An oap size-optimized aqp4 recombinant protein and application thereof in preparation of aqp4-igg antibody detection kit

Abstract

The application discloses an AQP4 recombinant protein with optimized OAP size and application thereof in preparation of an AQP4-IgG antibody detection kit, and belongs to the technical field of medical immunodetection and protein engineering. The recombinant protein is a mutant obtained by engineering modification on the N terminal of AQP4-M23 protein, and the average area of OAP aggregates of the mutant is larger than that of natural AQP4-M23; the membrane positioning expression efficiency of the mutant is equivalent to that of the natural AQP4-M23 protein; and the immunofluorescence end-point titer of the mutant in combination with AQP4-IgG is higher than that of the natural AQP4-M23 protein. The recombinant protein can also be used for preparing a kit for detecting AQP4-IgG antibody, solves the problems of unstable antigen OAP size and insufficient sensitivity of existing detection antigen OAP, and realizes the improvement of AQP4-IgG detection sensitivity.

Description

Technical Field

[0001] This invention relates to a recombinant AQP4 (aquaporin 4) protein with optimized OAP (orthogonal array) size and its application in the preparation of an antibody kit for detecting AQP4-IgG. Specifically, it involves a specific engineering modification of the N-terminus of the M23 isoform of AQP4 to obtain a recombinant protein with optimized OAP size, and the application of this recombinant protein in the preparation of an antibody kit for detecting AQP4-IgG. This invention belongs to the fields of medical immunoassay and protein engineering technology. Background Technology

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is a severe autoimmune inflammatory demyelinating disease of the central nervous system, with anti-aquaporin 4 antibody (AQP4-IgG) as its specific biomarker. Accurate detection of AQP4-IgG is crucial for the diagnosis, differential diagnosis, and assessment of disease activity in NMOSD.

[0003] AQP4 is expressed at the terminal podocytes of astrocytes and exists in two main isoforms: M1 (full length 323 amino acids) and M23 (N-terminal truncated 301 amino acids). The smallest unit of AQP4 assembly in the cell membrane is the tetramer. The lateral assembly of AQP4-M23 at its N-terminus, from amino acids 23 to 44, is driven by hydrophobic interactions, forming an orthogonal array (see Crane JM, et al. Live cell analysis of aquaporin-4 m1 / m23 interactions and regulated orthogonal array assembly in glial cells. Journal of Biological Chemistry, 2009, 284(50): 35806-35813; and Furman CS, et al. Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms. Proceedings of the National Academy of Sciences USA, 2003). 100(23): 13609-13614). Direct quantification of OAP size using super-resolution microscopy revealed that natural AQP4-M23 can form large OAPs containing up to 600 tetramers, while co-expression of AQP4-M1 physically blocks OAP expansion through its N-terminal extension, resulting in a reduction in OAP size (see Rossi A, et al. Super-resolution imaging of aquaporin-4 orthogonal arrays of particles in cell membranes. Journal of Cell Science, 2012, 125(Pt 19): 4445-4453).A mathematical model of OAP assembly was developed to quantitatively predict the relationship between the ratio of AQP4-M1 to AQP4-M23 and the size distribution of OAP, proving that pure AQP4-M23 forms the largest OAP (see Jin BJ, et al. Model of aquaporin-4 supramolecular assembly in orthogonal arrays based on heterotetrameric association of M1-M23 isoforms. Journal of Biological Chemistry, 2011, 286(1): 637-645).

[0004] Existing research indicates that the target epitopes of AQP4-IgG are located on the extracellular loops A, C, and E of AQP4 cells, rather than on the cytoplasmic N-terminus (see Nicchia GP, et al. Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies. Glia, 2009, 57(7): 670-679; and Crane JM, et al. Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1 / M23 isoforms and orthogonal arrays. Journal of Biological Chemistry, 2011, 286(19): 16516-16524). OAP assembly makes the loops A, C and E of adjacent tetramers spatially adjacent, forming new conformational epitopes and enhancing antibody affinity (see NicchiaGP, et al. Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies. Glia, 2009, 57(7): 670-679).OAP size was positively correlated with AQP4-IgG binding efficiency and complement-dependent cytotoxicity. The antibody affinity of AQP4-M23-OAP was significantly higher than that of AQP4-M23 monomer or AQP4-M1 (see Crane JM, et al. Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1 / M23 isoforms and orthogonal arrays. Journal of Biological Chemistry, 2011, 286(19): 16516-16524; and Phuan PW, et al. Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays (Complement-dependent cytotoxicity of neuromyelitis optica requires aquaporin-4 to be assembled in orthogonal arrays). Journal of Biological Chemistry, 2012, 287(17): 13829-13836.

