A fusion protein for detecting nmdar type encephalitis autoantibody and application thereof

Abstract

The application discloses a fusion protein for detecting NMDAR type encephalitis autoantibody and application thereof, and belongs to the technical field of medical detection, wherein the fusion protein comprises NR1 protein and at least one selected from NR2B-eGFP fusion protein or NR2B-mRFP fusion protein, the NR1 protein is an NR1 subunit, the NR2B-eGFP fusion protein is a fusion protein formed by replacing the amino acid sequence of an S2 site of an NR2B subunit with eGFP, and the NR2B-mRFP fusion protein is a fusion protein formed by replacing the amino acid sequence of the S2 site of the NR2B subunit with mRFP. Experiments prove that the fusion protein can effectively destroy the binding capacity of glutamate and NMDAR receptors, thereby eliminating the problem of glutamate-induced cytotoxicity, and can retain the overall spatial conformation of the NMDAR receptor and key autoantibody recognition epitopes, and significantly improve the sensitivity and specificity of NMDAR autoantibody detection based on living cells.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a fusion protein for detecting autoantibodies against NMDAR-type encephalitis and its application. Background Technology

[0002] Autoimmune encephalitis (AE) refers to a class of encephalitis mediated by autoimmune mechanisms. Due to its insidious onset and complex symptoms, it is easily misdiagnosed, posing a significant challenge to the clinical diagnosis and treatment of neurological diseases. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common type of AE, accounting for approximately 54% to 80% of all AE encephalitis cases. According to the "Expert Consensus on the Diagnosis and Treatment of Autoimmune Encephalitis in China (2022 Edition)" (Chinese Journal of Neurology, 2022, 55(9):931-948), the detection of NMDAR antibodies using cell-based substrates (CBA) is one of the necessary conditions for the diagnosis of NMDAR encephalitis. Studies have shown that NMDAR encephalitis autoantibodies mainly target the NR1 subunit of the NMDAR receptor. In brain tissue, NR1 often forms a functional NMDAR tetramer with NR2 (2A, 2B, 2C, and 2D) and NR3 (3A and 3B).

[0003] NMDAR autoantibody detection based on the fixed CBA (F-CBA) method primarily targets the NR1 subunit. Unlike NR2 or NR3, which form heterotetramers in vivo, the single NR1 subunit may not be able to form an antigenic epitope conformation that can fully bind to autoantibodies. Furthermore, since the single NR1 subunit cannot be localized to the cell membrane, it requires chemical fixation, which can also lead to the loss of the antigenic epitope. Therefore, some studies have indicated that F-CBA may result in missed detections (see Thouin et al. Comparison of N-methyl-D-aspartate receptor antibody assays using live or fixed substrates. J Neurol. 2021, 268(5):1818-1826.).

[0004] Live-cell antigenic absorptiometry (L-CBA) assay is a specialized CBA detection method primarily used for detecting membrane-localized extracellular antigenic epitopes. Because it does not involve the immobilization of chemical reagents, it effectively preserves the natural structure of the antigenic epitopes, thereby improving detection sensitivity and specificity. Tanaka et al. found that full-length co-expression of the NR1 and NR2 subunits enables membrane localization of NMDAR antigens and, to some extent, improves the detection sensitivity of NMDAR autoantibodies (see Tanaka et al. Evaluation of the concordance between GluN1-GluN2 heteromer live-cell-based assay and GluN1 monomer biochip kit assay on anti-NMDAR autoantibody detection. J Immunol Methods, 2021). However, since NR1 and NR2 constitute a physiologically active NMDAR receptor, this receptor can bind to glutamate in the culture medium, inducing cytotoxicity. Therefore, in practice, the NMDAR receptor blocker ketamine needs to be added to inhibit the cytotoxicity of glutamate production. However, such blockers are controlled substances, which greatly limits their large-scale application. Furthermore, the use of blockers also reduces the stability of NMDAR protein production.

