Antigen polypeptides based on beta-amyloid modification and uses thereof

Abstract

This invention discloses an antigenic peptide modified with β-amyloid protein and its applications. The antigenic peptide comprises three components: A, B, and C. Component a of component A is a β-amyloid protein monomeric amino acid fragment or a combination of several amino acid fragments; component b of component B is a small molecule compound capable of binding to β-amyloid protein monomers or oligomers; and component c of component C is a conjugated carrier protein. The modified antigenic peptide involved in this invention targets the lamellar regions of β-amyloid protein, blocking the formation of toxic oligomers from abnormally folded β-amyloid protein. Furthermore, the antibodies generated by antigen immunization specifically recognize β-amyloid protein oligomers and polymers bound to small molecule compounds—targeting immune complexes—and effectively clearing them. Ultimately, by preventing the aggregation of β-amyloid protein monomers and clearing β-amyloid protein aggregates, this invention prevents and alleviates the pathological abnormalities of β-amyloid protein in Alzheimer's disease (AD), thus producing preventive and therapeutic effects on AD.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to antigenic peptides based on β-amyloid protein modification and their applications. Background Technology

[0002] Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease with an insidious onset and a long course. Its main clinical feature is the decline in cognitive and memory functions caused by synapse and neuronal loss. Statistics show that the incidence of Alzheimer's disease in people aged 60 to 80 is approximately 4%, while the incidence in those over 80 years old is as high as 20% to 40%. According to the latest report from the World Health Organization, there are already more than 50 million people with dementia worldwide, and this number is projected to exceed 150 million by 2050. The costs of treatment and care for AD are expected to reach $2 trillion by 2030, placing a tremendous economic burden and pressure on patients' families and society. Currently, the few drugs available for treating AD can only alleviate symptoms to a certain extent and cannot achieve a cure. Given China's increasingly aging society, the level of research and treatment of Alzheimer's disease in China urgently needs to be improved.

[0003] The prevailing hypothesis regarding the pathogenesis of Alzheimer's disease (AD) is the amyloid cascade hypothesis. This hypothesis posits that the excessive production of β-amyloid (Aβ) through the hydrolysis of amyloid precursor protein (APP) in the brains of AD patients is the primary cause of neurotoxicity. Aβ can also induce hyperphosphorylation of Tau protein, leading to neurofibrillary tangles, ultimately resulting in synaptic damage and neuronal loss. Therefore, targeting Aβ as a therapeutic target has become a hot topic in current AD drug and vaccine development.

[0004] Immunotherapy for Alzheimer's disease (AD) is mainly divided into immunotherapies targeting Aβ and tau. Since various variants and aggregates of Aβ (Aβ40, Aβ42, Aβ35, Aβ oligomers, Aβ profibrillary oligomers, Aβ fibers, Aβ plaque deposits) are substances naturally present in the body, using endogenous Aβ peptides or proteins as antigens can trigger an autoimmune response, easily eliciting a Th1 cell subset response in CD4 T cells, leading to adverse reactions such as inflammation. Furthermore, using Aβ as an antigen to immunize the body can cause adverse reactions such as cerebral microbleeds, possibly due to excessive Aβ clearance caused by the resulting immune response.

[0005] Since the first use of human fibrotic Aβ42 peptide to immunize AD transgenic mice in 1999 and achieved some success, numerous Phase I clinical studies have also shown initial good results. However, subsequent Phase II clinical trials have resulted in meningitis caused by autotoxic T-cell immune responses. Furthermore, the active immunization vaccine targeting the B lymphocyte epitope Aβ1-15 of the Aβ42 peptide, which was selected for the Aβ42 peptide, has also shown side effects in clinical trials, ultimately leading to the termination of the trials.

[0006] Currently, other AD vaccines under development have also failed to achieve therapeutic effects in improving cognitive abilities. This may be because the antibodies produced after immunization with these vaccines cannot effectively block the abnormal folding of Aβ to form oligomers that are highly toxic to synaptic function and neurons, thus failing to improve cognitive abilities. Therefore, whether it is active or passive immunization or AD prevention, it should be required that the induced high levels of antibodies specifically block the abnormal folding of Aβ to form toxic Aβ oligomers, reduce the level of Aβ oligomers in the brain, protect neural synaptic function and cognitive abilities, thereby producing an effective immunoprophylactic effect. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides an antigenic polypeptide based on β-amyloid protein modification and its application in the prevention and treatment of Alzheimer's disease. This antigenic polypeptide can not only induce the body to produce antibodies to prevent the aggregation of Aβ monomers, but also cooperate with small molecule compounds to clear Aβ aggregates, thereby preventing and slowing down the Aβ pathological abnormalities of Alzheimer's disease, and realizing the prevention and treatment of Alzheimer's disease.

[0008] The specific technical solution is as follows:

[0009] This invention provides an antigenic polypeptide based on β-amyloid protein modification, the antigenic polypeptide comprising three components, A, B, and C, which are linked in any order by linking bonds or linking groups;

[0010] Component A is composed of m components a, component B is composed of n components b, and component C is composed of k components c; component a is a β-amyloid monomer amino acid fragment or a combination of several amino acid fragments; component b is a small molecule compound that can bind to β-amyloid monomers or oligomers; component c is a coupling carrier protein; m≥1, n≥1, k≥0.

[0011] Furthermore, when k≠0, the connection forms of A, B, and C are: ABC, CBA, ACB, CAB, BAC, and BCA; when k=0, the connection forms of A and B are AB, BA, ABA, or BAB.

[0012] Furthermore, the connection form of A, B, and C is CAB, where m = 1, n = 1, and k = 1; wherein, the C-terminal carboxyl group of component A is connected to the hydroxyl group of component B through an ester bond; a thiol group is attached to the N-terminus of component A, and a maleimide group is attached to the amino group on the surface of component C; and component A is connected to the maleamide group of component C through the N-terminal thiol group.

[0013] Furthermore, the β-amyloid monomer amino acid fragments in component a are continuous fragments located between positions 14 and 29 of the β-amyloid monomer, and the number of amino acids in the fragments is 3 to 7; the combination of amino acid fragments is an arbitrary sequence of N identical or different β-amyloid monomer amino acid fragments, where N ≥ 2.

[0014] The full amino acid sequence of the β-amyloid monomer mentioned above is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKNKGAIIGLMVGGVVIA; where the amino acid sequence from position 14 to 29 is HQKLVFFAEDVGSNKG.

