An active immunization animal model of neuromyelitis optica spectrum disorder and its construction method

Abstract

This invention discloses an active immunization animal model of neuromyelitis optica spectrum disorder and its construction method. The construction method includes: extracting a membrane protein retaining the native conformational epitope of AQP4 from cells overexpressing AQP4-M23 as an immunogen; emulsifying the immunogen with an adjuvant and then actively immunizing non-human mammals to induce the production of autoantibodies against the AQP4 conformational epitope, thereby constructing an active immunization animal model of neuromyelitis optica spectrum disorder. This invention uses an AQP4 membrane protein rich in conformational epitopes as an immunogen, overcoming the limitation of traditional linear peptides in inducing pathogenic autoantibodies in wild-type mice. It successfully constructs an active immunization animal model capable of mimicking the core pathological features of human neuromyelitis optica spectrum disorder (including AQP4 loss and astrocyte damage), providing an important experimental platform for research on the pathogenesis of neuromyelitis optica spectrum disorder and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active immunization animal model of neuromyelitis optica spectrum disease (NMOSD) and its construction method. Background Technology

[0002] Neuromyelitis optica spectrum disorders (NMOSD) are inflammatory demyelinating diseases of the central nervous system (CNS) primarily affecting the optic nerve and spinal cord, characterized by high relapse rates and high disability rates. The disease is typically characterized by recurrent optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM). Its pathological basis is autoantibody-mediated damage targeting astrocytes, rather than primary myelin sheath damage.

[0003] In the early 21st century, Lennon et al. discovered a specific biomarker in the serum of NMOSD patients—aquaporin-4 immunoglobulin G (AQP4-IgG). This discovery distinguished NMOSD from multiple sclerosis (MS). AQP4-IgG has a diagnostic specificity of over 90% and a sensitivity of approximately 70%, making it a core diagnostic criterion for NMOSD. Current research suggests that AQP4-IgG is the direct pathogenic antibody for NMOSD. When the blood-brain barrier (BBB) ​​is impaired, AQP4-IgG in the peripheral circulation enters the central nervous system and binds to AQP4, which is highly expressed on the terminal foot of astrocytes. Through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), it leads to astrocyte damage, which in turn causes oligodendrocyte damage, demyelination, and axonal damage, ultimately forming the characteristic pathological changes of NMOSD.

[0004] AQP4 is a six-transmembrane aquaporin, primarily existing in two isoforms: M1 and M23. AQP4-M23 is more likely to form highly ordered supramolecular structures on the cell membrane, namely orthogonal arrays of particles (OAPs). Studies have shown that patient-derived AQP4-IgG does not recognize linear epitopes of AQP4, but rather preferentially recognizes conformational epitopes formed by AQP4-M23, which are dependent on the higher-order structure of the AQP4 tetramer and its OAPs. These epitopes are composed of three extracellular loops: A, C, and E. The binding of AQP4-IgG to these OAP conformational epitopes is a key step in the subsequent activation of complement and the initiation of the CDC effect.

[0005] To further investigate the pathogenesis of NMOSD and screen therapeutic drugs, researchers have established various animal models. Currently, the main NMOSD animal models can be divided into two categories: passive transfer models and active immunization models. Passive transfer models observe the pathogenic effects by directly injecting AQP4-IgG (often combined with human complement or pro-inflammatory factors) into the brain of animals, such as the stereotactic injection model and the experimental autoimmune encephalomyelitis (EAE) combined with antibody injection model. The advantage of these models is that they can rapidly and directly verify the pathogenicity of AQP4-IgG, making them suitable for verifying antibody effector mechanisms and interventions. However, their limitations lie in that they bypass the immune activation process of autoantibody production, failing to simulate the complete immune response initiation stage of NMOSD from antigen presentation and lymphocyte activation to antibody production. Furthermore, the lesions are often limited to the injection site, making it difficult to comprehensively reflect the complex process of systemic autoimmune diseases. Active immunization models, on the other hand, immunize animals with AQP4 antigen, aiming to induce the production of AQP4-IgG, thereby more completely simulating the immune process of human diseases. Currently reported active immunization strategies mainly include the use of linear peptides of AQP4 (such as AQP4). 201-220 AQP4-encoding plasmids can be introduced via electroporation. However, these existing techniques have significant drawbacks: the linear AQP4 peptides or recombinant proteins used often fail to fold correctly, failing to fully preserve the conformational epitopes specific to AQP4 when forming OAPs on the cell membrane, which are recognized by pathogenic AQP4-IgG. Therefore, existing active immunization models generally suffer from weak pathogenicity of induced antibodies, unstable antibody titers, and atypical or mild central nervous system pathological changes (such as astrocyte damage, AQP4 deficiency, and complement deposition), resulting in a significant discrepancy with the pathological characteristics of human NMOSD. This makes existing active immunization models significantly inadequate in reproducing the core pathogenesis of NMOSD, limiting their application in studying key scientific questions such as disease initiation and the evolution of autoimmune responses.

