Recombinant protein for detecting anti-recoverin autoantibodies and use thereof

Abstract

The application discloses a kind of recombinant proteins for detecting anti-Recoverin autoimmune antibody and application thereof, belong to the technical field of biological medicine engineering.The recombinant protein is mutant Recoverin recombinant protein, it successively includes secretion signal peptide, mutant Recoverin protein partial sequence, transmembrane region and fluorescent label, the mutant Recoverin protein partial sequence is as shown in SEQ ID NO.1;Or include the mutant Recoverin recombinant protein and wild type Recoverin recombinant protein, the wild type Recoverin recombinant protein successively includes secretion signal peptide, wild type Recoverin protein partial sequence, transmembrane region and fluorescent label, the wild type Recoverin protein partial sequence is as shown in SEQ ID NO.2.The recombinant protein can be stablely expressed Recoverin antigen region on cell membrane surface, and can effectively distinguish the cross reaction of neuron calcium sensing protein family homologous protein, to significantly improve the specificity and sensitivity of detection.

Description

Technical Field

[0001] This invention relates to a recombinant protein for detecting anti-Recoverin autoantibodies and its application, specifically a Recoverin recombinant protein with reduced cross-reactivity for detecting anti-Recoverin autoantibodies by cellular immunofluorescence assay and its application, belonging to the field of biomedical engineering technology. Background Technology

[0002] Autoimmune retinopathy and paraneoplastic retinopathy are a group of degenerative diseases of retinal neurons mediated by autoantibodies, which can lead to sudden or subacute vision loss, visual field defects, and abnormal electroretinograms. Recoverin is a photoreceptor-specific calcium-binding protein that participates in the Ca²⁺ kinase of rhodopsin during light transmission. + Dependent regulation is the most important autoantigen in cancer-associated retinopathy (see Grazyna Adamus, et al. Autoantibodies against retinal proteins in paraneoplastic and autoimmune retinopathy, BMCOphthalmology, 2004(04):1-9; Alexandr V. Bazhin, et al. Recoverin as a cancer-retina antigen, Cancer Immunol Immunother, 2007(01):110-116).

[0003] Recoverin is abnormally expressed in various tumor cells, including lung cancer, melanoma, and gynecological tumors, and can induce the production of autoantibodies and cytotoxic T lymphocyte responses. Patients with cancer-associated retinopathy (CAR) may have better prognoses, suggesting a potential anti-tumor effect of anti-Recoverin immune responses. Therefore, developing highly sensitive and specific methods for detecting anti-Recoverin antibodies is of great significance for the auxiliary diagnosis of autoimmune retinopathy / CAR, immune monitoring of cancer patients, and prognostic assessment. Studies have shown that anti-Recoverin autoantibodies are present in the serum of some CAR patients, and antibody titers are closely related to the severity of visual symptoms. However, existing detection methods have significant limitations: Recoverin is an intracellular protein, and conventional fixed permeabilized cell immunofluorescence assays have high background signals and may disrupt the antigen conformation (see Grazyna Adamus, et al. Epitope recognition of recoverin in cancer associated retinopathy evidence for calcium-dependent conventional epitopes. Journal of Neuroscience Research, 1996 (45): 863-872).

[0004] However, it should be noted that although wild-type Recoverin does not share extremely high sequence homology with members of the neuronal calcium sensor (NCS) family, such as GCAP (retinal guanylate cyclase activator) and Hippocalcin (hippocampal calcium-binding protein, HPCA), their spatial conformations are highly similar (see JB Ames, et al. Molecular structure of membrane-targeting calcium sensors in vision: recoverin and guanylate cyclase-activating protein 2. Methods in Enzymology, 2000(316):121-126; JB Ames, et al. Structure and membrane-targeting mechanism of retinal Ca²⁺).+ -binding proteins, recoverin and GCAP-2, recoverin and GCAP-2 (Retinal calcium-binding proteins - the structure and membrane targeting mechanism of recoverin and GCAP-2). Adv Exp Med Biol (Advances in Experimental Medicine and Biology). 2002(514):333-348), which may lead to cross-reactivity of some antibodies targeting conformational epitopes, resulting in false positive results.

[0005] In antibody detection for neuroparaneoplastic syndromes, existing kits (such as patent CN119395290A) employ a multi-antibody co-detection strategy, simultaneously identifying 12 related autoantibodies, including Recoverin. This design significantly improves the comprehensiveness of the detection, helping to cover multiple pathogenic factors and avoiding missed diagnoses caused by single antibody detection. However, this kit uses wild-type antigen proteins with known sequences, making it impossible to fundamentally distinguish between specific anti-Recoverin antibodies and cross-reactive antibodies. False positive results are difficult to eliminate, severely impacting the clinical interpretation of the test results. Summary of the Invention

[0006] This invention aims to overcome the technical shortcomings of existing anti-Recoverin antibody detection methods, such as difficulty in detecting intracellular antigens, easy destruction of conformational epitopes, and difficulty in distinguishing cross-reactivity. It provides a recombinant protein for detecting anti-Recoverin autoantibodies and its applications. The recombinant protein can stably express the Recoverin antigen region on the cell membrane surface and can effectively distinguish cross-reactivity among homologous proteins of the neuronal calcium-sensing protein family, thereby significantly improving the specificity and sensitivity of the detection.