[0015] Furthermore, the β-amyloid monomeric amino acid fragment is one of HQK, KLV, FFA, AED, NKG, NKGKLV, HQKAED, CFFA, CAED, CNKG, CKLVFFA, and CLVFFAE.

[0016] Furthermore, the small molecule compounds are curcumin, demethoxycurcumin, didemethoxycurcumin, high-taurine, 3-sulfopropionic acid, epigallocatechin gallate, brasiliensis, cotton cellulose, oleuropein, quercetin, resveratrol, rosmarinic acid, 6-shogaol, tanshinone, vitamin A, and vitamin B. 12 One of vitamin D2, vitamin D3, and vitamin K3.

[0017] Furthermore, the small molecule compound is one of curcumin, curcumin derivatives, high taurine, and high taurine derivatives.

[0018] Furthermore, the curcumin derivative is one of demethoxycurcumin and bisdemethoxycurcumin; the high-taurine derivative is 3-sulfopropionic acid.

[0019] Furthermore, the coupling carrier protein is one of hemocyanin, bovine serum albumin, and ovalbumin.

[0020] Furthermore, the chemical structural formulas of the antigenic polypeptide are shown in formulas (1) to (14) below:

[0021]

[0022]

[0023]

[0024] KLH stands for hemocyanin.

[0025] The present invention also provides a method for synthesizing the above-mentioned antigenic polypeptide (taking the CAB linkage as an example, and m=1, n=1, k=1), comprising the following steps:

[0026] (1) The C-terminal carboxyl group of component A is connected to the hydroxyl group of component B through an esterification reaction, and a thiol group is introduced at the N-terminus of component A through cysteine ​​or mercaptopropionic acid molecules to obtain compound I.

[0027] (2) The amino groups on the surface of component C were modified by using succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups, thus obtaining compound II;

[0028] (3) The N-terminal thiol group of compound I is linked to the maleimide group of compound II via a Michael addition reaction to finally obtain the antigen polypeptide.

[0029] The present invention also provides the use of the β-amyloid protein-modified antigenic polypeptide in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease.

[0030] The present invention also provides the application of the antigenic polypeptide based on β-amyloid protein modification in the preparation of Alzheimer's disease vaccines.

[0031] Furthermore, the vaccine also includes an adjuvant; the adjuvant is Freund's adjuvant.

[0032] The present invention also provides the use of the β-amyloid protein-modified antigenic polypeptide in the preparation of products for clearing Aβ oligomers or multimers.

[0033] The present invention also provides the application of the β-amyloid protein-modified antigenic peptide in the preparation of products for improving the learning and memory abilities of Alzheimer's disease patients.

[0034] The present invention also provides the use of the β-amyloid protein-modified antigenic polypeptide in the preparation of products for reducing the content of Aβ oligomers and / or soluble Aβ in the brains of Alzheimer's disease patients.

[0035] The present invention also provides the application of the aforementioned β-amyloid protein-modified antigenic polypeptide in the preparation of products for detecting Aβ oligomers or polymers.

[0036] The present invention also provides the application of the aforementioned β-amyloid protein-modified antigenic peptide in the preparation of products for early risk assessment screening, disease diagnosis, or auxiliary diagnosis of Alzheimer's disease.

[0037] The present invention also provides an Alzheimer's disease vaccine comprising an antigenic polypeptide based on β-amyloid protein modification and an adjuvant, wherein the antigenic polypeptide is as described above.

[0038] Furthermore, the adjuvant is Freund's adjuvant.

[0039] The present invention also provides an Alzheimer's disease-targeting immune complex, including a vaccine comprising an antigenic peptide based on β-amyloid protein modification and an adjuvant, the antigenic peptide being as described above.

[0040] The Alzheimer's disease-targeting immune complex also includes small molecule compounds, namely curcumin, demethoxycurcumin, didemethoxycurcumin, high-taurine, 3-sulfopropionic acid, epigallocatechin gallate, brasiliensis, cotton cellulose, oleuropein, quercetin, resveratrol, rosmarinic acid, 6-shogaol, tanshinone, vitamin A, and vitamin B. 12 One of vitamin D2, vitamin D3, and vitamin K3.

[0041] The aforementioned small molecule compounds are administered as injectable or oral drugs, and are used in conjunction with vaccines.

[0042] To eliminate β-amyloid protein while avoiding autoimmune reactions, this invention designs a vaccine that uses a complex modified peptide formed by β-amyloid oligomers and small molecule compounds such as curcumin and homotaurine, which have high affinity for β-amyloid oligomers, as an antigen for the body's immunization. This allows the immune system to eliminate the toxic β-amyloid oligomers while clearing the complex, without causing an autoimmune reaction.

[0043] After this vaccine is administered to individuals at high risk of Alzheimer's disease (AD), the body acquires memory cells that target the complex formed by β-amyloid oligomers and small molecule compounds such as curcumin. Through AD disease progression or personalized medicine research, individuals susceptible to AD can be given specific doses and courses of treatment with small molecule compounds such as curcumin, which have a high affinity for β-amyloid oligomers. Because these compounds are taken exogenously, this active immunotherapy can be terminated by discontinuing the administration of these compounds, or by administering small amounts of these compounds to reduce the intensity of the immune response, thereby avoiding cerebral microbleeds induced by active immunotherapy.

[0044] Traditional vaccines primarily target proteins from exogenous organisms such as viruses or bacteria, recognizing antigenic determinants mainly as single exogenous proteins or recombinant peptides. A cutting-edge approach in infectious disease epidemiology involves using glycans as haptens, binding them to carrier proteins to form complete antigens that then immunize cells. In contrast, the antigenic peptide of this invention is a hapten composed of 3-7 amino acids linked to a small molecule compound. This method of modifying antigens with small molecules provides a novel strategy for future basic immunological research and for the immunoprophylaxis and clinical treatment of various diseases, including neurodegenerative diseases.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This invention modifies β-amyloid protein by binding small molecule compounds to sequence fragments, creating novel antigenic peptides. Furthermore, it conjugates these peptides with carrier proteins to enhance their antigenicity. Unlike traditional antigen designs based solely on β-amyloid protein sequences, the modified antigenic peptides of this invention target β-amyloid protein, blocking the formation of toxic oligomers from abnormally folded β-amyloid. Simultaneously, the antibodies generated by antigen immunization specifically recognize the β-amyloid oligomers and polymers bound to the small molecule compounds—targeting immune complexes—and effectively clearing them. Ultimately, by preventing the aggregation of β-amyloid monomers and clearing β-amyloid aggregates, this invention prevents and alleviates the pathological abnormalities of β-amyloid protein in Alzheimer's disease (AD), thus providing both preventative and therapeutic effects. Attached Figure Description

[0047] Figure 1 The results show the serum antibody titers and specific detection results of β-amyloid protein (Aβ) and Curcumin after immunization of A3 (Figure A), A7 (Figure B), A9 (Figure C), 7P (Figure D), and 8P (Figure E) for 15 days and 5 immunizations in Example 3.

