Antigen for detecting ryr1 antibody of myasthenia gravis based on cell method and application thereof

Abstract

This invention discloses an antigen for detecting RYR1 antibodies in myasthenia gravis based on a cell-based method and its application, belonging to the field of immunoassay in biotechnology. The antigen is designed by truncating specific functional domains (SPRY1 and / or SPRY3) in the RYR1 gene, overcoming the difficulty of expressing the long sequence of the complete RYR1 gene, while preserving the RYR1 structure and antigenic epitopes. It can be used for the diagnosis of myasthenia gravis (MG) and provides an efficient and reliable detection scheme for RYR1 autoantibody detection.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay in biotechnology, specifically relating to an antigen for detecting RYR1 antibody in myasthenia gravis based on a cell method and its application. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies, resulting in neuromuscular junction dysfunction. Its clinical features include fluctuating weakness and pathological fatigue. Studies have found that the pathogenesis of MG is closely related to the production of autoantibodies, with acetylcholine receptor antibodies being the most common pathogenic antibody. In addition, rynoceridine receptor (RYR) antibodies are also a contributing factor.

[0003] RyR receptors (RYRs) are calcium ion channels in the terminal cisternaechalimum of skeletal muscle. Antibodies against RYRs can block calcium ion release, leading to skeletal muscle inability to contract and resulting in muscle weakness. Clinically, MG patients with positive RYR antibodies usually have more severe conditions, such as significant bulbar palsy symptoms, generalized muscle weakness, and even requiring assisted ventilation to treat respiratory muscle weakness. RyR-Ab positive MG is mostly late-onset, and antibody titer levels are closely related to disease severity. In addition, RyR-Ab is highly correlated with thymoma and can serve as a marker of disease severity and thymoma. In terms of treatment, calmodulin inhibitors such as tacrolimus can enhance RyR-mediated calcium release from the sarcoplasmic reticulum and may be the first-line drug for RyR-Ab patients. In conclusion, accurate and efficient detection of RYR autoantibodies is of great significance for the diagnosis and treatment of MG.

[0004] There are three RYR types in mammals (RYR1, RYR2, and RYR3). RYR1 is mainly expressed in skeletal muscle, while RYR2 and RYR3 are mainly found in cardiac muscle and the brain, respectively. Therefore, RYR1 is a preferred detection target for patients with myasthenia gravis. The RYR1 gene consists of more than 15,000 nucleotides, encoding a 564 kDa protein. Given the large protein structure of RYR1, finding a good way to express the full-sequence RYR1 protein as a detection substrate presents a significant challenge.

[0005] Based on previous literature reports, the antigenic epitopes of RYR1 are widely distributed. The detection results of Takamori et al. showed that amino acid residues 4997-5017 at the C-terminus of RYR1 are its major antigenic epitopes, while 1191-1216 at the N-terminus are non-major antigenic epitopes (Takamori, M. et al. (2004). Anti-ryanodine receptor antibodies and FK506 in myasthenia gravis. Neurology, 62(10), 1894-1896.). Mygland et al.'s research showed that amino acid residues 4929-4949 at the C-terminus of cardiomyocytes are the main antigenic epitopes (Mygland, Å., et al. (1994). Anti-cardiac ryanodine receptor antibodies in thymoma-associated myasthenia gravis. Autoimmunity, 17(4), 327-331.). Conversely, the results of Skeie et al. show that the antigenic epitopes of RYR1 are now mainly the N-terminal amino acid residues 799-1172 and 2591-2939 (Skeie, GO, et al. (2003). Ryanodine receptor antibodies in myasthenia gravis: epitope mapping and effect on calcium release in vitro. Muscle & Nerve: Official Journal of the American Association of Electrodiagnostic Medicine, 27(1), 81-89.).According to literature reports, the RYR1 protein structure can be divided into NTD domain, SPRY domain, Handle domain, HD domain, Central domain, and Channel domain (Bauerová-Hlinková, V., et al. (2020). Structure and function of the human ryanodine receptors and their association with myopathies—present state, challenges, and perspectives. Molecules, 25(18), 4040.). Based on the research of Takamori, Mygland, and Skie, it is clear that important antigenic epitopes of RYR may exist in the SPRY domain, HD domain, and Channel domain. However, the specific RYR1 autoantigen fragments are still unclear. Therefore, screening for and identifying effective RYR1 autoantigen fragments is very urgent.