[0007] This invention is achieved through the following technical solution: a recombinant protein for detecting anti-Recoverin autoantibodies, wherein the recombinant protein is selected from any one of the following: (1) The mutant Recoverin recombinant protein, consisting of a secretion signal peptide, a partial sequence of the mutant Recoverin protein, a transmembrane region, and a fluorescent tag, from the N-terminus to the C-terminus, wherein the partial sequence of the mutant Recoverin protein is shown in SEQ ID NO.1; (2) A recombinant protein combination consisting of the mutant Recoverin recombinant protein and the wild-type Recoverin recombinant protein, wherein the wild-type Recoverin recombinant protein consists of a secretion signal peptide, a partial sequence of the wild-type Recoverin protein, a transmembrane region and a fluorescent tag from the N-terminus to the C-terminus, and the partial sequence of the wild-type Recoverin protein is shown in SEQ ID NO.2.

[0008] Preferably, the secretory signal peptide is human albumin signal peptide ALB.

[0009] Preferably, the amino acid sequence of the secretion signal peptide is shown in SEQ ID NO.3.

[0010] Preferably, the transmembrane region is the CD8a hinge region.

[0011] Preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO.4.

[0012] Preferably, the fluorescent tag is mCherry.

[0013] Preferably, the amino acid sequence of the fluorescent tag is as shown in SEQ ID NO.5.

[0014] Preferably, both the mutant Recoverin recombinant protein and the wild-type Recoverin recombinant protein further employ linker peptide A and linker peptide B; The linker peptide A is located between the partial sequence of the Recoverin protein and the transmembrane region, and its amino acid sequence is shown in SEQ ID NO.6; The linker peptide B is located between the transmembrane region and the fluorescent tag, and its amino acid sequence is shown in SEQ ID NO.7.

[0015] This invention also provides the application of the above-mentioned recombinant protein in the preparation of a kit for detecting anti-Recoverin autoantibodies, including the following two methods: (a) When the recombinant protein is a mutant Recoverin recombinant protein, the nucleic acid encoding the mutant Recoverin recombinant protein is transfected into a host cell to obtain a host cell expressing the mutant Recoverin recombinant protein. After the host cell expressing the mutant Recoverin recombinant protein is contacted with the sample to be tested, the binding signal on its surface is detected by immunofluorescence. (b) When the recombinant protein is a combination of recombinant proteins, host cells expressing mutant Recoverin recombinant protein and host cells expressing wild-type Recoverin recombinant protein are respectively brought into contact with the sample to be tested, and the binding signal on their respective surfaces is detected by immunofluorescence.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention designs a novel Recoverin mutant. By modifying the intracellular localization signal sequence of the natural N-terminus of Recoverin, adding a secretion signal peptide, and fusing the expression of the key antigen region with the transmembrane region, the intracellular protein Recoverin is stably displayed on the surface of the living cell membrane. It is suitable for live cell immunofluorescence detection, avoids the destruction of antigen conformation by traditional fixation and permeabilization operations, significantly reduces background signal, and improves the reliability and repeatability of detection results.

[0017] (2) The Recoverin mutant designed in this invention, by specifically modifying the non-critical antigenic region (amino acids 135-146) between EF-hand 3 and EF-hand 4, disrupts the conformational similarity with proteins such as GCAP and Hippocalcin while retaining the major antigenic epitopes (EF-hand 2 and EF-hand 3). This mutant can effectively distinguish between specific anti-Recoverin antibodies and cross-reactive antibodies against other neuronal calcium sensory protein family members, significantly reducing the false positive rate and improving the specificity of detection and the accuracy of clinical interpretation.

[0018] (3) The recombinant protein constructed in this invention can establish a stable monoclonal cell line, which is convenient for standardized and large-scale production and meets the needs of clinical testing.

[0019] (4) The present invention can obtain positive results of antibodies and epitope specificity information by using wild-type and mutant detection systems in combination, providing a more comprehensive basis for the auxiliary diagnosis of autoimmune retinopathy and cancer-related retinopathy, tumor immune monitoring and prognostic assessment. Attached Figure Description

[0020] Figure 1 A structural comparison of the natural full-length Recoverin protein, wild-type Recoverin recombinant protein, and mutant Recoverin recombinant protein.

[0021] Figure 2 Immunofluorescence images of positive samples for anti-Recoverin antibodies against Recoverin natural full-length protein, wild-type Recoverin recombinant protein, and mutant Recoverin recombinant protein.

