Human acetylcholine receptor dual-vector expression system based on split-GFP and antibody detection method

Abstract

This invention relates to the field of biotechnology, specifically to a human acetylcholine receptor dual-vector expression system based on split-GFP and an antibody detection method. The system consists of a first expression vector and a second expression vector: the first expression vector contains a first polycistronic transcription unit and a second polycistronic transcription unit; the second expression vector contains a third polycistronic transcription unit and a fourth transcription unit. This invention utilizes split-GFP technology to spatially and strongly bind the conditions for GFP fluorescence generation to the correct assembly of the AChR pentamer complex on the cell membrane, achieving self-reporting of structural integrity. It effectively solves the problems of subunit imbalance and transfection heterogeneity caused by traditional multi-plasmid co-transformation, fundamentally eliminating false-positive background interference. The detection substrate prepared using this system exhibits extremely high sensitivity, specificity, and batch-to-batch consistency in the detection of myasthenia gravis autoantibodies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a human acetylcholine receptor dual-vector expression system based on split-GFP and an antibody detection method. Background Technology

[0002] Myasthenia gravis is an autoimmune disease caused by acquired neuromuscular junction transmission disorders mediated by autoantibodies. Anti-acetylcholine receptor antibodies (AChR antibodies) are the most prevalent pathogenic antibodies in MG, with a positive rate as high as 80%-90% in patients with systemic MG. Accurate and highly sensitive detection of AChR antibodies in patient serum is crucial for the clinical diagnosis, disease grading, and treatment evaluation of MG.

[0003] Currently, cell-based assays are widely recognized as the "gold standard" for detecting conformation-dependent membrane protein antibodies such as AChR because they can provide antigens in their native physiological conformation. However, AChR is a highly complex multi-subunit transmembrane ion channel protein. In the adult form, the α1, β1, δ, and ε subunits assemble into a pentamer in a stoichiometric ratio of 2:1:1:1 (in the fetal form, the γ subunit replaces the ε subunit). Furthermore, it requires the assistance of the intracellular scaffold protein Rapsyn to achieve high-density aggregation of the receptor on the cell membrane. Therefore, the efficient and balanced recombinant expression and correct assembly of the complete AChR pentamer in in vitro mammalian cell lines (such as HEK293) is a core technological bottleneck in the preparation of high-quality CBA detection matrices.

[0004] In existing technologies, recombinant expression of such multi-subunit transmembrane protein complexes mainly relies on the following strategies, but all of them have significant inherent drawbacks: First, the multi-plasmid co-transfection strategy. This involves constructing each subunit (α, β, δ, ε) and the accessory protein Rapsyn on 5 to 6 separate expression plasmids and co-transfecting them into host cells. The fatal flaw of this method is its extremely low transfection efficiency, making it difficult to ensure that all necessary plasmids enter the same cell simultaneously. Furthermore, the copy number of each plasmid within the cell fluctuates wildly, leading to a severe imbalance in the expression ratio of each subunit and extremely high heterogeneity of the cell population. This not only generates a large number of false positives due to subunit deletions during assembly but also makes it extremely difficult to screen for stable engineered cell lines.

[0005] Second, the single-vector long cistron strategy. This involves tandemly linking the coding sequences of all essential subunits with self-cleaving peptides (such as P2A) and placing them downstream of a single promoter to construct an ultra-large expression vector. However, such extremely long transcripts easily lead to mRNA instability, and due to the decline in ribosome jumping efficiency, downstream gene translation efficiency drops sharply. Furthermore, this architecture forces all subunits to be expressed in equal molar amounts, failing to flexibly meet the physiological requirement of AChR's unique α-subunit double stoichiometry ratio (2:1:1:1).

[0006] Third, traditional fluorescent protein co-expression reporter systems. Whether using multiple plasmids or dual-vector systems, current technologies typically rely on the co-expression of free enhanced green fluorescent protein (e.g., IRES-eGFP) as an indicator of successful transfection. This traditional reporter mechanism merely indicates that the plasmid has entered the cell and transcribed, but free eGFP is completely unrelated to the physical assembly process of AChR. Cells with high eGFP expression may completely lack key subunits and fail to form a functional pentamer. This disconnect between structure and reporter leads to low co-localization rates and poor uniformity of detection results when cells selected by fluorescence screening are used for antibody detection, making it impossible to effectively distinguish between cells truly expressing the functional receptor and cells that have failed to assemble.

