Recombinant expression vector for inducing expression of clustered acetylcholine receptor, double stable cell strain and application thereof

Abstract

The application relates to the technical field of autoimmune disease in vitro diagnosis, and particularly relates to a recombinant expression vector for inducing expression of clustered acetylcholine receptors, a double-stable cell strain and application thereof. The recombinant expression vector is a tetracycline-inducible expression vector for single-carrier multi-subunit independent expression; the vector is integrated with acetylcholine receptor full-subunit coding sequence and Rapsyn protein coding sequence through an iterative enzyme cutting-linking mode, wherein each to-be-inserted subunit independent expression box is respectively provided with I-CeuI enzyme cutting sites and BstXI enzyme cutting sites at two ends, and each subunit and Rapsyn protein are driven to express by an independent Tet-On inducible promoter after iterative linking. Through the core design of single-carrier multi-subunit independent expression and Tet-On tetracycline induction regulation, the acetylcholine receptor pentamer assembly efficiency, the stable cell strain passage stability and the anti-AChR antibody detection performance are greatly improved.

Description

Technical Field

[0001] This application relates to the technical field of in vitro diagnostics of autoimmune diseases, and in particular to a recombinant expression vector for inducible expression of clustered acetylcholine receptors, a bistable transgenic cell line, and their applications. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that affects postsynaptic signaling at the neuromuscular junction (NMJ). Its core pathogenesis is closely related to autoantibodies targeting the acetylcholine receptor (AChR). MG is characterized by a wide age range of onset, high disability rate, frequent relapses, and poor long-term prognosis. It can lead to progressive weakness of skeletal muscles, and in severe cases, respiratory muscle involvement can trigger a crisis, posing a serious threat to patients' life, health, and quality of life. It is one of the key diseases for prevention and control in the field of clinical neuroimmunology.

[0003] Anti-AChR autoantibodies are core serological markers for the diagnosis and assessment of myasthenia gravis (MG). Clinical data show that these antibodies are detectable in approximately 85% of patients with systemic MG and approximately 50% of patients with ocular MG. The detection results are a core basis for the clinical diagnosis, classification, treatment monitoring, and prognosis of MG. With increasing clinical diagnostic needs, AChR autoantibody detection technologies have undergone several generations of development. Currently, the mainstream technologies mainly include radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and cell substrate methods.

[0004] The first type is the radioimmunoprecipitation assay (RIPA), which is the traditional reference method for MG antibody detection. Its technical principle is: using a radioactive isotope (such as ¹²) 5I) Labeled α-bungarotoxin (α-BTX) specifically binds to AChR protein, forming an isotopically labeled AChR antigen. The patient's serum is co-incubated with the labeled antigen, allowing anti-AChR antibodies in the serum to bind to the antigen. Subsequently, anti-human IgG antibodies are added to complete immunoprecipitation. The quantitative detection of anti-AChR antibodies in the serum is achieved by detecting the radioactivity intensity of the precipitate. This method has the advantages of high sensitivity and specificity, and can achieve quantitative antibody detection, and was long considered the gold standard for clinical testing. However, this method has unavoidable inherent drawbacks: First, it requires the use of radioactive isotopes, placing extremely high demands on laboratory qualifications, radiation safety protection, and radioactive waste disposal, greatly limiting its widespread application in primary healthcare institutions. Second, the operation procedure is cumbersome and the testing cycle is long, requiring 2-3 days to obtain results, which cannot meet the needs of rapid clinical diagnosis. Third, it can only detect total anti-AChR IgG antibodies and cannot distinguish IgG subtypes, failing to provide more reference information for pathogenic mechanism analysis and precision treatment.

[0005] The second type is the Enzyme-Linked Immunosorbent Assay (ELISA). Its technical principle is as follows: recombinantly expressed or cell-extracted AChR antigen is coated onto the surface of a solid-phase carrier in a microplate. After adding the serum to be tested, the anti-AChR antibody in the serum specifically binds to the solidified antigen. After washing to remove unbound substances, enzyme-labeled anti-human IgG secondary antibody is added. After substrate color development, the antibody is qualitatively and quantitatively detected by absorbance values. This method does not require the use of radioactive materials, is simple to operate, has a short detection cycle, and is easily automated and suitable for large-scale sample testing, thus having a certain range of clinical applications. However, the core drawback of this method is that the AChR antigen coated on the solid-phase carrier is prone to conformational changes, failing to retain the complete conformational epitopes of the natural membrane protein. This results in lower detection sensitivity and specificity than the RIPA method, especially for low-titer samples, which are prone to false negative results. Furthermore, it has extremely high requirements for the purity and conformational integrity of the AChR antigen, and batch-to-batch stability of the reagents is difficult to control.

[0006] The third type is the cell-based assay (CBA), which is currently the most widely used and most advantageous next-generation technology in clinical practice. Its technical principle is as follows: through genetic engineering, mammalian host cells (usually HEK293 series cells) express human AChR protein, allowing AChR to be localized on the cell membrane surface in its native conformation. The test serum is incubated with the cells, allowing the anti-AChR antibody in the serum to bind to the native AChR conformation on the cell surface. Then, a fluorescently labeled anti-human IgG secondary antibody is used for signal labeling, and finally, qualitative and quantitative detection of the antibody is achieved using fluorescence microscopy or flow cytometry. This method can completely preserve the native transmembrane conformation and conformational epitopes of AChR, and its detection sensitivity is significantly better than RIPA and ELISA methods. In particular, it can detect low-titer samples that are missed by RIPA and ELISA methods, and is currently the main research direction for MG antibody detection technology.

[0007] However, current commercial applications and existing publicly available CBA detection methods generally suffer from large batch-to-batch variations in reagents, insufficient consistency in test results, and poor reagent stability. The root cause lies in the inherent defects in the construction technology of the core raw material—the stable AChR cell line—which cannot achieve stable, efficient, and uniform expression of the AChR protein. AChR is a pentamer transmembrane receptor protein assembled from two subunits: α1, β1, δ, ε (adult type) / γ (fetal type). Balanced expression of each subunit within the host cell is a core prerequisite for the correct assembly, membrane localization, and formation of the native conformation of the pentamer. Therefore, constructing a stable cell line that can stably and evenly express all AChR subunits is the core key to ensuring the stability and reliability of CBA detection reagents.

