bone mass, and the amino acid sequence of the sclerostin is shown in SEQ NO. 1. Through synthesizing sclerostin heavy protein and a corresponding neutralizing antibody, and cell and zoology experimental verification, the sclerostin recombinant protein can accelerate the generation of Abeta protein outside brain tissues and accelerate the decline of cognitive function; after the neutralizing antibody is added, the generation of A beta protein is inhibited by obviously inhibiting the expression of BACE1 protein, and the effect of relieving Alzheimer's disease is achieved. The neutralizing antibody prepared based on sclerostin has the effect of treatment and/or prevention, can be used for preparing medicines or health-care products for treating and/or preventing Alzheimer's disease, provides a brand-new choice and idea for treating and/or preventing Alzheimer's disease, and widens the selection field for treating Alzheimer's disease.

Description

Sclerostin and application thereof in preparation of related products for treating or preventing Alzheimer's disease

Technical Field

The invention relates to a new application in the field of biological medicine, belongs to the field of research on related medicaments of Alzheimer's disease, and particularly relates to sclerostin (sclerostin) and an application thereof in preparation of related products for treating or preventing Alzheimer's disease.

Background

Alzheimer's Disease (AD) is a progressive neurodegenerative Disease that widely affects the cerebral cortex and hippocampal region of patients, leading to progressive memory impairment of patients, seriously affecting the cognitive function of normal elderly, bringing serious life burden to patients, and also bringing serious economic stress to society. By 2018, approximately 0.44 million people worldwide suffer from alzheimer's disease, and this figure will reach 1.5 million people in 2050, however, the specific mechanism is not well understood and there is no significantly improved drug treatment.

The current causative cause of alzheimer's disease may be related to the accumulation of plaque-like insoluble amyloid beta (a β) in the extracellular space and blood vessel walls and to the increase in neuro-tangles caused by the aggregation of microtubule Tau protein in neurons. The A.beta.protein is cleaved from the APP protein and released into the extracellular environment in the form of diffusible oligomers (A.beta.. O). A β O can be cleared from the brain by APOE-mediated clearance, and also can be taken up by astrocytes via low-density lipoprotein receptor-related protein 1(LRP1) to reduce a β O concentration in brain tissue. In addition, a β O can also aggregate in the intercellular spaces to form fibrillar structures, which are then assembled into plaques. The a β plaques can be cleared by macrophages and microglia through phagocytosis, and can also be degraded by astrocyte endoproteases (IDE, NEP, MMPs). However, it is now considered that in the preclinical phase of alzheimer's disease, a decrease in efficiency of abeta O clearance in cerebrospinal fluid may occur, increasing the cerebrospinal fluid abeta O concentration, which may be associated with dysfunction of these degrading proteases. In addition, not all a β O may be cleared, some oligomers dissociated from a β fibrils may not be cleared, and have some toxicity to neighboring synapses, promoting hyperphosphorylation and aggregation of microtubule-associated Tau proteins, causing them to lose their role in binding to and stabilizing microtubules, aggregating to form paired helical filaments, exacerbating the degeneration of cognitive function.