[0005] In the prior art, Chinese patent CN114144427A discloses a soluble N-methyl-D-aspartate receptor (NMDAR) protein construct, which includes one or more NMDAR autoantibody epitopes. Specifically, the construct includes the extracellular domain (ECD) of the NMDAR subunit GluN1 or a fragment thereof, and an ECD or a fragment thereof composed of at least one of the NMDAR subunits GluN2A, GluN2B, GluN2C, or GluN2D. According to the data provided in this patent, the soluble NMDAR protein construct (especially when present as an Fc fusion protein) can effectively detect autoantibodies in the serum of patients with NMDAR encephalitis. However, this construct is only a fusion protein of a partial NMDAR subunit fragment. Although it can recognize specific antigenic epitopes and provide a certain degree of specificity and accuracy in autoantibody detection, it only expresses a partial sequence of the NMDAR receptor, resulting in an incomplete protein structure that cannot fully mimic the spatial conformation of the natural receptor. Therefore, its detection rate is insufficient to meet the actual needs of clinical testing.

[0006] Based on the aforementioned technical deficiencies, there is an urgent need in this field to establish a novel live cell (CBA)-based method for detecting NMDAR autoantibodies in order to overcome the problems of incomplete fusion protein antigen structure and insufficient detection rate in existing methods. Summary of the Invention

[0007] This invention aims to address the technical shortcomings of existing human NMDAR encephalitis autoantibody detection materials, such as insufficient sensitivity, poor specificity, and the inability of antigen spatial conformation to fully mimic the natural receptor conformation. It provides a fusion protein for detecting NMDAR encephalitis autoantibodies and its applications. The fusion protein is constructed by fusing the NR1 subunit with an NR2B subunit modified with eGFP or mRFP (eGFP replacing its S2 site, or mRFP replacing its S2 site). This invention addresses the clinical diagnostic needs of NMDAR encephalitis and other antibody-related diseases, providing strong support for early screening, diagnosis, and disease monitoring, filling the gaps in existing detection technologies, and promoting the improvement of diagnosis and treatment of related autoimmune diseases.

[0008] This invention is achieved through the following technical solution: a fusion protein for detecting autoantibodies against NMDAR-type encephalitis, wherein the fusion protein comprises NR1 protein and at least one selected from NR2B-eGFP fusion protein or NR2B-mRFP fusion protein. The NR1 protein is an NR1 subunit; The NR2B-eGFP fusion protein is a fusion protein formed by replacing the S2 site amino acid sequence of the NR2B subunit with eGFP; The NR2B-mRFP fusion protein is a fusion protein formed by replacing the S2 site amino acid sequence of the NR2B subunit with mRFP.

[0009] The amino acid sequence of the NR1 protein is shown in SEQ ID NO: 1; The amino acid sequence of the NR2B-eGFP fusion protein is shown in SEQ ID NO: 2; The amino acid sequence of the NR2B-mRFP fusion protein is shown in SEQ ID NO: 3.

[0010] The present invention also provides a gene encoding the above-mentioned fusion protein, said gene comprising a nucleotide sequence encoding an NR1 subunit and at least one of a nucleotide sequence encoding an NR2B-eGFP fusion protein or an NR2B-mRFP fusion protein.

[0011] And a recombinant expression vector comprising a gene encoding the aforementioned fusion protein.

[0012] And a recombinant host cell comprising the above-described recombinant expression vector, or having a gene encoding the above-described fusion protein integrated into its genome.

[0013] Another technical solution of the present invention provides a product for detecting NMDAR-type encephalitis autoantibodies, the product comprising the fusion protein, the recombinant expression vector, or the recombinant host cell.

[0014] Preferably, the detection is based on a live cell detection method.

[0015] Preferably, the product is a testing reagent or a kit.

[0016] Preferably, the host cell is HEK-293 cell or CHO cell.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention accurately identifies the binding site of glutamate to the NMDAR receptor by systematically analyzing the S1 and S2 domains of the NR2B subunit. Experiments have shown that replacing the S2 fragment of the NR2B subunit can effectively disrupt the binding ability of the glutamate ligand to the receptor, thus solving the problem of glutamate cytotoxicity caused by the NMDAR receptor formed by the assembly of NR1 and NR2 from the source, significantly improving the stability of cell production based on the NMDAR receptor, and providing reliable core raw material support for the detection of NMDAR autoantibodies.

