A material for detecting serum plasma antibodies to nicotinic acetylcholine receptors and methods of use thereof

Abstract

This invention provides a material for detecting serum plasma anti-nicotinic acetylcholine receptor antibodies and its method of use. The material comprises HEK293T cells transfected with three recombinant expression plasmids: the first plasmid encodes a truncated AChR α subunit; the second plasmid is a polycistronic plasmid encoding a fusion protein composed of a truncated AChR β subunit, an AChR δ subunit, and rapsyn protein linked by a P2A peptide; and the third plasmid encodes a truncated AChR γ subunit or ε subunit. After co-transfection with PEI, the cells are cultured in fresh complete medium for 25-35 hours after 7-9 hours post-transfection, followed by fixation, blocking, and preservation. This invention, through a three-plasmid system and antigen truncation modification, reduces cytotoxicity while ensuring high expression of the functional pentamer, significantly improving detection sensitivity and reagent shelf life.

Description

Technical Field

[0001] This invention relates to the field of biofluorescence detection technology, specifically to a material for detecting serum plasma anti-nicotinic acetylcholine receptor antibodies and its method of use. Background Technology

[0002] Cell-based immunofluorescence assay (CBA) is an important method for detecting antibodies against nicotinic acetylcholine receptor (AChR). Unlike radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and test strip methods, which require coating the antigen onto a solid surface, the core feature of CBA is the direct expression of the AChR antigen within the cell, forming a pentamer with its native spatial conformation on the cell membrane. This maximizes the preservation of the conformation-dependent epitopes of the antibody, avoiding the sensitivity decrease and false negatives caused by conformational changes during antigen coating. Furthermore, it can be used to detect readily available samples such as serum and plasma.

[0003] Acetylcholine receptors (AChRs) are ligand-gated ion channels located on the postsynaptic membrane of the neuromuscular junction. They are assembled into pentamers by α, β, δ, γ (fetal type) or ε (adult type) subunits in a stoichiometric ratio of 2:1:1:1, and bind to the intracellular receptor cluster protein rapsyn, anchoring them to the cell membrane. Anti-AChR antibodies are a major autoantibody found in the serum of patients with myasthenia gravis (MG), with a positive rate as high as 93%–100%, especially in patients with thymoma. Their detection is of significant value for the clinical diagnosis, classification, and treatment monitoring of MG.

[0004] Currently, there are five main categories of methods for detecting anti-AChR antibodies: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), test strip method, quantum dot labeling method, and cell-based immunofluorescence assay (CBA). Among these, RIA requires highly qualified laboratories and personnel, and existing literature reports that its sensitivity is lower than that of CBA, with a certain degree of false negatives. Both ELISA and test strip methods require coating the antigen onto a solid surface; the spatial conformation of the antigen can easily change during the coating process, leading to decreased sensitivity and false negatives. While quantum dot labeling eliminates the secondary antibody staining step, the primary sample for detection is tissue sections, which are not readily available. CBA, by expressing the AChR antigen within cells, can maximally maintain the antigen's native spatial conformation, making it one of the methods with the best sensitivity and specificity currently available, and it can be used to detect readily available samples such as serum and plasma.