[0022] Figure 3 Immunofluorescence images of wild-type and mutant Recoverin recombinant protein detected in samples containing nonspecific antibodies. Detailed Implementation

[0023] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0024] 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.

[0025] To address the cross-reactivity issue caused by the high spatial conformational similarity between Recoverin and other members of the neuronal calcium sensor protein family, such as GCAP and Hippocalcin, this invention designs a novel Recoverin mutant. Based on a partial sequence of the wild-type Recoverin protein (as shown in SEQ ID NO.2), amino acids 135-146 are deleted, thereby obtaining the partial sequence of the mutant Recoverin protein described in this invention (as shown in SEQ ID NO.1).

[0026] Because the major antigenic epitopes of Recoverin are located in the EF-hand 2 and EF-hand 3 regions, while amino acids 135-146 are located between EF-hand 3 and EF-hand 4 and are not part of the major antigenic epitope region, and this region has high conformational similarity to proteins such as GCAP, deleting the 135-146 amino acid fragment slightly modulates the overall spatial conformation of Recoverin, but still retains the major antigenic epitope conformations of EF-hand 2 and EF-hand 3. Therefore, compared with wild-type Recoverin, the binding ability of this mutant to antibodies specifically recognizing epitopes in the EF-hand 2 or EF-hand 3 regions remains essentially unchanged, but its binding ability to antibodies recognizing the region between EF-hand 3 and EF-hand 4 or cross-reactive antibodies dependent on the intact spatial conformation is significantly reduced.

[0027] See Figure 1 As shown, the natural full-length protein of Recoverin is displayed. Figure 1 a) Wild-type Recoverin recombinant protein ( Figure 1 b) Mutant Recoverin recombinant protein ( Figure 1 The structure of c) in the middle. From Figure 1 It is evident that although the spatial structure of the mutant Recoverin recombinant protein of the present invention is slightly different, it still retains the conformational integrity of the major antigenic epitopes and has the ability to distinguish between specific and cross-reactive antibodies.

[0028] In an optional embodiment, the mutant Recoverin recombinant protein is obtained by sequentially linking a secretion signal peptide, a partial sequence of the mutant Recoverin protein (as shown in SEQ ID NO.1), a linker peptide A, a transmembrane region, a linker peptide B, and a fluorescent tag. The wild-type Recoverin recombinant protein is obtained by sequentially linking a secretion signal peptide, a wild-type Recoverin protein sequence (as shown in SEQ ID NO.2), a linker peptide A, a transmembrane region, a linker peptide B, and a fluorescent tag. As an example, in the above recombinant protein, the secretion signal peptide is the human albumin signal peptide ALB, whose amino acid sequence is shown in SEQ ID NO.3; the transmembrane region is the CD8a hinge region, whose amino acid sequence is shown in SEQ ID NO.4; the fluorescent tag is mCherry, whose amino acid sequence is shown in SEQ ID NO.5; the amino acid sequence of linker peptide A is shown in SEQ ID NO.6; and the amino acid sequence of linker peptide B is shown in SEQ ID NO.7.

[0029] In one optional implementation, nucleic acid encoding a mutant Recoverin recombinant protein is transfected into host cells to obtain host cells expressing the mutant Recoverin recombinant protein. These host cells are then contacted with a test sample, and the binding signal on their surface is detected using immunofluorescence. This provides diagnostic data for the auxiliary diagnosis of autoimmune retinopathy and cancer-related retinopathy. As an example, host cells expressing the mutant Recoverin recombinant protein can be used as a detection reagent in combination with other detection buffers (including but not limited to sample dilution buffer, phosphate buffer, and fluorescent secondary antibody) to form a kit for the auxiliary diagnosis of the aforementioned diseases.

[0030] In one optional implementation, host cells expressing mutant Recoverin recombinant protein are used in combination with host cells expressing wild-type Recoverin recombinant protein for detection. For example, during detection, wild-type Recoverin recombinant protein and mutant Recoverin recombinant protein are expressed in different host cells, and the reactivity of the test samples to the two antigens is detected in parallel. If the binding of the sample to mutant Recoverin recombinant protein is significantly reduced compared to wild-type Recoverin recombinant protein, it suggests that the antibody is a cross-reactive antibody or an antibody that recognizes a non-major epitope; if there is no significant change in binding, it suggests that the antibody is a specific antibody that recognizes the major antigenic epitope (EF-hand 2 or EF-hand 3).

[0031] The amino acid sequences involved in this invention are specifically shown in Table 1 below.

[0032] Table 1. Amino acid sequence of the present invention

[0033] 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.