[0006] In summary, there is currently a lack of an active immunization animal model that can stably induce the production of pathogenic AQP4-IgG and accurately reproduce the characteristic pathological changes of NMOSD. Therefore, developing a novel immunogen that can retain the native conformational epitopes of AQP4 and establishing an active immunization model of NMOSD that more closely resembles the human disease state is of significant scientific and clinical value for in-depth analysis of the immunopathogenesis of NMOSD, screening therapeutic drugs for different stages of the disease, and evaluating the effectiveness of novel treatment strategies. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a novel immunogen that can retain the natural conformational epitope of AQP4, and based on this, to construct an active immunization animal model of neuromyelitis optica spectrum disease that more closely resembles the human disease state, while also providing a method for constructing the model.

[0008] To achieve the above objectives, the active immunization animal model of neuromyelitis optica spectrum disorder provided by the present invention is obtained by the following construction method:

[0009] (1) Extract AQP4 membrane protein rich in conformational epitopes as an immunogen.

[0010] (2) The immunogen obtained in step (1) is mixed with adjuvant, emulsified, and then actively immunized in non-human mammals to induce them to produce autoantibodies against the AQP4 conformational epitope, thereby constructing an active immunization animal model of neuromyelitis optica spectrum disease.

[0011] Preferably, the AQP4 membrane protein rich in conformational epitopes in step (1) is a membrane protein that retains the native conformational epitopes of AQP4 and is extracted from cells that overexpress AQP4-M23.

[0012] Preferably, the concentration of AQP4 membrane protein in the emulsion obtained after emulsification in step (2) is 0.5 to 1 mg / mL.

[0013] Preferably, the adjuvant in step (2) is Freund's complete adjuvant, wherein the concentration of Mycobacterium tuberculosis in the Freund's complete adjuvant is 10 mg / mL.

[0014] Preferably, the non-human mammal in step (2) is a rodent; more preferably, the rodent is either a rat or a mouse.

[0015] Preferably, the active immunization method in step (2) is subcutaneous multi-point injection, with an immunization dose of 100 μL / animal; and pertussis toxin is administered to non-human mammals at the same time as or after active immunization.

[0016] Preferably, the active immunization in step (2) is a booster immunization, and the second immunization is performed 2 to 3 weeks after the first immunization.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention is the first to use AQP4 membrane protein rich in conformational epitopes as an immunogen, successfully constructing an active immunization animal model of neuromyelitis optica spectrum disease. This immunogen retains the orthogonal array particle conformational epitopes of AQP4, and can induce the production of functional AQP4 antibodies that recognize conformational epitopes, overcoming the technical bottleneck that traditional linear peptide immunogens are unable to induce pathogenic antibodies.

[0019] 2. The animal model constructed in this invention can produce functional antibodies with complement-dependent cytotoxic effects. At the behavioral level, it exhibits optic nerve dysfunction and motor impairment; at the histopathological level, it shows astrocyte damage, AQP4 expression loss, and myelin damage; and at the immunological level, it displays peripheral immune structural changes characterized by humoral immune activation. It comprehensively simulates the core clinical phenotypes and pathological features of neuromyelitis optica spectrum disorders in humans. The animal model constructed in this invention provides an important experimental platform for studying the pathogenesis, drug screening, and efficacy evaluation of neuromyelitis optica spectrum disorders, and is of great significance for promoting the development of targeted interventions for this disease. Attached Figure Description

[0020] Figure 1 It is AQP4 OAP Figure showing the results of membrane protein extraction and validation. (A) Detection of Vector proteins using the BCA method. M Membrane proteins and AQP4 OAP Membrane protein concentration; (B) Vector M Membrane proteins and AQP4 OAP Schematic diagram of membrane protein coating; (C) Detection results of serum from AQP4-IgG positive patients.