[0005] Currently, matrix-based indirect immunofluorescence assay (CBA) is the internationally recognized gold standard for AQP4-IgG detection. Existing CBA detection systems mainly use cells transfected with a single AQP4-M23 subtype, leveraging its tendency to form open apoptosis sites (OAPs) to improve detection sensitivity. Furthermore, human AQP4 monoclonal antibodies have been developed as positive standards for detection systems (see the authorized text of Chinese patent CN117304314B). However, existing technologies still have the following shortcomings: the OAP size of natural AQP4-M23 is affected by factors such as cell type, expression level, and culture conditions, resulting in significant batch-to-batch variations; some low antibody titer samples cannot be effectively detected due to insufficient OAP size, leading to potentially negative CBA test results in the serum of clinically diagnosed NMOSD patients. Summary of the Invention

[0006] The purpose of this invention is to provide an OAP-size-optimized recombinant AQP4 protein and its application in the preparation of a kit for detecting AQP4-IgG antibodies. The aim is to engineer the N-terminus of the AQP4-M23 isotype to obtain a recombinant protein with optimized OAP size. Compared to natural AQP4-M23, this recombinant protein has a larger average OAP aggregate area, comparable membrane localization efficiency, and higher AQP4-IgG binding affinity, resulting in lower antibody titer detection. This recombinant protein can also be used to prepare a kit for detecting AQP4-IgG antibodies, addressing the problems of unstable OAP size and insufficient sensitivity in existing antigen detection methods, thereby improving the detection sensitivity of AQP4-IgG.

[0007] This invention is achieved through the following technical solution: an OAP-size optimized recombinant AQP4 protein, wherein the recombinant protein is a mutant obtained by engineering modification of the N-terminus of the AQP4-M23 isotype, and the amino acid sequence of the mutant is shown in SEQ ID NO: 1. Compared with the natural AQP4-M23, the mutant satisfies the following characteristics: (1) The average area of ​​its OAP aggregates is greater than that of natural AQP4-M23 OAP aggregates; (2) Its membrane positioning efficiency is comparable to that of natural AQP4-M23; (3) Its immunoaffinity sensitivity to AQP4-IgG antibody is higher than that of natural AQP4-M23, that is, its immunofluorescence endpoint titer to AQP4-IgG is higher than that of natural AQP4-M23.

[0008] Preferably, the engineering modification involves replacing three leucine sites at positions 24, 28, and 31 of the N-terminus of the AQP4-M23 subtype.

[0009] Preferably, the substitution involves replacing leucine at position 24 with alanine, leucine at position 28 with alanine, and leucine at position 31 with glycine.

[0010] The present invention also provides the application of the above-mentioned AQP4 recombinant protein in the preparation of an AQP4-IgG antibody detection kit, wherein the recombinant protein is bound to the AQP4-IgG antibody for detection.

[0011] Preferably, the detection is performed using cell immunofluorescence assay.

[0012] Preferably, the kit is used for the auxiliary diagnosis of neuromyelitis optica spectrum disorders.

[0013] The present invention also provides a kit for detecting AQP4-IgG antibodies, comprising host cells expressing the recombinant protein.

[0014] Preferably, the host cell is a CHO cell or a HEK293T cell.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Based on the molecular mechanism of OAP assembly driven by N-terminal hydrophobic residues of the AQP4-M23 subtype, this invention selects three key leucine sites at positions 24, 28, and 31 for site-directed mutagenesis (L24A, L28A, L31G). By reducing the rigidity of the hydrophobic cluster and enhancing the flexibility of the N-terminus, the intrinsic restriction of the N-terminus on OAP expansion is removed. According to g-STED imaging analysis, the average area of ​​OAP aggregates of the obtained mutant (AQP4M23-OAPmax) is significantly larger than that of the natural AQP4 M23 (Mann-Whitney test, P < 0.001). The OAP size distribution frequency shows that the peak of the AQP4M23-OAPmax group shifts towards the larger size direction, thus achieving OAP size optimization.