[0048] Here, A3 / A7 / A9 / 7P / 8P(after) represent serum antibody detection of antigens A3 / A7 / A9 / 7P / 8P after 15 days of immunization and 5 immunizations, respectively; A3 / A7 / A9 / 7P / 8P(before) represents the negative group in each experiment, that is, the non-immunized group. Aβ(after) represents serum antibody detection of Aβ42 after 15 days of immunization and 5 immunizations. Curcumin was indicated by serum antibody detection after 15 days of immunization followed by 5 immunizations; the concentration of A3 / A7 / A9 / 7P / 8P / Aβ42 / Curcumin coated in the ELISA assay was 6.5 μM (100 μL); the dilutions of A3 / A7 serum antibodies were 10, 50, 250, 1250, 6250, and 31250; the dilutions of A9 / 7P / 8P were 100, 1000, 10000, and 100000; the dilution of HRP-labeled anti-mouse secondary antibody was 1:5000; and the detection wavelength of the microplate reader was 450 nm.

[0049] Figure 2 The results of serum antibody levels of A3 (Figure A), A7 (Figure B), A9 (Figure C), 7P (Figure D), and 8P (Figure E) in Example 3 were measured on day 7 after 55 days of immunization and 3 immunizations.

[0050] Among them, A3 / A7 / A9 / 7P / 8P(after) represent serum antibody detection of antigens A3 / A7 / A9 / 7P / 8P after 55 days of immunization and 3 immunizations, respectively; A3 / A7 / A9 / 7P / 8P(before) represents the negative group of each experiment, that is, the unimmunized group; Aβ(after) represents serum antibody detection of Aβ42 after 55 days of immunization and 3 immunizations; Curcumin represents serum antibody detection of curcumin after 55 days of immunization and 3 immunizations. The concentration of A3 / A7 / A9 / 7P / 8P / Aβ42 / Curcumin coated in the ELISA assay was 6.5 μM (100 μL); the dilution factors of A3 / A7 / A9 / 7P / 8P were 100, 1000, 10000, and 100000; the dilution factor of HRP-labeled anti-mouse secondary antibody was 1:5000; and the detection wavelength of the microplate reader was 450 nm.

[0051] Figure 3 The recognition of Aβ-Curcumin incubator and antibody was detected in the mixed immunization of A3, A7, and A9 (i.e., mixed serum antibody after immunization) (Figure A (15 days immunization) and Figure B (55 days immunization)) and the mixed immunization of 7P and 8P (Figure C (15 days immunization) and Figure D (55 days immunization)).

[0052] Wherein, Incubation Aβ:Cur 1:1 represents the Aβ to Curcumin complex with a 1:1 molar ratio of Aβ to Curcumin in positive serum after immunization; Negative serum represents the Aβ to Curcumin complex with a 1:1 molar ratio of Aβ to Curcumin in pro rataly diluted, non-immunized negative serum; the concentration of Incubation Aβ:Cur 1:1 coated in the ELISA assay is 6.5 μM (100 μL); A3 / A7 / A9 15 days, A3 / A7 / A9 55 days, 7P / 8P The dilutions of 55-day positive and negative serum antibodies were 100, 1000, 10000, and 100000; the dilutions of 7P / 8P 15-day positive and negative serum antibodies were 10, 100, 1000, and 10000; the dilution of HRP-labeled anti-mouse secondary antibody was 1:5000; and the wavelength of the ELISA reader was 450 nm.

[0053] Figure 4 The images show the microscopic observation results of staining analysis of Aβ and curcumin conjugates in vivo using 7P serum IgG (1:50, Figure A), 7P serum IgG (1:100, Figure B), and 4G8 (1:1000, Figure B) in Example 4.

[0054] In this study, A represents the immunohistochemical assay performed on brain sections of 4-month-old 5xFAD female mice that were intraperitoneally injected with 300 mg / ml curcumin (DMSO dissolved with curcumin) for 7 days and with the same volume of DMSO (without curcumin) for 7 days. B represents the results of immunohistochemical analysis on brain sections of 4-month-old 5xFAD female mice that were intraperitoneally injected with 300 mg / ml curcumin for 7 days, using 4G8 (1:1000) and 7P serum IgG (1:100). Each immunohistochemical result was observed and recorded under a microscope at 10×, 20×, and 40×.

[0055] Figure 5 The results of immunohistochemical assays in Example 5 showed changes in Aβ oligomers in the brains of 7P immunized 5×FAD mice after gavage administration of curcumin.

[0056] In this context, A indicates that immunohistochemical experiments were performed using A11 (specific anti-Aβ oligomeric antibody, 1:1000) and 4G8 (anti-Aβ17-24 fragment antibody, 1:1000) as primary antibodies to detect immunization (50ug / 20ul) in 5XFAD mice administered curcumin 300mg / kg by gavage (5XFAD mice / Alz813 immunization / Curcumin), 5XFAD mice administered curcumin 300mg / kg by gavage (5XFAD mice / Curcumin), and untreated 5XFAD mice (5XAFD mice). (control) Microscopic observation results of Aβ deposition in the hippocampus; B indicates that immunohistochemical experiments were conducted using A11 (specific anti-Aβ oligomeric antibody, 1:1000) and 4G8 (anti-Aβ17-24 fragment antibody, 1:1000) as primary antibodies to detect microscopic observation results of Aβ deposition in the cortex of immunized (50ug / 20ul) 5XFAD mice administered curcumin 300mg / kg by gavage (5XFAD mice / Alz813 immunization / Curcumin), 5XFAD mice administered curcumin 300mg / kg by gavage (5XFAD mice / Curcumin), and untreated 5XFAD mice (5XAFD mice control). C represents the results of immunohistochemical experiments using A11 (specific anti-Aβ oligomeric antibody, 1:1000) and 4G8 (anti-Aβ17-24 fragment antibody, 1:1000) as primary antibodies to detect Aβ deposition in the cerebellum of immunized (50ug / 20ul) 5XFAD mice (5XFAD mice / Alz813 immunization / Curcumin) administered by gavage, 5XFAD mice administered by gavage with 300mg / kg curcumin (5XFAD mice / Curcumin), and untreated 5XFAD mice (5XAFD mice control). D represents the statistical results of Aβ content in the hippocampus, cortex, and cerebellum detected by A11 in experiments A, B, and C. E represents the statistical results of Aβ plaque content in the hippocampus, cortex, and cerebellum detected by 4G8 in experiments A, B, and C.