[0021] Figure 2 This image shows the immunofluorescence results of different AQP4 mutants binding to serum from NMOSD patients. AQP4-M23 is the wild-type control; AQP4-HW... 230-231 GG and AQP4-TP 137-138 AA is a mutant of key extracellular amino acid residues; AQP4-OAP is a conformational membrane protein extracted in this invention.

[0022] Figure 3 It is AQP4 201-220 Mice and AQP4 OAPFigure 1 shows the results of antibody titer detection in mice. (A) shows the AQP4 antibody titer in the serum of different groups of mice 4 weeks after immunization, detected by the CBA method; (B) shows the quantitative display of AQP4 antibody titers in different groups of mice.

[0023] Figure 4 It is AQP4 201-220 Group mice and AQP4 OAP Figure 1 shows the results of the open field experiment on mice in each group. (A) shows the activity trajectory of mice in each group in the open field; (B) shows the quantitative analysis of the total distance and average speed of mice in different groups in the open field.

[0024] Figure 5 It is AQP4 OAP Figure 1 shows the results of the balance beam experiment for mice in different groups. (A) Schematic diagram of the balance beam experiment; (B) Quantitative analysis of the number of times the hind limbs of mice in different groups slipped off the 1.2 cm × 80 cm balance beam; (C) Quantitative analysis of the number of times the hind limbs of mice in different groups slipped off the 0.6 cm × 80 cm balance beam.

[0025] Figure 6 It is AQP4 201-220 Group mice and AQP4 OAP Figure 1 shows the gait analysis results of the mice in the group. (A) Schematic diagram of gait analysis; (B) Quantitative analysis of the average stride length of the left hind leg of mice in different groups and the stride width between the hind legs of mice in different groups; (C) Quantitative analysis of gait symmetry and forelimb support force of mice in different groups.

[0026] Figure 7 It is AQP4 OAP Mice and AQP4 201-220 In vivo imaging results of mice. (A) Representative in vivo images of mice after tail vein injection of Cy5-labeled probes in each group; (B) Quantitative analysis of fluorescence intensity in the central nervous system of mice in each group.

[0027] Figure 8 It is AQP4 OAP Figures showing the results of retinal and optic nerve function tests in mice. (A) Typical waveforms of light adaptation 3.0 response in each group of mice; (B) Quantitative analysis of the b-wave amplitude of light adaptation 3.0 response in each group of mice; (C) Typical waveforms of dark adaptation 3.0 response in each group of mice; (D) Quantitative analysis of the b-wave amplitude of dark adaptation 3.0 response in each group of mice; (E) Typical waveforms of dark adaptation OPs response in each group of mice; (F) Quantitative analysis of the Ops2 amplitude of dark adaptation in each group of mice; (G) Typical waveforms of FVEP in each group of mice; (H) Quantitative analysis of the P2 peak of FVEP in each group of mice; (I) Quantitative analysis of the P2 amplitude of FVEP in each group of mice.

[0028] Figure 9 It is AQP4 OAP Mice and AQP4201-220 Images showing myelin damage in mice. (A) Representative images of LFB staining in cross-sections of the spinal cord from different groups of mice; (B) Transmission electron micrographs of the white matter of the spinal cord from different groups of mice.

[0029] Figures 4-8 All data are expressed as Mean ± SEM. Statistical analysis was performed using one-way ANOVA. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these existing embodiments.

[0031] Example 1

[0032] 1. Extraction and validation of AQP4 membrane proteins rich in conformational epitopes

[0033] (1) Construction of AQP4 overexpression cell lines

[0034] Target plasmids: pCDH-CMV-AQP4-T2A-puro target plasmid and pCDH-CMV-T2A-puro control plasmid were used, both of which were preserved in our laboratory.