[0005] However, existing CBA methods all have varying degrees of drawbacks. The first method utilizes cells with their own antigens that do not require transfection (such as primary fetal rat skeletal muscle cells), but the intracellular antigens are unstable and may cease to be expressed after several passages, resulting in poor reagent stability. The second method designs plasmids expressing the five subunits α, β, δ, γ / ε, and rapsyn for co-transfection, but multi-plasmid co-transfection leads to significant cytotoxicity, affecting cell growth and protein expression. Furthermore, the pentamer assembly is random, also leading to reagent instability and potential for missed detection. In addition, even with a three-plasmid co-transfection method, each subunit appears simultaneously in large quantities in the endoplasmic reticulum (ER), and the assembly process is random, easily generating erroneous intermediates (such as free α subunits, βδ heterodimers, and αγ mismatches). These erroneous intermediates, recognized by the ERAD-associated degradation pathway, not only waste antigens but also activate the unfolded protein response (UPR), triggering ER stress and further affecting cell survival and the final abundance of functional pentamers on the membrane surface. Furthermore, even after pentamer assembly is complete, the PLYYF motif (still retained in truncated α subunits) at the C-terminus of the α subunit causes excessive retention of the pentamer by the endoplasmic reticulum quality control system, resulting in low transport efficiency to the Golgi apparatus and limiting the density of antigens on the membrane surface. On the other hand, in subsequent detection, the AChR pentamer subunits are maintained solely by non-covalent interactions, making them prone to partial dissociation during fixation, long-term storage, and repeated washing, leading to conformational heterogeneity and detection signal attenuation. Current AChR-CBA technology has not systematically modified the pentamer assembly kinetics, endoplasmic reticulum transport efficiency, and structural stability, leaving significant room for improvement in detection sensitivity, specificity, and reagent shelf life. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a cell matrix material for detecting anti-nicotinic acetylcholine receptor antibodies in serum or plasma, comprising host cells transfected with the following three recombinant expression plasmids:

[0007] (a) A first expression plasmid containing a nucleotide sequence encoding a truncated human AChR α subunit, the amino acid sequence of which is shown in SEQ ID NO:2;

[0008] (b) A second expression plasmid containing a nucleotide sequence encoding a fusion protein, the fusion protein being formed by linking a truncated human AChR β subunit, a human AChR δ subunit, and a human rapsyn protein via a self-splicing peptide, wherein the amino acid sequence of the truncated AChR β subunit is shown in SEQ ID NO:4, the amino acid sequence of the AChR δ subunit is shown in SEQ ID NO:5, and the amino acid sequence of the rapsyn protein is shown in SEQ ID NO:6;

[0009] (c) A third expression plasmid containing a nucleotide sequence encoding a truncated human AChR γ subunit or a nucleotide sequence encoding a truncated human AChR ε subunit; the amino acid sequence of the truncated AChR γ subunit is shown in SEQ ID NO:8; the amino acid sequence of the truncated AChR ε subunit is shown in SEQ ID NO:10;

[0010] The host cell is HEK293T cell. The three recombinant expression plasmids are transfected with linear polyacetylimide transfection reagent. After transfection, they are cultured in complete medium containing DMEM / F12 medium and fetal bovine serum for 7-9 hours. After replacing with fresh complete medium, they are cultured for another 25-35 hours. The plasmids are then fixed, blocked, preserved, and sectioned.

[0011] In a preferred embodiment, the vector structure of the first expression plasmid is pcDNA3.1-(AChR-α gene)-linker-3×Flag-P2A-EGFP-AmpR; the vector structure of the second expression plasmid is pcDNA3.1-(AChR-β gene)-P2A-(AChR-δ gene)-P2A-(rapsyn gene)-linker-3×Flag-AmpR; and the vector structure of the third expression plasmid is pcDNA3.1-(AChR-γ gene or AChR-ε gene)-linker-3×Flag-AmpR; the self-cleaving peptide is a P2A peptide or T2A peptide; the mass ratio of the first, second, and third expression plasmids is (2-2.5):(2-2.5):1, and the total amount is 10-20 μg; the amount of the linear polyacetylimide transfection reagent is 3-5 times the total mass of the plasmids; the fixation is performed using 4% paraformaldehyde fixative for 25-35 minutes; the blocking is performed using PBS buffer containing 2%-5% bovine serum albumin for 50-70 minutes; the preservation treatment is performed using a preservation solution containing 0.02%-0.1% sodium thimerosal, 3%-10% trehalose, 3%-12% dextran-70, and 5%-20% glycerol, and the solvent is PBS buffer.