[0016] (2) The N-terminal modification described in this invention follows four structural biology principles: spatial isolation, functional domain independence, conserved substitution, and membrane insertion independence. Through immunofluorescence detection analysis of permeable and non-permeable cells, the membrane localization efficiency of the mutant is comparable to that of natural AQP4-M23 (the density of AQP4 particles per unit membrane area is not significantly different from that of natural AQP4M23, P>0.05). Moreover, the N-terminal modification does not contact the extracellular LoopsA, C, and E epitope regions recognized by AQP4-lgG, and completely preserves the natural spatial conformation and immune recognition activity of the antigen. The above results indicate that the N-terminal modification does not affect the membrane localization efficiency.

[0017] (3) The present invention actively modulates the OAP size to the high affinity range of AQP4-IgG. The results of live cell immunofluorescence titer detection show that the median endpoint titer of the AQP4M23-OAPmax group (1:320) is significantly higher than that of the natural AQP4-M23 group (1:100). The detection rate of low titer samples (1:10) is increased from 90% to 100%, and about 10% of weak positive / borderline samples can be improved to clear positive, effectively reducing the risk of missed diagnosis.

[0018] (4) Unlike the natural OAP assembly which depends on uncontrollable factors such as cell type, expression level, and culture conditions, the present invention achieves stable regulation of OAP size by specific amino acid point mutations, eliminating the technical defects of significant batch-to-batch differences in natural antigens, and is more suitable for standardized production and clinical application. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the design principle of the engineered AQP4M23 antigen structure.

[0020] Figure 2 This is an immunofluorescence image of membrane-localized cells. Detailed Implementation

[0021] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] This invention addresses the technical problems of existing AQP4-IgG antibody detection antigens using natural AQP4-M23, such as uncontrollable OAP size, large batch-to-batch variability, and low detection rate of low-titer samples. It proposes a recombinant AQP4 protein with optimized OAP size, the application of this recombinant protein in the preparation of AQP4-IgG antibody detection kits, and a kit containing an AQP4-IgG antibody expressing the recombinant protein.

[0024] The detailed technical solution of this invention is as follows: AQP4 recombinant protein: This recombinant protein is a mutant obtained by engineering the N-terminus of the AQP4-M23 subtype (NCBI accession number: NM_004028.5). Specifically, site-directed mutations were performed on three leucine sites at positions 24, 28, and 31 of the N-terminus of AQP4-M23: leucine at position 24 was replaced with alanine (L24A), leucine at position 28 was replaced with alanine (L28A), and leucine at position 31 was replaced with glycine (L31G). The resulting combined mutant was named AQP4M23-OAPmax, and its amino acid sequence is shown in SEQ ID NO: 1.

[0025] SEQ ID NO: 1: MVAFKGVWTQAFWKAVTAEFLAMAIFVALSGGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0026] The recombinant protein designed in this invention aims to remove the inherent limitation of the N-terminus on OAP expansion by reducing the rigidity of the N-terminal hydrophobic cluster and enhancing the flexibility of the N-terminus, thereby obtaining an OAP array larger than that of the natural AQP4-M23. See [link to relevant documentation]. Figure 1 As shown.

[0027] Figure 1 Figure A shows the N-terminal conformational restriction of natural AQP4-M23 for OAP expansion. The blue circles represent AQP4 monomers, with four monomers forming a tetramer (dashed box). The red rectangles represent N-terminal leucine clusters, whose hydrophobic interactions (red arrows) restrict lateral assembly between tetramers, resulting in a limited OAP size (approximately 613 nm). 2 The red prohibition symbol indicates the restriction effect of the N-terminal on OAP extensions.

[0028] Figure 1 As shown in Figure B, the OAP size increases after the N-terminal mutation removes the restriction. The green circles represent the mutated AQP4 monomers. The L24A / L28A / L31G mutation shortens the N-terminus and reduces the rigidity of the hydrophobic cluster (light green short rectangles), removing the restriction on OAP expansion and allowing the tetramers to freely assemble into larger orthogonal arrays (OAP size approximately 1188 nm). 2 (Approximately 1.9 times more than natural); the green double-headed arrows indicate the direction of OAP expansion.

[0029] This design follows the following structural biology principles: (1) Spatial isolation principle: The N-terminus is located on the cytoplasmic side, and the AQP4-IgG epitope is located on the extracellular loops A, C and E. The N-terminal mutation does not directly contact the antibody binding region.

[0030] (2) Functional domain independence principle: The water channel is composed of 6 transmembrane helices and a conservative NPA motif, and the N end does not participate in the formation of the channel.