[0035] Construction of a stable AQP4 expression cell line: ① Observe the state of 293T cells under a microscope. When the cells reach approximately 90% confluence, change the medium to 8 mL of DMEM basal medium. ② Add the target plasmid and lentiviral helper plasmid to 500 μL of DMEM basal medium, vortex to mix, briefly centrifuge, then add PEI, vortex to mix, briefly centrifuge, and incubate for 10–12 min. Then transfer the cells to 8 mL of DMEM basal medium. ③ Discard the DMEM basal medium from the 293T cells and add prepared DMEM basal medium containing the plasmid. ④ After 4–6 h, change the medium to 8 mL of complete DMEM. ⑤ After 24 h, collect the cell supernatant, centrifuge at 800 rpm for 5 min, and transfer the supernatant to 40%–60% confluence cells for infection. ⑥ After 24 h, add 8 μg / mL puromycin for selection. ⑦ After 24 h, passage the cells using a medium containing 3… Cell lines stably expressing AQP4 were obtained by screening in DMEM complete medium containing μg / mL puromycin.

[0036] (2) AQP4 membrane protein rich in conformational epitopes was obtained by a mild extraction method.

[0037] Take AQP4 stable cell lines with approximately 90% confluence and extract membrane proteins according to the following steps: ① Discard the culture supernatant, slowly add 2 mL of 0.01 M PBS to each dish of cells, gently rinse the cell surface, and then discard the PBS; ② Add 1.5 mL of cell lysis buffer to each dish of cells (weigh 0.176 g of sodium chloride, 0.892 g of potassium chloride, 0.044 g of anhydrous calcium chloride, and 0.404 g of magnesium chloride hexahydrate, add triple-distilled water to 200 mL, mix well to obtain a cation complex solution; take a 15 mL centrifuge tube, add 6.0 mL of triple-distilled water and 6 tablets of protease inhibitor, dissolve thoroughly, invert and mix well to obtain 50 × protease inhibitor; take 19.4 mL of the cation complex solution, 200 μL of 5% NP-40 solution, and 400 μL of 50 × protease inhibitor, invert and mix well), and lyse on a shaker at 25 rpm for 5 min; ③ Collect the cell lysate and liquid in a 50 mL EP tube, and centrifuge at 650 rpm. ④ Centrifuge at 19000 g, 4℃ for 5 min, discarding cell nuclei and unlysed cells; ⑤ Collect the supernatant into new 2 mL EP tubes, 2 mL per tube, centrifuge at 19000 g, 4℃ for 5 min, discarding the supernatant; ⑥ Add 100 μL of cell lysis buffer to each precipitate, mix by pipetting with a 1 mL pipette tip, centrifuge at 19000 g, 4℃ for 5 min, discarding the supernatant; ⑦ Add 40 μL of 1.9 M sucrose aqueous solution and 10 μL of resuspension (1.2 mL of 0.8% NaCl, 12 μL of 5% NP-40 solution, and 24 μL of 50 × protease inhibitor, inverted and mixed), mix by pipetting with a pipette tip, centrifuge at 19000 g, 4℃ for 10 min; ⑧ Absorb the supernatant to obtain the AQP4 membrane protein containing the conformational antigen, named AQP4. OAP .

[0038] Simultaneously, a control membrane protein, named Vector, was extracted from control cells transfected with the empty vector using the same method. M .

[0039] (3) AQP4 membrane protein concentration determination and conformation verification

[0040] Vector detection using BCA method M Membrane proteins and AQP4 OAP Concentration of membrane proteins. Results showed that AQP4... OAP The concentration of membrane protein was 2.506 mg / mL, Vector M The concentration of the membrane protein was 3.334 mg / mL. Subsequent experiments standardized both concentrations to 1 mg / mL for animal immunization. Figure 1 A).

[0041] To verify AQP4 OAPWhether membrane proteins possess conformational epitopes will be determined by extracting AQP4. OAP Membrane proteins and vectors M Membrane proteins are coated onto nitrocellulose membranes. The coating method is as follows: Figure 1 As shown in B: AQP4 OAP Membrane proteins were coated at points A, B, and C of the NC membrane in the order of undiluted, 1.5-fold diluted, and 2.25-fold diluted; Vector M Membrane proteins were coated onto the E, F, and G regions of the NC membrane in the order of undiluted, 1.5-fold diluted, and 2.25-fold diluted; standard AQP4 was then added. OAP Membrane proteins and vectors M Membrane proteins were coated at the D and H positions of the NC membrane, respectively. Serum from AQP4-IgG positive patients was then incubated, and the test results were confirmed by a colorimetric reaction.