[0006] In addition, existing methods for detecting RYR1 antibodies include enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). However, both of these methodologies require the extraction of RYR1 antigen, a process that may alter the antigen conformation and thus affect the detection results. For example, the results of Skeie et al. showed that WB detection of crude extracts of skeletal muscle sarcoplasmic reticulum proteins caused some RYR1-positive samples to lose their immune response (Skeie, GO, et al. (2003). Ryanodine receptor antibodies in myasthenia gravis: epitope mapping and effect on calcium release in vitro. Muscle & Nerve: Official Journal of the American Association of Electrodiagnostic Medicine, 27(1), 81-89.). Furthermore, the protein raw materials for ELISA and Western blotting are derived from animal skeletal muscle cells, and the isolation and homogenization of the proteins also present considerable challenges.

[0007] In contrast, cell-based assays (CBA) allow RYR1 to be expressed in a native, highly clustered state, preserving its true conformation to the greatest extent. Therefore, it is essential to develop a cell-based method for detecting RYR1 autoantibodies. This method can not only reveal the antigenic epitope distribution of RYR1 autoantibodies, but also improve the detection sensitivity and specificity of RYR1 autoantibodies.

[0008] Chinese patent CN116143897B reports two effective antigens for detecting anti-RyR1 autoantibodies: MGTR1 and MGTR2. MGTR1 is a fusion protein composed of the N-terminal and middle sequences of RyR1, and MGTR2 is a fusion protein composed of the N-terminal, middle, and C-terminal sequences of RyR1. By transfecting cell lines with expression plasmids carrying the MGTR1 and MGTR2 genes, cell-based detection materials are prepared, which can be applied to the CBA method to efficiently detect anti-RyR1 antibodies in patients with thymoma MG, with a positive detection rate of 50%. Summary of the Invention

[0009] The purpose of this invention is to provide an antigen for detecting RYR1 antibodies against myasthenia gravis using a cell-based method. This antigen is designed by truncating specific functional domains (SPRY1 and / or SPRY3) of the RYR1 gene, overcoming the difficulties of expressing the long sequence of the complete RYR1 gene while preserving the RYR1 structure and antigenic epitopes. To this end, this invention also provides related applications of the antigen, such as its use in preparing reagents or kits for detecting RYR1 antibodies against myasthenia gravis, and further, cell-based detection reagents or kits.

[0010] This invention is achieved through the following technical solution: an antigen for detecting RYR1 antibodies against myasthenia gravis using a cell-based method, wherein the antigen is selected from one of the following groups:

[0011] (A) SPRY1 sequence, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0012] (B) SPRY3 sequence, the amino acid sequence of which is shown in SEQ ID NO: 3;

[0013] (C) A mixture of the SPRY1 sequence and the SPRY3 sequence.

[0014] This invention also includes the following applications:

[0015] The above-mentioned antigen is used in the preparation of reagents or kits for detecting RYR1 antibodies against myasthenia gravis, wherein the reagents or kits are cell-based detection reagents or kits.

[0016] The cell-based detection reagent is a cell line that overexpresses the antigen, obtained using gene transfection technology.

[0017] Preferably, the cell line is obtained by introducing a nucleic acid encoding the antigen into a host cell for expression, wherein the nucleic acid is selected from one of the following groups:

[0018] (a) A nucleotide sequence as shown in SEQ ID NO: 1, or its complementary sequence, or a nucleotide sequence having at least 80% homology with it;

[0019] (b) A nucleotide sequence as shown in SEQ ID NO: 3, or its complementary sequence, or a nucleotide sequence having at least 80% homology with it;

[0020] (c) A combination of the nucleic acids described in (a) and (b).

[0021] Preferably, the host cell is HEK293 cell, CHO cell or its derived cell line.

[0022] Specifically, for example: a detection reagent for detecting RYR1 antibodies against myasthenia gravis based on a cell method, wherein the detection reagent is a cell line overexpressing the antigen obtained by gene transfection technology.

[0023] For example, a detection kit for detecting RYR1 antibodies against myasthenia gravis based on a cell method, wherein the detection kit contains the antigen or contains a cell line overexpressing the antigen.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) This invention screens out a novel antigen fragment based on the RYR1 functional domain for the first time. This antigen fragment is designed and constructed by truncating the SPRY1 and / or SPRY3 sequences, which greatly reduces the molecular weight of the protein and overcomes the problem of the difficulty in expressing the large full-length RYR1 protein in vitro.