[0057] Figure 6 The results of the water maze test in Example 6 were used to detect the cognitive function of 7P immunized 5×FAD mice after gavage administration of curcumin.

[0058] In this table, A represents the statistical results of the water maze escape latency of untreated litter-controlled C57 mice (Littermates (WT)), Alz813 (50ug / kg) immunized and administered 50mg / kg curcumin 5XFAD mice (Alz813 / Curcumin), Alz813 (50ug / kg) immunized 5XFAD (Alz813) mice, 50mg / kg curcumin 5XFAD mice (Curcumin) administered by gavage, and untreated control 5XFAD mice (Control (5XFAD)) during the 1-4 day plateau hiding period; B represents the statistical results of the water maze escape latency of untreated litter-controlled C57 mice (Littermates (WT)), Alz813 (50ug / kg) immunized and administered 50mg / kg curcumin 5XFAD mice (Alz813 / Curcumin) after removing the plateau at 5 days. The results show the number of times mice immunized with Alz813 (50ug / kg) 5XFAD (Alz813), mice administered 50mg / kg curcumin via gavage, and untreated control 5XFAD mice (Control(5XFAD)) crossed the original safety platform. C represents the swimming trajectory of untreated litter control C57 mice (Littermates(WT)), mice immunized with Alz813 (50ug / kg) and administered 50mg / kg curcumin via gavage (Alz813 / Curcumin), mice immunized with Alz813 (50ug / kg) 5XFAD (Alz813), mice administered 50mg / kg curcumin via gavage, and untreated control 5XFAD mice (Control(5XFAD)) after the safety platform was removed on day 5. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0060] The materials used in the following examples were sourced as follows: 4-6 weeks old female Babl / c mice and 4 weeks old 5×FAD transgenic mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., Jiangsu Province. After purchase, the mice were housed in the SPF-grade foster room of the Animal Experiment Center of Guizhou Medical University, with the temperature maintained at (25±1)℃, and provided with 12 hours of light and SPF-grade feed and water daily. Freund's complete adjuvant (F5881) and Freund's incomplete adjuvant (F5506) were purchased from Sigma; Aβ42 (ab120301) was purchased from Abcam; curcumin (SC0299), TMB chromogenic solution (P0209), and TMB termination solution (P0215) were purchased from Beyotime; hemocyanin (KLH), Tween20 (T8220), citric acid repair solution (C1032), blocking-specific sheep serum (SL038), and SDS-PA were also used. GE gel preparation kit (P1200-1) was purchased from Solarbio; goat anti-mouse secondary antibody (M21001) was purchased from Abmart; Dapi staining solution (0100-20) was purchased from Southern Biotech; 4G8 (SIG-39220) antibody was purchased from Biolegend; A11 (AHB0052) was purchased from Thermo Fisher; DAB (ZLI-9018) was purchased from OriGene; Melon... TM The Gel IgG Spin Purification Kit (45206) was purchased from ThermoFisher, and the WB chromogenic solution (WBKLS0100) was purchased from Millipore.

[0061] Example 1: Antigenic polypeptide using curcumin (Cur) as a small molecule compound

[0062] Using curcumin (Cur) as the small molecule compound described in component b, and hemocyanin (KLH) as the coupling carrier protein described in component c, the antigenic polypeptide (m=1, n=1, k=1) is synthesized by free combination and linkage with the β-amyloid monomer amino acid fragment (abbreviated as polypeptide sequence) described in component a.

[0063] The specific combination forms are shown in Table 1 (KLH is hemocyanin).

[0064] Table 1. Chemical structural formulas of antigenic peptides using curcumin (Cur) as a small molecule compound.

[0065]

[0066] Taking KLH-CKLV-Cur as an example, the synthesis steps are as follows:

[0067] (I) The C-terminal carboxyl group of the polypeptide sequence is linked to the hydroxyl group of curcumin through an esterification reaction, and a thiol group is introduced at the N-terminus of the polypeptide sequence through a cysteine ​​molecule to obtain CKLV-Cur.

[0068] (II) The amino groups on the surface of hemocyanin were modified by using succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups;

[0069] (III) The N-terminal thiol group of cysteine ​​in CKLV-Cur is linked to the maleimide group of KLH(SMCC) via Michael addition reaction to finally obtain the antigen peptide KLH-CKLV-Cur.

[0070] The specific synthesis method is as follows:

[0071] (1) Dissolve 20 mg SMCC in 2 mL DMF;

[0072] (2) Add 0.8 mL of KLH to a 25 mL round-bottom flask and add PBS buffer (pH 7.2) to make the final protein concentration 15 mg / mL;

[0073] (3) Slowly add the dissolved SMCC solution dropwise to the 120mg KLH protein system and stir at room temperature for 1 hour;

[0074] (4) Dialyze with PBS solution (pH 7.4) at 4°C for 6 hours to remove free SMCC;

[0075] (5) Transfer 2.5 mg of the synthesized KLH(SMCC) solution to a 5 mL centrifuge tube;

[0076] (6) 460 mg of the synthesized CKLV polypeptide was dissolved in methanol and reacted with 368 mg of curcumin by heating for 4 h to precipitate the product CKLV-Cur;

[0077] (7) Dissolve 3.0 mg CKLV-Cur in 0.6 mL PBS solution (pH 7.2);

[0078] (8) Add the CKLV-Cur solution dropwise into KLH(SMCC) and mix the mixture with a vertical mixer at room temperature for 4 hours.

[0079] Example 2: Antigenic polypeptide using high taurine as a small molecule compound

[0080] Using high taurine (Hom) as a small molecule compound and hemocyanin (KLH) as a coupling carrier protein, antigenic polypeptides are synthesized by free combination and linkage with a portion of the amino acid sequence (referred to as polypeptide sequence) of β-amyloid monomer.