[0042] The results showed that the serum of AQP4-IgG positive patients exhibited characteristic color development, while the serum of AQP4-IgG negative patients did not show color development. Figure 1 C), indicating the extracted AQP4 OAP Membrane proteins retain complete conformational epitopes.

[0043] (4) Validation of AQP4 conformational epitopes using key residue mutants

[0044] To further validate the extracted AQP4 OAP Membrane proteins retained the correct conformational epitopes. Immunofluorescence staining was used to detect the binding ability of different AQP4 mutants to AQP4-IgG in the serum of NMOSD patients. The specific detection method was as follows: ① Cell transfection: 293T cells were transfected with the following plasmids: AQP4-M23; wild-type AQP4-M23; AQP4-HW... 230-231 GG: A key residue mutant located in the extracellular E loop of AQP4; AQP4-TP 137-138 AA: A key residue mutant located in the extracellular C-ring of AQP4; AQP4-OAP: AQP4 membrane protein rich in conformational epitopes extracted in this invention; ② Cell slide preparation: 48 h after transfection, cell slides were prepared, fixed with 4% paraformaldehyde for 15 min, and washed 3 times with PBS; ③ Serum incubation: 1:10 diluted serum from NMOSD patients (AQP4-IgG positive) was added and incubated overnight at 4℃; ④ Fluorescent secondary antibody incubation: After washing with PBS, anti-human IgG secondary antibody labeled with Alexa Fluor 488 was added and incubated at room temperature in the dark for 1 h; ⑤ Mounting observation: After washing with PBS, slides were mounted with mounting medium containing DAPI, observed and photographed under a laser confocal microscope.

[0045] The results are as follows Figure 2As shown: Cells transfected with AQP4-M23 exhibit typical speckled strong fluorescence signals, indicating that the OAPs structure formed by wild-type AQP4-M23 can be effectively recognized by AQP4-IgG in the serum of NMOSD patients. Transfected with AQP4-HW 230- 231 GG and AQP4-TP 137-138 The cellular fluorescence signal of AA was significantly reduced, almost invisible, indicating that TP located in the extracellular C-ring. 137-138 HW of site and E-ring 230-231 These sites are key residues for AQP4-IgG recognition; mutations at these sites lead to conformational epitope disruption and loss of antibody binding ability. Cells transfected with AQP4-OAP exhibit a speckled, strong fluorescence signal similar to AQP4-M23, indicating that the AQP4 extracted in this invention... OAP The membrane protein retains the same native conformational epitopes as wild-type AQP4-M23 and can be specifically recognized by AQP4-IgG in the serum of NMOSD patients.

[0046] The above results further confirm that the AQP4 obtained by the mild extraction method of this invention is effective. OAP The membrane protein successfully retained the native conformational epitope of AQP4, providing a reliable immunogen for the subsequent construction of an active immunization animal model.

[0047] 2. Construction of an active immunization animal model of neuromyelitis optica spectrum disease

[0048] (1) Laboratory animals

[0049] Female C57BL / 6J mice aged 8–10 weeks were selected as experimental subjects. All mice were raised under specific pathogen-free (SPF) conditions at an ambient temperature of 24 ± 2℃ and a day-night cycle of 12 h. The mice had free access to food and water.

[0050] (2) Experimental grouping

[0051] The experimental animals were randomly divided into the following groups: blank control group (WT group): no immunization treatment was given; control membrane protein immunization group (Vector group). M Group): Using Vector M Immunization with membrane proteins; AQP4 conformation membrane protein immunoassay (AQP4) OAP Group: AQP4 prepared in Example 1 OAP Immunization using membrane proteins; linear peptide immunization group (AQP4) 201-220 Group): Using AQP4 201-220 Linear peptides were used for immunization as a control.

[0052] (3) Preparation of immunogen

[0053] Preparation of Freund's complete adjuvant: Take 10 mL of Freund's incomplete adjuvant and 100 mg of Mycobacterium tuberculosis from a 4°C freezer and mix them evenly at a 1:1 ratio to make the final concentration of Mycobacterium tuberculosis 10 mg / mL.