[0007] In summary, there is an urgent need in this field for a novel expression system that can overcome the heterogeneity of multi-plasmid co-transformation and the inefficiency of single vectors, and break through the limitations of traditional reporter genes, achieving a leap from transfection-level reporting to self-reporting based on the structural integrity of the complex, thereby providing reliable cell-based raw materials for high-sensitivity, high-standardization, large-scale clinical CBA testing. Summary of the Invention

[0008] The purpose of this invention is to provide a human acetylcholine receptor dual-vector expression system based on split-GFP and an antibody detection method, in order to solve the technical problems mentioned in the background art regarding the high heterogeneity of multi-subunit transmembrane protein complex (such as acetylcholine receptor) recombinant expression, the low transcription efficiency of single vector, and the inability of traditional reporter genes to indicate the true assembly state of the complex.

[0009] To achieve the above objectives, on the one hand, the present invention provides a human acetylcholine receptor dual-vector expression system based on split-GFP, the system comprising a first expression vector and a second expression vector: The first expression vector contains two independent transcription units: The first polycistronic transcription unit, driven by the mEF-1α core promoter, contains, in sequence, the coding sequence for the human acetylcholine receptor α1 subunit (SEQ ID NO: 1), the self-cleaving peptide sequence (SEQ ID NO: 7), and the coding sequence for the β1 subunit (SEQ ID NO: 2). The second polycistronic transcription unit, driven by the rEF-1α core promoter, sequentially includes the coding sequence of the α1 subunit, the self-cleaving peptide sequence, the coding sequence of the δ subunit (SEQ ID NO: 3), and the coding sequence of the GFP11 fragment fused to the C-terminus of the δ subunit (SEQ ID NO: 9). The second expression vector contains two independent transcription units: The third polycistronic transcription unit, driven by the mEF-1α promoter, contains either the ε subunit coding sequence (SEQ ID NO: 4) or the γ subunit coding sequence (SEQ ID NO: 5), and the GFP1-10 fragment coding sequence fused thereto (SEQ ID NO: 8). The fourth transcription unit, driven by the rEF-1α promoter, contains the coding sequence for the independent scaffold protein Rapsyn (SEQ ID NO: 6).

