A fusion protein for detecting anti-ampar1 antibodies and uses thereof

Abstract

The present application relates to a kind of fusion protein for detecting anti-AMPAR1 antibody and purposes thereof.The present application specifically relates to a kind of fusion protein for detecting anti-AMPAR1 antibody, fusion protein includes extracellular amino-terminal domain, linker, extracellular ligand binding domain from N-terminal to C-terminal, the amino acid sequence of fusion protein is as shown in SEQ ID NO.1.The kit prepared from the fusion protein of the present application has the advantages of simple, accurate, high sensitivity and the like in the detection of anti-AMPAR1 antibody.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a fusion protein for detecting anti-AMPAR1 antibodies and its uses. Background Technology

[0002] Autoimmune encephalitis (AE) is an immune-mediated neurological disorder mediated by anti-neuronal autoantibodies. Clinically, it often presents with a variety of symptoms, including cognitive impairment, behavioral abnormalities, seizures, involuntary movements, and autonomic dysfunction. Since Dalmau first reported anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in 2007, a series of autoantibodies targeting neuronal cell surfaces or synaptic proteins have been discovered, advancing the understanding and diagnosis of this type of disease.

[0003] AMPA receptors, as a key glutamate receptor subtype in the central nervous system, are mainly distributed in brain regions closely related to learning and memory, such as the hippocampus. They are responsible for mediating excitatory neurotransmission and play a central role in synaptic plasticity, serving as an important molecular basis for cognitive function. AMPA autoimmune encephalitis is a relatively newly identified type of autoimmune encephalitis. Its pathogenesis is related to autoantibodies binding to AMPA receptors (especially the GluA1 subunit), causing receptor endocytosis and downregulation of surface expression. This essentially disrupts a key structure for interneuronal communication, leading to signal transduction disorders and triggering a series of neuropsychiatric symptoms.

[0004] AMPA autoimmune encephalitis typically presents with acute or subacute onset, and is generally characterized by involvement of the limbic system, including recent memory impairment such as amnesia, behavioral and mental abnormalities (such as anxiety, hallucinations, and aggression), and seizures. Clinically, AMPA encephalitis should be considered in patients exhibiting the above symptoms. For suspected cases of AMPA encephalitis, it is recommended to confirm the diagnosis by detecting anti-AMPAR1 antibodies using cell substrate-based assays in serum or cerebrospinal fluid samples.

[0005] Currently, routine detection relies on transfecting full-length wild-type GluA1 plasmid into cells, followed by antibody detection using cell-to-cell immunofluorescence assay (CBA). The CBA method mainly takes two forms: one uses live cells, which helps maintain the native conformation of the antigen and improves detection sensitivity and accuracy; the other involves fixing the transfected cells and storing them in buffer or preparing frozen stem cell slides to extend the shelf life of the detection matrix. However, the fixation or freeze-drying process may damage the protein conformation, thereby affecting antigenicity and reducing detection sensitivity.

[0006] Existing antibody detection methods heavily rely on cell culture and subsequent processing procedures, including plasmid transfection, plate coating, cell fixation, and lyophilization, which are cumbersome and time-consuming. Deviations in any step can severely affect the reliability of the detection results or even lead to detection failure. In addition, GluA1, as a cation channel, can cause persistent channel opening due to overexpression on the cell membrane, resulting in leakage current even in the absence of agonists. Trace amounts of glutamate in the culture medium or glutamate released by the cells themselves can also activate a large number of receptors, leading to the influx of sodium and calcium ions, which in turn causes cell swelling, vacuolization, and even death, severely affecting cell state and detection stability.

[0007] In summary, existing CBA detection methods based on full-length GluA1 have significant limitations, and there is an urgent need to establish a novel detection strategy to reduce the over-reliance on cell systems for GluA1 autoantibody detection and improve the stability, reproducibility, and applicability of the detection. Summary of the Invention

[0008] In view of this, the main objective of the present invention is to provide a simple and accurate method for detecting AMPAR1 antibodies.