[0018] (2) This invention breaks through the cognitive limitations of the prior art. For the first time, it targets the characteristic of the glutamate binding site being formed by the folding of S1 and S2 polypeptides into a "clam shell" structure. Through targeted and exclusive experimental verification, the S2 sequence of NR2B was identified as the modification target from a novel truncation division. The inventive site replacement design enables the constructed fusion protein (NR2B-eGFP or NR2B-mRFP) to avoid the problem of glutamate cytotoxicity while retaining the structural integrity and antigenic epitope effectiveness of NMDAR, significantly improving the sensitivity and specificity of NMDAR autoantibody detection.

[0019] (3) This invention provides a reliable technical tool for early screening, clinical diagnosis, disease progression assessment, treatment plan adjustment and efficacy judgment of NMDAR encephalitis and related autoimmune diseases. It makes up for the shortcomings of traditional detection methods in disease monitoring, helps to improve the whole process of diagnosis and treatment of such diseases, and has important clinical application value. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the construction of NR2B-eGFP3 according to the present invention.

[0021] Figure 2 This is a schematic diagram illustrating the detection results of normal healthy human serum (negative sample) in HEK-293 cell lines overexpressing NR1 and different types of NR2B-eGFP constructs in an embodiment of the present invention.

[0022] Figure 3 This is a schematic diagram illustrating the detection results of serum (titer 1:32) from NMDAR-positive AE patients in HEK-293 cell lines overexpressing NR1 and different types of NR2B-eGFP constructs in an embodiment of the present invention.

[0023] Figure 4 This is a schematic diagram illustrating the detection results of serum (titer 1:320) from NMDAR-positive AE patients in HEK-293 cell lines overexpressing NR1 and different types of NR2B-eGFP constructs in an embodiment of the present invention.

[0024] Figure 5 This diagram illustrates the detection results of serum from normal healthy individuals (negative samples) and NMDAR-positive AE patients in HEK-293 cell lines overexpressing NR1 and NR2B-mRFP construct combinatorial proteins in an embodiment of the present invention. Detailed Implementation

[0025] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0026] 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.

[0027] This invention provides a fusion protein for detecting autoantibodies against NMDAR-type encephalitis and its applications. The design concept of this fusion protein is to fuse the NR1 subunit of the NMDAR receptor with an NR2B subunit modified with fluorescent protein (eGFP or mRFP). The modification of the NR2B subunit targets its S2 site, a key region essential for glutamate binding. By replacing the entire amino acid sequence of the S2 site of the NR2B subunit with eGFP, or by replacing the entire amino acid sequence of the S2 site with mRFP, this invention successfully constructed two types of fusion proteins: NR2B-eGFP and NR2B-mRFP. Experiments have shown that the above modifications effectively disrupt the binding ability of glutamate to the NMDAR receptor, thereby eliminating glutamate-induced cytotoxicity. Simultaneously, when the modified NR2B subunit is co-expressed with the NR1 subunit, the overall spatial conformation of the NMDAR receptor and key autoantibody recognition epitopes are still preserved, significantly improving the sensitivity and specificity of NMDAR autoantibody detection based on live cells.

[0028] In the natural NMDAR receptor, the glutamate binding site is formed by the folding of the S1 and S2 polypeptides of the NR2 subunit into a "clamshell" structure, with S1 located in the N-terminal extracellular region and S2 located in the extracellular region between transmembrane regions 3 and 4. Direct co-expression of the full-length NR1 and NR2 subunits, while achieving membrane localization of the receptor and providing a complete antigenic epitope, can lead to cytotoxicity due to glutamate binding, necessitating the addition of controlled substances such as ketamine during the procedure, significantly limiting its clinical application. On the other hand, expressing only the NR1 single subunit or a soluble fragment (such as CN114144427A) avoids the toxicity problem, but the incomplete antigen conformation results in detection rates that are insufficient to meet clinical needs.

[0029] This invention specifically selects the S2 site of the NR2B subunit as the modification target. By replacing it with a fluorescent protein (eGFP or mRFP), the glutamate binding site is precisely disrupted while preserving the overall NR2B framework and its ability to assemble with NR1, thereby eliminating cytotoxicity at its source. Simultaneously, the introduction of the fluorescent protein not only reports the expression and localization of the fusion protein but also helps maintain the natural epitope recognized by NMDAR autoantibodies due to its spatial stabilization of the NR2B extracellular conformation. This design overcomes the limitations of existing technology (which believes that a complete S1 / S2 structure is required to maintain antigenicity), and the feasibility and superiority of this invention have been successfully demonstrated through targeted and exclusive experimental verification.