[0010] When the first expression vector and the second expression vector are co-transfected into the same host cell, the GFP11 fragment and the GFP1-10 fragment will generate membrane-localized fluorescence through spatial complementary recombination if and only if the α1 subunit, β1 subunit, δ subunit and ε subunit or γ subunit are assembled into a complete transmembrane pentamer. Preferably, in the second polycistronic transcription unit, the coding sequence of the δ subunit is connected to the coding sequence of the GFP11 fragment via a GS linker; in the third polycistronic transcription unit, the coding sequence of the ε subunit or γ subunit is connected to the coding sequence of the GFP1-10 fragment via a GS linker. Preferably, the amino acid sequence corresponding to the coding sequence of the GFP1-10 fragment is amino acids 1-214 of enhanced green fluorescent protein; the amino acid sequence corresponding to the coding sequence of the GFP11 fragment is amino acids 215-230 of enhanced green fluorescent protein. The self-cleaving peptide sequence is the P2A sequence. In a second aspect, the present invention provides an engineered cell that recombinantly expresses the complete human acetylcholine receptor protein, wherein the engineered cell is co-transfected with the above-described split-GFP-based human acetylcholine receptor dual-vector expression system. In a third aspect, the present invention provides an antibody detection kit comprising the aforementioned engineered cells as a detection substrate. In a fourth aspect, the present invention provides the use of the above-described dual-vector expression system or the above-described engineered cells in the preparation of a diagnostic kit for detecting autoantibodies associated with myasthenia gravis. A fifth aspect of the present invention provides a method for in vitro detection of acetylcholine receptor antibodies, the method not for disease diagnosis, comprising: obtaining a cell suspension or cell adherent culture containing the engineered cells described above; performing pre-screening under a fluorescence microscope and recording cells that generate GFP positive fluorescence signals as target cells; incubating the target cells with a serum sample to be tested; adding fluorescently labeled anti-human IgG secondary antibody for secondary incubation; acquiring and interpreting images; if the cell surface where the GFP positive fluorescence signal is located also shows a secondary antibody signal and the two are spatially co-located, then it is determined to be positive for acetylcholine receptor antibody binding.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This split-GFP-based human acetylcholine receptor dual-vector expression system and antibody detection method combines split-GFP technology with a complex AChR pentamer expression system, anchoring the GFP11 and GFP1-10 fragments to the extracellular domains of specific subunits. This design ensures that GFP fluorescence not only indicates successful transfection but also strictly adheres to the correct spatial folding and assembly of the transmembrane pentamer (α2βδε or α2βδγ) on the cell membrane. Untransfected cells, cells with missing subunits, or cells with incorrect assembly naturally show no fluorescent signal, fundamentally eliminating false-positive interference from "successful transfection but failed assembly." To address the subunit expression imbalance caused by six-plasmid co-transfection (Comparative Example 2), this invention utilizes four independent transcription units in a dual-vector design, cleverly pre-loading a near-physiological stoichiometric ratio (α, β, δ, ε ≈ 2:1:1:1) at the DNA molecule design level. Simultaneously, it avoids the mRNA instability and transcriptional attenuation that may result from a single-vector long cistron strategy (Comparative Example 1), ensuring efficient expression of each subunit and product uniformity. Only two plasmids need to be co-transfected, significantly increasing the probability of all necessary multi-gene plasmids entering the same cell simultaneously. The split-GFP system acts as a built-in quality control mechanism, ensuring that only correctly assembled receptors emit light, naturally filtering out incompletely expressed cell clones. This solves the problems of poor transfection reagent compatibility, competitive uptake of various plasmids, and complex screening markers in multi-plasmid co-transfection. In antibody detection applications, the complementary fluorescence signal spatially overlaps significantly with the AChR antibody binding site. Parallel experiments show that the co-localization rate of the tag protein and antibody in the system of this invention is as high as approximately 95%, far exceeding that of traditional single-vector co-expression of free eGFP (approximately 80%) or dual-vector systems without split-GFP (approximately 50%). In dilution detection of positive serum samples for myasthenia gravis, even at high dilutions of 1:500 and even 1:1000, this invention still maintains excellent positive detection rates (14 / 20 and 8 / 20, respectively), with signal-to-noise ratio and sensitivity comprehensively superior to existing conventional comparison schemes.

[0012] The complementary GFP fluorescence generated by this invention is extremely stable, and can be used not only for live cell pre-screening but also for direct detection in unfixed live cells after adding serum. Compared to traditional fixation methods, the Live CBA method avoids protein cross-linking and conformational changes caused by fixatives, preserving the natural physiological conformation of AChR as a complex transmembrane channel protein to the greatest extent, thereby significantly improving the binding efficiency against conformation-dependent antibodies. Attached Figure Description

[0013] Figure 1 The plasmid map of the first expression vector constructed in the embodiments of the present invention; Figure 2 The plasmid map of the second expression vector constructed in this embodiment of the invention is a human-type plasmid map; Figure 3 The plasmid map of the fetal type of the second expression vector constructed in this embodiment of the invention; Figure 4 These are fluorescence microscope images comparing the transfection efficiency after transfection in an embodiment of the present invention. Figure 5 These are fluorescence microscope images comparing the transfection efficiency after transfection in Comparative Example 1 of this invention. Figure 6 These are fluorescence microscope images comparing the transfection efficiency after transfection in Comparative Example 2 of this invention. Figure 7 These are fluorescence microscope images comparing the transfection efficiency after transfection in Comparative Example 3 of this invention. Figure 8 Bright-field microscope images showing the cell growth status under the same transfection conditions in an embodiment of the present invention. Figure 9 Bright-field microscope images showing the cell growth status of Comparative Example 1 of this invention under the same transfection conditions. Figure 10 Bright-field microscope images showing the cell growth status of Comparative Example 2 of this invention under the same transfection conditions. Figure 11 Bright-field microscope images showing the cell growth status of Comparative Example 3 of this invention under the same transfection conditions. Figure 12 This is a combined fluorescence micrograph comparing the co-localization of the tag protein eGFP and the antibody in an embodiment of the present invention; Figure 13 This is a combined fluorescence micrograph comparing the co-localization of the tag protein eGFP and the antibody in Comparative Example 1 of this invention; Figure 14 This is a combined fluorescence micrograph comparing the co-localization of the tag protein eGFP and the antibody in Comparative Example 2 of this invention; Figure 15 This is a combined fluorescence micrograph comparing the co-localization of the tag protein eGFP with the antibody in Comparative Example 3 of this invention. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0015] Example: Construction, cell transfection and functional validation protocol of a dual-vector split-GFP system for expressing the complete human acetylcholine receptor.