[0009] The specific technical solution of the present invention is as follows.

[0010] This invention provides a fusion protein for detecting anti-AMPAR1 antibodies. The fusion protein includes an extracellular N-terminal domain, a linker, and an extracellular ligand-binding domain from the N-terminus to the C-terminus.

[0011] The amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0012] In some embodiments, the C-terminus of the fusion protein includes a PDGFR β transmembrane peptide, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] In some embodiments, the N-terminus of the fusion protein includes a κ-chain signal peptide, the amino acid sequence of which is shown in SEQ ID NO.4.

[0014] The present invention also provides an isolated or synthesized nucleic acid molecule that encodes the fusion protein of the present invention.

[0015] In some implementations, the sequence of the nucleic acid molecule is shown in SEQ ID NO.6.

[0016] The present invention also provides a carrier comprising the nucleic acid molecule of the present invention.

[0017] In some implementations, the vector is an expression vector, which may be a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

[0018] The present invention also provides a host cell, which includes the nucleic acid molecule of the present invention or the vector of the present invention.

[0019] The present invention also provides the use of the aforementioned fusion protein in the preparation of a kit for detecting anti-AMPAR1 antibodies.

[0020] The present invention also provides the use of the aforementioned fusion protein in the preparation of a kit for diagnosing autoimmune encephalitis.

[0021] In some embodiments, the kit of the present invention is an immunoblotting strip kit.

[0022] The beneficial effects of this invention are as follows.

[0023] This invention successfully screened the key antigenic regions of the GluA1 protein and prepared fusion proteins for membrane display and secretory expression, respectively. Validation with real clinical samples showed that the fusion protein detection method based on this invention can be effectively applied in clinical testing.

[0024] This invention, through innovative screening of core antigen regions, successfully constructs a highly efficient and universal detection scheme, overcoming the technical bottleneck of applying membrane proteins to autoantibody detection. The strategy of this invention breaks away from the traditional reliance on cell-based CBA methods, providing two optional detection pathways: CBA and immunoblotting membrane strip methods. This not only facilitates methodological comparison and optimization for different membrane protein targets, allowing for the selection of the optimal detection scheme, but also, due to its flexibility, allows for broad adaptation to the platform conditions of different types of testing institutions, possessing significant value for widespread application. Attached Figure Description

[0025] Figure 1 The expression of wild-type and various truncated mutants in cells.

[0026] Figure 2 Comparison of GluA1 wild-type and Mutant positive sample test results.

[0027] Figure 3 The SDS-PAGE electrophoresis results of the recombinant protein of the fusion protein of the present invention are shown.

[0028] Figure 4 The results of the recombinant protein membrane strip method for detecting the fusion protein of the present invention are shown. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The following definitions supplement those in the art and relate to this application, but are not extrapolated to any relevant or unrelated circumstances, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0031] In this invention, the terms "comprising," "including," and "having" are open-ended descriptions that include the specified steps described, as well as other steps that do not substantially affect them, and are optional and not excluded.

[0032] As mentioned earlier, GluA1 antibody is a characteristic serological marker for diagnosing anti-AMPA receptor encephalitis, and it is currently commonly detected using cell-based immunofluorescence assays (CBA). However, the expression of full-length GluA1 in cells is often inhibited by feedback regulation mechanisms, resulting in limited expression levels and thus affecting the sensitivity of CBA detection. In practice, it is necessary to precisely balance protein expression time with cell viability; otherwise, overexpression or poor cell condition can easily affect the detection results.