Sclerostin (sclerostin) is a glycoprotein encoded and translated by a sost gene, and is found to be secreted by osteocytes, liver cells, heart cells and vascular endothelial cells before the adult, and after the adult, the sost gene is mainly expressed by osteocytes and chondrocytes and secreted in blood to regulate the peripheral bone mass. Previous researches find that sclerostin is a specific inhibitor of Wnt signals, can be combined with cell surface LRP5/6, activates downstream GSK-3 beta activity, increases phosphorylation level of beta-catenin, promotes protein degradation of the beta-catenin, and plays a role in inhibiting the Wnt signals. Meanwhile, the Wnt signal pathway plays an important role in regulating and controlling neuronal synaptic plasticity and memory function in the adult brain, previous researches find that in familial AD patients with familial PS1 mutation, the expression of beta-catenin protein is obviously reduced, and LRP6 receptor is specifically knocked out in forebrain neuron cells, so that synaptic plasticity can be obviously damaged, the expression of A beta protein can be increased, and the expression of BACE1 protein can be activated and the formation of A beta protein shearing bodies can be promoted when the Wnt signal is inhibited. Thus, it was suggested that sclerostin may play an important role in the pathogenesis of alzheimer's disease as well as in the production of a β protein.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides sclerostin (sclerostin) and application thereof in preparing related products for treating or preventing Alzheimer's disease, wherein the amino acid sequence of the sclerostin is shown as SEQ NO.1, and the sclerostin is applied to preparing drugs for treating and/or preventing neutralizing antibodies of Alzheimer's disease, shows better effects of reducing the expression of BACE1 in neuronal cells and inhibiting the generation of alpha beta protein, provides a brand new choice and thought for treating and/or preventing Alzheimer's disease, and widens the selection field of treating Alzheimer's disease.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

sclerostin (sclerostin), wherein the amino acid sequence of the sclerostin is shown in SEQ NO. 1.

In an improvement, the nucleotide sequence for coding the sclerostin is shown as SEQ NO. 2.

A recombinant plasmid containing the nucleotide sequence of the sclerostin.

A recombinant strain, which is characterized in that the strain expresses the recombinant plasmid.

The sclerostin, the recombinant plasmid or the recombinant strain are applied to the preparation of a neutralizing antibody for treating or preventing Alzheimer's disease.

A neutralizing antibody characterized by comprising the above-mentioned sclerostin, recombinant plasmid or recombinant strain as an active ingredient.

A product for treating or preventing alzheimer's disease, comprising the above sclerostin, recombinant plasmid, recombinant strain or neutralizing antibody as an active ingredient; the product is a pharmaceutical composition or a health product.

The improved dosage form of the pharmaceutical composition is injection powder, injection solution or powder.

Has the advantages that:

compared with the prior art, the sclerostin (sclerostin) is used as a protein for regulating bone mass secreted by bone cells, the inventor synthesizes recombinant protein by copying nucleotide sequence of the sclerostin, and verifies through experiments of cells and zoology that the sclerostin protein can accelerate the generation of A beta protein outside brain tissues and the decline of cognitive function, and meanwhile, after a synthesized neutralizing antibody is added, the expression of BACE1 protein is obviously inhibited, so that the sclerostin (sclerostin) can inhibit the generation of the A beta protein and has the effect of relieving Alzheimer's disease. Therefore, the neutralizing antibody prepared based on sclerostin has the effect of treatment and/or prevention, can be used for preparing medicines or health-care products for treating and/or preventing the Alzheimer's disease, provides a brand-new choice and idea for treating and/or preventing the Alzheimer's disease, and widens the selection field for treating the Alzheimer's disease.

Drawings

FIG. 1 shows that increasing bone-derived sclerostin significantly increases amyloid deposition and accelerates cognitive function decline in mice with Alzheimer's disease, (A) the level of sclerostin in the serum of mice is significantly increased after the mice are injected with bone-targeted overexpressed sost virus; (B-C) increasing the serum sclerostin protein concentration, significantly increasing the amyloid deposition pattern (B) and statistical (C) in the alzheimer mouse model APP/PS1 mouse; (D-G) increasing the concentration of sclerostin protein in serum, significantly increasing production of abeta 1-40, 1-42 proteins in cerebral cortex and hippocampus in the alzheimer mouse model APP/PS1 mouse; (H) the water maze detection finds that in an Alzheimer mouse model APP/PS1 mouse, the concentration of sclerostin protein in serum is increased, and the spatial memory capacity of the Alzheimer mouse is remarkably damaged; (I) the cognitive detection of new things discovers that in an Alzheimer's disease mouse model APP/PS1 mouse, the concentration of sclerostin protein in serum is increased, and the capability of the Alzheimer's disease mouse for exploring new things is remarkably damaged;