[0016] I. Materials Construction of expression carrier: The first expression vector (pVITRO-A2B1D1-GFP11, Figure 1 Based on the commercially available pVITRO dual-promoter expression vector, it contains a kanamycin resistance gene for bacterial selection and includes the following two independent mammalian cell expression units: The first polycistronic transcription unit is located at the multiple cloning site A (MCS A) of the vector, driven by the mEF-1α core promoter, and inserted into the coding sequence of the human AChRα1 subunit - P2A self-cleaving peptide sequence - β subunit (SEQ ID NO: 1-SEQ ID NO:7-SEQ ID NO:2).

[0017] The second polycistronic transcription unit is located at the multiple cloning site B (MCS B) of the vector. It is driven by the rEF-1α core promoter and inserts the α1 subunit coding sequence - P2A sequence - δ subunit coding sequence - GS linker - GFP11 coding sequence (-SEQ ID NO:1-SEQ ID NO:7-NO:3-SEQ ID NO:9).

[0018] The second expression vector (pVITRO-E1R1-GFP1-10, Figure 2 Also based on the pVITRO dual promoter vector backbone, containing the kanamycin resistance gene, and constructing the following expression units: The third polycistronic transcription unit (MCS A): driven by the mEF-1α promoter, inserts the ε subunit coding sequence -GSlinker-GFP1-10 (SEQ ID NO:4-SEQ ID NO:8).

[0019] Fourth transcription unit (MCS B): Driven by the rEF-1α promoter, it inserts into the Rapsyn protein coding sequence (SEQ ID NO: 6). Note: GFP1-10 are amino acids 1-214 of enhanced GFP (EGFP), containing the first 10 of the 11 complete β-sheets. They are non-fluorescent on their own and need to be complementary to GFP11.

[0020] Variants of the second expression vector ( Figure 3 The ε subunit can be used to construct the adult acetylcholine receptor (α1,β1,δ1,ε), and the γ subunit can be used to replace the ε subunit to construct the fetal acetylcholine receptor (α1,β1,γ,δ1) (third polycistronic transcription unit SEQ ID NO:5-SEQ ID NO:8).

[0021] Supplement to the principles of split-GFP technology: GFP1-10 (214aa): Forms a stable β-barrel structure, but lacks a C-terminal α-helix, the fluorophore is incomplete, and there is no green fluorescence.

[0022] GFP11 (16aa): Contains only amino acids 215-230, is a disordered peptide, and has no structure or fluorescence when alone.

[0023] Complementation mechanism: When GFP1-10 and GFP11 meet in the same cytoplasmic space (distance <5nm, concentration >1μM), GFP11 with 16 amino acids can be inserted into the gap of GFP1-10, restoring the complete β-barrel structure, spontaneously forming a fluorophore, and producing green fluorescence (Ex 488nm / Em 509nm).

[0024] In this invention, GFP11 is anchored to the C-terminal extracellular domain of the δ subunit, while GFP10 and the ε subunit are expressed in the same vector. Only when the δ subunit is successfully integrated into the membrane and the ε subunit is present to form a complete pentamer, is GFP1-10 exposed to the perimembrane space, complementing GFP11 on the membrane and producing membrane-localized fluorescence.

[0025] Cells and culture media: HEK293 cell line; DMEM high glucose complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin); Opti-MEM serum-free medium.

[0026] Transfection reagent: lipo2000.

[0027] Cell fixative: 4% paraformaldehyde.