[0026] (2) The antigen fragment of the present invention can retain the correctness of the RYR1 antigen structure and its antigen epitopes to the maximum extent, thereby greatly improving the probability of capturing RYR1 autoantibodies in patient serum and the detection sensitivity. Compared with using a single small linear peptide, it can better simulate the natural conformation epitope and effectively avoid the risk of missed detection due to epitope selection bias.

[0027] (3) By transfecting the antigen-encoding gene into cells such as HEK-293, this invention can obtain high-level antigen-expressing cells with membrane surface expression. These cells retain natural conformational epitopes and can be directly used as a cell-based assay (CBA) reagent for the diagnosis of myasthenia gravis (MG), providing an efficient and reliable detection scheme for RYR1 autoantibody detection.

[0028] (4) This invention provides a standardized antigen for detection such as immunoblotting (a recombinant protein purified from cells that overexpress the antigen, which has high purity and high immunoreactivity, and is used as a standardized antigen). It can be widely used in linear epitope detection platforms such as Western Blot and Dot Blot, overcoming the disadvantages of the cumbersome process, low yield and large batch-to-batch differences in extracting RYR1 protein from natural tissues, and realizing the standardization and high-throughput production of detection reagents. Attached Figure Description

[0029] Figure 1 The results are obtained from the detection of normal human serum in HEK-293 cell lines that overexpress different RYR1 antigen fragments.

[0030] Figure 2 The results show the detection of serum (RYR titer 1:32) from patients with thymoma in HEK-293 cell lines overexpressing different RYR1 antigen fragments.

[0031] Figure 3 The results show the detection of serum (RYR titer 1:320) from patients with thymoma in HEK-293 cell lines overexpressing different RYR1 antigen fragments.

[0032] Figure 4 The results of CBA detection were obtained from serum samples (RYR titer 1:32) from patients with thymoma, using a cell line containing an equal proportion of SPRY1 and SPRY3 plasmids. Detailed Implementation

[0033] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0034] 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.

[0035] The complete RYR1 gene consists of over 15,000 nucleotides, encoding a large protein that presents challenges for in vitro expression of the full-length RYR1 protein. Furthermore, the RYR1 antigenic epitopes are not yet fully elucidated, making the construction of overexpression plasmids containing the correct epitopes uncertain. To overcome these technical bottlenecks, this invention proposes extracting an effective self-antigen fragment from the complete gene based on the reported RYR1 protein structure, and designing truncated variants targeting each functional domain of this antigenic fragment. This method avoids the difficulties of expressing the full-length protein while preserving the structural integrity of RYR1 and the effectiveness of its antigenic epitopes.

[0036] According to literature reports, the RYR1 protein structure can be divided into multiple functional domains, including the NTD domain, SPRY domain, Handle domain, HD domain, Central domain, and Channel domain. Based on the research of Takamori, Mygland, and Skie, important antigenic epitopes of RYR may reside in the SPRY domain, HD domain, and Channel domain. To preserve the native conformation and antigenic epitopes of RYR1, this invention, for the first time, designs expression vectors for different gene fragments targeting multiple different domains of RYR1, including the SPRY domain (SPRY1: 642-1060 aa; SPRY2: 1050-1360 aa; SPRY3: 1350-1700 aa), the HD domain (HD1: 2115-2401 aa; HD2: 2391-2824 aa, HD3: 2814-3249 aa, HD4: 3240-3698 aa), and the Channel domain (Channel: 4231-5038 aa). Details are as follows:

[0037] The SPRY1 sequence (642-1060aa) has the amino acid sequence shown in SEQ ID NO: 1;

[0038] The SPRY2 sequence (1050-1360aa) has the amino acid sequence shown in SEQ ID NO: 2;

[0039] The SPRY3 sequence (1350-1700 aa) has the amino acid sequence shown in SEQ ID NO: 3;

[0040] The HD1 sequence (2115-2401aa) has the amino acid sequence shown in SEQ ID NO: 4;

[0041] The HD2 sequence (2391-2824aa) has the amino acid sequence shown in SEQ ID NO: 5;

[0042] The HD3 sequence (2814-3249aa) has the amino acid sequence shown in SEQ ID NO: 6;

[0043] The HD4 sequence (3240-3698aa) has the amino acid sequence shown in SEQ ID NO: 7;

[0044] The Channel sequence (4231-5038aa) has the amino acid sequence shown in SEQ ID NO: 8.