[0033] GluA1 protein (Uniprot: P42261) is a four-transmembrane protein composed of 906 amino acids. Its structure can be divided into four functionally defined regions: extracellular N-terminal domain (ATD): mediates subunit assembly and receptor anchoring on the membrane; extracellular ligand-binding domain (LBD): composed of two segments, S1 and S2, which fold in space to form a "clam shell"-like structure, responsible for binding glutamate and regulating the opening of ion channels; transmembrane domain (TMD): contains four transmembrane regions, M1-M4, which together form the ion channel pore; intracellular C-terminal domain (CTD): participates in synaptic anchoring and intracellular transport of receptors.

[0034] Given that autoantibodies typically target extracellular regions, based on the above structural analysis, the inventors focused on the ATD and LBD extracellular domains. Through extensive research and screening of fragments containing ATD and LBD, the inventors constructed a series of truncated GluA1 extracellular regions of varying lengths, preserving their native conformation to the greatest extent possible. Using systematic screening of GluA1 antibody-positive samples from multiple patients, they ultimately obtained an effective full-length GluA1 substitute antigen region, thus completing this invention.

[0035] The first aspect of the present invention provides a fusion protein for detecting anti-AMPAR1 antibodies, wherein the fusion protein includes an extracellular amino-terminal domain, a linker, and an extracellular ligand-binding domain from the N-terminus to the C-terminus, and wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0036] SEQ ID NO.1:

[0037] QEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKE RLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKG RRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKP QKSKPGVFSGGGSGGGSGGGSAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDK

[0038] In this invention, a fusion protein is constructed that outperforms full-length GluA1 in expression level, producing a positive signal intensity no less than the latter. The increased expression effectively compensates for potential sensitivity loss due to the deletion of partial antigenic epitopes. Furthermore, compared to other constructed fusion proteins, the fusion protein of this invention exhibits better detection accuracy.

[0039] In some embodiments, the C-terminus of the fusion protein includes a PDGFR β transmembrane peptide, the amino acid sequence of which is shown in SEQ ID NO.2.

[0040] SEQ ID NO.2:

[0041] VVISAILALVVLTIISLIILI

[0042] In some implementations, the nucleotide sequence of the PDGFR β transmembrane peptide is shown in SEQ ID NO.3.

[0043] SEQ ID NO.3:

[0044] GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT

[0045] In some embodiments, the N-terminus of the fusion protein includes a κ-chain signal peptide, the amino acid sequence of which is shown in SEQ ID NO.4.

[0046] SEQ ID NO.4:

[0047] METDTLLLWVLLLWVPGSTGD

[0048] In some implementations, the nucleotide sequence of the κ chain signal peptide is shown in SEQ ID NO.5.

[0049] SEQ ID NO.5:

[0050] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC

[0051] In some embodiments, the amino acids of the fusion protein can be conserved variant sequences that do not affect its function, generated by mutation based on the above-described amino acid sequence. For example, it can be a conserved substitution of one or more amino acids in the above-described amino acid sequence, or the addition of one or more amino acids that do not affect its function (e.g., adding a linker peptide, protein tag sequence, etc.) to the N-terminus or C-terminus of the above-described amino acid sequence.

[0052] In some implementations, the protein tag sequence includes, but is not limited to, HA tag, Myc tag, His tag, GST tag, MBP tag, etc.

[0053] In some implementations, the linker can be a flexible linker peptide rich in GS. For example, the linker can be GGGSGGGSGGGS.

[0054] In some embodiments, the amino acids of the fusion protein can be derived sequences obtained by one or more modifications based on the above-mentioned amino acid sequence. For example, these modifications may include phosphorylation, PEGylation, amidation, glycosylation, biotinylation, and coupling or fusion with antibodies, vectors, ligands, albumin, Fc fragments, etc.

[0055] A second aspect of the present invention provides an isolated or synthesized nucleic acid molecule that encodes the fusion protein of the present invention.

[0056] Based on the amino acid sequence and codon rules of the fusion protein provided by this invention, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned fusion protein. Due to the degeneracy of codons, the nucleotide sequence encoding a single amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the above-mentioned fusion protein are within the protection scope of this invention.