[0028] Antibody detection reagent: Fluorescently labeled goat anti-human IgG (cy3-anti-human-IgG) Patient serum samples: Serum from patients with clinically diagnosed myasthenia gravis.

[0029] II. Methods Step 1: Vector Construction and Validation The above gene fragments were sequentially cloned into the designated MCS site of the pVITRO vector using standard molecular cloning techniques (such as homologous recombination and restriction endonuclease ligation).

[0030] The constructed plasmid was sequenced to verify its accuracy, ensuring that the gene sequences, reading frames, adapter sequences, and split-GFP fusion sites were correct.

[0031] High-purity plasmid DNA was extracted using an endotoxin-free plasmid extraction kit for transfection.

[0032] Step 2: Cell transfection and transient expression The day before transfection, HEK293 cells were loaded at a density of 3 × 10⁶ cells per well. 5 Seeds were placed at a density of [number] cells per well in 6-well plates. Transfection was performed when cell confluence reached 70-80%. For each well, two sterile tubes were prepared: Tube A (DNA Mixture): Add 1.2 μg of the first expression vector and 0.8 μg of the second expression vector (adult or fetal second expression vector) to 150 μL of Opti-MEM and mix gently. Tube B (Transfection Reagent): Add 6 μL of Lipo2000 to 150 μL of Opti-MEM, vortex to mix, and incubate at room temperature for 5 minutes.

[0033] Add all the liquid from tube B into tube A, immediately vortex to mix, and let stand at room temperature for 15 minutes to form a complex.

[0034] Add 300 μL of the complex dropwise to each well and gently shake to mix. Return the cells to the incubator (37°C, 5% CO2). Replace with fresh complete culture medium 6 hours after transfection.

[0035] Live cell imaging observation (24-48 h post-transfection): Observe the GFP complementary signal directly under a fluorescence microscope (no fixation required).

[0036] The appearance of clear green fluorescence on the cell membrane indicates that the two plasmids have been successfully co-transformed and a complete pentamer structure has been formed.

[0037] Control settings: Single transfection of the first expression vector (without GFP11) → no fluorescence; Single transfection of the second expression vector (without GFP1-10) → no fluorescence.

[0038] Step 3: Establishment of stable cell lines (for standardized production) Forty-eight hours after transfection, the cells were passaged at a ratio of 1:5 into complete selection medium containing 600 μg / mL G418.

[0039] split-GFP auxiliary screening strategy: Days 5-7: Label strong GFP fluorescent clones under a fluorescence microscope and pick them into 96-well plates using a cloning loop.

[0040] Flow cytometry sorting: Direct sorting of GFP + The cell population enrichment efficiency is 3-5 times higher than that of traditional G418 screening, and it naturally excludes false positive clones with incomplete expression.

[0041] Change the selection medium every 3-4 days and continue culturing for 10-14 days until all untransfected control group cells die and resistant clones are visible.

[0042] High-expressing cell populations were sorted by flow cytometry based on GFP fluorescence intensity (no additional reporter gene is needed, as split-GFP itself serves as the selection marker). The sorted cells were then cultured and expanded in medium containing 300 μg / mL G418 to establish a stable cell line (named HEK293-AChR-splitGFP).

[0043] Step 4: Preparation of cells for detection: Mode A: Transient transfection cell preparation Directly for detection: After transfection and expression, cells can be directly used for live cell detection (no fixation required) and proceed to the detection process (step 5). This method has a short preparation cycle and retains the native conformation of AChR, making it suitable for flexible, small-batch detection needs. Furthermore, cells transfected 24 hours post-transfection can be passaged. During passage, cells transfected with the human-type second expression vector and cells transfected with the fetal-type second expression vector are mixed, achieving the effect of accommodating both antigen types during detection. Passaged cells can be fixed for long-term preservation.

[0044] Mode B: Preparation of Mixed Stable Cell Lines The selected stable monoclonal cell lines were amplified to prepare a sufficient number of working cell libraries and master cell libraries, and partially cryopreserved in liquid nitrogen to ensure the long-term consistency and traceability of the detection matrix.

[0045] Cell plating and mixing before detection: Resuscitation and amplification: Before detection, HEK293-AChR-splitGFP cells and untransfected wild-type HEK293 cells were resuscitated from the working cell bank and then subjected to routine culture and amplification.