[0031] (3) Conservative substitution principle: The substitution of leucine with alanine or glycine is a conservative substitution that retains hydrophobicity and does not change the overall physicochemical properties of the N-terminus.

[0032] (4) Membrane insertion independence principle: The membrane positioning of AQP4 is completed by its transmembrane helix in cooperation. The N end does not participate in the membrane insertion process. Therefore, the modification of the N end does not affect the membrane positioning efficiency.

[0033] The above design principles together ensure that the mutant retains its membrane localization efficiency and native antigen conformation completely while increasing the OAP size.

[0034] Application of AQP4 recombinant protein in AQP4-IgG detection: For example, a kit for detecting AQP4-IgG antibodies can be prepared. This kit contains host cells expressing the recombinant protein, preferably CHO cells or HEK293T cells. During detection, the sample to be tested is brought into contact with the host cells. If AQP4-IgG antibodies are present in the sample, the antibodies specifically bind to the recombinant protein expressed on the cell membrane surface. Detection is performed using a cell immunofluorescence assay (CBA method). The detection results are used for the auxiliary diagnosis of neuromyelitis optica spectrum disorders.

[0035] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Example 1: Construction of mutants and initial screening of stable cell lines N-terminal site-directed mutagenesis was performed based on the human AQP4M23 sequence (NCBI accession number: NM_004028.5). Site-directed mutagenesis was performed at three leucine sites: position 24 was replaced with alanine (L24A), position 28 with alanine (L28A), and position 31 with glycine (L31G), resulting in the mutant AQP4M23-OAPmax, whose amino acid sequence is shown in SEQ ID NO: 1.

[0037] 1.1 Construction of mutant expression vector Using human AQP4-M23 cDNA as a template, three mutations at L24A, L28A, and L31G were introduced using overlap extension PCR (SOE-PCR). After double digestion (NheI / XbaI), the resulting clones were cloned into the lentiviral expression vector pLVX-Puro to construct the recombinant plasmid pLV-AQP4M23-OAPmax-Puro. The plasmid was transformed into DH5α competent cells, and the mutations were verified to be correct by colony PCR and Sanger sequencing. Wild-type control plasmids pLV-AQP4M23-Puro and pLV-AQP4M1-Puro were constructed using the same method.

[0038] 1.2 Lentiviral Packaging and Titer Validation HEK293T cells were cultured to 70–80% confluence. Recombinant plasmid (10 μg), psPAX2 (7.5 μg), and pMD2.G (2.5 μg) were co-transfected using liposome transfection. The medium was changed 6 hours after transfection, and the cells were cultured for another 48–72 hours. The supernatant was collected and filtered through a 0.45 μm filter to obtain the lentiviral stock solution. A small amount of the stock solution was used to infect HEK293T cells, and the titer was verified by puromycin (2 μg / mL) resistance selection. The remainder was aliquoted and stored at -80°C.

[0039] 1.3 Establishment and screening of stable cell lines CHO-K1 cells were infected with lentiviral stock solution (containing 5 μg / mL Polybrene) at an MOI ≈ 5. The medium was changed 24 hours after infection, and the cells were cultured for 48 hours. Puromycin (7 μg / mL) was added, and selection was performed for 10–14 days to obtain a mixed clonal cell pool. Cells were seeded into 96-well plates (approximately 1 cell / well) using a limiting dilution method. After 7–10 days of culture, single-clone wells were labeled, and the cells were subsequently expanded to 24-well plates, 6-well plates, and T25 culture flasks. Single clones with high expression and good membrane localization were selected by Western blot and immunofluorescence and named CHO-AQP4M23-OAPmax, which were then cryopreserved in liquid nitrogen. Simultaneously, CHO-AQP4M23 and CHO-AQP4M1 control cell lines were constructed.

[0040] Example 2: Membrane localization verification and g-STED imaging analysis 2.1 Verification of membrane positioning efficiency Immunofluorescence assays (permeabilized and non-permeabilized) were used to verify the membrane localization efficiency of the mutant. Anti-AQP4 C-terminal antibody (permeabilized CBA method) and anti-AQP4 extracellular loop antibody (permeabilized non-permeabilized CBA method) were used, respectively. Cells were seeded in glass-bottomed culture dishes and cultured for 48 hours. After fixation with 4% paraformaldehyde at room temperature for 15 minutes, the permeabilized group was treated with 0.1% Triton X-100 at room temperature for 10 minutes; this step was omitted for the non-permeabilized group. Cells were blocked with 5% BSA at room temperature for 30 minutes, incubated with primary antibody (1:200) at 37°C for 1 hour, incubated with Alexa Fluor 488 fluorescent secondary antibody (1:500) at room temperature in the dark for 45 minutes, and stained with DAPI for 5 minutes. Cells were observed and photographed under a fluorescence microscope, and quantitative analysis was performed based on the AQP4 fluorescence intensity per unit membrane area. If there was no significant difference in fluorescence signal intensity between the permeabilized and non-permeabilized groups, it indicated that the mutant was correctly located on the cell membrane and its topological structure was intact.