[0008] The conventional procedure for constructing stable AChR cell lines is as follows: using a self-cleaved 2A peptide sequence, the five subunits of AChR are split and constructed into two lentiviral vectors carrying different eukaryotic resistance selection markers; the two recombinant vectors are packaged into lentiviruses and co-infected with host cells; after selection by combined pressure with two antibiotics, single clones are picked using limiting dilution method to finally obtain positive cell lines that can constitutively express the AChR receptor.

[0009] However, the existing technical solutions mentioned above have two major technical defects, which directly result in the performance of stable cell lines failing to meet the industrial production requirements of clinical testing reagents:

[0010] First, uneven subunit expression leads to low pentamer assembly efficiency. Existing methods use 2A peptide sequences to achieve tandem expression of multiple genes, but the self-cleavage efficiency of 2A peptides has an upper limit, and the expression levels of genes downstream of the 2A peptide sequence are generally significantly lower than those upstream, making it impossible to achieve balanced expression of multiple AChR subunits. Furthermore, the correct assembly of AChR pentamers requires the simultaneous expression of four subunits at a fixed stoichiometric ratio. Uneven subunit expression directly leads to a significant decrease in pentamer assembly efficiency, resulting in low expression levels of functional AChRs on the cell membrane surface. Ultimately, this causes decreased detection sensitivity and large batch-to-batch variations in detection results.

[0011] Second, constitutive expression leads to high cytotoxicity and extremely poor passage stability. Existing technologies generally use constitutive promoters to drive the continuous expression of the AChR subunit. Host cells must continuously endure endoplasmic reticulum stress and cytotoxicity caused by high expression of exogenous membrane proteins, resulting in poor cell growth and low survival rates after cryopreservation and thawing. At the same time, the continuous pressure of exogenous gene expression can lead to the silencing or loss of exogenous genes in host cells during passage, causing AChR expression levels to decrease significantly with increasing passage number. This results in extremely poor passage stability of stable cell lines, making it impossible to achieve large-scale, standardized industrial production. This is the fundamental reason for the insufficient batch-to-batch stability of existing CBA detection reagents.

[0012] In summary, existing AChR stable cell line construction technologies suffer from core technical bottlenecks such as uneven subunit expression, low receptor assembly efficiency, high cytotoxicity, and poor passage and storage stability. These limitations fail to meet the industrial and standardized production requirements of MG anti-AChR antibody CBA detection reagents. Therefore, developing an expression system that can achieve balanced AChR subunit expression, efficient assembly, stability, controllability, and no significant cytotoxicity is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0013] This application provides a recombinant expression vector for inducible expression of clustered acetylcholine receptors, a bistable transgenic cell line, and their applications. Through a core design combining independent expression of multiple subunits in a single vector and Tet-On tetracycline-induced regulation, this application achieves efficient, balanced, and controllable expression of acetylcholine receptors, significantly improving receptor pentamer assembly efficiency, stable cell line passage stability, and anti-AChR antibody detection performance. Simultaneously, it simplifies the stable cell line construction process, making it suitable for industrial-scale production.

[0014] In a first aspect, this application provides a recombinant expression vector for inducibly expressing clustered acetylcholine receptors, employing the following technical solution:

[0015] A recombinant expression vector for inducible expression of clustered acetylcholine receptors, wherein the recombinant expression vector is a tetracycline-inducible expression vector with independent expression of multiple subunits of a single vector;

[0016] The vector is based on pACEtetO as its backbone, and its multiple cloning site region contains a BstXI restriction endonuclease single-enzyme cleavage site. The vector integrates the full subunit coding sequence of the acetylcholine receptor and the coding sequence of the Rapsyn protein through an iterative enzyme digestion-ligation method. Each subunit to be inserted has an independent expression cassette with an I-CeuI homing endonuclease site and a BstXI restriction endonuclease site at both ends. After iterative ligation, each subunit and the Rapsyn protein are expressed by an independent Tet-On inducible promoter, and the transcription and translation processes of each subunit do not interfere with each other.

[0017] The acetylcholine receptor subunits are the adult subunit combination CHRNA1, CHRNB1, CHRND, and CHRNE, or the fetal subunit combination CHRNA1, CHRNB1, CHRND, and CHRNG.

[0018] Optionally, the expression of the Tet-On inducible promoter is controlled by doxycycline. Without doxycycline induction, the target gene is silenced, and the target gene is efficiently expressed after the addition of doxycycline.

[0019] Optionally, the vector may further comprise a bleomycin resistance selection marker, a WPRE transcriptional enhancement element, an expression packaging element, and a prokaryotic amplification element.

[0020] Optionally, the specific method of the iterative digestion-ligation is as follows: the vector is linearized by single digestion with BstXI, the independent expression cassette of the subunit to be inserted is double digested with I-CeuI and BstXI, the vector and the inserted fragment are ligated by T4 DNA ligase, and a unique I-CeuI restriction site is re-formed at the ligation site; the above steps are repeated to integrate all subunits and independent expression cassettes of Rapsyn protein into the same vector in sequence.

[0021] Optionally, the recombinant expression vector is the adult-type plasmid pACEtetO-AChR-α1-β-δ-ε-rapsyn.

[0022] Optionally, the recombinant expression vector is the fetal plasmid pACEtetO-AChR-α1-β-δ-γ-rapsyn.

[0023] Optionally, the NCBI reference sequence accession number for CHRNA1 is NM_001039523.3.

[0024] Optionally, the NCBI reference sequence accession number of CHRNB1 is NM_000747.2.

[0025] Optionally, the NCBI reference sequence accession number of the CHRND is NM_000751.3.

[0026] Optionally, the NCBI reference sequence accession number of the CHRNE is NM_000080.4.

[0027] Optionally, the NCBI reference sequence accession number of the CHRNG is NM_005199.5.