[0057] In some implementations, the sequence of the nucleic acid molecule is shown in SEQ ID NO.6.

[0058] SEQ ID NO.6:

[0059]

[0060] In some implementations, nucleic acid molecules include DNA or RNA.

[0061] The present invention also provides a carrier comprising the nucleic acid molecule of the present invention.

[0062] In some implementations, the vector is an expression vector, which may be a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

[0063] In some embodiments, the viral vector includes, but is not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Preferably, the vector can transfer the nucleic acid molecules of the present invention into cells, such as T cells.

[0064] The present invention also provides a host cell comprising the nucleic acid molecule or vector of the present invention.

[0065] In some implementations, the host cell is a mammalian cell. For example, the host cell is a human cell. However, the host cell can be any cell type, can originate from any type of tissue, and can be a cell at any developmental stage.

[0066] In some implementations, methods for transfecting host cells include PEI transfection, liposome-mediated transfection (lipofectamin 2000 transfection, lipofectamin 3000 transfection, other liposome transfections), electroporation, or other suitable transfection methods.

[0067] The present invention also provides the use of the above-mentioned fusion protein in the preparation of a kit for detecting anti-AMPAR1 antibodies.

[0068] In some embodiments, the kit includes the fusion protein of the present invention linked to a detectable marker.

[0069] In some implementations, the kit includes additional antigens or a second antibody.

[0070] In some implementations, the kit also includes a blocking solution and a working concentrate.

[0071] In some implementations, the blocking solution is goat serum and / or fetal bovine serum.

[0072] In some implementations, an additional antigen or secondary antibody is attached with a detectable marker.

[0073] In some implementations, the detectable marker is selected from luciferin or enzyme.

[0074] In some implementations, the second antibody is selected from one or more of the following: goat anti-human antibody, rat anti-human antibody, mouse anti-human antibody, pig anti-human antibody, donkey anti-human antibody, sheep anti-human antibody, chicken anti-human antibody, horse anti-human antibody, rabbit anti-human antibody, hamster anti-human antibody, dog anti-human antibody, or bovine anti-human antibody.

[0075] In some embodiments, the fluorescein is selected from one or more of fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, Texas Red fluorescent dye, phycoerythrin, propidium iodide, Alexa Fluor series fluorescent dyes, Dylight series fluorescent dyes, or iFluor series fluorescent dyes.

[0076] In some implementations, the working concentrate is selected from one or more of PBS, Triton 100, and Tween 20.

[0077] The present invention also provides the use of the above-mentioned fusion protein in screening anti-AMPAR1 antibodies.

[0078] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0079] Example 1: Construction and transfection of GluA1 truncated mutant plasmid

[0080] Step 1: Synthesis of the full-length GluA1 gene

[0081] The GluA1 gene plasmid synthesized by General Electric.

[0082] GluA1 was synthesized into 906aa cells according to the Uniprot: P42261 sequence. An HA tag was added to the C-terminus of the pEGFP-N1 vector, and the GFP tag at the C-terminus was removed using NheI and NotI restriction enzymes. After successful sequencing, the plasmid was extracted for subsequent transfection.

[0083] Step 2: Construction of the GluA1 truncated mutant plasmid

[0084] By selecting the ATD and LBD domains, a series of different truncated mutant plasmids were designed, as shown in Table 1.

[0085] Table 1: Plasmids of different truncated mutants of GluA1

[0086]

[0087] Among them, the specific amino acid sequences of each mutant are as follows.