FIG. 2(A) is a diagram showing the change of intracellular BACE1 mRNA levels after 24 hours and 48 hours by adding sclerostin recombinant protein to the neuronal cell line N2 a;

FIG. 2(B) is the expression of BACE1 protein in neuronal cells N2a after addition of 200ng/ml of recombinant sclerostin in N2a cells;

FIG. 2(C) is a graph showing that the addition of 200ng/ml recombinant sclerostin can significantly promote the change of luciferase activity of BACE1 in N2a cells;

FIG. 2(D) shows that 200ng/ml sclerostin recombinant protein was added to the cells of the Alzheimer's neuron cell line N2a-APP, which significantly promoted the protein levels of the protein fragments sAPP β and β -CTF during the production of A β protein;

FIG. 2(E) shows the production of Abeta 1-40 protein by adding 200ng/ml sclerostin recombinant protein to the cells of the Alzheimer's disease neuron cell line N2 a-APP;

FIG. 2(F) shows the production of Abeta 1-42 protein by adding 200ng/ml sclerostin recombinant protein to the cells of the Alzheimer's disease neuron cell line N2 a-APP;

FIG. 3(A) shows BACE1 protein production in neuronal cells N2a by adding sclerostin neutralizing antibody in coculture of neuronal cell line N2a cells and bone cells MLO-Y4;

FIG. 3(B) is the protein fragments sAPP β and β -CTF protein levels during the production of A β protein in N2a-APP cells by adding sclerostin neutralizing antibody in coculture of neuronal cell line N2a-APP cells and bone cells MLO-Y4 in the Alzheimer's disease cell model;

FIG. 3(C) is the change in the levels of A β 1-40 protein in the culture medium by the addition of sclerostin neutralizing antibody in coculture of neuronal cell line N2a-APP cells and osteocytes MLO-Y4 in the Alzheimer's disease cell model;

FIG. 3(D) is the change in the levels of A.beta.1-42 protein in the culture medium when sclerostin neutralizing antibody was added in coculture of the neuronal cell line N2a-APP cell and the osteocyte MLO-Y4 in the Alzheimer's disease cell model.

Detailed Description

The invention is further described below with reference to the accompanying drawings and specific embodiments.

Example 1Sclerostin preparation

The amino acid sequence of the chemically synthesized mouse sclerostin (sclerostin) with the purity of more than 98 percent is shown in SEQ NO. 1:

MQPSLAPCLICLLVHAAFCAVEGQGWQAFRNDATEVIPGLGEYPEPPPENNQTMNRAENGGRPPHHPYDAKDVSEYSCRELHYTRFLTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRVKWWRPNGPDFRCIPDRYRAQRVQLLCPGGAAPRSRKVRLVASCKCKRLTRFHNQSELKDFGPETARPQKGRKPRPGARGAKANQAELENAY

dissolving the synthesized sclerostin powder in double distilled water to obtain solution with concentration of 1 μ g/μ l, packaging, and storing at-80 deg.C. When in use, the extract is diluted to corresponding concentration by double distilled water and stored for later use, and in the embodiment, the extract is diluted to 200 ng/ml.

Example 2 construction of sclerostin neutralizing antibodies based on sclerostin recombinant proteins

According to the amino acid sequence of the mouse sclerostin (sclerostin), a neutralizing antibody which can be injected into a mouse is constructed, and the specific construction method is as follows:

a material preparation stage: immunogen validation

Preparing immunogen: the mouse sclerostin constructed according to example 1 was used as an immunogen, and protein antigens were examined by SDS-PAGE. It is responsible for coupling it to immunolipoplus as an immunogen for breaking the immune tolerance of mice.

Quality Control (QC): the purity was > 75% by polyacrylamide gel electrophoresis (SDS-PAGE).