[0028] Optionally, the NCBI reference sequence accession number for the gene RAPSN, which encodes the Rapsyn protein, is NM_005055.4.

[0029] Secondly, this application provides a bistable transgenic cell line that stably expresses clustered acetylcholine receptors, using the following technical solution:

[0030] A bistable transgenic cell line stably expressing clustered acetylcholine receptors, wherein the bistable transgenic cell line is a mammalian host cell co-expressing pcDNA6TR vector and the above-mentioned recombinant expression vector, and the bistable transgenic cell line is resistant to both blast fungicide and bleomycin, and expresses the native conformation of clustered acetylcholine receptors on the cell membrane surface by doxycycline induction.

[0031] Optionally, the mammalian host cell is a HEK293T cell, a HEK293 cell, or a CHO-K1 cell.

[0032] Optionally, the bistable transgenic cell line is the adult-type bistable transgenic cell line 6TR-α1-β-δ-ε-rapsyn.

[0033] Optionally, the bistable transgenic cell line is the fetal bistable transgenic cell line 6TR-α1-β-δ-γ-rapsyn.

[0034] Optionally, after induction with doxycycline at a final concentration of 1 μg / mL for 48 h, the bistable transgenic cell line forms high-density, patchy clusters of acetylcholine receptors on the cell membrane surface.

[0035] Thirdly, this application provides a method for constructing a bistable transgenic cell line, employing the following technical solution:

[0036] A method for constructing a bistable transgenic cell line, the method specifically comprising the following steps:

[0037] (1) Construction of stable pcDNA6TR cell line: The pcDNA6TR plasmid was transfected into host cells, and after selection by blast fungicide pressure and monoclonalization, a 6TR cell line stably expressing TetR tetracycline repressor protein was obtained.

[0038] (2) Transfection of recombinant expression vector and screening of bistable transgenic cells: After linearizing the above recombinant expression vector, the 6TR cell line obtained in step S1 was transfected and screened under pressure with blastomycin and bleomycin, and then monoclonalized to obtain bistable transgenic cell lines.

[0039] (3) Functional verification: The monoclonal cell line was induced to express and verified. The qualified standard was that the cell membrane surface could form a high-density cluster of acetylcholine receptors. The target bistable transgenic cell line was obtained by screening.

[0040] Optionally, the screening concentration of blast fungicide in step (1) is 7.5 μg / mL; the screening concentration of double resistance in step S2 is 7.5 μg / mL blast fungicide + 300 μg / mL bleomycin, and the concentration of bleomycin in monoclonal maintenance culture is halved to 150 μg / mL.

[0041] Optionally, in step (2), the recombinant expression vector is linearized with I-SceI restriction endonuclease and transfected using Lipofectamine 3000 reagent.

[0042] Optionally, in step (3), the function is verified by indirect immunofluorescence assay using fluorescently labeled α-cobra venom.

[0043] Fourthly, this application provides a detection kit for anti-acetylcholine receptor antibodies in myasthenia gravis, employing the following technical solution:

[0044] A kit for detecting anti-acetylcholine receptor antibodies in myasthenia gravis, the kit comprising the above-mentioned bistable transgenic cell line, or an antigen sheet prepared from the bistable transgenic cell line.

[0045] Optionally, the kit contains both adult bistable transgenic cell lines and fetal bistable transgenic cell lines.

[0046] Optionally, the kit may further include fluorescently labeled α-cobra venom, fluorescently labeled anti-human IgG secondary antibody, cell fixative, blocking solution, positive control serum, and negative control serum.

[0047] Optionally, the antigen sheet is a unit cell climbing sheet prepared by inducing expression, fixing, blocking, and cutting of bistable transgenic cell lines, and then pasting it onto a glass slide with reaction wells.

[0048] Fifthly, this application provides the use of the above-mentioned recombinant expression vector and the above-mentioned bistable transgenic cell line in the preparation of a detection reagent or kit for anti-acetylcholine receptor antibodies against myasthenia gravis.

[0049] In summary, this application includes at least one of the following beneficial technical effects:

[0050] (1) Completely solves the core problem of uneven subunit expression, and significantly improves pentamer assembly efficiency.

[0051] This application employs a single-vector iterative assembly design with each subunit driven by an independent promoter, completely avoiding the randomness of dual plasmid co-transfection and the problem of restricted downstream gene expression in 2A peptide tandem. This ensures that the gene copy number of each subunit in each cell is completely consistent and the expression level is highly balanced. The correct assembly efficiency of AChR pentamer is improved by more than 60% compared with the prior art, and the CV value of cell membrane surface receptor expression uniformity is less than 5%, completely solving the problem of large batch-to-batch differences in the prior art.

[0052] (2) Inducible expression completely eliminates cytotoxicity, and the stability of stable transfected cells achieves a qualitative breakthrough.

[0053] The Tet-On inducible expression system integrated in this application completely silences the target gene without an inducer, and the host cells are not under continuous exogenous protein expression pressure. The growth status is no different from wild-type cells, and the cryopreservation and thawing survival rate is ≥95%. After 20 consecutive passages, tests were conducted at passages 1, 5, and 20, and the receptor expression level and clustering efficiency did not decrease significantly. This completely solves the inherent defects of existing constitutive expression systems such as passage silencing, gene loss, and high cytotoxicity, and fully meets the needs of industrialized and standardized production of diagnostic reagents.

[0054] (3) The detection sensitivity and coverage have been significantly improved, and the clinical false negative rate has been greatly reduced.

[0055] The bistable transfected cell line of this application can stably express clustered AChRs in their native conformation and can detect weakly positive samples with titers as low as 1:10, which is more than 12% higher than the positive detection rate of existing transient transfection antigen slides. At the same time, the combined detection of adult and fetal subtypes can cover pathogenic antibodies against both subtypes of AChR, reducing the clinical false negative rate by more than 80% and achieving 100% detection specificity.

[0056] (4) The process of constructing stable transgenic lines has been greatly simplified and the preparation efficiency has been significantly improved.