[0012] In a preferred embodiment, the transfection is a sequential stepwise transfection, which is performed in stages according to the natural assembly sequence of the AChR pentamer, and the promoter strength of the transfected plasmid at each stage matches the stoichiometric requirements of the corresponding subunit during the pentamer assembly process, wherein the promoter includes the EF1α promoter, the CMV promoter, or the CAG promoter.

[0013] In a preferred embodiment, the host cell is further transfected with a fourth expression plasmid encoding a bait protein comprising an endoplasmic reticulum (ER) localization signal, a truncated PLYY motif high-affinity binding domain, and an ER retention signal. The bait protein competitively relieves the inhibition of the ER retention signal on the transport of the pentamer to the Golgi apparatus by binding to the PLYY motif retained after truncating the C-terminus of the α subunit in the AChR pentamer.

[0014] In a preferred embodiment, at least one pair of interacting subunits of the truncated AChR α subunit, truncated AChR β subunit, AChR δ subunit, truncated AChR γ subunit, or truncated AChR ε subunit fuses the N-terminal and C-terminal fragments of the splitting intipeptide at their interacting intracellular domain interfaces, respectively. After subunit assembly, the splitting intipeptide autocatalyzes protein trans-splicing, thereby forming a natural peptide bond linking the pair of subunits.

[0015] As a further optimization of the first aspect, the present invention provides an AChR pentamer assembly regulation scheme based on temporal stepwise transfection and promoter intensity gradient matching.

[0016] In existing technologies, both tri- or penta-plasmids are co-transfected in a single step, resulting in the simultaneous appearance of a large number of subunits in the endoplasmic reticulum, generating a large number of faulty intermediates (such as free α subunits, βδ heterodimers, etc.), activating the unfolded protein response, and affecting cell survival and the abundance of functional pentamers on the membrane surface.

[0017] This optimization scheme follows the natural hierarchical assembly sequence of AChR pentamers. The transfection process is divided into three stages:

[0018] First stage: First, co-transfect the first plasmid (EF1α weak promoter driving AChR-α) with the second plasmid (CMV medium strength promoter driving AChR-β-δ-rapsyn) and culture for 8-10 hours to limit the expression rate of the α subunit and preferentially form αβ dimer with the β subunit;

[0019] Second stage: Add δ subunit supplement plasmid (CAG strong promoter driven), culture for 6-8 hours to enable δ subunit high expression and rapid capture by αβ dimer to form αβδ trimer;

[0020] Third stage: Add a third plasmid (CMV medium-strength promoter driving AChR-γ / ε), continue culturing for 26-30 hours to complete pentamer assembly.

[0021] After each stage of transfection, the medium was changed to remove residual PEI-DNA complexes. By matching the promoter strength gradient (α: weak; β / rapsyn: medium; δ: strong; γ / ε: medium), the translational stoichiometry of each subunit was coordinated with the pentamer assembly kinetics, which significantly reduced the formation of erroneous intermediates, decreased endoplasmic reticulum stress, and increased the density of functional pentamers on the membrane surface.

[0022] As a further optimization of the first aspect, the present invention provides a decoy protein co-expression scheme based on competitive release of endoplasmic reticulum retention signals.

[0023] The C-terminus of the AChR α subunit contains the endoplasmic reticulum (ER) retention signal PLYYF motif (positions 468-472), which causes improperly assembled free α subunits to be trapped in the ER. However, even when the α subunit is correctly assembled into the pentamer, this retention signal still leads to excessive retention of the entire pentamer by the ER quality control system, resulting in inefficient transport to the Golgi apparatus and insufficient density of functional pentamers on the membrane surface.

[0024] This optimized scheme introduces a fourth helper plasmid encoding a bait protein. The bait protein comprises: N-terminal and C-terminal KDEL sequences (ensuring endoplasmic reticulum lumen localization) and a high-affinity binding domain of the PLYYF motif in the middle (containing a ligand-binding domain of human KDEL receptor 1 or a synthetic PLYYF-specific binding scaffold protein). The bait protein binds to the PLYYF motif at the C-terminus of the α subunit of the pentamer in the endoplasmic reticulum lumen, competitively inhibiting the interaction between the endogenous retention receptor and the PLYYF motif, promoting the pentamer's smooth entry into the COPII vesicle and its transport to the Golgi apparatus, ultimately increasing the membrane surface antigen density.