[0081] The specific combinations are shown in Table 2.

[0082] Table 2. Chemical structural formulas of antigenic peptides using high-taurine as a small molecule compound.

[0083]

[0084]

[0085] Taking KLH-CKLV-Hom as an example, the synthesis steps are as follows:

[0086] (I) The C-terminal carboxyl group of the polypeptide sequence is linked to the hydroxyl group of high taurine through an esterification reaction, and a thiol group is introduced at the N-terminus of the polypeptide sequence through a cysteine ​​molecule to obtain CKLV-Hom.

[0087] (II) The amino groups on the surface of hemocyanin were modified by succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups, thus obtaining KLH(SMCC);

[0088] (III) The N-terminal thiol group of cysteine ​​in CKLV-Hom is linked to the maleimide group of KLH(SMCC) via a Michael addition reaction to finally obtain the antigen peptide KLH-CKLV-Hom.

[0089] The specific synthesis method is as follows:

[0090] (1) Dissolve 20 mg SMCC in 2 mL DMF;

[0091] (2) Add 0.8 mL of KLH to a 25 mL round-bottom flask and add PBS buffer (pH 7.2) to make the final protein concentration 15 mg / mL;

[0092] (3) Slowly add the dissolved SMCC solution dropwise to the 120mg KLH protein system and stir at room temperature for 1 hour;

[0093] (4) Dialyze with PBS solution (pH 7.4) at 4°C for 6 hours to remove free SMCC;

[0094] (5) Transfer 2.5 mg of the synthesized KLH(SMCC) solution to a 5 mL centrifuge tube;

[0095] (6) 460 mg of the synthesized CKLV polypeptide was dissolved in methanol and reacted with 139 mg of high taurine by heating for 4 h to precipitate the product CKLV-Hom;

[0096] (7) Dissolve 3.0 mg CKLV-Hom in 0.6 mL PBS solution (pH 7.2);

[0097] (8) Add the CKLV-Hom solution dropwise into KLH(SMCC) and mix the mixture with a vertical mixer at room temperature for 4 hours.

[0098] Example 3

[0099] I. Experimental Objective

[0100] KLH-CFFA-Cur (denoted as A3), KLH-CAED-Cur (denoted as A7), KLH-CNKG-Cur (denoted as A9), KLH-CKLVFFA-Cur (denoted as 7P), and KLH-CLVFFAE-Cur (denoted as 8P) from Example 1 were selected to prepare a vaccine, and immune serum antibodies were obtained and tested.

[0101] II. Experimental Methods

[0102] 1. Vaccine production

[0103] The specific method is as follows: A certain concentration of antigenic peptide and Freund's adjuvant (divided into complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)) are mixed at a volume ratio of 1:1 using an ultrasonic homogenizer to thoroughly homogenize the Freund's adjuvant and antigenic peptide solution, thus preparing a water-in-oil emulsion. The ultrasonic homogenizer is used under the following conditions: frequency 20 kHz, power 750 watts, ultrasonic frequency of 3 seconds followed by a 3-second pause, and the number of ultrasonic cycles 3-10 depending on the characteristics and volume of the sample. This process requires preventing the exothermic effects of ultrasound on the peptides. The vaccine is prepared immediately before use and the entire process is performed on ice.

[0104] 2. Vaccine-immunized mice (4-6 month old female Babl / c) experiment

[0105] 1) 15-day short-term immunization:

[0106] (1) Use 10 μg of A3 (or A7 / A9 / 7P / 8P) to synthesize short peptides and mix them with an equal amount of Freund's complete adjuvant to prepare an oil-in-water emulsion (20 μL), and immunize mice by multiple injections into the footpads.

[0107] (2) Every 3 days thereafter, use the same dose of A3 (or A7 / A9 / 7P / 8P) to synthesize short peptides and mix them with an equal amount of Freund's incomplete adjuvant. Repeat this process 4 times.

[0108] (3) On day 15, blood (serum) was collected from the inner canthus of the eye to measure the titer.

[0109] 2) 55-day long-term immunization:

[0110] (1) Use 50 μg of A3 (or A7 / A9 / 7P / 8P) to synthesize short peptides and mix them with an equal amount of Freund's complete adjuvant to prepare a water-in-oil emulsion (20 μL), and immunize mice by multiple injections into the footpads.

[0111] (2) Every 20 days thereafter, mix equal amounts of the same dose of A3 (or A7 / A9 / 7P / 8P) synthetic short peptide Freund's incomplete adjuvant and inject subcutaneously at multiple points, for a total of two injections.

[0112] (3) On day 55, blood (serum) was collected from the inner canthus of the eye to measure the titer.

[0113] Blood extracted from the inner canthal vein of the eye was left at room temperature for 1-3 hours or overnight at 4°C. After centrifugation at 4000 rpm for 15 minutes at 4°C, the serum was aliquoted and stored at -80°C.

[0114] 3. Indirect ELISA experiment

[0115] Dilute the antigen peptide or control (Aβ42, Curcumin) to 6.5 μM with PBS, add 100 μL to each well of a 96-well plate, seal with ELISA sealing film, and incubate overnight at 4°C. Wash the plate with the P3 program of Bio-rad immunowash 1575, blot dry on paper, add 140 μL of 1% blocking-specific goat serum prepared with PBST (0.05% Tween 20), and incubate at 37°C for 1.5 hours. Dilute the extracted immune serum serially at 10, 100, 1000, 10000, and 100000. After blocking, wash the plate with the P3 program of Bio-rad immunowash 1575, blot dry on paper, add the serially diluted serum as primary antibodies to each well at 50 μL, and incubate at 37°C for 1.5 hours. After the primary antibody reaction was complete, the plate was washed using the P4 program of Bio-rad immunowash 1575, patted dry on paper, and 100 μL of HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution) was added to each well. The plate was incubated at 37°C for 1 hour. After the secondary antibody reaction was complete, the plate was washed again using the P4 program of Bio-rad immunowash 1575, patted dry on paper, and 100 μL of TMB chromogenic buffer was added to each well. After incubation at room temperature for 20 min, 100 μL of TMB stop solution was added. Immediately after adding the stop solution, the OD value was measured using a microplate reader. The washing buffer used throughout the process was PBST (0.05% Tween 20).