[0057] The single-vector design of this application requires only two rounds of transfection and double-antibody screening to obtain the target stable transfected strain, eliminating the need for multiple rounds of multi-antibiotic screening. This shortens the experimental cycle by more than 50%, increases the positive clone yield by more than 3 times, and significantly reduces the cost and difficulty of large-scale preparation. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the vector used for the assembly and expression of multiple subunits of multimeric receptor-like proteins in this application.

[0059] Figure 2 A schematic diagram of the pACEtetO-AChR-α1-β-δ-γ-rapsyn plasmid constructed for this application.

[0060] Figure 3 A schematic diagram of the pACEtetO-AChR-α1-β-δ-ε-rapsyn plasmid constructed for this application.

[0061] Figure 4 This is a fluorescence image showing the detection results of different AChR antigen tablets on positive serum with different titers of anti-AChR antibodies in Example 4 of this application.

[0062] Figure 5 The image shows the fluorescence results of different titers of anti-AChR antibody-positive serum detected by 6TR-α1-β-δ-ε-rapsyn bistable transgenic cell lines of adult AChR-Rapsyn from different passages in Example 5 of this application. Detailed Implementation

[0063] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0064] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0065] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0066] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0067] This application provides a recombinant expression vector for inducible expression of clustered acetylcholine receptors, a bistable transgenic cell line, and their applications.

[0068] To address the core technical challenges of existing technologies, such as uneven AChR subunit expression, low pentamer assembly efficiency, cumbersome screening procedures for stable transfectants due to dual plasmid co-transfection, and high cytotoxicity, poor passage stability, and large batch-to-batch variability caused by constitutive expression systems, this application proposes the following targeted design for recombinant expression vectors:

[0069] On the one hand, this application selects the pACEtetO expression vector as the basic framework. This vector is an expression vector specifically designed for the assembly and expression of multiple subunits of multimeric receptor proteins. A schematic diagram of the vector is shown below. Figure 1 As shown, the vector's multiple cloning site region is designed with a BstXI restriction endonuclease single-restriction site. Each subunit to be inserted into an independent expression cassette contains both I-CeuI homing endonuclease and BstXI restriction endonuclease cleavage sites at both ends of the gene expression cassette (including the promoter, multiple cloning site, polyA tail, and other elements). This makes the vector suitable for the iterative assembly and expression of multiple subunits of multimeric proteins. The principle is as follows: the vector is linearized by BstXI single-restriction digestion; the insert fragment is then double-digested with I-CeuI and BstXI to obtain subunit-independent expression cassettes with sticky ends. After ligation of the vector and insert fragment with T4 DNA ligase, a unique I-CeuI restriction site is re-formed at the ligation point. Repeating the above digestion-ligation steps allows all subunits of the multimeric protein to be inserted into the same expression vector, with each subunit carrying an independent Tet-On inducible promoter, multiple cloning site, and polyA tail signal, forming independent expression units. The transcription and translation processes of each subunit do not interfere with each other.

[0070] The advantages of the above design are:

[0071] (1) It completely solves the inherent defects of existing multi-plasmid co-transfection technology: avoids the uncontrollability caused by traditional multi-plasmid co-transfection when expressing multimeric proteins. Its uncontrollability is usually due to the fact that co-transfection cannot solve the randomness of plasmid entry into the cell, resulting in low assembly efficiency of multimers in each cell and a significant reduction in expression level. Traditional multi-plasmid co-transfection has the randomness of plasmid entry into the cell and cannot guarantee that the gene copy number of each subunit in each cell is consistent, resulting in low assembly efficiency of AChR pentamer and large batch-to-batch differences. However, the single-vector multi-subunit independent expression design of this application does not require co-transfection. All subunits are integrated into the same genomic site of the same vector. The gene copy number of each subunit in each cell is completely consistent, and the expression level is highly balanced, which greatly improves the correct assembly efficiency and cell membrane surface localization efficiency of AChR pentamer, and enables the target receptor protein to be expressed in large quantities.

[0072] (2) The process of constructing stable transgenic cells is greatly simplified and the screening efficiency is improved: The design of this assembly expression vector improves the convenience of subsequent stable transgenic cell construction, avoids the complicated experimental process of screening positive clones with multiple antibiotics required by traditional multi-plasmid co-transformation, and only single antibiotic screening is required to obtain positive cells that integrate all target genes, shortening the experimental cycle and improving the efficiency of stable transgenic cell construction.

[0073] On the other hand, this application designs and adds a Tet-on tetracycline-induced expression system based on the above-mentioned vector. The principle of this system is that the expression of the target protein is strictly controlled by the Tet-on system. That is, the target protein is expressed in large quantities and efficiently when tetracycline or its derivative doxycycline (Dox, preferred in this application) is present, and the target protein is hardly expressed when no inducer is added.

[0074] The advantages of the above design are:

[0075] (1) It eliminates the dependence of target protein expression on high gene copy number: The protein expression level of traditional stable cells is limited by the final copy number of the target gene integrated into the chromosome, and often requires a cumbersome screening process to obtain clone cells with higher copy numbers. However, the target protein expression level of stable cells based on the Tet-on system can be precisely controlled by the concentration of the inducer and the induction time, which means that even clone cells with low gene copy numbers can obtain a large amount of target protein expression under the induction of the inducer, which greatly reduces the workload of screening single clone cells.

[0076] (2) Completely solves the cytotoxicity and stability defects of constitutive expression: Traditional constitutive expression stable cells usually face a series of problems such as loss of target gene, incorrect expression of target protein and cytotoxicity of target protein caused by cell cryopreservation, thawing and passage. These problems usually lead to unstable expression of clones or clone death due to protein toxicity. However, clones based on the Tet-on induction system have sufficient spatiotemporal controllability, completely avoid various cellular stresses caused by constitutive expression, and can start induction expression when the cells are in the best growth state, avoiding endoplasmic reticulum stress and cytotoxicity caused by continuous expression of exogenous membrane proteins to the greatest extent. This not only improves the passage stability of stable cells, cryopreservation and thawing survival rate and batch uniformity, but also obtains a higher level of target protein expression.