The first stage is as follows: animal immunization SC2033-PF

Selecting a conventional immunization scheme, and immunizing 3 BALB/C mice and 3C 57 mice by adopting a target protein-immunolplus;

conventional protocol immunization schedule the table below is presented.

Blood sampling detection: 7 days after each immunization, the level of immune response was determined by measuring the serum of the immunized animals by indirect ELISA.

Feeding: mouse feeding

After the end of the first stage of conventional immunization, if the immunized animal is able to reach the level of immune response to the immunogen (OD >1.0, titer 1:8,000), the second stage is entered.

And a second stage: cell fusion and screening

Cell fusion and plating: 2 rounds of cell fusion were performed using the electrofusion method. The average fusion efficiency was that about 2500 spleen cells were used to fuse to generate 1 hybridoma, and thus it was expected that the number of hybridoma cells could reach about 2X 10⁴And (4) respectively. All cells from each round of fusion will be plated in 96-well platesIn, at most 15 96-well plates were plated.

Primary screening: supernatants from the fusion cells were screened by indirect ELISA and supernatants positive for the target protein were selected.

The specific screening scheme is as follows:

and (3) confirmation screening: and screening all positive clones obtained in the primary screening stage by adopting an indirect ELISA method, wherein the positive clones have a better blocking effect on a blocker receptor.

Cloning, expanding culture and freezing storage:

positive mother clone cells were transferred to 24-well plates for expanded culture, up to 10 strains. 2ml of supernatant was collected from each expanded clone for indirect ELISA detection. All specifically positive clonal cells were cryopreserved to avoid clonal loss.

And a third stage: subcloning, scale-up culture and cryopreservation

Subcloning: positive parent clones were subcloned by limiting dilution (up to 5 strains were selected) to ensure that they were derived from individual parent clone cells. Up to 3 rounds of subcloning were performed. The success rate of subcloning was typically 80%. Loss of positive may occur during the subcloning phase. (if the resulting positive parent clone fails to meet the specificity requirements, additional selection of other parent clones will be performed for recloning).

Subclone screening: subclone screening was performed by indirect ELISA and blocking ELISA methods.

Cryopreservation of monoclonal cells: 2 stable daughter clones were selected for each parent clone for amplification and cryopreservation. 5ml of supernatant was collected from each subclone prior to cryopreservation. Each subclone provided 2 tubes of cells.

A fourth stage: small-scale antibody production

Antibody production: and (3) carrying out small-scale antibody production by adopting the fixed strain cells, and carrying out in vitro culture.

Antibody purification: the antibody was purified by Protein A/G affinity chromatography, and the purified antibody was stored in Phosphate Buffered Saline (PBS) by dialysis.

Quality Control (QC): QC detection was performed by polyacrylamide gel electrophoresis (SDS-PAGE), indirect ELISA and blocking ELISA, and antibody concentrations were determined at NanoDrop 2000.

Example 3

A sost overexpression plasmid is constructed according to the amino acid sequence of sclerostin (sclerostin), and is combined with the existing mature Bone-targeted virus AAV9-DSS-Nlter (standard reference: Yang, Y.S., et al (2019). "Bone-targeting AAV-mediated diagnosis of Schnurri-3 precursors Bone loss in osteoporosis." Nat Commun 10(1):2958.), and the construction is called Bone-targeted overexpression sost virus which is used for injecting into mice for relevant experimental verification.

Obtaining a target gene through PCR or enzyme digestion, constructing a target vector through connection or homologous recombination/assembly, and transforming E.coli DH5 alpha competent cells. Transformants were screened by colony PCR and positive clones were sequenced. Plasmid extraction was performed on clones verified to be error-free. In this example, PCR was used for construction.

The target gene and the sequences of the upstream and downstream are inquired from GenBank, and primer design is carried out by using VectorNTI software, wherein the sequences of the primers are shown as follows:

the forward primer is shown as SEQ NO. 2: CGCAAATGGGCGGTAGGCGTG, respectively;

the reverse primer is shown as SEQ NO. 3: CATAGCGTAAAAGGAGCAACA, respectively;

the gene of interest was amplified using the high fidelity PrimeSTAR enzyme, and the reaction system and conditions were as follows.