[0034] The experimental materials and instruments involved in the following embodiments are as follows: [Experimental Materials]: Human embryonic kidney cells HEK293T cells and Chinese hamster ovary cells CHO-K1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. DMEM high glucose medium, fetal bovine serum (FBS), 0.25% trypsin-EDTA digestion solution, PBS buffer, and Opti-MEM serum-depleted medium were purchased from Gibco (Thermo Fisher Scientific). Puromycin (product number P8833): purchased from Sigma-Aldrich; Plasmid mini-prep kit: purchased from Omega; Goat anti-human IgG-488 fluorescent secondary antibody (catalog number 109-545-190): purchased from Jackson Immuno Research; Bovine serum albumin (catalog number A8020): purchased from Solarbio. CaCl2 (analytical grade): purchased from Sinopharm Chemical Reagent Co., Ltd. Cell culture plates (6-well plates, 96-well plates, culture dishes): purchased from Jetbio Co., Ltd. Transfection reagent Lipofectamine 3000 (catalog number L3000015): purchased from Invitrogen.

[0035] [Experimental Apparatus]: Fluorescence microscope (model CKX53): Olympus Corporation; CO2 constant temperature incubator (model QP-80): Biobase Corporation; Biosafety cabinet (model BSC-1100II A2-X): Biobase Corporation; Micropipettes (Eppendorf Research® plus series): Eppendorf.

[0036] Example 1: Construction of wild-type Recoverin recombinant protein expression vector A partial sequence of the human Recoverin gene was ligated with DNA fragments encoding secretion signal peptides, linker peptide A, CD8a transmembrane region, linker peptide B, and mCherry, and cloned into a lentiviral transfer plasmid to construct a lentiviral vector that stably expresses wild-type Recoverin recombinant protein. The specific steps are as follows: (1) Sequence design: Based on the human Recoverin protein sequence (Gene ID: 4697) in the NCBI database, the full sequence encoding the wild-type Recoverin recombinant protein was designed. The sequence contains, in sequence: human albumin secretion signal peptide ALB (SEQ ID NO.3), a partial sequence of wild-type Recoverin protein (SEQ ID NO.2), linker peptide A (SEQ ID NO.6), CD8a transmembrane region (SEQ ID NO.4), linker peptide B (SEQ ID NO.7) and mCherry fluorescent tag (SEQ ID NO.5). The overall structure is: ALB-Recoverin(23-200)-linker peptide A-CD8a-linker peptide B-mCherry.

[0037] (2) Gene synthesis and sequencing identification: Based on the sequence design, the full-sequence gene was synthesized and codons were optimized (optimized for CHO cell expression). The synthesized gene was cloned into a lentiviral vector, and single colonies were selected for subsequent identification. The constructed recombinant plasmid was verified by full-sequence Sanger sequencing to confirm that the inserted sequence was correct, the reading frame was complete, and there were no mutations or deletions. Finally, the wild-type Recoverin recombinant protein expression plasmid was obtained and named pLVX-Rec-WT-CD8a-mCherry.

[0038] Example 2: Construction of a mutant Recoverin recombinant protein expression vector Using the wild-type Recoverin gene as a template, the nucleotide sequence encoding amino acids 135-146 was deleted, and a lentiviral vector was constructed according to the method in Example 1. The specific steps are as follows: (1) Sequence design: Based on the wild-type Recoverin protein sequence (SEQ ID NO.2) in Example 1, a mutant sequence was designed that deletes amino acids 135-146 (sequence: PEDVKLLPDDEN, a total of 12 amino acids, corresponding to 36 nucleotides), while the remaining domains (ALB signal peptide, linker A, CD8a transmembrane region, linker B, mCherry) remain unchanged. The mutated sequence structure is: ALB-Recoverin(23-200, Δ135-146)-linker A-CD8a-linker B-mCherry.

[0039] (2) Gene synthesis and sequencing identification: Refer to Example 1. Finally, the mutant Recoverin recombinant protein expression plasmid was obtained and named pLVX-Rec-Δ135-146-CD8a-mCherry.

[0040] Comparative Example 1: Construction of a full-length Recoverin protein expression vector (without transmembrane crossing) Based on the full-length human Recoverin gene, a DNA fragment encoding amino acids 1-200 was synthesized, ligated to a DNA fragment encoding linker peptide B and mCherry, and cloned into a lentiviral transfer plasmid to construct a lentiviral vector that stably expresses the full-length Recoverin protein. The specific steps are as follows: (1) Sequence design: Based on the human Recoverin gene sequence (Gene ID: 4697) in the NCBI database, a sequence encoding the full-length Recoverin protein (amino acids 1-200, retaining the N-terminal myristoylation modification site) was designed, and a linker peptide B (SEQ ID NO. 7) and an mCherry fluorescent tag (SEQ ID NO. 5) were sequentially linked downstream of it. The overall structure is: Recoverin(1-200)-linker peptide B-mCherry. Compared with Example 1, it does not contain the secretion signal peptide ALB, linker peptide A, and CD8a transmembrane region, and is an intracellular expression vector.