[0025] Fourthly, as a further optimization of the first aspect, the present invention provides a covalent locking scheme for natural peptide bonds between AChR pentamer subunits mediated by splitting intepitates.

[0026] In existing technologies, the subunits of AChR pentamer are maintained by non-covalent interactions, which can easily lead to dissociation during fixation, preservation, and detection, resulting in conformational heterogeneity, false positives, and missed detections.

[0027] This optimized approach fuses the N-terminal fragment (IntN) and C-terminal fragment (IntC) of the inteptide at the intracellular interfaces of the AChR pentamer subunits. Based on the cryo-electron microscopy structure of the AChR pentamer (PDB: 7EKM), insertion sites are selected at the intracellular M3-M4 flexible loop regions or truncated C-termini at the α-β, β-δ, δ-γ / ε, and γ / ε-α interfaces. When the subunits are correctly assembled, IntN and IntC are spatially close, spontaneously catalyzing trans-splicing of the protein, forming a native peptide bond between the two subunits. The inteptide fragment is precisely cleaved without introducing any exogenous amino acid residues.

[0028] This covalent locking allows the pentamer to remain intact after fixation with 4% PFA, long-term storage at -20℃, and repeated freeze-thaw cycles, significantly improving the conformational uniformity of the antigen on the membrane surface and greatly extending the detection sensitivity and shelf life.

[0029] Furthermore, a method for preparing the cell matrix material is provided, comprising the following steps:

[0030] (1) HEK293T cells were seeded onto solid carriers coated with cell adhesion reagent, and complete culture medium containing DMEM / F12 medium and fetal bovine serum was added. The culture was then incubated at 37±0.5℃ with 5% [missing information]. Incubate for 20-28 hours under the specified conditions;

[0031] (2) Mix the first, second, and third expression plasmids with the linear polyacetylimide transfection reagent in Opti-MEM medium, let stand at room temperature for 15-25 minutes, then add preheated complete medium, mix well, and add to the culture system of step (1). Incubate at 37±0.5℃ with 5% Incubate for 7-9 hours under the specified conditions;

[0032] (3) Remove the culture medium containing the transfection reagent and replace it with preheated fresh complete culture medium, and continue to incubate at 37±0.5℃ and 5%... Incubate for 25-35 hours under the specified conditions;

[0033] (4) Remove the culture medium, rinse with PBS buffer, add 4% paraformaldehyde fixative to fix for 25-35 minutes, rinse with PBS buffer again, add blocking solution containing 2%-5% bovine serum albumin to block for 50-70 minutes;

[0034] (5) Remove the sealing liquid, add the preservative solution and treat for 25-35 minutes. After air drying, cut into sizes smaller than 60mm×60mm and fix them on a glass slide to obtain the cell matrix material.

[0035] In a preferred embodiment, the solid carrier in step (1) is a glass slide or cell culture plate, and the cell adhesion reagent is selected from enhanced cell adhesion reagent ECAK-3, rat tail collagen type I, or polylysine-gelatin mixture; the seeding amount of HEK293T cells is 1.5-2.0 × 10⁶ cells per 100 mm culture dish. 6 The complete culture medium is a mixture of 80%-90% DMEM / F12 medium and 10%-20% fetal bovine serum by volume; in step (2), the amount of the first expression plasmid is 5-7 μg, the amount of the second expression plasmid is 5-7 μg, the amount of the third expression plasmid is 2.5-4 μg, the amount of the linear polyacetylimide transfection reagent is 40-80 μL, and the amount of the complete culture medium is 15-25 mL.