[0030] For more details, see Figure 1 As shown, Figure 1 A in the diagram illustrates the combination of the wild-type NR1 subunit and the wild-type NR2 subunit to form the NMDAR receptor. Figure 1 As shown in Figure A, the S1 polypeptide of the NR1 subunit and the S2 polypeptide of the NR2 subunit fold together to form a "clam shell" structure that can bind to glutamic acid (Glu). Figure 1 B is a schematic diagram illustrating the NMDAR receptor formed by the combination of the wild-type NR1 subunit and the NR2-S2-eGFP fusion protein. Figure 1 As shown in Figure B, the S2 polypeptide of the NR2 subunit is replaced by the eGFP protein, thereby losing its ability to fold with the S1 polypeptide of the NR1 subunit to form a "clam shell" structure, resulting in glutamate being unable to bind to the NMDAR receptor. Figure 1 In this context, ATD stands for amino acid terminal domain; LBD for ligand-binding domain; TMD for transmembrane domain; I for ifendil (NMDAR receptor antagonist); P for neurotransmitter binding site; and C for channel blocker.

[0031] Based on the above design, the present invention specifically provides the following fusion protein components: NR1 protein: NR1 subunit, whose amino acid sequence is shown in SEQ ID NO: 1.

[0032] NR2B-eGFP fusion protein: formed by replacing the entire S2 site amino acid sequence of the NR2B subunit with eGFP, and its amino acid sequence is shown in SEQ ID NO: 2.

[0033] NR2B-mRFP fusion protein: formed by replacing the entire S2 site amino acid sequence of the NR2B subunit with mRFP, and its amino acid sequence is shown in SEQ ID NO: 3.

[0034] It should be noted that "eGFP" in this invention refers to enhanced green fluorescent protein; and "mRFP" refers to monomeric red fluorescent protein. In practical implementation, the above-mentioned fusion proteins can be used alone (e.g., composed of NR1 protein and NR2B-eGFP fusion protein, or composed of NR1 protein and NR2B-mRFP fusion protein), or they can be used in combination (containing both of the aforementioned NR2B modified forms) to adapt to the needs of different detection platforms or signal readout methods.

[0035] To achieve stable expression of the above-mentioned fusion protein, the present invention also provides the corresponding gene, recombinant expression vector and recombinant host cell.

[0036] Encoding gene: Contains at least one of the nucleotide sequences encoding the NR1 subunit (as shown in SEQ ID NO: 4) and the nucleotide sequences encoding the NR2B-eGFP fusion protein or the NR2B-mRFP fusion protein (as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively). The above sequences may be located on the same expression vector or may be constructed separately in different expression vectors and then co-transfected.

[0037] Recombinant expression vector: Contains a gene expressing the fusion protein described in this invention, and may use a cell expression vector known in the art, and contains necessary regulatory elements, such as promoters, enhancers, etc.

[0038] Recombinant host cells: Mammalian cells are preferred as host cells, such as HEK-293 cells or CHO cells, which have efficient protein expression and post-translational modification capabilities and can correctly locate the fusion protein on the cell membrane, thereby mimicking the membrane presentation of the natural NMDAR receptor.

[0039] In the application stage, the fusion protein, recombinant expression vector, or recombinant host cell of the present invention can be used to prepare products for detecting NMDAR-type encephalitis autoantibodies, including but not limited to detection reagents or kits. Preferably, the detection method can employ a live cell-based assay (L-CBA), because the native conformation of membrane proteins is optimally preserved in the live cell state, and chemical fixation is not required, avoiding epitope loss. After co-incubating the host cells expressing the fusion protein with the serum sample to be tested, the NMDAR autoantibodies in the sample can be quantitatively or qualitatively detected by flow cytometry or immunofluorescence microscopy using a fluorescently labeled secondary antibody.

[0040] In summary, the core difference between this invention and existing technologies lies in: (1) Breaking through the limitations of existing technology: This invention is the first to use the S2 site of the NR2B subunit as the key modification target, rather than simply deleting the entire extracellular domain or directly using the natural subunit, thus achieving precise destruction of the glutamate binding site.