[0116] III. Experimental Results

[0117] 1. Mouse serum antibody titer and specific recognition

[0118] Figure 1The titers of A3, A7, A9, 7P, and 8P after immunization for 15 days and 5 immunizations were specifically detected for Aβ and Curcumin. Figure 2 The results show the serum antibody levels of A3, A7, A9, 7P, and 8P on day 7 after immunization for 55 days and 3 immunizations.

[0119] like Figures 1-2 As shown, the synthetic short peptides A3, A7, A9, 7P, and 8P can induce the body to produce corresponding antibodies. The antibody titers for short-term immunization (15 days) ranged from 1:1000 to 1:100000, while those for long-term immunization (55 days) ranged from 1:10000 to 1:100000. This indicates that the antibody titers induced by the five synthetic peptides plus an adjuvant (Freudian adjuvant) are relatively high. There was no significant difference in antibody titers produced by the five peptides. Furthermore, the serum antibodies against the five peptides only recognized the synthetic antigenic peptides and not Aβ or Curcumin alone.

[0120] 2. Detection of Aβ-Curcumin incubation and antibody recognition after mixed immunization with A3, A7, A9, 7P, and 8P.

[0121] To further verify whether the Aβ complex antigen obtained by chemical synthesis can mimic the binding of aberrant Aβ42 monomers and aggregates to curcumin under natural conditions, we detected serum antibodies using an overnight incubation of Aβ and curcumin. Figure 3 As shown, sera from A3 / A7 / A9 immunizations over a short duration of 15 days showed weak recognition of the incubator; the titer of 7P / 8P antibodies in sera from a short duration of 15 days was 1000–10000. In a long duration of 55 days, the titer of A3 / A7 / A9 antibodies in sera detecting the incubator was 1000:10000, while the titer of 7P / 8P antibodies in sera from a long duration of 55 days was 10000–100000. Based on the repeatability and stability of the experiments and the titer of antibodies in sera detecting the incubator, the immunization efficacy of 7P and 8P antigen peptides was superior to that of A3 / A7 / A9.

[0122] Example 4

[0123] I. Experimental Objective

[0124] Serum antibody detection of antigen peptide 7P: in vivo complex of curcumin with Aβ abnormal monomers and aggregates.

[0125] II. Experimental Methods

[0126] 1. 7P IgG was used to test 5×FAD mice aged 4–6 months that had been fed 300 mg / kg curcumin for 7 days;

[0127] The treatments were divided into two groups: a 5×FAD mouse group (Positive) receiving intraperitoneal injection of 300 mg / kg curcumin and a 5×FAD mouse group (Control) receiving intraperitoneal injection of the same volume of DMSO. The injections were administered once every 24 hours for 7 consecutive days. On the 7th day, 4 hours after the injection, the mice were anesthetized, and the thoracic cavity was cut open with scissors and forceps to expose the heart. A 1 ml syringe needle was inserted into the left ventricle, and an incision was made in the atrial appendage. The heart was then perfused with a 10 ml syringe filled with PBS. After perfusion, the mouse skull was opened, the whole brain was removed, and the whole brain was divided into two hemispheres with a scalpel. One hemisphere was placed at -80°C for frozen sectioning, and the other hemisphere was placed in 4% paraformaldehyde for paraffin sectioning.

[0128] In the 4-week-old Babl / c mouse immunization experiment, 7P immunization of Babl / c mice yielded positive 7P serum. The positive 7P serum was then analyzed using Melon... TM Gel IgG Spin Purification Kit was used to purify 7P IgG (7P sera-derived IgG); unimmunized 4-week-old Babl / c mice were used as negative control serum, which was purified using Melon... TM The IgG Spin Purification Kit was used to purify IgG into negative IgG. Positive 7P serum (7P sera-derived IgG) was used as the primary antibody to detect Aβ abnormal monomers and aggregates and curcumin complexes in the brains of positive mice (with curcumin) and control mice (without curcumin). The detection was then performed using goat anti-mouse secondary antibody combined with the primary antibody.

[0129] 2. Purification of serum IgG:

[0130] Serum IgG was purified using Melon. TMGel IgG Spin Purification Kit. Serum samples were diluted 1:10 with MeonGel Purification Buffer. The kit was incubated at room temperature for 15 min. Purification Support was inverted and mixed, and 500 μL of Purification Support was added to the centrifuge column. The column was centrifuged at 4000 rpm for 1 min, and the waste liquid was discarded. 300 μL of Purification Buffer was added to the centrifuge column, and the column was centrifuged briefly at 4000 rpm for 10 s. The waste liquid was discarded, and the washing process was repeated once. After capping the centrifuge column, 100–500 μL of serum diluent was added, and the mixture was inverted and mixed for 5 min. The purified IgG was collected by centrifuging at 4000 rpm for 1 min in a new centrifuge tube.

[0131] 3. Immunohistochemical experiments;

[0132] Remove paraffin-embedded sections of mouse brain from each group and dewax at 70℃ for 40 min. Dewax in xylene I and xylene II for 15 min each. Wash with 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each. Rinse once with distilled water for 5 min each time. Autoclave with citric acid retrieval buffer for 5 min, then wash three times with PBS for 3 min each time. Add 80 μL of 3% H2O2 and incubate at room temperature in the dark for 10 min, then wash three times with PBS for 3 min each time. Add 80 μL of 5% blocking-specific goat serum (diluted with PBS) and incubate at 37℃ for 30 min. Add 50 μL of primary antibody (P7 IgG (1:50, 1:100), 4G8 (1:1000)) and incubate overnight at 4℃. Rewarm at room temperature for 30 min, then wash three times with PBS for 3 min each time. Add 50 μL of goat anti-rabbit secondary antibody (1:200 dilution), incubate at 37°C for 1 h, wash 3 times with PBS, 3 min each time. Add 50 μL of DAB chromogenic solution, wash 3 times with PBS, 3 min each time. Stain with hematoxylin for 10 s, immediately place in double-distilled water, and blue under running water for 5 min. Stain with 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 5 min each. Then place the slide in xylene I and xylene II for 15 min each for clearing. Add a drop of neutral resin, cover with a coverslip, observe and record under a microscope.

[0133] The primary antibody used was 4G8 (to verify the presence of abundant Aβ in 5×AFD mice as an AD mouse model), diluted 1:1000. 4G8 (Biolegend Cat#800701) is an IgG2b protein, an anti-Aβ17-24 antibody. The secondary antibody for 4G8 was a goat anti-rabbit antibody, diluted 1:200. After DAB staining, hematoxylin was used for nuclear staining. Finally, images were observed and recorded at 4×, 10×, and 40× using a Nikon Ci-E microscope.