[0054] Preparation of antigen emulsion: Add 4 glass beads to a 2 mL centrifuge tube; add 1 mL Freund's complete adjuvant and 1 mL antigen (AQP4). OAP Membrane proteins, Vector M Membrane protein or AQP4 201-220 The peptides were all at a concentration of 2.5 mg / mL. They were then ground using a small grinder at 4°C for 3 min (30 s / time), followed by a large grinder for 1 min (30 s / time), and finally ground again using a small grinder at 4°C for 3 min (30 s / time). The ground emulsion was then dropped into double-distilled water; if it did not diffuse within 30 s, the emulsification was considered complete.

[0055] (4) Immunization schedule

[0056] ① Anesthesia: Anesthetize the mice by intraperitoneal injection of 1% sodium pentobarbital, based on their body weight; ② First immunization: Inject a total of 100 μL of emulsion subcutaneously into four points on the back and groin of the mice; simultaneously, inject 200 ng of pertussis toxin intraperitoneally; ③ Booster immunization: On the second day after the first immunization, inject 200 ng of pertussis toxin intraperitoneally again; two weeks after the first immunization, administer the second immunization using the same method.

[0057] 3. Identification and Validation of Animal Models

[0058] (1) Serum AQP4 antibody detection (CBA method)

[0059] Four weeks after immunization, mouse serum was collected, and the AQP4 antibody titer was detected using a cell-based assay (CBA). The specific detection method was as follows: ① Preparation: Serum was diluted 1:10 with working PBST; anti-mouse secondary antibody was diluted 1:200 with working PBST; 293T cell slides transfected with AQP4-M23 were removed from -20℃ and allowed to warm to room temperature for 10 min; ② Washing: 1 mL of PBST was added to the reaction area of ​​the slide and washed for 10 min; ③ Primary antibody incubation: PBST was aspirated, and diluted serum was immediately added and incubated at room temperature for 45 min; ④ Washing: Serum was aspirated, and the slides were washed three times with PBST; ⑤ Secondary antibody incubation: 50 μL of diluted anti-mouse secondary antibody was added and the slides were incubated at room temperature in the dark for 30 min; ⑥ Washing: Liquid was aspirated, and the slides were washed three times with PBST; ⑦ Color development: 1 mL of PBST was added, and the slides were developed under 20x red light and observed under a fluorescence microscope.

[0060] The results showed that the WT group and AQP4 201-220Groups and Vectors M None of the mice in the group produced AQP4-IgG, while AQP4 OAP The mice in this group could produce AQP4-IgG, with antibody titers reaching up to 1:1000. Figure 3 A, 3B).

[0061] (2) Behavioral assessment

[0062] Open field test: Mice were placed in the test area for 2 hours to acclimatize; the open field box was cleaned with 75% alcohol to ensure no odor residue; the mice were gently placed in the center of the open field box, and their movement trajectory was recorded over 10 minutes. The total movement distance and average speed were analyzed using Smart software. Results showed that, compared with the WT group, AQP4... 201-220 The total distance and speed of movement in the group of mice were significantly reduced; compared with Vector M Compared to the previous group, AQP4 OAP The total distance and speed of movement in the group of mice were significantly reduced; AQP4 OAP The movement distance and speed of the mice in the group were compared with those in AQP4. 201-220 The group showed a downward trend, but there was no significant difference. Figure 4 The results indicate that the motor abilities of both mouse models were reduced.

[0063] Balance beam experiment: Mouse motor coordination was assessed using balance beams of 1.2 × 80 cm and 0.6 × 80 cm widths. A camera was attached to the beam 50 cm above the ground. Mice were trained to walk on the thicker beam until they became proficient. The time taken for the mice to cross the balance beam and the number of times their hind limbs slipped were recorded per minute. Results showed that on the 1.2 cm × 80 cm balance beam, AQP4 levels... OAP The number of times the hind limbs of the mice in the group slipped was significantly higher than that of the Vector group. M Groups and AQP4 201-220 Group; on a 0.6 cm × 80 cm balance beam, AQP4 OAP The number of times the hind limbs of the mice in the group slipped was significantly higher than that of the Vector group. M Group( Figure 5 (AQP4 prompt) OAP The group of mice showed a more significant decline in motor coordination.

[0064] Gait analysis: Gait analysis was performed using a Mouse Specifics DigiGait instrument. Mice were acclimatized to the treadmill environment and trained starting at a low speed of 5 cm / s. The treadmill speed was set to 10 cm / s, and stable running videos of the mice were recorded. Parameters such as stride length, stride width, gait symmetry, and forelimb support force were automatically extracted. Results showed that, compared with the WT group, AQP4... 201-220 Groups and AQP4 OAPThe stride length of all mice in the group was significantly shortened, but there were no significant differences in stride width, gait symmetry, and forelimb support force. Figure 6 This indicates that both mouse models exhibit motor dysfunction.