Preparing a PCR reaction solution according to the following components:

carrying out Agarose Gel electrophoresis on the PCR product to detect the amplification effect, cutting a target gene band from the Gel after the Agarose Gel electrophoresis, using TaKaRa miniBEST Agarose Gel DNA Extraction Kit Ver.3.0 as Gel for recovery, and obtaining a sost overexpression plasmid sequence shown as SEQ NO. 4:

ATGCAGCCCTCACTAGCCCCGTGCCTCATCTGCCTACTTGTGCACGCTGCCTTCTGTGCTGTGGAGGGCCAGGGGTGGCAAGCCTTCAGGAATGATGCCACAGAGGTCATCCCAGGGCTTGGAGAGTACCCCGAGCCTCCTCCTGAGAACAACCAGACCATGAACCGGGCGGAGAATGGAGGCAGACCTCCCCACCATCCCTATGACGCCAAAGATGTGTCCGAGTACAGCTGCCGCGAGCTGCACTACACCCGCTTCCTGACAGACGGCCCATGCCGCAGCGCCAAGCCGGTCACCGAGTTGGTGTGCTCCGGCCAGTGCGGCCCCGCGCGGCTGCTGCCCAACGCCATCGGGCGCGTGAAGTGGTGGCGCCCGAACGGACCGGATTTCCGCTGCATCCCGGATCGCTACCGCGCGCAGCGGGTGCAGCTGCTGTGCCCCGGGGGCGCGGCGCCGCGCTCGCGCAAGGTGCGTCTGGTGGCCTCGTGCAAGTGCAAGCGCCTCACCCGCTTCCACAACCAGTCGGAGCTCAAGGACTTCGGGCCGGAGACCGCGCGGCCGCAGAAGGGTCGCAAGCCGCGGCCCGGCGCCCGGGGAGCCAAAGCCAACCAGGCGGAGCTGGAGAACGCCTAC

thirdly, constructing the target gene into a linearized expression vector by using a seamless assembly kit (a plurality of target gene insertion fragments are suitable), namely adding the target gene fragment and the linearized vector into a centrifuge tube in a molar ratio of 1:1 for recombination reaction:

the optimum amount of the insert used was [0.02X number of bases of insert ] ng (0.03pmol),

the optimum amount of linearized vector used was [0.02x number of linearized vector bases ] ng (0.03pmol),

after mixing, the cells were incubated at 37 ℃ for 30 minutes, and then transferred to ice for 5 minutes. Direct transformation or thawing transformation when the transformation is needed, such as storage at-20 ℃.

Example 4

To verifyWhether sclerostin is involved in the pathogenesis of alzheimer's disease, we will construct a bone-targeted over-expression sost plasmid virus through the tail vein at 4 x 10¹¹The concentration is injected into APP/PS1 mice of 5 months of age, and APP/PS1 mice are raised in a single cage for 3 months for relevant experimental analysis. Mainly comprises the concentration of sclerostin in serum, the concentration of Abeta protein in brain tissues of mice, behavioral tests (including water maze and new thing cognition) of the mice, synaptic plasticity, integrity tests and the like, so as to determine whether the generation of Abeta protein can be influenced to change the cognitive function of the mice after the sclerostin in the serum is increased.