[0134] III. Experimental Results:

[0135] like Figure 4 As shown, 4G8 is an antibody against Aβ17–24. Immunohistochemical staining of 4G8 against Aβ shows patchy staining. In this experiment, 4G8 served as a positive control to verify that the mice were the correct AD mouse model. Results showed that in the 7P IgG (1:50) detection experiments in 5×FAD mice treated with and without Curcumin, the immunohistochemical results of mice treated with Curcumin were strongly positive compared to mice treated without Curcumin. 7P IgG (1:100) detection in 5XFAD mice treated with Curcumin was also positive.

[0136] In summary, 7P IgG can specifically bind to the complex of curcumin and abnormal Aβ monomers and aggregates in the mouse brain—a targeted immune complex.

[0137] Example 5

[0138] I. Experimental Objective

[0139] A 5×FAD mouse model was established after immunization with a synthetic short peptide. Curcumin was then fed to produce a targeted immune complex between curcumin and Aβ in vivo. The Aβ oligomers were cleared through the specific binding of the antibody.

[0140] II. Experimental Methods

[0141] 1. Prevention and treatment of 5×FAD mouse vaccine

[0142] The experimental treatments were divided into: control group; curcumin group; and curcumin immunization group (Alz813immunization / Curcumin), with 3-5 littermate 5×FAD mice in each group.

[0143] Curcumin group: Starting 15 days after the start of the experiment, mice were fed 300 mg / kg of curcumin every other day for a total of 75 days by gavage. Mice were sacrificed at 100 days, and the entire experiment lasted 100 days. Finally, immunohistochemistry was used to detect Aβ oligomers in 5×FAD.

[0144] Control group: No treatment was given. Mice were sacrificed after 100 days. The entire experiment lasted 100 days. Finally, immunohistochemistry was used to detect Aβ oligomers in 5×FAD.

[0145] Curcumin immunization (Alz813 immunization / Curcumin): Four-week-old 5×FAD female mice were immunized five times every 3 weeks (20-day intervals) with 50 μg (20 uL) of Alz813 (i.e., 7P, chemical structure detailed in Example 1). Starting 15 days after the first immunization, mice were administered curcumin (300 mg / kg) by gavage every other day for 75 days. Mice were sacrificed 15 days after the last immunization. The entire experiment lasted 100 days. Finally, immunohistochemistry was used to detect Aβ oligomers in 5×FAD.

[0146] 2. Obtaining mouse tissue

[0147] Under aseptic conditions, the mouse's thoracic cavity was opened with a scalpel to expose the heart. A 1ml syringe needle was inserted into the left ventricle, and an incision was made in the auricle. The heart was perfused with a 10ml syringe filled with PBS. After perfusion, the mouse's skull was opened, the whole brain was removed, and the whole brain was divided into two hemispheres with a scalpel. One hemisphere was placed at -80℃, and the other hemisphere was placed in 4% paraformaldehyde for paraffin sectioning.

[0148] 3. Immunohistochemical experiment

[0149] Remove paraffin-embedded sections of mouse brain from each group and dewax at 70℃ for 40 min. Dewax in xylene I and xylene II for 15 min each. Run the slides down the slides for 5 min each in 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol. Wash once with distilled water for 5 min each time. Perform autoclaving with citric acid retrieval buffer for 5 min, followed by washing three times with PBS for 3 min each time. Add 80 μl of 3% H2O2 and incubate at room temperature in the dark for 10 min, followed by washing three times with PBS for 3 min each time. Add 80 μl of 5% blocking-specific goat serum (diluted with PBS) and incubate at 37℃ for 30 min. Add 50 μl of primary antibody (4G8 (1:1000), A11 (1:1000)) and incubate overnight at 4℃. Rewarm at room temperature for 30 min, followed by washing three times with PBS for 3 min each time. Add 50 μl of goat anti-mouse or goat anti-rabbit secondary antibody (1:200 dilution), incubate at 37°C for 1 h, wash 3 times with PBS, 3 min each time. Add 50 μl of DAB chromogenic solution, wash 3 times with PBS, 3 min each time. Stain with hematoxylin for 10 s, immediately place in double-distilled water, and blue under running water for 5 min. Stain with 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 5 min each. Place the slide in xylene I and xylene II for 15 min each. Add a drop of neutral resin, cover with a coverslip, observe and record under a microscope.

[0150] 4. Statistical Analysis

[0151] Images were processed using ImageJ, data were analyzed using GraphPad Prism 9, and one-way AN OVA was used for comparisons between multiple groups. P < 0.05 was considered statistically significant, *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001. Mice within the same group were represented using mean ± SEM.

[0152] III. Experimental Results

[0153] like Figure 5 As shown, the content of Aβ oligomers in the hippocampus, cortex, and cerebellum of 5×FAD mice in each experimental group was detected by immunohistochemistry. A11 (Invitrogen Cat#AHB0052) is an IgG protein, a specific Aβ oligomer antibody (https: / / www.thermofisher.cn / cn / zh / antibody / product / Oligomer-A11-Antibody-Polyclonal / AHB0052); 4G8 (Biolegend Cat#800701) is an IgG2b protein, an anti-Aβ17-24 antibody. Primary antibodies were A11 and 4G8, both diluted 1:1000.

[0154] The secondary antibodies for A11 and 4G8 were goat anti-mouse antibody and goat anti-rabbit antibody, respectively, at a dilution ratio of 1:200. After DAB staining, hematoxylin was used for nuclear staining. The results showed that, compared with the curcumin group and the control group, the hippocampus of the curcumin-immunized group had significantly reduced Aβ oligomers, but there was no significant difference in the frontal cortex and cerebellum.

[0155] Example 6

[0156] I. Experimental Objective

[0157] A 5xFAD mouse model was established after immunization with a synthetic short peptide. After feeding the mice with curcumin, the cognitive function of the 5xFAD mice was examined to see if it improved.

[0158] II. Test Methods

[0159] 1. Prevention and treatment of 5×FAD mouse vaccine

[0160] The experiment was divided into 5 groups: Littermates (WT); Alz813 / Curcumin; Alz813; Curcumin; Control (5×FAD). Each group contained 3-12 mice.