[0045] The nucleic acids encoding the SPRY1, SPRY2, SPRY3, HD1, HD2, HD3, HD4, and Channel sequences are as follows:

[0046] The nucleotide sequence encoding SPRY1 (642-1060aa) is shown in SEQ ID NO: 9;

[0047] The nucleotide sequence encoding SPRY2 (1050-1360aa) is shown in SEQ ID NO: 10;

[0048] The nucleotide sequence encoding SPRY3 (1350-1700aa) is shown in SEQ ID NO: 11;

[0049] The nucleotide sequence encoding HD1 (2115-2401aa) is shown in SEQ ID NO: 12;

[0050] The nucleotide sequence encoding HD2 (2391-2824aa) is shown in SEQ ID NO: 13;

[0051] The nucleotide sequence encoding HD3 (2814-3249aa) is shown in SEQ ID NO: 14;

[0052] The nucleotide sequence encoding HD4 (3240-3698aa) is shown in SEQ ID NO: 15;

[0053] The nucleotide sequence encoding Channel (4231-5038aa) is shown in SEQ ID NO: 16.

[0054] This invention specifically selects functional fragments containing RYR1 autoantigen epitopes and uses them as specific antigens for detecting RYR1 autoantibodies in cell substrate-based assays (CBA). These specific antigens are:

[0055] The SPRY1 sequence (642-1060aa) has the amino acid sequence shown in SEQ ID NO: 1;

[0056] Or the SPRY3 sequence (1350-1700aa), whose amino acid sequence is shown in SEQ ID NO: 3;

[0057] Or it could be a mixture of the SPRY1 and SPRY3 sequences.

[0058] Specifically, the amino acid sequences of the SPRY1 sequence (642-1060 aa) and SPRY3 sequence (1350-1700 aa) described in this invention comprise natural amino acid sequences, variants with high homology to the natural sequences (e.g., ≥80%, preferably ≥90%), and functional fragments containing their key antigenic epitopes. These sequences can be chemically modified as necessary (e.g., covalent modification or isotopic labeling, such as methylation, glycosylation, phosphorylation, acetylation, hydroxylation, etc.) to suit CBA detection, as long as the modification does not significantly affect its binding activity with the RYR1 antibody.

[0059] Furthermore, the present invention also provides the application of the above-mentioned antigen in the preparation of reagents or kits for detecting RYR1 antibodies against myasthenia gravis.

[0060] Specifically, the reagent or kit is a detection reagent or kit for use in cell-based assays (cell substrate assay, CBA assay), wherein the detection reagent can be a cell line overexpressing the antigen composition obtained by gene transfection technology.

[0061] For example, in one possible implementation, the cell line is obtained by introducing a nucleic acid encoding the antigen into a host cell for expression; the nucleic acid may be selected from:

[0062] (a) The nucleotide sequence encoding SPRY1 (632-1060aa) shown in SEQ ID NO: 9;

[0063] Or (b) the nucleotide sequence encoding SPRY3 (1351-1700aa) shown in SEQ ID NO: 11;

[0064] Or (c) a combination of the nucleic acids described in (a) and (b).

[0065] The nucleic acid can be its complementary sequence, or a nucleotide sequence that has at least 80% homology with it and can encode the antigen.

[0066] Optionally, in the gene transfection technology, the host cells used can be common protein-expressing cell lines, such as HEK293 cells, CHO cells, or their derivative cell lines; the expression vector used can be a commonly used protein expression vector, including pcDNA3.1(+), pCMV, or pENTER, more preferably pcDNA3.1(+); the transfection reagents used include lipo 3000 and PEI reagent, more preferably 25 kDa PEI reagent, and further, the transfection concentration is controlled at 500–4000 µg / mL, such as 700 µg / mL, 1000 µg / mL, 1500 µg / mL, 2000 µg / mL, 2500 µg / mL, 3500 µg / mL, or 3800 µg / mL, etc.

[0067] Furthermore, based on the above applications, this invention also provides a detection reagent for detecting RYR1 antibodies against myasthenia gravis using a cell-based method. This detection reagent is a cell line overexpressing the antigen composition, obtained using gene transfection technology. Secondly, a detection kit for detecting RYR1 antibodies against myasthenia gravis using a cell-based method is also provided. This detection kit contains the above-mentioned antigen composition, or contains a cell line overexpressing the above-mentioned antigen composition.