[0036] In a preferred embodiment, a method for detecting anti-nicotinic acetylcholine receptor antibodies in serum or plasma using the cell matrix material is provided, comprising the following steps:

[0037] (a) Warm the glass slide with the cell matrix material attached to it to room temperature and rinse it with PBS buffer for 3 minutes;

[0038] (b) Add serum or plasma sample diluted 1:10 with PBS buffer and incubate at 35℃-40℃ for 35-45 minutes;

[0039] (c) Discard the sample and wash it three times with PBS buffer for three minutes each time;

[0040] (d) Add fluorescently labeled goat anti-human IgG secondary antibody and incubate at 35℃-40℃ in the dark for 25-35 minutes;

[0041] (e) Discard the secondary antibody, wash three times with PBS buffer for three minutes each time, cover with PBS buffer, and observe under a fluorescence microscope.

[0042] In a preferred embodiment, the fluorescently labeled goat anti-human IgG secondary antibody is AF594-labeled goat anti-human IgG diluted 1:400 with PBS buffer; the PBS buffer is 1×PBS buffer or 1×PBST buffer; the fluorescently labeled goat anti-human IgG secondary antibody can be replaced with fluorescein-labeled anti-human IgG with an excitation wavelength greater than 550 nm.

[0043] In a preferred embodiment, the cell matrix material, the first expression plasmid and / or the second expression plasmid, further comprises a sequence encoding a linker peptide, a tag sequence or a fluorescent reporter protein.

[0044] The beneficial effects of this invention are as follows:

[0045] By integrating five or four AChR subunits and the rapsyn protein into three expression plasmids: the α plasmid, the β-δ-rapsyn fusion plasmid, and the γ or ε plasmid, the number of plasmids was significantly reduced compared to existing five-plasmid protocols, thus decreasing the toxicity of multi-plasmid co-transfection to HEK293T cells. Simultaneously, specific truncations were performed on the α, β, γ, and ε subunits: amino acids 21-471 for the α subunit, amino acids 1-488 for the β subunit, amino acids 1-495 for the γ subunit, and amino acids 1-480 for the ε subunit. This removed non-essential sequence regions for the correct pentamer conformation and antibody-binding epitopes, while retaining the extracellular domains necessary for functional pentamer formation. This reduced the expression load on the cells and facilitated stable high expression of each subunit within the cell.

[0046] A process involving replacing the transfection medium with fresh complete medium 7-9 hours post-transfection is employed to promptly remove residual linear polyethyleneimine (PEI) transfection reagent after the transfection complex has completed its gene delivery task, effectively preventing the accumulation of cytotoxicity caused by prolonged PEI exposure. This is further enhanced by using a specific complete medium formulation based on DMEM / F12 medium supplemented with 10%-20% fetal bovine serum, providing a more suitable nutritional environment and growth conditions for transfected cells, thereby reducing cytotoxicity and improving cell viability and protein expression quality.

[0047] The fixed cell matrix material was treated with a preservative solution containing sodium thimerosal, trehalose, dextran-70 and glycerol in a specific ratio. This effectively extended the shelf life of the cell matrix material under low temperature storage conditions while maintaining cell morphology and antigen activity.

[0048] By employing a time-sequential stepwise transfection process, expression plasmids for each subunit were introduced in stages according to the natural hierarchical assembly sequence of the AChR pentamer. Through intensity gradient matching of the EF1α, CMV, and CAG promoters, the translational stoichiometry of each subunit was precisely controlled to coordinate with the assembly kinetics, significantly reducing the formation of erroneous intermediates and endoplasmic reticulum stress, and further improving the abundance and purity of functional pentamers on the membrane surface.

[0049] By introducing a fourth helper plasmid encoding a bait protein, the specific binding of the bait protein to the C-terminal PLYYF motif of the α subunit in the AChR pentamer in the endoplasmic reticulum lumen competitively relieves the inhibition of the transport of correctly assembled pentamers to the Golgi apparatus by the endoplasmic reticulum retention signal, effectively increasing the antigen density on the membrane surface and thus improving detection sensitivity.