[0041] (2) Gene synthesis and sequencing identification: Based on the sequence design, the full-sequence gene was synthesized and codons were optimized (optimized for CHO cell expression). The synthesized gene was cloned into a lentiviral vector, and single colonies were selected for subsequent identification. The constructed recombinant plasmid was verified by full-sequence Sanger sequencing to confirm that the inserted sequence was correct, the reading frame was complete, and there were no mutations or deletions. Finally, the full-length Recoverin protein expression plasmid was obtained and named pLVX-Rec-Full-mCherry.

[0042] Comparative Example 2: Construction of the Recoverin Recombinant Protein-1 Expression Vector (deletion of amino acids 140-143) Using the wild-type Recoverin gene as a template, the nucleotide sequence encoding amino acids 140-143 was deleted, and a lentiviral vector was constructed according to the method in Example 1. The specific steps are as follows: (1) Sequence design: Based on the wild-type Recoverin protein sequence (SEQ ID NO.2) in Example 1, a mutant sequence was designed that deletes amino acids 140-143 (sequence: TTPE, a total of 4 amino acids, corresponding to 12 nucleotides), while the remaining domains (ALB signal peptide, linker A, CD8a transmembrane region, linker B, mCherry) remain unchanged. The mutated sequence structure is: ALB-Recoverin(23-200, Δ140-143)-linker A-CD8a-linker B-mCherry.

[0043] (2) Gene synthesis and sequencing identification: Refer to Example 1. The Recoverin recombinant protein-1 expression plasmid was finally obtained and named pLVX-Rec-Δ140-143-CD8a-mCherry.

[0044] Comparative Example 3: Construction of the Recoverin Recombinant Protein-2 Expression Vector (Mutation of amino acids 135-146) Using the wild-type Recoverin gene as a template, amino acids 135-146 were mutated, and the lentiviral vector was constructed according to the method in Example 1. The specific steps are as follows: (1) Sequence design: Based on the wild-type Recoverin protein sequence (SEQ ID NO.2) in Example 1, amino acids 135-146 (sequence: PEDVKLLPDDEN, 12 amino acids in total) were mutated to a flexible linker peptide sequence of the same length (sequence: GGGGSGGGGSGA, 12 amino acids in total), while the remaining domains (ALB signal peptide, linker peptide A, CD8a transmembrane region, linker peptide B, mCherry) remained unchanged. The sequence structure after mutation is: ALB-Recoverin(23-200, Mut135-146)-linker peptide A-CD8a-linker peptide B-mCherry.

[0045] (2) Gene synthesis and sequencing identification: Refer to Example 1. The Recoverin recombinant protein-2 expression plasmid was finally obtained and named pLVX-Rec-Mut135-146-CD8a-mCherry. Comparative Example 4: Construction of the Recoverin recombinant protein-3 expression vector (deleting amino acids 95-110, where the major antigenic epitope is located). Using the wild-type Recoverin gene as a template, the nucleotide sequence encoding amino acids 95-110 was deleted, and a lentiviral vector was constructed according to the method in Example 1. Details are as follows: (1) Sequence design: Based on the wild-type Recoverin protein sequence (SEQ ID NO.2) in Example 1, a mutant sequence was designed that deletes amino acids 95-110 (sequence: AGKTNQKLEWAFSLYD, a total of 16 amino acids). This region is located in the EF-hand 2 domain and is the major antigenic epitope region. The remaining domains (ALB signal peptide, linker peptide A, CD8a transmembrane region, linker peptide B, mCherry) remain unchanged. The sequence structure after mutation is: ALB-Recoverin(23-200, Δ95-110)-linker peptide A-CD8a-linker peptide B-mCherry.

[0046] (2) Gene synthesis and sequencing identification: Refer to Example 1. The Recoverin recombinant protein-3 expression plasmid was finally obtained and named pLVX-Rec-Δ95-110-CD8a-mCherry.

[0047] The amino acid sequences of the recombinant proteins involved in each of the above pairs are shown in Table 2 below.

[0048] Table 2. Amino acid sequences of the recombinant proteins described in each comparative example.

[0049] Example 3: Establishment of a stable expression cell line The lentiviral vectors constructed in Examples 1, 2, and Comparative Examples 1 to 4 were transfected into CHO cells. After selection with puromycin and cloning using the limiting dilution method, monoclonal cell lines stably expressing wild-type and mutant Recoverin recombinant proteins were obtained. mCherry expression was observed using fluorescence microscopy, and high-expression clones were screened for subsequent experiments. The specific steps are as follows: (1) Lentiviral Packaging: Lentiviral packaging was performed. The lentiviral transfer plasmids constructed in Examples 1, 2, and Comparative Examples 1 to 4 were co-transfected with packaging plasmids into HEK293T cells. The viral supernatant was collected, concentrated, and high-titer lentiviral particles (titer ≥ 1 × 10⁻⁶) were obtained. 8 TU / mL).