[0068] 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.

[0069] The experimental materials used in the following examples include: 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 secondary antibody (Thermo Fisher Scientific, catalog number A20000).

[0070] Example 1: Construction of an antigen overexpression cell line

[0071] (a) Obtaining the RYR1 gene fragment

[0072] (1) Linearize the pcDNA3.1(+) vector by XbaI single enzyme digestion;

[0073] (2) The myc-6×His tag sequence was obtained by amplifying the pcDNA6TR vector using primers MCS-Myc-F / BGH-CX-R;

[0074] (3) Homologous recombination of the vectors and fragments in (1) and (2) was performed using a One Step Cloning Kit to construct a new vector pCDNA3.1NS-mycHis;

[0075] (4) The pCDNA3.1NS-mycHis vector was digested with KpnI and NotI double enzymes;

[0076] (5) Using primers: 3.1NS-RYR23-F / 3.1NS-RYR3K-R; 3.1NS-RYR34-F / 3.1NS-RYR4K-R; 3.1NS-RYR45-F / 3.1NS-RYR5K-R; 3.1NS-RYRHD1-F / 3.1NS-RYRHD1-R; 3.1NS-RYRHD2-F / 3.1NS-RYRHD2-R; 3.1NS-RYRHD3-F / 3.1NS-RYRHD3-R; 3.1NS-RYRHD4-F / 3.1NS-RYRHD4-R and 3.1NS-RYRC-F / 3.1NS-RYRC-R, the human RYR1 gene was amplified to obtain the SPRY1 fragment, SPRY2 fragment, SPRY3 fragment, HD1 fragment, HD2 fragment, HD3 fragment, HD4 fragment, and Channel fragment;

[0077] (6) The vectors in (4) and (5) above are used to construct new vectors SPRY1, SPRY2, SPRY3, HD1, HD2, HD3, HD4 and Channel by homologous recombination with each fragment using a One Step Cloning Kit (see amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 8).

[0078] (II) Construction of HEK293 cell line overexpressing RYR1 fragment

[0079] (1) Soak the glass slide in 75% ethanol, clean it with PBS phosphate buffer, and treat it with laminin at 37°C for 30 min.

[0080] (2) HEK-293 cells were cultured at a concentration of 3.5 × 10⁻⁶. 5 Density seeding is applied to the glass slides prepared in the first step;

[0081] (3) After culturing for 24 hours, the vector plasmids (transfection concentration of 2000 µg / mL) of SPRY1, SPRY2, SPRY3, HD1, HD2, HD3, HD4, Channel and SPRY1, SPRY3 dual plasmids were mixed in equal proportions and transfected into the cells using 25 kDa PEI reagent.

[0082] (4) After 6 hours, change the medium to HEK-293 cell culture medium (DMEM medium containing 10% fetal bovine serum) and continue culturing for 36-48 hours before subsequent detection.

[0083] The primer sequences described in this embodiment are shown in Table 1 below.

[0084] Table 1 Primer sequences of Example 1

[0085]

[0086] Example 2: Application of antigen overexpression cell lines (cell-based detection reagents or kits)

[0087] This embodiment involves using the antigen-overexpressing cell line obtained in Example 1 as a detection reagent, or including it in a detection kit, to perform the following detection process:

[0088] (1) The antigen composition overexpressing cell line obtained 36-48 hours after transfection was fixed with acetone for 10 min;

[0089] (2) Collect serum from healthy individuals and patients, and dilute them at a ratio of 1:10 respectively;

[0090] (3) Mix the diluted serum with the cells treated in (1) and incubate at 37°C for 60 min;

[0091] (4) After incubation, rinse with PBS 3 times, 5 min each time;

[0092] (5) Incubate with the anti-human Alexa 488 secondary antibody and the incubation product in (4) at 37°C for 30 min;

[0093] (6) After incubation, rinse three times with PBS, each time for 5 minutes;

[0094] (7) Observe and photograph under a fluorescence microscope.

[0095] Using the antigen-overexpressing cell line obtained in Example 1 as a detection reagent, immunofluorescence detection was performed on serum samples from normal individuals, thymoma patients, and RYR-positive patients (see Example 2). Analysis of the detection results led to the following conclusions:

[0096] (a) Results of serum sample testing in normal individuals (see) Figure 1 )

[0097] Depend on Figure 1It is evident that during the detection of normal human serum, no green fluorescence was detected in SPRY1, SPRY2, SPRY3, HD1, HD2, HD3, HD4, and the Channel functional domain truncated variant, indicating that normal human bodies do not contain autoantibodies against RYR1.