[0077] The combination of the above-mentioned single-vector multi-subunit independent expression design with the Tet-On inducible expression system can significantly improve the assembly efficiency of AChR pentamer while significantly enhancing the expression level, conformational integrity and passage stability of the receptor protein. These properties are the core quality control key of anti-AChR antibody detection reagents, and directly determine the sensitivity, specificity and batch stability of the detection reagents.

[0078] The constructed plasmid is pACEtetO-AChR-α1-β-δ-γ-rapsyn (fetal type, structural diagram shown below). Figure 2 ) or pACEtetO-AChR-α1-β-δ-ε-rapsyn (adult type, structural diagram as shown) Figure 3 ).

[0079] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0080] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0081] The present application will be further described in detail below with reference to the embodiments and test results.

[0082] Example 1

[0083] This embodiment provides a method for constructing a stable pcDNA6TR cell line (hereinafter referred to as 6TR cell line) that stably expresses tetracycline repressor protein (TetR).

[0084] The host cells used in this embodiment and subsequent embodiments were all HEK293T cells, purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee; the complete culture medium used was DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; all cell culture operations were performed in a cell culture incubator at 37°C and 5% CO2 saturated humidity.

[0085] Specifically, the following steps are included:

[0086] (a) Screening for the optimal concentration of antibiotics

[0087] Blastidin is a commonly used antibiotic for screening stable mammalian cell lines, with an effective screening concentration range of 1-50 μg / mL. In this example, an optimal screening concentration was determined through a concentration gradient preliminary experiment: HEK293T cells were seeded at the same density into 6-well plates, and blastidin was added at final concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, and 10 μg / mL, respectively. After 7 days of continuous culture, the lowest concentration that caused complete cell death was determined as the optimal screening concentration. The optimal screening concentration for this experiment was ultimately determined to be 7.5 μg / mL.

[0088] (ii) Transform into pcDNA6TR plasmid to construct a stable expression cell line.

[0089] (1) On day 1, HEK293T cells were seeded into two 10cm cell culture dishes at a passage ratio of 1:50. 10mL of complete culture medium was added to each dish. One culture dish was used for pcDNA6TR plasmid transfection, and the other culture dish served as a blank negative control without plasmid transfection.

[0090] (2) On the second day, when the cell confluence reaches 70%-80%, one culture dish is transfected with 12 μg pcDNA6TR plasmid using Lipofectamine 3000 transfection reagent, and the other culture dish is only added with the same amount of transfection reagent, without adding plasmid.

[0091] (3) On the third day, 24 hours after transfection, the selection medium containing blast fungicide was replaced in the two culture dishes. The final concentration of antibiotic was 7.5 μg / ml.

[0092] (4) Observe the cell status under a microscope every day and replace the screening medium with fresh medium every 2-3 days to ensure that all cells in the negative control culture dish without plasmid die within the first week.

[0093] (5) After 1-2 weeks, resistant monoclonal cells began to appear in the transfected culture dish. Use a cloning loop to separate 3-5 independent, morphologically uniform monoclonal cells and transfer each monoclonal cell to a single well of a 24-well plate for further culture.

[0094] (6) For monoclonal cells in 24-well plates, maintain the concentration of blast fungicide in the culture medium at 7.5 μg / ml and continue culturing. After the cells have grown to confluence, transfer them to 6-well plates for further culture and maintain the concentration of blast fungicide in the culture medium at 7.5 μg / ml.

[0095] (7) After the cell expansion culture is completed, the expression of tetR (tetracycline repressor protein) gene in the single clone cell line is detected. PCR, Western blot or qPCR can be used. The standard for passing the test is that the target band is obtained by PCR amplification and the specific TetR protein band can be detected by Western blot. After passing the test, the pcDNA6TR stable cell line is successfully constructed, referred to as the 6TR cell line.

[0096] Example 2

[0097] This embodiment provides a method for constructing a 6TR-α1-β-δ-γ-rapsyn bistable transgenic cell line (hereinafter referred to as the fetal bistable transgenic cell line) that stably expresses TetR and fetal AChR-Rapsyn.

[0098] Specifically, the following steps are included:

[0099] (a) Screening for the optimal concentration of antibiotics

[0100] Bleomycin (Zeocin) is a commonly used antibiotic for screening stable mammalian cell lines, with an effective screening concentration range of 50-1000 μg / mL. In this example, the optimal screening concentration was determined through a concentration gradient preliminary experiment: 6TR cells constructed in Example 1 were seeded at the same density into 6-well plates, and Zeocin was added at final concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, respectively. The cells were cultured continuously for 14 days, and the lowest concentration that caused all cells to die was determined to be the optimal screening concentration. The optimal screening concentration for this experiment was ultimately determined to be 300 μg / mL.

[0101] (ii) Transform the pACEtetO-AChR-α1-β-δ-γ-rapsyn recombinant plasmid to construct a stable expression cell line.

[0102] (1) On the first day, the 6TR cells obtained in Example 1 were seeded into two 10cm culture dishes at a passage ratio of 1:100, and 10mL of complete culture medium was added to each dish.

[0103] (2) On the second day, when the cell confluence reached 70%-80%, one culture dish was transfected with 12 μg of pACEtetO-AChR-α1-β-δ-γ-rapsyn plasmid that had been linearized with I-SceI restriction endonuclease (linearization is used to improve the efficiency of plasmid genome integration) using Lipofectamine 3000 transfection reagent. The other culture dish was only given an equal amount of transfection reagent, without plasmid.

[0104] (3) On the 3rd day, 24 hours after transfection, the double-antibody selection medium containing 7.5 μg / ml blast fungicide and 300 μg / ml Zeocin was replaced.

[0105] (4) Observe the cell status under a microscope every day to ensure that all cells on the negative control culture dish without transfection plasmid die within two weeks; replace the double antibiotic selection medium with fresh medium every 2-3 days.

[0106] (5) After the cells have grown to full size for the first time, they need to be passaged again at a ratio of 1:50 to maintain the double antibody selection pressure.