We examined the serum of APP/PS1 Alzheimer's disease mice injected with bone-targeted sost overexpression virus via tail vein and found that the serum of mice shows a significant increase in sclerostin (sclerostin concentration) after the injection of sost overexpression virus (see FIG. 1 (A)). Then, Α β protein deposition in the cortex and hippocampus of the mice was detected, and it was found that in APP/PS1 mice, increasing the concentration of sclerostin protein in serum significantly increased the phenomenon of amyloid deposition in the cortex and hippocampus (fig. 1 (B-G)). We performed behavioral tests on different groups of mice (test methods and criteria refer to Vorhees, c.v. and m.t.williams (2006). "Morris water size: procedure for assessing spaciality and related for learning and memory." Nat protocol 1(2):848-858 Leger, m., et al (2013). "Object registration test in mice" Nat protocol 8(12):2531 and 2537.), and increasing the serostin in the serum can significantly destroy the spatial memory of the mice (fig. 1(H)), new recognizers (fig. 1 (I)). This indicates that increasing sclerostin protein in serum accelerates the decline of cognitive function in alzheimer mice by promoting amyloid formation and deposition.

Example 5

200ng/ml of sclerostin recombinant protein was added to the cell culture medium of the neuronal cell line N2a, and the content of BACE 19. beta. protein in the binding of T-cell-4. beta. protein in BACE 623-1 was examined (examination methods refer to Liu, C.C., et al. (2014). "Deficiency in LRP6-mediated Wtoksignalings conjugates to synthetic antigens and amyloidal Pathology in Alzheimer's disease" Neuron 84(1):63-77.Parr, C., et al. (2015). "Activation of the/Wnt a-gene pathway compression of the transfection of the a-amyloidal precursor protein cleavage BACE (1): EB 3. beta. protein in

EB

1, 1. beta. protein in the binding of T-cell-4. beta.," BACE 7-beta. protein in 1. Then, the N2a cells are transformed into a beta-catenin overexpression plasmid and a BACE1 promoter plasmid, and Luciferase activity detection is carried out after sclerostin is added, so that the transcription condition of sclerostin on BACE1 mRNA is determined.

We used neuron cell line N2a cell to carry out cell detection, recombinant Sclerostin (Sclerostin) is added into the cell, and the change of BACE1 mRNA level in the cell is detected after 24 hours and 48 hours respectively, and the addition of Sclerostin protein with different concentrations can significantly increase BACE1 mRNA transcription level as shown in figure 2(A), and it can be seen from the figure that the addition of Sclerostin recombinant protein with 100ng/ml and 200ng/ml can significantly promote BACE1 mRNA level Sclerostin recombinant protein can significantly promote BACE1 protein expression and promote A beta protein production. Then, N2a intracellular protein was detected, and it is further clarified that sclerostin can significantly reduce the intracellular β -catenin protein level and increase BACE1 protein expression (FIG. 2 (B)). We later confirmed that sclerostin increased BACE1 transcript levels by Luciferase (FIG. 2 (C)). We then constructed the alzheimer's disease cell line N2a-APP and found that the addition of sclerostin recombinant protein significantly promoted protein levels of the protein fragments sAPP β and β -CTF during Α β protein production (fig. 2 (D)). The culture medium is detected, and the concentration of the Α beta 1-40 and Α beta 1-42 protein in the culture medium is obviously increased after the sclerostin recombinant protein is added.

The above-described method of construction using the N2a-APP cell line used was as follows:

firstly, experimental materials:

1. cell lines and lentiviruses:

virus (pRLenti-CMV-MCS-3FLAG-PGK-Puro, and meta-construct: GL 102);

virus (pRLenti-CMV-APP695swe (K595N/M596L) -3FLAG-PGK-Puro, and Yuan Jian: H13319); neuro-2a cells are derived from stock,

culture medium: MEM + 10% FBS, 37 ℃, 5% CO₂And (5) culturing.

2. Main reagent consumables:

MEM (Hyclone, SH30023.01B); fetal bovine serum (Gibco: 10099-141); polybrene (Sigma: H9268); puromycin (Sigma: P8833); 24-well plates (Corning: 3524); 6 well plates (Corning: 3516); 60mm dish (Corning: 430166); 100mm discoring 430167)

3. The main apparatus comprises:

fluorescence microscopy (DMI8, LEICA); CO 2₂An incubator (311, Thermo); biological safety cabinet (BSC-II-A2, Shanghai purification).