[0161] 5XFAD mice are created by introducing three human APP mutant genes (Swedish (L670N, M671L), Florida I716V, London V717I) and two PS1 mutant genes (M146L, L286V) into the C57 mouse genetic background. This results in a large accumulation of Aβ in the brain of C57 mice, hence the name 5xFAD mice. They are widely used as a mouse model of autism (AD). Studies have shown that, compared to C57 mice, 5XFAD mice exhibit cognitive impairment at 5-6 months of age.

[0162] Alz813 / Curcumin: Four-week-old 5×FAD female mice were immunized six times with 50 μg (20 uL) of Alz813 every 3 weeks (20 days apart). Starting 15 days after the first immunization, mice were administered curcumin (50 mg / kg) by gavage daily for 99 days. The water maze test was started 7 days after the last immunization and lasted for 7 days. The entire experiment lasted 120 days.

[0163] Alz813: Every 3 weeks (20 days apart), littermate 4-week-old 5×FAD female mice were immunized 6 times with 50 μg (20 uL) of Alz813. The water maze test was started 7 days after the last immunization (113 days) and lasted for 7 days. The entire experiment lasted 120 days.

[0164] Curcumin: Starting 15 days after the start of the experiment, 4-week-old 5xFAD mice from the same littermate were given 50 mg / kg of curcumin daily by gavage for a total of 99 days. After the gavage ended (113 days), the water maze experiment was started and lasted for 7 days. The entire experiment lasted 120 days.

[0165] Control (5×FAD): 4-week-old 5xFAD mice from the same littermate were not treated during the period. The water maze test was started at 113 days and lasted for 7 days. The entire experiment lasted for 120 days.

[0166] Littermates (WT): 4-week-old C57 mice, without any treatment, started the water maze test at 113 days, the water maze test lasted for 7 days, and the entire experiment lasted 120 days.

[0167] 2. Water Maze Experiment: The water pool was divided into four quadrants (1, 2, 3, and 4). The escape platform was placed in the middle of quadrant 1. Different icons were placed around the pool to help the mice locate it. The experiment lasted for 6 days. Day 0 was the visibility plateau period, with the water level set 1 cm below the top of the escape platform. Mice were placed in the pool to familiarize themselves with the environment. Days 1-4 were the platform hiding period, with the water level set 1 cm above the top of the escape platform. Four points were selected as release points for the mice. Each day, each mouse entered the pool from one of the four release points. Each mouse was given 60 minutes to find and memorize the hidden escape platform. After reaching the platform, the mouse was given 15 seconds to memorize the platform and its surroundings. The interval between each release was greater than 30 minutes. On day 5, the safety platform was removed, and the quadrant opposite the platform was selected as the release point. The number of times the mouse crossed the original safety platform was recorded. The entire experiment was recorded using Smart v3.0 software.

[0168] 3. Statistical Analysis: Data were processed and exported using Smart v3.0, analyzed using GraphPad Prism 9, and compared across multiple groups using One-way ANOVA. P < 0.05 was considered statistically significant, with *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001. Mice within the same group were represented using mean ± SEM.

[0169] II. Experimental Results

[0170] The results are as follows Figure 6As shown in Figure A, on day 1, the time it took for each group to find the escape platform was roughly the same. With increasing training time, the time taken for Littermates (WT) and Alz813 / Curcumin to find the platform decreased, and the time taken for Curcumin to find the platform also decreased, but not as significantly as the Littermates (WT) and Alz813 / Curcumin groups. Finally, the time taken for Alz813 and Control (5×FAD blank group) was the longest, indicating impairment in learning and memory. At day 6, compared to Alz813, Curcumin, and Control (5×FAD), the Littermates (WT) and Alz813 / Curcumin groups found the platform more times, approaching the number of times the WT group mice crossed it. Compared to the Alz813 and Curcumin groups, Alz813 / Curcumin showed a significant difference. Figure C shows the trajectory of each group of mice crossing the platform. The above results indicate that after Alz813 immunization and feeding with curcumin, cognitive and memory abilities were significantly improved and nearly consistent with those of normal mice. Feeding with curcumin also had a certain improving effect on the cognitive function of 5×FAD, but the effect was not as good as that of the combined immunization group.

Claims

1. An antigenic polypeptide based on β-amyloid protein modification, characterized in that, Composed of A, B, and C, wherein the connection of A, B, and C is CAB, where A is CKLVFFA, B is curcumin, and C is hemocyanin. The C-terminal carboxyl group of A is connected to the hydroxyl group of B through an ester bond; A has a thiol group attached to its N-terminus, and the amino group on the surface of C has a maleimide group attached to it. A is connected to the maleamide group of C through the N-terminal thiol group.

2. The use of the β-amyloid protein-modified antigenic polypeptide as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of Alzheimer's disease.

3. The application of the antigenic polypeptide based on β-amyloid protein modification as described in claim 1 in the preparation of Alzheimer's disease vaccines.

4. The application as described in claim 3, characterized in that, The vaccine also includes an adjuvant; the adjuvant is Freund's adjuvant.

5. The use of the β-amyloid protein-modified antigenic polypeptide as described in claim 1 in the preparation of products for detecting Aβ oligomers or polymers.

6. The use of the β-amyloid protein-modified antigenic peptide as described in claim 1 in the preparation of products for early risk assessment screening, disease diagnosis, or auxiliary diagnosis of Alzheimer's disease.

7. An Alzheimer's disease vaccine, characterized in that, It includes an antigenic peptide modified with β-amyloid protein and an adjuvant, said antigenic peptide as described in claim 1.

8. The Alzheimer's disease vaccine as described in claim 7, characterized in that, The adjuvant is Freund's adjuvant.

9. An Alzheimer's disease-targeting immune complex, comprising a vaccine, characterized in that, The vaccine comprises an antigenic polypeptide modified with β-amyloid protein and an adjuvant, the antigenic polypeptide being as described in claim 1.

10. The Alzheimer's disease-targeting immune complex as described in claim 9, characterized in that, It also includes small molecule compounds, namely curcumin, demethoxycurcumin, didemethoxycurcumin, high-taurine, 3-sulfopropionic acid, epigallocatechin gallate, brasiliensis, cotton cellulose, oleuropein, quercetin, resveratrol, rosmarinic acid, 6-shogaol, tanshinone, vitamin A, and vitamin B. 12 One of vitamin D2, vitamin D3, and vitamin K3.

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