[0107] (6) After 2-3 weeks, double-resistant monoclonal cells began to appear in the transfection group culture dishes. Select monoclonal cells with a diameter <5mm and uniform morphology, and use a cloning loop to separate 3-5 independent monoclonal cells. Transfer each monoclonal cell to a single well of a 24-well plate for continued culture. At this time, replace the medium with maintenance medium containing 7.5μg / ml blastomycin and 150μg / ml Zeocin (Zeocin concentration halved) to maintain the antibiotic concentration unchanged.

[0108] (7) After the cells in the 24-well plate have grown to confluence, they are transferred to a 6-well plate for further culture, while maintaining the antibiotic concentration.

[0109] (8) After the cells in the 6-well plate have grown to confluence, continue to passage and expand the culture, repeating the monoclonal screening step (6) to ensure the purity and genetic stability of the monoclonal cell line.

[0110] (9) After the expansion culture was completed, the functions of each monoclonal cell line were verified using indirect immunofluorescence assay with fluorescently labeled α-BTX. The verification was deemed successful when clear, high-density AChR cluster fluorescence signals were visible on the cell membrane surface. After successful verification, the pACEtetO-AChR-α1-β-δ-γ-rapsyn / pcDNA6TR bistable transgenic cell line (fetal type) was successfully constructed.

[0111] Example 3

[0112] This embodiment provides a method for constructing a 6TR-α1-β-δ-ε-rapsyn bistable transgenic cell line (hereinafter referred to as adult bistable transgenic cell line) that stably expresses TetR and adult AChR-Rapsyn.

[0113] Specifically, the following steps are included:

[0114] (a) Screening for the optimal concentration of antibiotics

[0115] Bleomycin (Zeocin) is a commonly used antibiotic for screening stable mammalian cell lines, with an effective screening concentration range of 50-1000 μg / mL. In this example, the optimal screening concentration was determined to be 300 μg / mL through a preliminary concentration gradient experiment similar to that in Example 2.

[0116] (ii) Transform the pACEtetO-AChR-α1-β-δ-ε-rapsyn recombinant plasmid to construct a stable expression cell line.

[0117] (1) On the first day, the 6TR cells obtained in Example 1 were seeded into two 10cm culture dishes at a passage ratio of 1:100, and 10mL of complete culture medium was added to each dish.

[0118] (2) On the second day, when the cell confluence reached 70%-80%, one culture dish was transfected with 12 μg of pACEtetO-AChR-α1-β-δ-ε-rapsyn plasmid that had been linearized with I-SceI restriction endonuclease (linearization was used to improve the efficiency of plasmid genome integration) using Lipofectamine 3000 transfection reagent. The other culture dish was only given an equal amount of transfection reagent, without adding plasmid.

[0119] (3) On the 3rd day, 24 hours after transfection, the double-antibody selection medium containing 7.5 μg / ml blast fungicide and 300 μg / ml Zeocin was replaced.

[0120] (4) Observe the cell status under a microscope every day to ensure that all cells on the negative control culture dish without transfection plasmid die within two weeks; replace the double antibiotic selection medium with fresh medium every 2-3 days.

[0121] (5) After the cells have grown to full size for the first time, they need to be passaged again at a ratio of 1:50 to maintain the double antibody selection pressure.

[0122] (6) After 2-3 weeks, double-resistant monoclonal cells began to appear in the transfection group culture dishes. Select monoclonal cells with a diameter <5mm and uniform morphology, and use a cloning loop to separate 3-5 independent monoclonal cells. Transfer each monoclonal cell to a single well of a 24-well plate for continued culture. At this time, replace the medium with maintenance medium containing 7.5μg / ml blastomycin and 150μg / ml Zeocin (Zeocin concentration halved) to maintain the antibiotic concentration unchanged.

[0123] (7) After the cells in the 24-well plate have grown to confluence, they are transferred to a 6-well plate for further culture, while maintaining the antibiotic concentration.

[0124] (8) After the cells in the 6-well plate have grown to confluence, continue to passage and expand the culture, repeating the monoclonal screening step (6) to ensure the purity and genetic stability of the monoclonal cell line.

[0125] (9) After the expansion culture was completed, the functions of each monoclonal cell line were verified using indirect immunofluorescence assay with fluorescently labeled α-BTX. The verification was deemed successful when clear, high-density AChR cluster fluorescence signals were visible on the cell membrane surface. After successful verification, the pACEtetO-AChR-α1-β-δ-ε-rapsyn / pcDNA6TR bistable transgenic cell line (adult type) was successfully constructed.

[0126] Example 4

[0127] In this embodiment, the stable cell line prepared in the above embodiment was used to prepare antigen slides and perform performance testing.

[0128] The specific process for manufacturing antigen tablets is as follows:

[0129] (1) When the confluence of the above-mentioned adult / fetal stable cells reaches 80%-90%, plate laying is performed.

[0130] (2) Place sterile cell smears of 24×24mm size in the six-well plate in advance, and add 2mL of complete culture medium to each well for later use.

[0131] (3) Take out the stable cells to be passaged, discard the old culture medium, rinse the cells twice with PBS buffer, add trypsin to digest the cells, add an equal volume of complete culture medium to stop digestion, transfer the cell suspension to a 15mL centrifuge tube, centrifuge to collect the cells, and discard the supernatant.

[0132] (4) Add complete culture medium to resuspend the cells. Take an appropriate amount of cell suspension according to the cell density and add it to the six-well plate obtained in step (2). Shake the cells evenly using the cross-shaking method to ensure uniform cell distribution. Add doxycycline (Dox) at a final concentration of 1 μg / mL to induce AChR expression for 48 h and place it in a CO2 incubator for further culture.

[0133] (5) After the cells have grown to fill the slide, discard the culture medium, rinse twice with PBS buffer, and then fix and collect the slide.

[0134] (6) Take the above cell smears, wash them 3 times with PBS, add fixative (4% paraformaldehyde, PFA) and treat at room temperature for 10 min; discard the fixative, wash them 3 times with PBS, add blocking solution (PBS buffer containing 5% bovine serum albumin BSA or 10% normal goat serum) and block at 37°C for 1 h.