[0041] (2) Functional synergy and structural preservation: By introducing eGFP or mRFP, this invention not only eliminates the cytotoxicity caused by glutamate binding, but also fully preserves the overall spatial conformation of the NMDAR receptor and its key autoantibody recognition epitopes, thereby ensuring the sensitivity and specificity of autoantibody detection based on live cells.

[0042] 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.

[0043] The experimental materials used in the following examples included: restriction endonuclease XbaI (NEB, catalog number R0145V); high-fidelity DNA polymerase (Novizan, catalog number P505); One Step Cloning Kit (Novizan, catalog number C117-01); restriction endonuclease KpnI (NEB, catalog number R3142V); restriction endonuclease NotI (NEB, catalog number R3189V); glass slides (Mervid); high-glucose DMEM (Gibco, catalog number 11965092); 10% fetal bovine serum (Gibco, catalog number 10099158); anti-human Alexa 488 (Thermo Fisher Scientific, catalog number A20000) or Alexa 594 (Thermo Fisher Scientific, catalog number A11012) secondary antibody; all primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd.; the initial vector pcDNA3.1(+) was a conventional vector provided by Shaoxing University.

[0044] Example 1: Construction of a fusion protein expression vector The complete NR1 gene fragment was obtained. Based on the protein structure of the NR2B subunit, and while ensuring the integrity of the transmembrane region, several gene fragments with different transmembrane domain modifications were designed, including: NR2B-S1-eGFP, NR2B-S2-eGFP1, NR2B-S2-eGFP2, NR2B-S2-eGFP3, NR2B-S2-eGFP4, and NR2B-S2-mRFP. The polypeptides encoded by these gene fragments not only contain the natural sequence but also utilize their functional variants.

[0045] The amino acid and nucleotide sequences of each fragment are as follows: NR2B-S1-eGFP: formed by replacing the entire S1 site amino acid sequence of the NR2B subunit with eGFP, its amino acid sequence is shown in SEQ ID NO: 7, and its nucleotide sequence is shown in SEQ ID NO: 8.

[0046] NR2B-S2-eGFP1: It is formed by inserting eGFP into the N-terminus of the amino acid sequence at the S2 site of the NR2B subunit. Its amino acid sequence is shown in SEQ ID NO: 9, and its nucleotide sequence is shown in SEQ ID NO: 10.

[0047] NR2B-S2-eGFP2: It is formed by inserting eGFP into the C-terminus of the amino acid sequence at the S2 site of the NR2B subunit. Its amino acid sequence is shown in SEQ ID NO: 11, and its nucleotide sequence is shown in SEQ ID NO: 12.

[0048] NR2B-S2-eGFP3 (i.e., the NR2B-eGFP fusion protein of the present invention): formed by replacing the entire S2 site amino acid sequence of the NR2B subunit with eGFP, the amino acid sequence of which is shown in SEQ ID NO: 2 and the nucleotide sequence of which is shown in SEQ ID NO: 5.

[0049] NR2B-S2-eGFP4: The partial amino acid sequence (aa648-aa700) of the S2 site of the NR2B subunit replaced by eGFP, the amino acid sequence of which is shown in SEQ ID NO: 13 and the nucleotide sequence of which is shown in SEQ ID NO: 14.

[0050] NR2B-S2-mRFP (i.e., the NR2B-mRFP fusion protein of the present invention): formed by replacing the entire S2 site amino acid sequence of the NR2B subunit with mRFP, the amino acid sequence of which is shown in SEQ ID NO: 3 and the nucleotide sequence of which is shown in SEQ ID NO: 6.

[0051] The above-mentioned NR2B modified fragment and NR1 overexpression vectors were constructed using homologous recombination, and the specific steps are as follows: S1. Linearize the pcDNA3.1(+) vector by XbaI single enzyme digestion.

[0052] S2. The pcDNA6TR vector was amplified using primers MCS-Myc-F / BGH-CX-R to obtain the myc-6×His tag sequence fragment.

[0053] S3. The linearized vector obtained in step S1 and the fragment obtained in step S2 are subjected to homologous recombination using the One Step Cloning Kit to construct a new vector pCDNA3.1NS-mycHis.

[0054] S4. Digest the pCDNA3.1NS-mycHis vector with NheI and NotI for later use.