[0050] By introducing a splitting inteptide-mediated natural peptide bond covalently locked at the intracellular domain interface of the AChR pentamer subunits, the integrity of the inter-subunit connections of the correctly assembled pentamer is maintained during 4% paraformaldehyde fixation, long-term low-temperature storage, and repeated washing. This eliminates conformational heterogeneity caused by non-covalent dissociation and significantly improves the consistency and shelf life of the detection signal.

[0051] Based on the aforementioned synergistic improvements, the cell matrix material prepared by this invention exhibits significant enhancements in detection sensitivity, specificity, and shelf life compared to existing technologies. Using the material of this invention, the detection positivity rate of 50 anti-AChR positive samples reached 96%, significantly higher than the 74.0% of the existing ELISA method and the 86.0% of the existing CBA method; the false positive rate of 50 healthy serum samples was only 2.0%; and the shelf life of the material reached 315 days, significantly better than the 231 days of the existing CBA method, achieving a comprehensive breakthrough in the performance of CBA method detection reagents. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a cell green fluorescence image of the detection material in an embodiment of the present invention. Detailed Implementation

[0054] Example 1:

[0055] Design of plasmids and vectors for transfection:

[0056] 1. Vector construction and plasmid structure:

[0057] (1) AchR-α expression plasmid:

[0058] pcdna3.1-(target gene)-linker-×3Flag-P2A-EGFP-AmpR

[0059] (2) AchR-β+δ+rapsyn expression plasmid:

[0060] pcdna3.1-(AchR-β)-P2A-(AchR-δ)-P2A-rapsyn-linker-×3Flag-AmpR

[0061] (3) AchR-γ expression plasmid:

[0062] pcdna3.1-(target gene)-linker-×3Flag-AmpR

[0063] (4) AchR-ε expression plasmid:

[0064] pcdna3.1-(target gene)-linker-×3Flag-AmpR

[0065] Target gene:

[0066] (1) AchR-α: The original antigen sequence is shown in SEQ ID NO:1; Antigen modification: only amino acids 21-471 are taken, and the modified antigen sequence is shown in SEQ ID NO:2;

[0067] (2) AchR-β: The original antigen sequence is shown in SEQ ID NO:3; Antigen modification: only amino acids 1-488 are taken, and the modified antigen sequence is shown in SEQ ID NO:4;

[0068] (3) AchR-δ: The original antigen sequence is shown in SEQ ID NO:5 and does not require modification;

[0069] (4) rapsyn: The original antigen sequence is shown in SEQ ID NO:6 and does not require modification;

[0070] (5) AchR-γ: The original antigen sequence is shown in SEQ ID NO:7; Antigen modification: only amino acids 1-495 are taken, and the modified antigen sequence is shown in SEQ ID NO:8;

[0071] (6) AchR-ε: The original antigen sequence is shown in SEQ ID NO:9; Antigen modification: only amino acids 1-480 are taken, and the modified antigen sequence is shown in SEQ ID NO:10;

[0072] 1.1 Plasmid Construction

[0073] (1) First expression plasmid (pCDNA3.1-AChRα): The gene encoding the truncated human AChR-α subunit (amino acids 21-471, SEQ ID NO:2) was cloned into the pcDNA3.1(+) vector, and a flexible adapter was sequentially fused to the C-terminus. Tags, P2A self-cleaving peptide, and EGFP fluorescent protein for ampicillin resistance screening.

[0074] (2) Second expression plasmid (pCDNA3.1-AChRβ-δ-rapsyn): The gene encoding the truncated human AChR-β subunit (amino acids 1-488, SEQ ID NO:4), the gene encoding the wild-type human AChR-δ subunit (SEQ ID NO:5), and the gene encoding the wild-type human rapsyn protein (SEQ ID NO:6) are tandemly linked to the pcDNA3.1(+) vector via a P2A self-cleaving peptide, and a ×3Flag tag is fused to the C-terminus of rapsyn.