[0065] (3) Detection of inflammatory infiltration in the central nervous system

[0066] In vivo imaging of small animals was used to detect inflammatory infiltration in the central nervous system. Cy5-labeled probes were injected via the tail vein; in vivo imaging analysis was performed using the small animal in vivo imaging system; and fluorescence intensity in the central nervous system was quantitatively analyzed. Results showed that, compared with the WT group, AQP4... 201-220 Groups and AQP4 OAP The fluorescence signal in the central nervous system of mice in both groups was significantly enhanced, but there was no significant difference between the two groups. Figure 7 This suggests that both immunization methods can effectively induce inflammatory infiltration of the central nervous system.

[0067] (4) Visual function testing

[0068] Electroretinography (ERG) was performed on mice after 12 hours of dark adaptation. Mice were anesthetized under weak red light by intraperitoneal injection of 1% sodium pentobarbital (0.25 mL / kg) combined with 10% sedative (0.01 mL / mouse). Mydriasis was achieved using compound tropicamide eye drops, and ocular surface anesthesia was performed using oxybuprofen hydrochloride eye drops. Recording electrodes were AgCl corneal ring electrodes, reference electrodes were stainless steel buccal needle electrodes, and grounding electrodes were stainless steel tail needle electrodes. Light adaptation 3.0 response, dark adaptation 3.0 response, and dark adaptation OPs response were measured. Results showed that in the light adaptation 3.0 response, AQP4... 201-220 Groups and AQP4 OAP The amplitude of the b-wave in all mice in the group was significantly reduced ( Figure 8 A, 8B); In the dark adaptation 3.0 reaction, AQP4 OAP The amplitude of the b-wave in the >group mice was significantly reduced, while the AQP4 group mice showed a significantly reduced amplitude. 201-220 No significant changes were observed in the group ( Figure 8 C, 8D); In dark-adapted Ops2 wavelet detection, AQP4 OAP The amplitude values ​​of the AQP4 group mice were significantly reduced, while the amplitude values ​​of the AQP4 group were significantly reduced. 201-220 No significant changes were observed in the group ( Figure 8 E, 8F). The results show that AQP4 OAP The mice in the group showed significant retinal dysfunction, while AQP4... 201-220 The retinal function of the mice in the group was basically normal.

[0069] Flash visual evoked potential (FVEP) testing: The FVEP testing method is the same as the ERG testing method, used to assess the conduction function from the retina to the visual cortex of the brain. Results showed that, compared with Vector... M Compared to the previous group, AQP4OAP The FVEP response in the mice group was delayed in P2 peak time and decreased in amplitude, while AQP4... 201-220 No significant changes were observed in the FVEP response of the mice in the group. Figure 8 GI). The results showed that AQP4 OAP The mice in this group exhibited visual pathway conduction dysfunction.

[0070] (5) Histopathological examination

[0071] Animal perfusion and tissue collection: After anesthetizing the mice, the eyeballs were removed and blood was collected; the mice were fixed on the dissection table, the heart was exposed, the syringe needle was inserted into the left ventricle, the right atrial appendage was cut open, and about 50 mL of pre-cooled PBS buffer was perfused; the brain tissue, spinal cord lumbar enlargement and optic nerve were quickly removed for subsequent testing.

[0072] Immunofluorescence staining was used to detect AQP4 and GFAP expression: Frozen sections were prepared after tissue fixation, with a thickness of 6 μm. After thawing, the sections were washed with PBS to remove the embedding medium. The sections were permeated with ice-cold acetone for 15 min. Blocking was performed with PBS solution containing 3% BSA and 0.3% Triton X-100 for 60 min. Primary antibodies (anti-AQP4 antibody and anti-GFAP antibody) were added, and the sections were incubated overnight at 4°C. After washing with PBS, fluorescein-conjugated secondary antibody was added, and the sections were incubated at room temperature in the dark for 60 min. The sections were mounted with DAPI-containing mounting medium and observed under a fluorescence microscope. The results showed that AQP4... OAP The expression of AQP4 and GFAP was significantly reduced in the optic nerve and spinal cord tissues of the mice in the group, while the expression of AQP4 was significantly reduced. 201-220 No significant loss of AQP4 and GFAP was observed in the mice group.