[0046] Proportional Mixing and Plate Coding: On the day of or the day before testing, mix HEK293-AChR-splitGFP cells with wild-type HEK293 cells at a predetermined ratio (e.g., 1:1) and seed them at an appropriate density in 96-well plates or climbing slides. Purpose of Mixing: To simultaneously provide positive and negative control cells within the microscope's field of view, facilitating result interpretation and internal quality control.

[0047] Adherent culture: Incubate the mixed cells at 37°C in a 5% CO2 incubator for 4-24 hours to allow the cells to fully adhere and form a monolayer. This method produces a homogeneous and stable matrix, suitable for high-throughput, standardized clinical testing.

[0048] Step 5: CBA Testing Process The following procedure is applicable to cells prepared using the above-mentioned methods and can be performed on 96-well plates or slides: Live cell pre-screening GFP fluorescence was observed directly under an inverted fluorescence microscope to confirm that the cells were in good condition and that membrane expression was uniform.

[0049] Recording GFP + Cell ratio is used as a batch quality control parameter.

[0050] Antigen presentation and processing: Depending on the testing requirements, either the live cell method or the fixation method can be selected. (1) Live cell method (Live CBA, preferred method): After aspirating the culture medium and gently washing with PBS, cell fixation is not required. Directly add PBS containing 5% BSA and block at room temperature or 4°C to maintain the native conformation of the protein to the greatest extent.

[0051] (2) Fixation and permeabilization: Discard the culture medium and wash gently once with PBS. Add 4% paraformaldehyde for fixation for 15 minutes. If detecting surface antibodies, fixation alone is sufficient; if detecting intracellular targets, permeabilize with 0.1% Triton X-100 for 10 minutes. Wash three times with PBS.

[0052] Blocking: Add PBS containing 5% BSA and block at room temperature for 60 minutes.

[0053] Primary antibody incubation: Discard the blocking buffer. Serially dilute the serum samples to be tested (patients and controls) with blocking buffer and add them to the wells. Incubate overnight at 4°C or for 2 hours at room temperature. Wash 3 times with PBS.

[0054] Secondary antibody incubation: Add fluorescently labeled anti-human IgG secondary antibody (cy3-anti-human-IgG) diluted with blocking buffer, and incubate at room temperature in the dark for 60 minutes. Wash three times with PBS.

[0055] Counterstaining and mounting: Counterstain cell nuclei with DAPI staining solution for 5 minutes, then wash with PBS. For 96-well plates, PBS can be added directly to prevent drying, and a coverslip can be placed before imaging; for cell smears, they need to be removed, cell side up, and mounted on a glass slide with anti-quenching mounting medium.

[0056] Image acquisition and analysis: Observation was performed using a fluorescence microscope or a high-content imaging system.

[0057] Channel settings: DAPI channel (blue, cell nucleus), GFP channel (green, split-GFP complementation signal, indicating complete AChR assembly), TRITC / Cy3 channel (red, serum antibody signal).

[0058] Result interpretation: GFP-positive cells exhibit a red fluorescent signal on their surface, and the signal intensity is positively correlated with the GFP fluorescence intensity (reflecting receptor density), thus indicating a positive result. GFP-negative cells show no red signal, naturally eliminating false positives.

[0059] This design provides an excellent internal negative control without requiring additional labeling of engineered cells.

[0060] III. Results and Advantages Highly efficient co-expression and assembly verification: After transfection, membrane-localized GFP fluorescence can be observed under a fluorescence microscope. Positive serum samples bind to cell antigens, and Cy3-anti-human IgG fluorescent secondary antibody is added. Co-localization is observed under a fluorescence microscope, confirming the assembly of split-GFP strictly reporter functional receptor.

[0061] Excellent detection performance: When using known positive sera for detection, it still produces strong specific signals at high dilutions (e.g., 1:500), while the signal from healthy control sera is close to background. Split-GFP pre-screening ensures that all detected cells are high-expression clones, with batch-to-batch CV <10%.