[0041] Cell immunofluorescence detection results as follows Figure 2 As shown, both AQP4M23-OAPmax and native AQP4M23 exhibited clear fluorescence signals on the cell membrane. The fluorescence intensity ratios of the permeabilized group and the non-permeabilized group were 0.97±0.05 and 0.96±0.04, respectively, with no significant difference between the two groups (P>0.05), indicating that the membrane localization expression efficiency of the mutant is comparable to that of native AQP4M23.

[0042] It should be noted that the phrase "membrane localization expression efficiency comparable to natural AQP4-M23" in this invention means that, under the same experimental conditions, the ratio of AQP4 fluorescence signal intensity after permeabilization treatment (0.1% Triton X-100 at room temperature for 10 minutes) to that after non-permeabilization treatment in cells expressing the mutant is not statistically significantly different (P > 0.05) from the corresponding ratio in the natural AQP4M23 control group. In this invention, the deviation of this ratio typically does not exceed ±10% of the ratio in the natural AQP4M23 control group.

[0043] 2.2 g-STED Imaging and OAP Analysis Transfected cells were seeded onto coverslips and cultured for 48 hours. They were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes. The cells were then double-labeled with anti-AQP4 antibody (1:200) and anti-GFAP antibody (1:500), incubated with Alexa Fluor 488 / 594 fluorescent secondary antibody (1:500) at room temperature in the dark for 45 minutes, stained with DAPI for 5 minutes, and mounted with anti-fluorescence quenching mounting medium.

[0044] g-STED imaging was used (following the method of Zhu et al., 2022, J. Neurosci.). OAP analysis was performed using the ImageJ software package Fiji. Cell outlines were delineated using GFAP markers to exclude extracellular noise. After converting the images to binary images, the area of ​​connected pixels (i.e., OAP area) was calculated using the Particle Analyzer plugin, and the ratio of the major and minor axes (elongation) of the fitted ellipse was calculated using the FracLac plugin to exclude non-square structures. At least 200 independent OAP aggregates were randomly selected from each group for statistical analysis.

[0045] The results of OAP size measurement are shown in Table 1: Comparison of OAP size parameters for each AQP4 conformation.

[0046] Table 1: Comparison of OAP size parameters for each AQP4 conformation

[0047] *P<0.01, compared with the natural AQP4M23 group, Mann-Whitney U test.

[0048] The above results indicate that the average area of ​​OAP in the AQP4M23-OAPmax group is 1188±56 nm. 2 It is approximately the natural AQP4-M23 group (613±28 nm) 2 The area of ​​the OAP (Optical Area Per Scale) is 1.9 times larger than that of the OAP, and the peak value of the OAP size distribution shifts significantly towards larger sizes, with the largest OAP area reaching 4385 nm. 2 The proportion of OAPs increased to 87%. Meanwhile, the AQP4 particle density per unit membrane area was not significantly different from the natural AQP4-M23 group (P>0.05), indicating that membrane localization expression efficiency was not affected. Therefore, the OAP size optimization was deemed successful.

[0049] Example 3: Comparison of clinical AQP4-IgG positive serum titer detection and sensitivity This implementation directly verifies the sensitivity advantage of AQP4M23-OAPmax in actual detection through endpoint titer testing.

[0050] Serum samples were collected from 87 clinically diagnosed NMOSD patients, including 67 high-titer samples and 20 low-titer samples, as well as 90 healthy control serum samples and 30 multiple sclerosis control serum samples. All serum samples were confirmed by AQP4-IgG detection using a commercial CBA kit (EUROIMMUN AQP4-IgG CBA kit).

[0051] The three cell groups constructed in Example 1, namely CHO-AQP4M23-OAPmax, CHO-AQP4M23 and CHO-AQP4M1, were subjected to serial dilution titers with serum from the same batch of confirmed NMOSD patients.