II, an experiment step:

1. cell plating:

neuro-2a cells were seeded at 30% confluence onto 6cm dishes.

1) Neuro-2a cells were made up to 0.33X 10⁵cells/ml cell suspension, ready for plating.

2) Each dish is filled with 3ml, namely 1X 10⁶cells/well, 6cm petri dishes.

2. Infection with lentivirus:

infecting virus after 12-20 hours

The method for calculating the virus addition amount comprises the following steps: (cell number. times. MOI value/Virus stock titer). times.10³Biviral dose (mul)

Viral load, as shown in the table below.

1) Adding polybrene: adding 15 mu l of 1mg/ml polybrene into each dish, and finally, the final concentration of polybrene in the cell sample is 5 mu g/ml;

2) medium change 12-20 hours after infection: the medium was discarded and 3ml of fresh medium was added to each dish.

3. Stable strain screening

1) After 72h, puromycin was added to a final concentration of 1. mu.g/ml; changing a puromycin fresh culture medium with the final concentration of 1 mu g/ml every 2-3 days;

2) taking a fluorescent photograph about two weeks after drug screening;

3) and (5) freezing and storing the stable cell strain.

Example 6

An Alzheimer disease cell model and an N2a-APP cell line are constructed, 200ng/ml sclerostin recombinant protein is added into a neuron cell line N2a cell culture medium for culture, N2a-APP cell protein is extracted, protein fragments such as beta-CTF and sAPP beta protein in the alpha beta protein production process are detected, and the concentrations of the A beta 1-40 protein and the A beta 1-42 protein in the culture medium are detected (the detection adopts ELISA of R & D company, the product numbers are DAB142 and DAB 140B).

Example 7

The bone cell line MLO-Y4 and the neuron cell line N2a are co-cultured, 200ng/ml sclerostin neutralizing antibody is added into a culture medium (the culture medium is specifically: alpha MEM culture medium + 10% fetal bovine serum + 2% penicillin + streptomycin), after the culture medium is placed into an incubator and incubated for 24 hours, N2a cell protein is extracted, the expression condition of BACE1 protein is detected by using a Western Blot technology, and the result is shown in FIG. 3(A), and the addition of the sclerostin neutralizing antibody can obviously reduce the expression of BACE1 protein in N2a cells.

Then, the bone cell line MLO-Y4 is co-cultured with the constructed Alzheimer disease cell model and the N2a-APP cell line, 200ng/ml sclerostin neutralizing antibody is added into a culture medium (the culture medium is specifically alpha MEM culture medium + 10% fetal bovine serum + 2% penicillin + streptomycin), and after 24 hours of incubation, N2a-APP cell protein is extracted to detect BACE1 protein expression and protein fragments such as beta-CTF and sAPP beta protein in the production process of the alpha beta protein (detection uses Western Blot technology), and A beta 1-40 and A beta 1-42 protein concentration in the culture medium (detection uses Elisa technology, product company R & D), so that the influence of the sclerostin neutralizing antibody on the production process of the alpha beta protein is determined. Protein fragments sAPP beta and beta-CTF protein levels in the production process of Α beta protein can be significantly inhibited after the sclerostin neutralizing antibody is added, and as a result, as shown in figure 3(B), simultaneous detection of the culture medium shows that the concentrations of Α beta 1-40 (figure 3(C)) and Α beta 1-42 (figure 3D) proteins in the culture medium are significantly reduced after the sclerostin neutralizing antibody is added.