[0050] (2) CHO-K1 cell infection: 24 h before transfection, CHO-K1 cells were infected at a rate of 2×10⁶ cells / year. 5Inoculate lentiviruses at a density of 10 cells / well into 6-well plates and culture in DMEM / F12 medium containing 10% FBS. At infection, add lentivirus particles (MOI=10) and polybrene (5 μg / mL) and incubate at 37°C with 5% CO2. Replace with fresh complete medium 24 h after infection.

[0051] (3) Puromycin screening: 48 h after infection, the culture medium was replaced with a selection medium containing puromycin (7 μg / mL), and the medium was changed every 2 or 3 days for 14 days. Cell death was observed regularly during the period. All cells in the uninfected control group died within 5 to 7 days of screening.

[0052] (4) Limiting dilution cloning: After puromycin screening, resistant cells were seeded at a density of 1 cell / well in 96-well plates (CHO complete medium containing 10% FBS + 2 μg / mL puromycin). The growth of single clones was observed under a microscope after 10 to 14 days. The single clone wells were labeled, and when the cells expanded to cover 80% of the bottom of the well, they were transferred to 24-well plates for further culture.

[0053] (5) Expression identification and clone screening: The expression intensity of each clone of mCherry was observed by fluorescence microscopy, and high expression clones were selected; the mCherry positivity rate was further detected by flow cytometry (it should be >95%), and high expression monoclonal cell lines were screened. The qualified clones were gradually expanded into 6-well plates and T25 culture flasks and frozen in liquid nitrogen for later use.

[0054] Example 4: Cellular Immunofluorescence Experiment Cells stably expressing Recoverin recombinant protein were seeded into 96-well plates. After cell adhesion, serum samples were diluted with DMEM basal medium (containing 1% BSA) with 2 mM CaCl2 and added to the cell wells. The plates were incubated at 37°C for 1 hour. After washing with PBS, 488-labeled goat anti-human IgG secondary antibody was added and incubated at room temperature for 45 minutes. After washing with PBS, the samples were read under a fluorescence microscope.

[0055] The specific steps are as follows: (1) Cell plating: The stable expression cells screened in Example 3 (from Example 1, Example 2, and Comparative Examples 1 to 4, respectively) were plated at 5 × 10⁻⁶ cells per cell. 3 The cells were seeded at a density of 1 cell per well in black transparent 96-well plates and cultured at 37°C and 5% CO2 for 24 h until the cells adhered.

[0056] (2) Sample dilution and incubation: Remove the culture medium and block the sample with DMEM basal medium (containing 1% BSA, pH 7.4) containing 2 mM CaCl2 at room temperature for 30 min. Dilute the serum samples to be tested in gradients of 1:10, 1:32, 1:100, 1:320, 1:1000, and 1:3200, add them to the cell wells, and incubate at 37°C for 1 h.

[0057] (3) Washing and secondary antibody incubation: Remove serum, wash 3 times with PBS, 5 min each time. Add 488-labeled goat anti-human IgG secondary antibody (1:200 dilution, Jackson Immuno Research, catalog number 109-545-190), and incubate at room temperature in the dark for 45 min.

[0058] (4) Observation and interpretation: After washing three times with PBS, observe and photograph under an Olympus CKX53 fluorescence microscope. Judgment criteria: The antibody titer of the sample is the reciprocal of the highest dilution that shows obvious green fluorescence (488) and is higher than the background of the negative control. A titer ≥1:10 is considered positive, and no obvious fluorescence should be seen at any dilution of the negative control.

[0059] Example 5: Parallel detection of recombinant Recoverin protein This embodiment is used to evaluate the ability of the recombinant protein described in this invention to distinguish between specific antibodies and cross-reactive antibodies, and to verify the rationality of each pair ratio design.

[0060] The stable expression cells obtained in Example 3 were seeded into different wells of a 96-well plate. The test samples were serially diluted at 1:10, 1:32, 1:100, 1:320, 1:1000, and 1:3200 using DMEM basal medium (containing 2 mM CaCl2 and 1% BSA) and added to different cell wells simultaneously. Immunofluorescence was used for detection. The cells were incubated at 37°C for 1 hour, washed with PBS, and then incubated with 488-labeled goat anti-human IgG secondary antibody at room temperature for 45 minutes. After washing with PBS, the antibody titer was read under a fluorescence microscope. The reciprocal of the highest dilution showing obvious green fluorescence was taken as the antibody titer. The results are shown in Table 3. Figure 2 .in, Figure 2 The image shows immunofluorescence patterns of positive samples for anti-Recoverin antibodies against Recoverin natural full-length protein, wild-type Recoverin recombinant protein, and mutant Recoverin recombinant protein.

[0061] Experimental Groups: (1) Example 1: Wild-type Recoverin recombinant protein; (2) Example 2: Mutant Recoverin recombinant protein; (3) Comparative Example 1: Full-length Recoverin protein (no transmembrane region, intracellular expression); (4) Comparative Example 2: Recoverin recombinant protein-1 (amino acids 140-143 deleted); (5) Comparative Example 3: Recoverin recombinant protein-2 (mutated amino acids 135-146); (6) Comparative Example 4: Recoverin recombinant protein-3 (amino acids 95-110 deleted, major antigenic epitope region).