[0088] SEQ ID NO.7:

[0089] ANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTIL

[0090] SEQ ID NO.8:

[0091] QEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILED

[0092] SEQ ID NO.9:

[0093] EDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFS

[0094] SEQ ID NO.10:

[0095] EDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSGGGSGGGSGGGSAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDK

[0096] SEQ ID NO.11:

[0097] ANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFS

[0098] SEQ ID NO.12:

[0099] ANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSGGGSGGGSGGGSAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDK

[0100] SEQ ID NO.13:

[0101] QEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIIDHYKWQKFVYIYDADRGLSVLQ KVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDW KRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQN RTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFS

[0102] Subcloning was performed on the truncated mutant GluA1 gene from Table 1. The κ-chain signal peptide was used to replace the GluA1 signal peptide, and a transmembrane region of PDGFRβ was added to the C-terminus of GluA1. The restriction enzyme sites were SmaI and SalI. The inserted truncated mutant GluA1 gene had a Myc tag at the N-terminus and an HA tag at the C-terminus. After successful sequencing, plasmids were extracted for subsequent transfection.

[0103] Step 3: Cell transfection of GluA1 truncated mutant plasmid

[0104] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0105] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-60%.

[0106] The GluA1 wild-type, truncated mutant expression plasmids, and pMcherry-N1 (empty vector control) plasmids were mixed with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortexed, and allowed to stand for 10 min before being transfected into the prepared cells. The cells were then cultured at 37°C and 5% CO2 for 48 h.

[0107] Step 4: Observe fluorescence expression under a microscope

[0108] 24 hours after cell transfection, cells were fixed with 1% PFA for 5 minutes, washed three times with PBS, and the fixed cells were then used to verify expression using anti-HA monoclonal antibody. The results are as follows. Figure 1 As shown.

[0109] from Figure 1 As can be seen, the plasmids of each mutant can be successfully expressed, and the expression effect signal is strong.

[0110] Example 2: Antigenicity verification of GluA1 truncated mutant

[0111] Step 1: Transfect cells with GluA1 truncated mutant plasmid:

[0112] The cultured 293T cells were digested with trypsin and the digestion was terminated with DMEM complete medium containing 10% serum. The digested cells were transferred to centrifuge tubes and centrifuged at 800 to 1000 rpm for 3 min. The supernatant was discarded and DMEM complete medium containing 10% serum was added. The cells were gently mixed by pipetting to prepare a cell suspension.

[0113] Place the autoclaved glass slides into a cell culture dish, then treat with poly-L-lysine (PDL). After the slides have dried, add the prepared cell suspension to the dish and mix gently. Incubate overnight at 37°C in a 5% CO2 incubator. Observe the cells the next day; transfection is indicated when the cell density reaches 40-60%.

[0114] Mutant1-8 and GluA1 WT plasmids, and pMcherry-N1 (empty vector control) plasmid were mixed with PEI transfection reagent at a mass-to-volume ratio of 1:2, vortexed, and incubated for 10 min before being transfected into prepared cells. Cells were then cultured at 37°C and 5% CO2 for 48 h. 24 to 48 h after transfection, cells were fixed with 1% PFA for 5 min, washed three times with PBS, and used to prepare the assay cell matrix for later use.

[0115] Step 2: Screening of clinical samples

[0116] Five GluA1-positive samples and ten GluA1-negative samples were retrieved from the sample bank of the Neuroimmunology Center of Xuanwu Hospital. These samples were detected using cell matrix expressing GluA1 WT. Cells of the newly identified GluA1 truncated mutants (Mutant 1-8) were then re-screened. Blood samples were diluted 1:10 (cerebrospinal fluid samples were used directly), and cell slides were incubated for 0.5 to 1 hour. Cells were washed three times with PBS for 2 minutes each time. Cells were then incubated with the fluorescently labeled secondary antibody Alexa Fluor 488 (Thermofisher) for 30 minutes, followed by three washes with PBS for 2 minutes each time. The results were observed and photographed under a microscope. The detection results for each truncated mutant are shown in Table 2.

[0117] Table 2: Detection results of each truncated mutant plasmid

[0118]

[0119] As shown in Table 2, the detection results indicate that both the ATD and LBD domains contribute to the antigenicity of GluA1. Among the mutants, Mutant 6 and 7 showed the best performance, with all 5 positive samples being detectable.