[0098] (ii) Serum test results of patients with thymoma (see Figure 2 and Figure 3 )

[0099] Depend on Figure 2 As can be seen, serum tests in patients with thymoma and low RYR titers (1:32) showed that the SPRY1 truncated variant exhibited significant green fluorescence, while the SPRY3 truncated variant showed a weak green fluorescence signal, and the remaining truncated variants did not show any green fluorescence. This indicates that both the SPRY1 and SPRY3 truncated variants possess antigenic epitopes, and that SPRY1 has a higher number of antigenic epitopes and greater affinity for antibodies than SPRY3.

[0100] Depend on Figure 3 As can be seen, serum testing results from thymoma patients with high RYR titers (1:320) showed that the SPRY1 truncated fragment exhibited very strong green fluorescence, while the SPRY3 truncated fragment showed a weak green fluorescence signal, and the remaining truncated fragments showed no green fluorescence. This result further indicates that the SPRY1 and SPRY3 truncated fragments possess antigenic epitopes, and that SPRY1 has higher antigenicity and affinity than SPRY3, making it a better fragment for detection.

[0101] It should be noted that although the SPRY2 truncated variant contains partial linear sequences of SPRY1 and SPRY3, no fluorescent signal was observed. It is speculated that this is because the selection of the truncation site disrupts the spatial conformation of the protein, causing the original epitopes to be unable to form correctly or to be masked, thereby affecting antibody binding.

[0102] (III) Results of dual plasmid mixture detection (see) Figure 4 )

[0103] Depend on Figure 4 It is evident that serum test results from thymoma patients with a titer of 1:32 showed that transfected cell lines with an equal ratio of SPRY1 and SPRY3 plasmids could detect stronger green fluorescence than those with SPRY1 or SPRY3 alone, suggesting a possible synergistic effect that could improve detection sensitivity.

[0104] (iv) Sample positive detection rate

[0105] Immunofluorescence was performed on serum samples from 45 RYR-positive patients. The detection rates of truncations in each functional domain are shown in Table 2 below.

[0106] Table 2 Comparison of serum sample detection rates in RYR-positive patients

[0107]

[0108] As shown in Table 2, the detection rates of SPRY1 and SPRY3 were 60.00% and 55.56%, respectively, both higher than those of other functional domain fragments. When SPRY1 and SPRY3 were mixed, the detection rate was further increased to 82.22%. This result indicates that the mixing of two plasmids has higher detection sensitivity and comprehensive recognition ability.

[0109] In summary, the SPRY1 and SPRY3 truncated variants provided by this invention can serve as specific recognition targets for thymoma-associated autoantibodies. SPRY1 exhibits superior antigenicity, and the dual plasmid mixing strategy can enhance the detection signal, providing experimental basis for the establishment of subsequent autoantibody detection methods.

[0110] 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 polypeptide for use in detecting Ry RyRl antibodies, characterized in that: The polypeptide is selected from the group consisting of: (A) SPRY1, the amino acid sequence of which is shown as SEQ ID NO: 1; (B) SPRY3, the amino acid sequence of which is shown as SEQ ID NO: 3; (C) a mixture of the SPRY1 and the SPRY3.

2. Use of a polypeptide according to claim 1 for the manufacture of a reagent or kit for the detection of RYR1 antibodies in myasthenia gravis, characterized in that: The reagent or kit is a cell-based detection reagent or a detection kit.

3. Use according to claim 2, characterized in that: The cell-based detection reagent is a cell strain overexpressing the polypeptide obtained by gene transfection technology.

4. Use according to claim 3, characterized in that: The host cell of the cell strain is HEK293 cell, CHO cell or a cell line derived therefrom.

5. A detection reagent for detecting RyRl antibodies in myasthenia gravis based on a cell method, characterized by: The detection reagent is a cell strain overexpressing the polypeptide of claim 1 obtained by gene transfection technology.

6. A test kit for detecting RyRl antibodies in myasthenia gravis based on a cellular method, characterized by: The detection kit comprises the polypeptide of claim 1, or a cell strain overexpressing the polypeptide of claim 1.

Images