[0062] Positive control setup: Specific antibody positive control: Clinically validated anti-Recoverin positive patient serum (n=3); Cross-reactive antibody positive control: Serum from patients who were positive for anti-GCAP-1 / GCAP-2 or anti-Hippocalcin as confirmed by commercial kits (n=3).

[0063] Negative control: serum from healthy individuals (n=5).

[0064] Table 3. Parallel detection results of Recoverin recombinant protein

[0065] According to the test results: (1) Specific antibody detection: The titers of specific anti-Recoverin antibodies against wild-type recombinant protein (Example 1) and mutant recombinant protein (Example 2) were consistent. This result demonstrates that after the deletion of amino acids 135-146, the major antigenic epitopes (EF-hand 2 and EF-hand 3) of the Recoverin protein were completely preserved, and the binding ability of specific antibodies to mutant recombinant protein was not affected.

[0066] (2) Cross-reactive antibody differentiation: The cross-reactive antibodies were positive for wild-type recombinant protein (Example 1) (titer 1:10-1:32), but turned negative for mutant recombinant protein (Example 2) (<1:10). This proves that the mutant recombinant protein of the present invention can effectively eliminate the false positive signal of cross-reactive antibodies and achieve effective differentiation between specific antibodies and cross-reactive antibodies.

[0067] (3) Verification of the rationality of the proportional design: Comparative Example 1 (full-length intracellular expression): The specific antibody titer was significantly lower than that of the membrane-displayed type (positive control 1: 1:32 vs wild-type recombinant 1:320, a 10-fold decrease), demonstrating that membrane surface display (Example 1) can significantly improve detection sensitivity; and it was still 1:32 positive for cross-reactivity control 1, and could not distinguish cross-reactivity.

[0068] Comparative Example 2 (Δ140-143): The titer was consistent with that of the wild-type recombinant protein (Example 1), and it also had no ability to distinguish cross-reactive antibodies (cross-reactive control 1 was still 1:32 positive), proving that amino acids at positions 140-143 are not a key region for conformational similarity. Deleting this region does not affect antibody binding, nor can it eliminate cross-reactivity.

[0069] Comparative Example 3 (Mut135-146): The specific antibody titer was significantly lower than that of the wild-type recombinant protein (positive control 1: 1:100 vs 1:320, a decrease of 3.2-fold; positive control 2: 1:32 vs 1:100, a decrease of 3.2-fold), and it was still positive for cross-reactive antibodies (cross-reactive control 1: 1:32). This proves that the simple mutation of amino acids 135-146 not only failed to eliminate cross-reactivity, but also disrupted the antigen conformation and reduced the specific antibody binding ability. Therefore, deletion is better than mutation.

[0070] Comparative Example 4 (Δ95-110): all specific antibodies turned negative (<1:10), proving that deleting the major antigenic epitope region (EF-hand 2) would result in a complete loss of detection capability, which in turn proves the rationality of the design of deleting positions 135-146 (non-major epitope region) in this invention, which both preserves the major epitope and destroys the cross-reactivity conformation.

[0071] (4) The mutant recombinant protein of the present invention (Example 2) achieves the dual effect of "preserving specificity and eliminating cross-reactivity" by deleting amino acids 135-146. The specific antibody titer is completely consistent with that of wild-type recombinant protein (without loss), and the cross-reactive antibody is completely eliminated (turning negative), thereby effectively distinguishing between specific antibody and cross-reactive antibody and significantly improving detection specificity.

[0072] Example 6: Clinical Sample Testing This embodiment is used to evaluate the diagnostic efficacy of the recombinant protein combination described in this invention in the detection of actual clinical samples, and to verify the clinical value of the combined detection strategy of wild-type and mutant types.

[0073] Serum samples were collected from 18 patients clinically diagnosed with cancer-associated retinopathy (CAR) and 60 healthy individuals. Following the method described in Example 5, parallel assays were performed using cells from Example 1 (wild-type Recoverin recombinant protein) and Example 2 (mutant Recoverin recombinant protein). Serum samples were serially diluted at 1:10, 1:32, 1:100, 1:320, 1:1000, and 1:3200, and interpreted under a fluorescence microscope. The reciprocal of the highest dilution exhibiting obvious green fluorescence was used as the antibody titer.

[0074] The test results are as follows: Serum from CAR patients (18 cases): Wild-type Recoverin recombinant protein detection: 12 cases were positive, with titers ranging from 1:32 to 1:3200, and a median titer of 1:320; Detection of mutant Recoverin recombinant protein: 12 cases were positive, with titers ranging from 1:10 to 1:3200 and a median titer of 1:320.