[0055] S5. Amplify the three regions (upstream homologous arm, fluorescent protein coding region, and downstream homologous arm) corresponding to each NR2B modified fragment. The primer combinations used are shown in Table 1 below: Primer combinations used for each NR2B modified fragment.

[0056] Table 1: Primer combinations used for each NR2B modified fragment

[0057] S6. The vector obtained from the double enzyme digestion in step S4 and each NR2B modified fragment obtained in step S5 were homologously recombinated using OneStepCloningKit to construct the following overexpression vectors: NR2B-S1-eGFP, NR2B-S2-eGFP1, NR2B-S2-eGFP2, NR2B-S2-eGFP3, NR2B-S2-eGFP4, and NR2B-S2-mRFP.

[0058] The primer sequences used in this embodiment are shown in Table 2 below: Primer sequences for constructing the overexpression vector.

[0059] Table 2: Primer sequences for constructing overexpression vectors

[0060] Example 2: Construction of HEK-293 cell line In this embodiment, the NR1 expression plasmid constructed in Example 1 was co-transfected with different NR2B modified fragment plasmids into the HEK-293 cell line to obtain cell lines overexpressing the corresponding fusion proteins. The specific steps are as follows: A1. Soak the glass slide in 75% ethanol, clean it with PBS phosphate buffer, and treat it with laminin at 37°C for 30 minutes.

[0061] A2. HEK-293 cells were cultured at a rate of 2 × 10⁻⁶. 6 cells / 25cm 2 The seeds are inoculated at a density on the prepared glass slides as described above.

[0062] A3. After culturing for 24 hours, the NR1 expression plasmid was mixed with each of the NR2B modified plasmids (NR2B-S1-eGFP, NR2B-S2-eGFP1, NR2B-S2-eGFP2, NR2B-S2-eGFP3, NR2B-S2-eGFP4 or NR2B-S2-mRFP) at a 1:1 ratio, with a total transfection concentration of 3000 ng / mL. The cells were then transfected using 25 kDa PEI reagent.

[0063] A4. Six hours after transfection, replace with HEK-293 cell culture medium (DMEM medium containing 10% fetal bovine serum) and continue culturing for 36 to 48 hours before use for subsequent detection.

[0064] Example 3: Immunofluorescence detection and fusion protein performance evaluation based on fixed cells This embodiment aims to evaluate the detection efficacy of different NR2B modified fusion proteins co-expressed with NR1 against NMDAR autoantibodies by immunofluorescence staining, and to compare the differences in sensitivity and specificity between the NR2B-S2-eGFP3 (NR2B-eGFP) and NR2B-S2-mRFP (i.e., NR2B-mRFP) described in this invention and other control constructs.

[0065] The specific steps for immunofluorescence detection are as follows: B1. 36 to 48 hours after transfection, fix the cells with acetone for 10 minutes.

[0066] B2. Dilute the serum of healthy individuals and the serum of NMDAR encephalitis patients at a ratio of 1:10.

[0067] B3. Mix the diluted serum with the fixed cells and incubate at 37°C for 60 minutes.

[0068] B4. After incubation, rinse three times with PBS, each time for 5 minutes.

[0069] B5. The above-mentioned incubation products were co-incubated with secondary antibodies labeled with anti-human Alexa 594 or Alexa 488 at 37°C for 30 minutes.

[0070] B6. After incubation, rinse three times with PBS, each time for 5 minutes.

[0071] B7. Observe and photograph under a fluorescence microscope.

[0072] The test results are as follows: (1) Specificity verification (negative serum) Figure 2 The results show the detection of normal human serum (negative sample) in HEK-293 cells co-transfected with NR1 and different NR2B-eGFP constructs.

[0073] The results showed that during the detection of normal human serum, no obvious red fluorescence binding to autoantibodies was detected in cells co-transfected with different NR1 and NR2B-eGFP constructs, but background-level green eGFP fluorescent protein expression was observed in all cells, indicating that NMDAR autoantibodies are not present in normal human bodies. This demonstrates that the detection system constructed in this invention has good specificity.

[0074] (2) Sensitivity verification (serum from low-titer patients) Figure 3 The results show the detection of serum (titer 1:32) from NMDAR-positive AE patients in HEK-293 cells co-transfected with NR1 and different NR2B-eGFP constructs.