[0073] LFB myelin staining: Paraffin sections were dewaxed to water, and myelin staining solution A (preheated for 30 min) was added. The sections were incubated at 65°C for 2 h, allowed to cool naturally, and then washed with water until colorless. Myelin staining solutions B and C were used alternately for color separation until the white matter turned blue and the gray matter turned white. After dehydration and clearing, the sections were mounted and observed under a microscope. Results showed that no obvious demyelinating lesions were observed in any group of mice. Figure 9 A).

[0074] Transmission electron microscopy observation of myelin sheath ultrastructure: Mice were perfused with PBS and then fixed with cardiac perfusion containing 2.5% glutaraldehyde and 4% PFA; the lumbar enlargement of the spinal cord was removed and cut into 1 mm sections. 3 Small pieces were fixed with 2.5% glutaraldehyde at 4℃ for 2 h and 1% osmium tetroxide at 4℃ for 2 h; after gradient dehydration, they were embedded to prepare 50 nm ultrathin sections; after staining with uranium acetate and lead citrate, they were observed under a transmission electron microscope. The results showed that the WT group and Vector group... M In the AQP4 group of mice, the axons in the spinal cord white matter were regularly arranged, and the myelin sheath lamina were dense and continuous; while in the AQP4 group... 201-220 Groups and AQP4OAP All mice in the group showed loosening, separation, and vacuolar changes in the myelin sheath lamina, with significant thinning or even breakage of the myelin sheath around some axons, exhibiting typical demyelination ultrastructural characteristics. Figure 9 B). The results showed that both immunization methods could cause significant damage to the myelin sheath structure of the mouse spinal cord white matter.

[0075] The above experimental results show that the present invention uses AQP4 rich in conformational epitopes. OAP Membrane proteins were used as immunogens to successfully construct an active immunization animal model of neuromyelitis optica spectrum disorder (NMDS). This model produced AQP4-specific antibodies that recognize conformational epitopes and exhibited complement-dependent cytotoxic effects. Functionally, it displayed optic nerve dysfunction and motor impairment; histopathologically, it showed astrocyte damage, AQP4 expression loss, and myelin sheath damage; and immunologically, it exhibited characteristics of humoral immune activation. This model comprehensively simulates the core clinical phenotypes and pathological features of human NMDS, providing an important experimental platform for research on the pathogenesis of this disease and drug screening.

Claims

1. A method for constructing an active immunization animal model of neuromyelitis optica spectrum disease, characterized in that, Includes the following steps: (1) Extract AQP4 membrane protein rich in conformational epitopes as an immunogen; (2) The immunogen obtained in step (1) is mixed with adjuvant, emulsified, and then actively immunized in non-human mammals to induce them to produce autoantibodies against the AQP4 conformational epitope, thereby constructing an active immunization animal model of neuromyelitis optica spectrum disease.

2. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, The AQP4 membrane protein rich in conformational epitopes mentioned in step (1) is a membrane protein that retains the native conformational epitopes of AQP4 and is extracted from cells that overexpress AQP4-M23.

3. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, In the emulsion obtained after emulsification in step (2), the concentration of AQP4 membrane protein is 0.5 to 1.0 mg / mL.

4. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, The adjuvant mentioned in step (2) is Freund's complete adjuvant, and the concentration of Mycobacterium tuberculosis in the Freund's complete adjuvant is 5 to 15 mg / mL.

5. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, The non-human mammals mentioned in step (2) are rodents.

6. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 5, characterized in that, The rodents mentioned are either rats or mice.

7. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, The active immunization method described in step (2) is subcutaneous multi-point injection, with an immunization dose of 100 μL / animal; and pertussis toxin is administered to non-human mammals at the same time as active immunization or 48 h later.

8. The method for constructing an active immunization animal model of neuromyelitis optica spectrum disorder according to claim 1, characterized in that, The active immunization mentioned in step (2) is a booster immunization, and the second immunization is performed 2 to 3 weeks after the first immunization.

9. An active immunization animal model of neuromyelitis optica spectrum disorder obtained by the construction method according to any one of claims 1 to 8.

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