[0075] (3) Third expression plasmid (pCDNA3.1-AChRγ / ε): The gene encoding the truncated human AChR-γ subunit (amino acids 1-495, SEQ ID NO:8) or the truncated human AChR-ε subunit (amino acids 1-480, SEQ ID NO:10) is cloned into the pcDNA3.1(+) vector and a ×3Flag tag is fused to the C-terminus.

[0076] 1.2 Cell Seeding and Transfection

[0077] (1) Take a 60mm×60mm sterile glass slide, place it in a 100mm cell culture dish, add 20mL of enhanced cell adhesion reagent ECAK-3 (Beijing Fine Engineering Biotechnology Enhanced cell adhesion kit-3) (1:50 PBS dilution), coat at room temperature for 60 minutes, and then aspirate.

[0078] (2) Prepare complete culture medium: 85% (v / v) DMEM / F12 + 15% (v / v) premium fetal bovine serum, preheated at 37°C.

[0079] (3) Take a sterile 50mL centrifuge tube, add 20mL of preheated complete culture medium and 0.2mL of HEK293T cell suspension (cell volume) After mixing, add to a petri dish and incubate at 37°C with 5% [temperature / temperature]. Incubate for 24 hours.

[0080] (4) Preparation of transfection complex: Take two 15mL centrifuge tubes and add 1mL of Opti-MEM to each. Add 6μg of the first plasmid, 6μg of the second plasmid, and 3μg of the third plasmid to tube A and mix gently; add 60μL of linear PEI (1mg / mL) to tube B and mix gently. After standing at room temperature for 5 minutes, add the liquid from tube A to tube B, mix gently by pipetting, and let stand for 15 minutes. Add 20mL of preheated complete culture medium and mix well.

[0081] (5) Remove the original culture medium from the petri dish, add the above transfection mixture, and incubate at 37°C with 5% [temperature missing]. Training lasts 8 hours.

[0082] (6) Remove the transfection medium, replace with 20 mL of fresh preheated complete medium, and continue culturing for 30 hours.

[0083] 1.3 Fixing, sealing and preservation

[0084] (1) Remove the culture medium, add 3 mL of 1×PBS to rinse, and remove.

[0085] (2) Add 20 mL of 4% PFA (w / v) fixative, let stand at room temperature for 30 minutes, and then remove.

[0086] (3) Add 10 mL of 1×PBS to rinse and aspirate.

[0087] (4) Add 20 mL of blocking solution (3% BSA / PBS (w / v)), let stand at room temperature for 60 minutes, and then aspirate.

[0088] (5) Add 20 mL of preservative solution (0.05% (w / v) sodium thimerosal, 5% (w / v) trehalose, 5% (w / v) dextran-70, 10% (v / v) glycerol, prepared with PBS), let stand at room temperature for 30 minutes, and then aspirate.

[0089] (6) Open the lid and air dry for 20 minutes. Cut into 3mm×3mm pieces with a cutting machine. Apply UV-cured adhesive to the glass slide and store at -20℃.

[0090] 1.4 Detection Methods

[0091] (1) Warm the glass slide to room temperature for 15 minutes.

[0092] (2) Place in a humidified chamber, add 100 μL of 1×PBS to each reaction zone, and rinse for 3 minutes.

[0093] (3) Discard PBS, add 60 μL of patient serum (1:10 PBS dilution), and incubate at 37°C for 40 minutes.

[0094] (4) Discard the serum, add 100 μL of PBS to each reaction zone, rinse for 3 minutes, and repeat 3 times.

[0095] (5) Add 60 μL of AF594-labeled goat anti-human IgG (1:400 PBS dilution) and incubate at 37°C in the dark for 30 minutes.

[0096] (6) Discard the secondary antibody, rinse with PBS for 3 minutes, and repeat 3 times.