[0075] The results showed that only co-transfection of NR1 with NR2B-S2-eGFP3 (NR2B-eGFP) resulted in detectable red fluorescence. Other control constructs (NR2B-S2-eGFP1, NR2B-S2-eGFP2) showed completely absent signals; while NR2B-S2-eGFP4 and NR2B-S1-eGFP showed weak signals, their intensity was significantly reduced. These results demonstrate that the NR1 and NR2B-S2-eGFP3 combination described in this invention can still efficiently detect NMDAR autoantibodies in low-titer serum, exhibiting excellent sensitivity.

[0076] (3) Serum detection in patients with high titers Figure 4 The results show the detection of serum (titer 1:320) from NMDAR-positive AE patients in HEK-293 cells co-transfected with NR1 and different NR2B-eGFP constructs.

[0077] The results showed that the NR1 and NR2B-S2-eGFP3 groups still exhibited the strongest red fluorescence signal, while the signal intensity of other control constructs was lower than that of this combination. This further confirms that the fusion protein described in this invention also possesses stable and reliable detection capabilities in samples with high antibody titers.

[0078] (4) Validation of the red fluorescent protein tag (mRFP) system Figure 5The results are for detecting serum from normal individuals or NMDAR-positive AE patients in HEK-293 cells co-transfected with NR1 and NR2B-S2-mRFP (NR2B-mRFP) constructs.

[0079] The results showed that no obvious green fluorescence binding to autoantibodies was detected in normal human serum (negative samples), but background-level expression of red mRFP fluorescent protein was observed, further verifying the good specificity of the detection system constructed in this invention. In the serum of NMDAR-positive AE patients (titers 1:32 and 1:320), bright green fluorescence was detected in HEK-293 cells co-transfected with NR1 and NR2B-S2-mRFP (NR2B-mRFP), indicating that the NR2B-mRFP fusion protein constructed in this invention can also efficiently recognize NMDAR autoantibodies, and the detection signal is not affected by the type of fluorescent protein tag.

[0080] Based on the above detection results, it is evident that the combination of NR1 constructed in this invention with NR2B-S2-eGFP3 (NR2B-eGFP) or NR2B-S2-mRFP (NR2B-mRFP) exhibits extremely high sensitivity and good specificity in cell-based immunofluorescence detection. In particular, the NR2B-S2-eGFP3 construct can still detect a clear positive signal in low-titer (1:32) patient serum, significantly superior to other control modification methods (such as S1 site replacement, N-terminal or C-terminal insertion of the S2 site, partial S2 site replacement, etc.), demonstrating the superiority of using full-length S2 site replacement. For reference, the wild-type full-length sequence of the NR2B subunit described in this invention is shown in SEQ ID NO: 32.

[0081] 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 fusion protein for detecting autoantibodies against NMDAR-type encephalitis, characterized in that: The fusion protein includes NR1 protein, and at least one selected from NR2B-eGFP fusion protein or NR2B-mRFP fusion protein. The NR1 protein is an NR1 subunit, and its amino acid sequence is shown in SEQ ID NO: 1; The NR2B-eGFP fusion protein is a fusion protein formed by replacing the S2 site amino acid sequence of the NR2B subunit with eGFP, and its amino acid sequence is shown in SEQ ID NO: 2; The NR2B-mRFP fusion protein is a fusion protein formed by replacing the S2 site amino acid sequence of the NR2B subunit with mRFP, and the amino acid sequence is shown in SEQ ID NO:

3.

2. The gene encoding the fusion protein of claim 1, characterized in that: The gene includes at least one of a nucleotide sequence encoding the NR1 subunit and a nucleotide sequence encoding either the NR2B-eGFP fusion protein or the NR2B-mRFP fusion protein.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene as described in claim 2.

4. A recombinant host cell, characterized in that: The recombinant host cell comprises the recombinant expression vector of claim 3, or has the gene of claim 2 integrated into its genome.

5. A product for detecting autoantibodies against NMDAR-type encephalitis, characterized in that: The product comprises the fusion protein of claim 1, the recombinant expression vector of claim 3, or the recombinant host cell of claim 4.

6. The product according to claim 5, characterized in that: The detection method is based on live cells.

7. The product according to claim 5, characterized in that: The product is a testing reagent or kit.

8. The product according to claim 5, characterized in that: The host cells are HEK-293 cells or CHO cells.

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