[0120] Between Mutant 6 and Mutant 7, Mutant 6 has a higher signal intensity, which can more effectively detect low-titer samples and thus improve the detection sensitivity. Therefore, Mutant 6 was chosen for subsequent experiments.

[0121] The immunofluorescence results of Mutant 6 cells were compared with those of wild-type cells, and the results are as follows: Figure 2 As shown, based on the fluorescence signal, Mutant 6 performs better overall than wild-type full-length GluA1. Figure 2 The results showed that the truncated GluA1 protein can completely replace the full-length membrane protein, and the positive signal detected was stronger than that of the full-length protein. This indicates that the truncated protein is more advantageous in expression or membrane application, and can bind better to antibodies in positive samples, thereby producing a stronger fluorescence signal.

[0122] Example 3: Obtaining the Neoantigen GluA1

[0123] The Mutant 6 gene sequence from Example 2 was subcloned into the eukaryotic expression vector pCDNA3.1, using the κ chain signal peptide instead of its own signal peptide, with a His tag at the C-terminus. After successful sequencing, the plasmid was extracted for subsequent transfection.

[0124] The specific transfection method is as follows:

[0125] Expi29 cells were cultured to a density of 2.5 to 3.0 × 10⁻⁶. 6Cells / mL, ready for transfection;

[0126] Add the Mutant 6 expression plasmid to 3 mL of OptiMEM and mix well;

[0127] Add 120µL of PEI MAX 40K (1mg / mL) to the above OptiMEM and mix well. Let stand at room temperature for 10 to 15 minutes.

[0128] Add the well-mixed plasmid and PEI mixture to 30 mL of Expi293 cells and transfect by shaking culture at 37°C.

[0129] 24 hours after transfection, VPA and glucose solution were added to final concentrations of 1 mM and 2 g / L, respectively.

[0130] Continue shaking culture for 4 days, then harvest the culture.

[0131] The purification method for Mutant 6 protein is as follows:

[0132] Centrifuge the collected Expi293 cell culture at 15000 rpm and 4°C for 10 minutes, and collect the supernatant to remove dead cells and cell debris.

[0133] Add the appropriate volume of Protein A beads to the supernatant according to the instructions for use of Protein A beads, and rotate the supernatant and Protein A beads to combine and incubate.

[0134] After incubation for 1 hour, rinse Protein A beads with Wash buffer 3-5 times to remove impurities that are weakly bound to beads or antibodies.

[0135] The antibodies bound to Protein A beads were then eluted with acidic glycine buffer (pH 3.0-3.5), and the eluent was then neutralized with 1 M Tris-HCl (pH 9.0) at a volume equal to 1 / 10 of the acidic glycine buffer.

[0136] After Protein A affinity chromatography, the eluent was concentrated to a volume of about 0.5 mL using an Amicon Ultra 4 mL ultrafiltration tube. Then, a second molecular sieve chromatography step was performed using a BioCore SEC-300 column. The protein peak flow-through was collected and concentrated by ultrafiltration to obtain about 1 mL of Mutant 6 protein.

[0137] Samples of Mutant 6 protein (3 μg each) were prepared by adding SDS-loading buffer with and without β-ME. Samples were then electrophoresed using a SurePAGE™ Bis-Tris SDS-PAGE 4-12% precast gel at 100V for 1 hour. After removing the gel, the samples were stained with Coomassie Brilliant Blue for 20 minutes. Mutant 6 protein bands were then visible. Results are shown below. Figure 3 As shown.

[0138] Mutant 6 protein contains 637 amino acids and has a protein size of 56 kDa. Figure 3 As can be seen, the electrophoresis results are consistent with the expected size.

[0139] Example 4: Mutant 6 antigen used for membrane strip detection

[0140] To expand the application scope of the novel GluA1 antigen, we further used the Mutant 6 recombinant protein, which can be used for CBA detection, for membrane strip preparation.