[0062] This embodiment is the first to combine split-GFP technology with a dual-vector expression system, achieving structural integrity self-reporting. Compared with traditional eGFP co-expression, the fluorescence generation of split-GFP strictly depends on the correct folding and membrane integration of the δ subunit, thus: Exclude false-positive transfected cells: cells that are single-transfected, have missing subunits, or are incorrectly assembled do not have GFP signals and are naturally not included in subsequent detection; Provides real-time readings of assembly efficiency: GFP fluorescence intensity is positively correlated with pentamer integrity, which can be used to optimize culture conditions; Simplified detection process: No additional staining is needed to distinguish engineered cells from control cells; GFP itself serves as the cell identification identifier. Preservation of live cell detection capability: The complementary GFP is stable (t1 / 2>24h), allowing for preliminary screening in live cell state before fixation for antibody detection.

[0063] This design provides a practical solution for the standardized, large-scale production of highly active diagnostic cell substrates, while offering more stringent quality control standards than traditional reporting systems.

[0064] Proportional Design Comparative Example 1: Single-vector long cistron strategy (without split-GFP) The coding sequences of all five essential subunits (α, β, δ, ε, rapsyn) were tandemly linked by a P2A sequence and placed downstream of a single strong promoter to construct a single expression vector, which co-expressed free eGFP as a reporter gene.

[0065] defect: (1) Long transcripts may lead to reduced transcription or translation efficiency and mRNA instability.

[0066] (2) All subunits are expressed in equal molar amounts, and the stoichiometry of the α subunit cannot be flexibly adjusted.

[0067] Comparative Example 2: Six-plasmid co-transfection strategy (without split-GFP) The five subunits were cloned into five independent vectors, and the sixth plasmid carried the helper plasmids rapsyn and eGFP. The six plasmids were co-transfected.

[0068] defect: (1) The transfection efficiency is low, making it difficult to ensure that all plasmids enter the same cell at the same time.

[0069] (2) The copy number of each plasmid fluctuates randomly, and the expression ratio of subunits is severely unbalanced.

[0070] (3) eGFP has no physical connection with the AChR subunit: cells with high eGFP may lack key subunits, resulting in low screening efficiency.

[0071] Comparative Example 3: Dual-vector system but using conventional co-expression of eGFP (non-split-GFP) The same dual-vector quadruple-unit design as the present invention is used, but IRES-eGFP is used instead of split-GFP in the second expression vector.

[0072] defect: (1) IRES-driven efficiency is low, and the eGFP expression level is only 10-30% of that of cap-dependent translation, resulting in a weak signal.

[0073] (2) eGFP and AChR assembly are not coupled: eGFP positivity does not mean complete AChR assembly, and the co-localization rate of green fluorescent tag and antibody binding is low.

[0074] (3) It is impossible to distinguish between cells that are "successfully transfected but fail to assemble", and the uniformity of antibody binding results is poor.

[0075] IV. Parallel Experiments and Comparative Analysis of Results The experimental group of this invention and comparative examples 1-3 were conducted in parallel under the same conditions: Table 1. Comparison of transfection efficiency:

[0076] Table 2 Cell growth status:

[0077] Table 3. Co-localization rate of the tag protein eGFP with antibodies:

[0078] Table 4. Detection performance: positive detection rate and signal-to-noise ratio (20 positive samples)

[0079] The above systematic comparative experiments clearly demonstrate that: Compared to all comparative examples, the dual-vector four-unit split-GFP system of this invention exhibits significant advantages in four dimensions: transfection efficiency, assembly fidelity, cell uniformity, and detection performance. The split-GFP technology upgrades the reporter gene into a structural integrity tag, providing a novel quality control paradigm for the recombinant expression and diagnostic applications of membrane protein complexes.

[0080] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A human acetylcholine receptor dual-vector expression system based on split-GFP, characterized in that, The system consists of a first expression vector and a second expression vector: The first expression vector contains two independent transcription units: The first polycistronic transcription unit, driven by the mEF-1α core promoter, sequentially contains a human acetylcholine receptor α1 subunit coding sequence, a self-cleaving peptide sequence, and a β1 subunit coding sequence, wherein the α1 subunit coding sequence is shown in SEQ ID NO:1, the self-cleaving peptide sequence is shown in SEQ ID NO:7, and the β1 subunit coding sequence is shown in SEQ ID NO:

2. The second polycistronic transcription unit, driven by the rEF-1α core promoter, sequentially includes the α1 subunit coding sequence, the self-cleaving peptide sequence, the δ subunit coding sequence, and the GFP11 fragment coding sequence fused to the C-terminus of the δ subunit. The δ subunit coding sequence is shown in SEQ ID NO:3, and the GFP11 fragment coding sequence is shown in SEQ ID NO:

9. The second expression vector contains two independent transcription units: The third polycistronic transcription unit, driven by the mEF-1α promoter, sequentially contains either an ε subunit or a γ subunit coding sequence, and a GFP1-10 fragment coding sequence fused to the ε subunit or γ subunit coding sequence. The ε subunit coding sequence is shown in SEQ ID NO:4, the γ subunit coding sequence is shown in SEQ ID NO:5, and the GFP1-10 fragment coding sequence is shown in SEQ ID NO:

8. The fourth transcription unit, driven by the rEF-1α promoter, contains an independent scaffold protein coding sequence for Rapsyn, as shown in SEQ ID NO: 6; When the first expression vector and the second expression vector are co-transfected into the same host cell, the GFP11 fragment and the GFP1-10 fragment will generate membrane-localized fluorescence through spatial complementary recombination if and only if the α1 subunit, β1 subunit, δ subunit and ε subunit or γ subunit are assembled into a complete transmembrane pentamer.

2. The human acetylcholine receptor dual-vector expression system based on split-GFP according to claim 1, characterized in that, In the second polycistronic transcription unit, the coding sequence of the δ subunit is connected to the coding sequence of the GFP11 fragment via a GS linker; In the third polycistronic transcription unit, the coding sequence of the ε subunit or γ subunit is connected to the coding sequence of the GFP1-10 fragment via a GS linker.

3. The human acetylcholine receptor dual-vector expression system based on split-GFP according to claim 1, characterized in that, The amino acid sequence corresponding to the coding sequence of the GFP1-10 fragment is amino acids 1-214 of enhanced green fluorescent protein; The amino acid sequence corresponding to the GFP11 fragment coding sequence is amino acid positions 215-230 of enhanced green fluorescent protein.

4. The human acetylcholine receptor dual-vector expression system based on split-GFP according to claim 1, characterized in that, The self-cleaving peptide sequence is the P2A sequence.

5. An engineered cell that recombinantly expresses a complete human acetylcholine receptor protein, characterized in that: The engineered cells are co-transfected with the human acetylcholine receptor dual-vector expression system based on split-GFP as described in any one of claims 1 to 4.

6. The engineered cells expressing the complete human acetylcholine receptor protein according to claim 5, characterized in that, The host cell for the engineered cells is HEK293 cells.

7. An antibody detection kit, characterized in that: Engineered cells comprising the recombinant human acetylcholine receptor protein as described in claim 6 are used as the detection substrate.

8. The antibody detection kit according to claim 7, characterized in that, It also contains fluorescently labeled anti-human IgG secondary antibody, cell fixation solution, blocking solution, and cell nuclear staining solution.

9. The use of the split-GFP-based human acetylcholine receptor dual-vector expression system according to any one of claims 1 to 4, or the engineered cells expressing the recombinant human acetylcholine receptor protein according to any one of claims 5 to 6, in the preparation of a diagnostic kit for detecting myasthenia gravis-related autoantibodies.

10. A method for in vitro detection of acetylcholine receptor antibodies, characterized in that, Includes the following steps: Step 1: Obtain a cell suspension or cell adherent culture containing the engineered cells of claim 5; Step 2: Pre-screening is performed under a fluorescence microscope to record cells with GFP-positive fluorescence signals generated by complementary recombination of GFP11 fragment and GFP1-10 fragment, which are then used as target cells; Step 3: Incubate the serum sample to be tested with the target cells; the incubation can be performed in the unfixed live cell state or in the fixed cell state. Step 4: Add fluorescently labeled anti-human IgG secondary antibody and incubate for a second time; Step 5: Acquire images and interpret the results: If the cell surface where the GFP positive fluorescent signal is located also shows a fluorescently labeled anti-human IgG secondary antibody signal, and the two fluorescent signals are spatially co-localized, then it is determined to be positive for acetylcholine receptor antibody binding.

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