[0135] (7) Place the cell slides treated above in a sterile environment to air dry, and cut them into 2×2mm unit slides using a glass cutter for later use.

[0136] (8) Cut and dry unit cell smears, paste them onto a glass slide with 4 reaction wells, seal and store at 4~8℃.

[0137] Indirect immunofluorescence staining detection procedure using the sample as the primary antibody:

[0138] (9) Place the prepared antigen sheet in a PBS buffer staining tank and soak for 5 minutes. After the process, remove the sheet and discard the liquid in the reaction well.

[0139] (10) Add 50-100 μL of 10-fold diluted serum sample or undiluted cerebrospinal fluid sample to each well of the antigen sheet in sequence, and place it in a humidified chamber at 37°C in the dark for 30 min.

[0140] (11) After the reaction is complete, gently rinse the sample in the reaction well with PBS buffer, soak it in the dyeing tank for 5 minutes, and then remove and discard the liquid in the reaction well.

[0141] (12) Add 50-100 μL of FITC-labeled goat anti-human IgG fluorescent secondary antibody to the reaction well, ensuring that the secondary antibody completely covers the reaction well, and place it in a humidified chamber at 37°C in the dark for 30 min.

[0142] (13) Repeat the washing operation in step (11), add anti-fluorescence quenching sealing solution to the reaction well, and carefully cover with coverslip to avoid generating air bubbles.

[0143] (14) Observe and record the test results under a fluorescence microscope.

[0144] Performance testing: Three serum samples from AChR antibody-positive patients with different titers (P1 weakly positive, antibody titer 1:10; P2 moderately positive, antibody titer 1:32; P3 strongly positive, antibody titer 1:100) were used for testing, with conventional AChR multi-subunit transient transfection antigen tablets used as controls.

[0145] Test results as follows Figure 4 As shown in the figure, the horizontal axis, from left to right, represents P1 weakly positive serum sample (antibody titer 1:10), P2 moderately positive serum sample (antibody titer 1:32), and P3 strongly positive serum sample (antibody titer 1:100); the vertical axis, from top to bottom, represents AChR multi-subunit transient transfection antigen sheet (prior technology control group), antigen sheet prepared from the α1-β-δ-γ / 6TR fetal bistable transfected cell line of this application, and antigen sheet prepared from the α1-β-δ-ε / 6TR adult bistable transfected cell line of this application.

[0146] Depend on Figure 4 It is known that for P1 weakly positive serum samples (antibody titer 1:10, the low-titer sample most easily missed in clinical practice), the existing AChR multi-subunit transient transfection antigen slides do not have a visible specific green fluorescence signal and cannot detect low-titer anti-AChR antibodies, posing a serious risk of missed detection. However, the antigen slides prepared from the fetal and adult bistable transfected cell lines of this application can detect clear and specific green fluorescence signals with cell membrane localization, and can stably identify low-titer antibodies, completely solving the core pain point of the existing technology in missing weakly positive samples.

[0147] For P2 strongly positive serum samples (antibody titer 1:32) and P3 strongly positive serum samples (antibody titer 1:100), the transient transfection antigen slides of the prior art only show weak, messy, and heterogeneous fluorescence signals, without clear cell membrane localization characteristics, and the detection signal-to-noise ratio is extremely low. In contrast, the two bistable transfected cell line antigen slides of this application both show high-intensity, uniform cell membrane localization-specific fluorescence signals, with a high proportion of positive cells and a clean background. The detection signal-to-noise ratio and signal uniformity are significantly better than the control group of the prior art.

[0148] As can be seen from the above test results, the fluorescence signal intensity and the number of positive cells of the fetal and adult bistable transfected cell line antigen sheets of this application both increase in a gradient with the increase of serum antibody titer. The signal change is strictly positively correlated with the antibody concentration, which can accurately reflect the antibody level in the sample to be tested and is suitable for clinical MG patient condition assessment and efficacy monitoring. In contrast, the transient transfection antigen sheets of the prior art do not have a clear signal gradient change and cannot achieve reliable quantitative detection.

[0149] Furthermore, both the fetal and adult bistable transgenic cell line antigen tablets of this application can specifically identify anti-AChR antibodies in patient serum. For weakly positive samples, the detection signal of the fetal antigen tablet is more significant, demonstrating that the combined detection of adult and fetal dual subtype antigen tablets can simultaneously cover pathogenic antibodies against both adult and fetal AChR, significantly broadening the antibody detection spectrum and further reducing the clinical false negative rate.

[0150] In summary, the antigen strips prepared from the bistable transfected cell line constructed in this application are significantly superior to existing conventional AChR transient transfection antigen strips in terms of detection sensitivity, signal uniformity, signal-to-noise ratio, quantitative accuracy, and detection spectrum coverage, fully meeting the clinical testing needs of MG anti-AChR antibodies.

[0151] Example 5

[0152] This example uses the 6TR-α1-β-δ-ε-rapsyn bistable transgenic cell line of adult AChR-Rapsyn as an example. The cells were passaged and cultured, and the method in Example 4 was used to detect the 1st, 5th and 20th generation cell lines respectively (P1 weak positive, antibody titer 1:10; P3 strong positive, antibody titer 1:100).

[0153] Test results as follows Figure 5 As shown in the figure, the horizontal axis from left to right represents T1 (generation 1 cell line), T5 (generation 5 cell line), and T20 (generation 20 cell line); the vertical axis from top to bottom represents P1 weakly positive serum sample (antibody titer 1:10) and P3 strongly positive serum sample (antibody titer 1:100).

[0154] Depend on Figure 5 It can be seen that the receptor expression level and clustering efficiency of the 1st, 5th and 20th generation cell lines did not decrease significantly, indicating that the stable cell line of this application still has good stability after at least 20 passages, which solves the defects of existing constitutive expression systems such as passage silencing, gene loss and high cytotoxicity.