[0052] The serum to be tested was serially diluted 3.2-fold starting from 1:10 (1:10, 1:32, 1:100, 1:320). Each group of cells was incubated with each dilution of serum at 37°C for 1 hour, washed with PBS, and then incubated with Alexa488-anti-human IgG secondary antibody (1:500) for 45 minutes. The endpoint titer was determined.

[0053] The titer test results are shown in Table 2: AQP4-IgG titer test results for each AQP4 conformation.

[0054] Table 2: AQP4-IgG titer detection results for each AQP4 conformation

[0055] The above results indicate that: (1) In high-titer positive samples (>1:10), the detection rates of natural AQP4M23 and AQP4M23-OAPmax were both 100% (67 / 67), significantly higher than that of natural AQP4M1 (83.6% (56 / 67)) (chi-square test, P<0.01). This result demonstrates that the AQP4M23 subtype has a higher antibody binding efficiency than the M1 subtype, which is consistent with the mechanism by which M23 easily forms OAP, thereby enhancing antibody affinity.

[0056] (2) In the positive low-titer samples (1:10), the detection rate of natural AQP4M23 was 90% (18 / 20), which was 25 percentage points higher than that of natural AQP4M1 (65%, 13 / 20); the detection rate of AQP4M23-OAPmax reached 100% (20 / 20), which was 10 percentage points higher than that of natural AQP4M23. Although the difference in detection rates between M23 and M1, and between OAPmax and M23 in the low-titer samples was not statistically significant due to the small sample size (n=20) (P>0.05), AQP4M23-OAPmax changed the detection rate of M23 in 2 low-titer samples from non-detection to detection, achieving complete coverage of low-titer samples.

[0057] (3) In terms of the overall positive detection rate, the natural AQP4M23 was 97.7% (85 / 87), which was significantly higher than the natural AQP4M1's 79.3% (69 / 87) (Chi-square test, P<0.01); AQP4M23-OAPmax reached 100% (87 / 87), which was 2 more cases detected than natural AQP4M23, achieving complete detection of serum from NMOSD patients.

[0058] (4) No false positives were found in any of the three conformations in healthy controls (90 cases) and multiple sclerosis controls (30 cases), indicating that all three conformations have good detection specificity.

[0059] Sensitivity assessment criteria: The median endpoint titer, detection rate of each subgroup, and overall detection rate were used as core indicators for assessment. The median endpoint titer of the AQP4M23-OAPmax group (1:320) was significantly higher than that of the natural AQP4M23 group (1:100) and the natural AQP4M1 group (1:32) (paired Wilcoxon signed-rank test, P < 0.01); the detection rate of the AQP4M23-OAPmax group for low-titer samples (100%) was higher than that of the natural AQP4M23 group (90%), and the overall detection rate reached 100%. These results indicate that the OAP-size optimized AQP4 recombinant protein described in this invention can significantly improve the sensitivity of AQP4-IgG detection, especially for low-titer samples, and can improve approximately 2.3% of borderline samples to a clear positive result, effectively reducing the risk of missed diagnoses.

[0060] Comparative Example 1: Leucine mutations at other N-terminal sites To verify the specificity of the selected mutation sites (positions 24, 28, and 31) in this invention, single mutants at other N-terminal leucine sites were constructed in this comparative example: L21A (as shown in SEQ ID NO: 2), L29A (as shown in SEQ ID NO: 3), and L44A (as shown in SEQ ID NO: 4). Mutants were constructed and OAP size analysis was performed according to the methods of Examples 1 and 2.

[0061] SEQ ID NO: 2: MVAFKGVWTQAFWKAVTAEFAAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0062] SEQ ID NO: 3: MVAFKGVWTQAFWKAVTAEFLAMLIFVLASLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0063] SEQ ID NO: 4: MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPAPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0064] The results of OAP size determination are shown in Table 3: Comparison of OAP size parameters of mutants with different N-terminal leucine sites.

[0065] Table 3: Comparison of OAP size parameters of mutants with different N-terminal leucine sites

[0066] The above results indicate that single mutations at other N-terminal leucine sites (L21, L29, L44) had no significant effect on the average area, median area, maximum area, or proportion of OAPs (P > 0.05), showing no statistical difference compared to the natural AQP4M23 group. However, the L24A / L28A / L31G combined mutation (AQP4M23-OAPmax) described in this invention increased the average area of ​​OAPs by approximately 1.9 times and increased the proportion of OAPs to 87%, with a significant difference (P < 0.01). These results demonstrate that positions 24, 28, and 31 are key sites driving OAP assembly, while other leucine sites do not participate or only weakly participate in the regulation of OAP expansion. The mutation sites selected in this invention exhibit high specificity.