In conclusion, Sclerostin (Sclerostin) can significantly promote the production of a β protein in both animal and cellular levels, accelerating the decline of cognitive function. Meanwhile, the neutralizing antibody constructed according to the sclerostin recombinant protein can reduce the level of BACE1 protein in neuron cells in a cell layer, further inhibit the generation of A beta protein, and has the effect of relieving and/or treating Alzheimer's disease.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

<110> Jianqing

<120> sclerostin and application thereof in preparation of related products for treating or preventing Alzheimer's disease

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgcaaatggg cggtaggcgt g 21

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<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catagcgtaa aaggagcaac a 21

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgcagccct cactagcccc gtgcctcatc tgcctacttg tgcacgctgc cttctgtgct 60

gtggagggcc aggggtggca agccttcagg aatgatgcca cagaggtcat cccagggctt 120

ggagagtacc ccgagcctcc tcctgagaac aaccagacca tgaaccgggc ggagaatgga 180

ggcagacctc cccaccatcc ctatgacgcc aaagatgtgt ccgagtacag ctgccgcgag 240

ctgcactaca cccgcttcct gacagacggc ccatgccgca gcgccaagcc ggtcaccgag 300

ttggtgtgct ccggccagtg cggccccgcg cggctgctgc ccaacgccat cgggcgcgtg 360

aagtggtggc gcccgaacgg accggatttc cgctgcatcc cggatcgcta ccgcgcgcag 420

cgggtgcagc tgctgtgccc cgggggcgcg gcgccgcgct cgcgcaaggt gcgtctggtg 480

gcctcgtgca agtgcaagcg cctcacccgc ttccacaacc agtcggagct caaggacttc 540

gggccggaga ccgcgcggcc gcagaagggt cgcaagccgc ggcccggcgc ccggggagcc 600

aaagccaacc aggcggagct ggagaacgcc tac 633

<210> 5

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<213> Amino acid sequence (Amino acid sequence)

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Met Gln Pro Ser Leu Ala Pro Cys Leu Ile Cys Leu Leu Val His Ala

1 5 10 15

Ala Phe Cys Ala Val Glu Gly Gln Gly Trp Gln Ala Phe Arg Asn Asp

20 25 30

Ala Thr Glu Val Ile Pro Gly Leu Gly Glu Tyr Pro Glu Pro Pro Pro

35 40 45

Glu Asn Asn Gln Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro

50 55 60

His His Pro Tyr Asp Ala Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu

65 70 75 80

Leu His Tyr Thr Arg Phe Leu Thr Asp Gly Pro Cys Arg Ser Ala Lys

85 90 95

Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu

100 105 110

Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly Pro

115 120 125

Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu

130 135 140

Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val

145 150 155 160

Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu

165 170 175

Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gln Lys Gly Arg Lys

180 185 190

Pro Arg Pro Gly Ala Arg Gly Ala Lys Ala Asn Gln Ala Glu Leu Glu

195 200 205

Asn Ala Tyr

210

Claims

1. The sclerostin is characterized in that the amino acid sequence of the sclerostin is shown in SEQ NO. 1.

2. The sclerostin according to claim 1, wherein the nucleotide sequence encoding the sclerostin is shown in SEQ No. 2.

3. A recombinant plasmid comprising a plasmid having a nucleotide sequence encoding the sclerostin protein of claim 1.

4. A recombinant strain, characterized by expressing the recombinant plasmid of claim 3.

5. Use of the sclerostin according to claim 1, the recombinant plasmid according to claim 3 or the recombinant strain according to claim 4 for the preparation of a neutralizing antibody for the treatment or prevention of alzheimer's disease.

6. A neutralizing antibody characterized in that the active ingredient thereof is the sclerostin according to claim 1, the recombinant plasmid according to claim 3 or the recombinant strain according to claim 4.

7. A product for treating or preventing Alzheimer's disease, comprising the sclerostin of claim 1, the recombinant plasmid of claim 3, the recombinant strain of claim 4, or a neutralizing antibody of claim 6 as an active ingredient; the product is a pharmaceutical composition or a health product.

8. The product for treating or preventing Alzheimer's disease according to claim 7, wherein the pharmaceutical composition is in the form of powder for injection, injectable powder or powdered powder.