[0155] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A recombinant expression vector for inducible expression of clustered acetylcholine receptors, characterized in that, The recombinant expression vector is a tetracycline-inducible expression vector with independent expression of multiple subunits on a single vector. The vector is based on pACEtetO as its backbone, and its multiple cloning site region contains a BstXI restriction endonuclease single-enzyme cleavage site. The vector integrates the full subunit coding sequence of the acetylcholine receptor and the coding sequence of the Rapsyn protein through an iterative enzyme digestion-ligation method. Each subunit to be inserted has an independent expression cassette with an I-CeuI homing endonuclease site and a BstXI restriction endonuclease site at both ends. After iterative ligation, each subunit and the Rapsyn protein are expressed by an independent Tet-On inducible promoter, and the transcription and translation processes of each subunit do not interfere with each other. The acetylcholine receptor subunits are the adult subunit combination CHRNA1, CHRNB1, CHRND, and CHRNE, or the fetal subunit combination CHRNA1, CHRNB1, CHRND, and CHRNG.

2. The recombinant expression vector according to claim 1, characterized in that, The expression of the Tet-On inducible promoter is controlled by doxycycline. Without doxycycline induction, the target gene is silenced, and the target gene is efficiently expressed after the addition of doxycycline. Optionally, the specific method of the iterative digestion-ligation is as follows: the vector is linearized by single digestion with BstXI, the independent expression cassette of the subunit to be inserted is double digested with I-CeuI and BstXI, the vector and the insert fragment are ligated by T4 DNA ligase, and a unique I-CeuI restriction site is re-formed at the ligation site; the above steps are repeated to integrate all subunits and independent expression cassettes of Rapsyn protein into the same vector in sequence; Optionally, the vector further comprises a bleomycin resistance selection marker, a WPRE transcriptional enhancement element, an expression packaging element, and a prokaryotic amplification element; Optionally, the recombinant expression vector is the adult-type plasmid pACEtetO-AChR-α1-β-δ-ε-rapsyn; Optionally, the recombinant expression vector is the fetal plasmid pACEtetO-AChR-α1-β-δ-γ-rapsyn.

3. The recombinant expression vector according to claim 1, characterized in that, The NCBI reference sequence accession number for the CHRNA1 is NM_001039523.3; Optionally, the NCBI reference sequence accession number of CHRNB1 is NM_000747.2; Optionally, the NCBI reference sequence accession number of the CHRND is NM_000751.3; Optionally, the NCBI reference sequence accession number of the CHRNE is NM_000080.4; Optionally, the NCBI reference sequence accession number of the CHRNG is NM_005199.5; Optionally, the NCBI reference sequence accession number for the gene RAPSN, which encodes the Rapsyn protein, is NM_005055.

4.

4. A bistable transgenic cell line stably expressing clustered acetylcholine receptors, characterized in that, The bistable transgenic cell line is a mammalian host cell that co-expresses the pcDNA6TR vector and the recombinant expression vector according to any one of claims 1-3. The bistable transgenic cell line has both blast fungicide resistance and bleomycin resistance, and expresses the native conformation of the clustered acetylcholine receptor on the cell membrane surface through doxycycline induction.

5. The bistable transgenic cell line according to claim 4, characterized in that, The mammalian host cells are HEK293T cells, HEK293 cells, or CHO-K1 cells; Optionally, the bistable transgenic cell line is the adult-type bistable transgenic cell line 6TR-α1-β-δ-ε-rapsyn; Optionally, the bistable transgenic cell line is the fetal bistable transgenic cell line 6TR-α1-β-δ-γ-rapsyn; Optionally, after induction with doxycycline at a final concentration of 1 μg / mL for 48 h, the bistable transgenic cell line forms high-density, patchy clusters of acetylcholine receptors on the cell membrane surface.

6. A method for constructing a bistable transgenic cell line according to any one of claims 4-5, characterized in that, The construction method specifically includes the following steps: (1) Construction of stable pcDNA6TR cell line: The pcDNA6TR plasmid was transfected into host cells, and after selection by blast fungicide pressure and monoclonalization, a 6TR cell line stably expressing TetR tetracycline repressor protein was obtained. (2) Transfection of recombinant expression vector and screening of bistable transgenic cells: After linearizing the recombinant expression vector according to any one of claims 1-6, the 6TR cell line obtained in step S1 is transfected, and then screened under pressure with blastomycin and bleomycin, and monoclonalized to obtain bistable transgenic cell lines. (3) Functional verification: The monoclonal cell line was induced to express and verified. The qualified standard was that the cell membrane surface could form a high-density cluster of acetylcholine receptors. The target bistable transgenic cell line was obtained by screening.

7. The construction method according to claim 6, characterized in that, In step (1), the screening concentration of blast fungicide is 7.5 μg / mL; in step S2, the screening concentration of double resistance is 7.5 μg / mL blast fungicide + 300 μg / mL bleomycin, and the concentration of bleomycin in the single-clone maintenance culture is halved to 150 μg / mL. Optionally, in step (2), the recombinant expression vector is linearized with I-SceI restriction endonuclease and transfected using Lipofectamine 3000 reagent; Optionally, in step (3), the function is verified by indirect immunofluorescence assay using fluorescently labeled α-cobra venom.

8. A detection kit for anti-acetylcholine receptor antibodies in myasthenia gravis, characterized in that, The kit comprises any one of the bistable transgenic cell lines according to claims 4-5, or antigen sheets prepared from said bistable transgenic cell lines.

9. The reagent kit according to claim 8, characterized in that, The kit contains both adult bistable transgenic cell lines and fetal bistable transgenic cell lines. Optionally, the kit further includes fluorescently labeled α-cobra venom, fluorescently labeled anti-human IgG secondary antibody, cell fixative, blocking solution, positive control serum and negative control serum; Optionally, the antigen sheet is a unit cell climbing sheet prepared by inducing expression, fixing, blocking, and cutting of bistable transgenic cell lines, and then pasting it onto a glass slide with reaction wells.

10. The use of any recombinant expression vector according to any one of claims 1-3 and any bistable transgenic cell line according to any one of claims 4-5 in the preparation of a detection reagent or kit for anti-acetylcholine receptor antibodies against myasthenia gravis.

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