[0067] Comparative Example 2: Mutations at the same site into different types of amino acids To verify the specific effects of the selected amino acid substitutions (alanine and glycine) in this invention, different types of amino acid substitution mutants at the same sites were constructed in this comparative example (mutations at positions 24, 28, and 31 were replaced with other amino acids): L24V / L28V / L31A (as shown in SEQ ID NO: 5), L24I / L28I / L31S (as shown in SEQ ID NO: 6), and L24F / L28F / L31W (as shown in SEQ ID NO: 7). The mutants were constructed and OAP size analysis was performed according to the methods of Examples 1 and 2.

[0068] SEQ ID NO: 5: MVAFKGVWTQAFWKAVTAEFLAMVIFVVLSAGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0069] SEQ ID NO: 6: MVAFKGVWTQAFWKAVTAEFLAMIIFVILSSGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0070] SEQ ID NO: 7: MVAFKGVWTQAFWKAVTAEFLAMFIFVFLSWGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0071] The results of OAP size determination are shown in Table 4: Comparison of OAP size parameters of mutants with different amino acid substitutions at the same site.

[0072] Table 4: Comparison of OAP size parameters of mutants with different amino acid substitutions at the same site

[0073] *P<0.01, compared with the natural AQP4M23 group, Mann-Whitney U test.

[0074] The above results indicate that mutating positions 24, 28, and 31 to other types of amino acids (such as valine, isoleucine, phenylalanine, etc., residues with stronger hydrophobicity or greater steric hindrance) did not significantly change the average area, median area, maximum area, or proportion of OAPs (P > 0.05), showing no statistical difference from the natural AQP4M23 group. However, the conservative substitution of alanine and glycine (L24A / L28A / L31G) in this invention increased the average area of ​​OAPs by approximately 1.9 times and the proportion of OAPs to 87%, with a highly significant difference (P < 0.01). This demonstrates that smaller side chains of alanine and glycine can effectively reduce the rigidity of the N-terminal hydrophobic cluster and enhance its flexibility, thereby removing the restriction on OAP expansion; while substitutions with larger side chains or stronger hydrophobic amino acids cannot achieve the same effect. The type of amino acid substitution selected in this invention is highly specific.

[0075] Comparative Example 3: Partial Site Mutations (Two-Point Mutations and Single-Point Mutations) To verify the synergistic effect of the three-site combined mutations of the present invention, this comparative example constructed single-site mutants: L24A (as shown in SEQ ID NO: 8), L28A (as shown in SEQ ID NO: 9), L31G (as shown in SEQ ID NO: 10) and two-site mutants: L24A / L28A (as shown in SEQ ID NO: 11), L24A / L31G (as shown in SEQ ID NO: 12), L28A / L31G (as shown in SEQ ID NO: 13). The mutants were constructed and OAP size analysis was performed according to the methods of Example 1 and Example 2.

[0076] SEQ ID NO: 8: MVAFKGVWTQAFWKAVTAEFLAMAIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0077] SEQ ID NO: 9: MVAFKGVWTQAFWKAVTAEFLAMLIFVALSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0078] SEQ ID NO: 10: MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSGGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0079] SEQ ID NO: 11: MVAFKGVWTQAFWKAVTAEFLAMAIFVALSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0080] SEQ ID NO: 12: MVAFKGVWTQAFWKAVTAEFLAMAIFVLLSGGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0081] SEQ ID NO: 13: MVAFKGVWTQAFWKAVTAEFLAMLIFVALSGGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFT IFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV.

[0082] The results of OAP size measurement are shown in Table 5: Comparison of OAP size parameters of mutants at some sites.

[0083] Table 5: Comparison of OAP size parameters for mutants at certain sites

[0084] *P<0.01, compared with the natural AQP4M23 group, Mann-Whitney U test.