[0141] First, the recombinant antigen protein was diluted to an appropriate concentration using the corresponding buffer solution, and then spotted onto the front side of a nitrocellulose (NC) membrane along with the positive control band using a Biodot-AD 3220 membrane sprayer. The spotted NC membrane was then sealed for several hours and thoroughly dried at 37°C. Finally, the membrane was cut into 2.5 mm wide strips using a Goldlabel Biotech ZQ3500 CNC high-speed cutter for subsequent testing.

[0142] The testing process is as follows:

[0143] Add 1 mL of dilution buffer to the incubation tank to ensure complete coverage of the membrane strip, and make sure the NC membrane is facing upwards during incubation;

[0144] Add 5µL of positive control material or sample to each reaction vessel, with a final dilution ratio of 1:200;

[0145] Mix carefully, then incubate on a shaker at room temperature (18-25°C) for 30 minutes.

[0146] Wash with washing buffer: Carefully aspirate or gently pour out the liquid from the incubator using a pipette, then add 1 mL of washing buffer and vortex for 30 seconds. Repeat the above steps 5 times;

[0147] Add 1 mL of enzyme conjugate to each incubation tank;

[0148] Incubate on a shaker for 30 minutes at room temperature;

[0149] Wash with washing buffer: Carefully aspirate or gently pour out the liquid from the incubator using a pipette, then add 1 mL of washing buffer and vortex for 30 seconds. Repeat the above steps 5 times;

[0150] Add 1 mL of substrate solution to each incubation tank;

[0151] Incubate at room temperature for 15 to 20 minutes until the bands on the membrane strip are clearly visible, using a positive control membrane strip as a reference.

[0152] Terminate the reaction with deionized water: Carefully aspirate or gently pour out the liquid from the incubation tank using a pipette, then add 1 mL of deionized water to each incubation tank and shake for 30 seconds. Repeat the above steps 5 times.

[0153] Place the membrane strip on absorbent paper and allow it to dry completely before reading the results.

[0154] In this experiment, 4 positive samples and 5 negative samples (different from the samples in Example 2, but also from the sample bank of the Neuroimmunology Center of Xuanwu Hospital) were selected for verification. The results are as follows: Figure 4 As shown. From Figure 4 As can be seen, the membrane strip method based on Mutant6 can accurately detect all four CBA-positive serum samples, while producing no non-specific signals for the five healthy control serum samples, indicating perfect detection accuracy (100% sensitivity and specificity). The detection results of the membrane strip method and the CBA method show a high degree of consistency. This successfully verifies that the truncated GluA1 protein can effectively replace the full-length membrane protein in autoantibody detection.

[0155] Therefore, the truncated GluA1 protein has solved the technical bottleneck of the difficulty in expressing full-length membrane proteins, laying a solid foundation for the development of stable and reliable membrane strip detection kits.

[0156] In summary, this invention significantly expands the detection technology pathway for anti-AMPA1 receptor antibodies, breaks through the technical barriers of existing methods, and provides diversified and high-performance solutions for testing institutions of different levels and types, greatly improving the application accessibility and industry standards of this detection project.

[0157] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A fusion protein for detecting anti-AMPAR1 antibodies, characterized in that, The fusion protein consists of an extracellular N-terminal domain, a linker, and an extracellular ligand-binding domain from the N-terminus to the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. A nucleic acid molecule that is isolated or synthesized, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

6.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2 or 3.

5. The carrier according to claim 4, characterized in that, The vector is an expression vector, which is a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, virus, or viral particle.

6. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 2 or 3 or the vector of claim 4 or 5.

7. Use of the fusion protein of claim 1 in the preparation of a kit for detecting anti-AMPAR1 antibodies.

8. Use of the fusion protein of claim 1 in the preparation of a kit for diagnosing autoimmune encephalitis.

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