[0075] Serum from healthy controls (60 cases): Wild-type Recoverin recombinant protein detection: 2 positive cases, titers 1:10 to 1:32; Detection of mutant Recoverin recombinant protein: 0 positive cases.

[0076] Figure 3 Immunofluorescence patterns of wild-type and mutant Recoverin recombinant proteins in samples containing non-specific antibodies are shown. The results indicate that in the serum of 18 patients with cancer-related retinopathy, 12 were positive for both wild-type and mutant recombinant proteins in parallel detection. Among these, two patients showed significantly reduced binding signals to the mutant compared to the wild-type, suggesting possible cross-reactivity of their Recoverin antibodies. The remaining 10 patients showed no significant difference in binding between the wild-type and mutant recombinant proteins, suggesting that their antibodies are specific to the EF-hand 2 or EF-hand 3 regions. Two healthy controls were positive for the wild-type recombinant protein but negative for the mutant recombinant protein, indicating that the mutant recombinant protein has better specificity.

[0077] Example 7: Sensitivity Experiment This embodiment is used to evaluate the advantage of the recombinant protein of the present invention in terms of detection sensitivity compared with conventional intracellular expression (Comparative Example 1).

[0078] Serum from 12 patients who tested positive for Recoverin antibody in Example 6 was selected and tested in parallel using stably expressing cells from Example 1 (wild-type Recoverin recombinant protein, membrane surface display), Example 2 (mutant Recoverin recombinant protein, membrane surface display), and Comparative Example 1 (full-length Recoverin protein, intracellular expression, no transmembrane region). The serum samples were serially diluted at 1:10, 1:32, 1:100, 1:320, 1:1000, and 1:3200 and interpreted under a fluorescence microscope. The highest dilution showing obvious green fluorescence was taken as the antibody titer.

[0079] Detection method: Membrane display group (Examples 1 and 2): Live cells were directly incubated and detected according to the method in Example 4; Intracellular expression group (Comparative Example 1): Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and then detected using the same method.

[0080] The test results are shown in Table 4.

[0081] Table 4. Serum titer range and median titer in patients with positive Recoverin antibody.

[0082] The test results showed that the median titer of both the membrane-displayed wild-type (Example 1) and the mutant (Example 2) was 1:320, significantly higher than the 1:100 of traditional intracellular expression (Comparative Example 1), demonstrating that the membrane-displayed strategy can improve detection sensitivity by approximately 3.2 times. The upper limit of titer for both reached 1:3200, while intracellular expression only reached a maximum of 1:320, further confirming that membrane display avoids the disruption of antigen conformation caused by immobilization and permeation. The mutant type had comparable sensitivity to the wild-type, but effectively excluded cross-reactive antibodies, significantly improving detection specificity while maintaining high sensitivity.

[0083] 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. A recombinant protein for detecting anti-Recoverin autoantibodies, characterized in that: The recombinant protein is selected from any of the following: (1) The mutant Recoverin recombinant protein consists of a secretion signal peptide, a partial sequence of the mutant Recoverin protein, a linker peptide A, a transmembrane region, a linker peptide B, and a fluorescent tag, from the N-terminus to the C-terminus. (2) A recombinant protein combination, consisting of the mutant Recoverin recombinant protein and the wild-type Recoverin recombinant protein, wherein the wild-type Recoverin recombinant protein, from the N-terminus to the C-terminus, comprises a secretion signal peptide, a partial sequence of the wild-type Recoverin protein, a linker peptide A, a transmembrane region, a linker peptide B, and a fluorescent tag. The partial sequence of the mutant Recoverin protein is shown in SEQ ID NO.1; The partial sequence of the wild-type Recoverin protein is shown in SEQ ID NO.2; The secretory signal peptide is human albumin signal peptide ALB, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane region is the CD8a hinge region, and its amino acid sequence is shown in SEQ ID NO.4; The fluorescent tag is mCherry, and its amino acid sequence is shown in SEQ ID NO.5; The amino acid sequence of the linker peptide A is shown in SEQ ID NO. 6; The amino acid sequence of the linker peptide B is shown in SEQ ID NO.

7.

2. The use of the recombinant protein as described in claim 1 in the preparation of a kit for detecting anti-Recoverin autoimmune antibodies, characterized in that: (a) When the recombinant protein is a mutant Recoverin recombinant protein, the nucleic acid encoding the mutant Recoverin recombinant protein is transfected into a host cell to obtain a host cell expressing the mutant Recoverin recombinant protein. After the host cell expressing the mutant Recoverin recombinant protein is contacted with the sample to be tested, the binding signal on its surface is detected by immunofluorescence. (b) When the recombinant protein is a combination of recombinant proteins, host cells expressing mutant Recoverin recombinant protein and host cells expressing wild-type Recoverin recombinant protein are respectively brought into contact with the sample to be tested, and the binding signal on their respective surfaces is detected by immunofluorescence.

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