[0085] The above results indicate that both single-site mutations (L24A, L28A, L31G) and two-site mutations (L24A / L28A, L24A / L31G, L28A / L31G) can increase the average area of ​​the OAP compared to the natural AQP4M23, but the increase is limited (single-site mutations increase by approximately 0.2-0.5%, and two-site mutations increase by approximately 0.7-1.1%), and neither reaches a statistically significant level (P>0.05). However, the three-site combination mutation (L24A / L28A / L31G, AQP4M23-OAPmax) described in this invention increases the average area of ​​the OAP by approximately 1.9 times (1188±56 nm). 2 The proportion of OAP increased to 87%, with a highly significant difference (P < 0.01). This result demonstrates that the three leucine sites at positions 24, 28, and 31 have a synergistic effect in the regulation of OAP assembly, and none of them can be omitted. Mutations at individual or partial sites cannot effectively remove the restriction on OAP expansion at the N-terminus. The three-site combination mutation of this invention has a significant synergistic advantage.

[0086] Example 4: AQP4-IgG detection reagent based on AQP4M23-OAPmax and its application This embodiment provides an AQP4-IgG detection reagent based on AQP4M23-OAPmax and its application in cellular immunofluorescence detection.

[0087] CHO-K1 cells stably expressing AQP4M23-OAPmax obtained in Example 1 were revived and passaged in F-12K medium containing 10% FBS. Cells in logarithmic growth phase were then cultured at 2 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well black transparent-bottom cell culture plates and cultured at 37°C and 5% CO2 for 48 hours until the cell confluence reached 80-90%. The culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed three times with PBS to obtain the cell matrix for AQP4-IgG detection.

[0088] Detection of the sample to be tested: The specific detection steps are as follows: The serum to be tested was serially diluted 3.2 times starting from 1:10 (1:10, 1:32, 1:100, 1:320), with 50 μL of diluted serum added to each well, and incubated in a humidified chamber at 37°C for 1 hour; washed three times with PBS, and 50 μL of Alexa Fluor 488-labeled anti-human IgG secondary antibody (1:500) was added to each well, and incubated at 37°C in the dark for 45 minutes; washed three times with PBS, and DAPI was added to stain the nuclei for 5 minutes. After washing with PBS, the samples were observed and photographed under a fluorescence microscope. Using the results of the commercial CBA kit (EUROIMMUN) as a reference, a positive control (known AQP4-IgG positive serum), a negative control (healthy human serum), and a blank control (PBS-substituted serum) were simultaneously set up.

[0089] Result judgment: Positive result: The cell membrane shows a clear, continuous ring-shaped green fluorescent signal, and the fluorescence intensity is higher than the negative control cut-off value (mean fluorescence intensity of the negative control + 3 standard deviations). The endpoint titer ≥ 1:32 is judged as positive.

[0090] Negative result: No green fluorescence signal on the cell membrane or fluorescence intensity below the Cut-off value, and an endpoint titer <1:32 is considered negative.

[0091] Example 5: AQP4-IgG Detection Kit Based on AQP4M23-OAPmax This embodiment provides a kit for preparing AQP4-IgG antibodies. The kit is used for in vitro qualitative or semi-quantitative detection of AQP4-IgG antibodies. The detection results are used as an auxiliary diagnostic tool for neuromyelitis optica spectrum disorders, but should not be the sole basis for diagnosis. Clinicians should make a comprehensive judgment based on the patient's clinical symptoms and other laboratory test results.

[0092] Specifically, the kit contains at least the following components: detection matrix (as described in Example 4), blocking solution, positive control, negative control, washing solution, and fluorescent secondary antibody.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. An OAP-size optimized recombinant AQP4 protein, characterized in that: The recombinant protein is a mutant obtained by engineering the N-terminus of the AQP4-M23 isotype, and the amino acid sequence of the mutant is shown in SEQ ID NO:

1. Compared with the natural AQP4-M23, the mutant satisfies the following characteristics: (1) The average area of ​​its OAP aggregates is greater than that of natural AQP4-M23 OAP aggregates; (2) Its membrane localization expression efficiency is comparable to that of natural AQP4-M23; (3) Its immunoaffinity sensitivity to binding with AQP4-IgG antibody is higher than that of natural AQP4-M23.

2. The application of the AQP4 recombinant protein according to claim 1 in the preparation of an antibody kit for detecting AQP4-IgG, characterized in that: The detection was performed by binding the recombinant protein to the AQP4-IgG antibody.

3. The application according to claim 2, characterized in that: The detection was performed using cell immunofluorescence assay.

4. The application according to claim 2, characterized in that: The kit is used for the auxiliary diagnosis of neuromyelitis optica spectrum disorders.

5. A kit for detecting AQP4-IgG antibodies, characterized in that: A host cell expressing the recombinant protein of claim 1.

6. The reagent kit according to claim 5, characterized in that: The host cells are CHO cells or HEK293T cells.

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