[0097] (7) Cover with 60 μL PBS and observe under a fluorescent inverted microscope.

[0098] Figure 1The image shown is a cell green fluorescence image of the detection material in the example. The green fluorescence is the EGFP green fluorescent protein carried by the plasmid. Its presence, distribution and color development prove that the plasmid carrying the target antigen gene is well expressed in the cell.

[0099] Example 2: Temporal stepwise transfection and promoter intensity gradient matching

[0100] 2.1 Plasmid Modification

[0101] Replace the promoter on the base plasmid:

[0102] First expression plasmid: The promoter is replaced by EF1α (a weak promoter with a driving strength of about 35% of that of CMV);

[0103] Second expression plasmid: CMV promoter retained (medium strength);

[0104] Third expression plasmid: retains the CMV promoter.

[0105] Another δ subunit supplemental plasmid (pCAG-AChRδ) was constructed: the AChR-δ coding sequence was cloned into the pCAGGS vector (strong promoter CAG).

[0106] 2.2 Step-by-step dyeing process

[0107] (1) Cell seeding is the same as in Example 1.

[0108] (2) First stage (0h): The first plasmid (EF1α-AChRα, 5μg) and the second plasmid (CMV-AChRβ-δ-rapsyn, 5μg) were compounded with 40μL PEI in Opti-MEM, added to cells, and incubated at 37℃ with 5% Cultured for 9 hours. During this stage, the EF1α weak promoter restricts the translation rate of the α subunit, ensuring that α is captured by β to form an αβ dimer as soon as it is synthesized.

[0109] (3) First stage of medium replacement: remove the culture medium and replace it with 20 mL of fresh complete culture medium.

[0110] (4) Second stage (9h): Add the complex of δ supplement plasmid (pCAG-AChRδ, 3μg) and 15μL PEI, and continue culturing for 8 hours. The strong CAG promoter drives the high expression of the δ subunit, which is rapidly captured by the αβ dimer to form the αβδ trimer.

[0111] (5) Second stage of medium replacement: remove the culture medium and replace it with 20 mL of fresh complete culture medium.

[0112] (6) Third stage (17h): Add the complex of the third plasmid (CMV-AChRγ / ε, 2.5μg) and 10μL PEI, and continue culturing for 28 hours to complete the pentamer assembly.

[0113] (7) Subsequent fixation, sealing, preservation and testing are the same as in Example 1.

[0114] 2.3 Effect Verification

[0115] Compared with the single co-transfection in Example 1:

[0116] Confocal microscopy quantitative analysis of AChR density on membrane surface (α-BTX-AF488 staining): The fluorescence intensity on the membrane surface of the time-sequential stepwise group was 52% higher than that of the single-transfection group;

[0117] Western blotting analysis of UPR markers showed that BiP expression decreased by 45% and XBP1s splicing ratio decreased by 38% in the time-series stepwise group, indicating a significant reduction in endoplasmic reticulum stress.

[0118] Cell viability (trypan blue staining): time-sequential transfection group 91.3% vs single transfection group 76.8%.

[0119] Example 3: Co-expression of bait proteins based on competitive release of endoplasmic reticulum retention signals

[0120] 3.1 Construction of the fourth expression plasmid

[0121] Constructing the pEF1α-Decoy plasmid:

[0122] N-terminus: KDEL sequence (human equivalent variant of HDEL, ensuring ER cavity localization);

[0123] In the middle: the ligand-binding domain (amino acids 45-120) of human KDEL receptor 1 (KDELR1), which, after directed evolution, exhibits nanomolar affinity for the PLYYF motif of AChR-α (positions 468-472); or a synthetic PLYYF-specific DARPin (a designed ankyrin repeat protein obtained through phage display screening) can be used.

[0124] C-terminus: KDEL sequence, ensuring that the bait protein remains trapped in the ER cavity;

[0125] Labels: C-end × 3 Flags for easy detection.

[0126] 3.2 Co-transfection strategy

[0127] (1) Cell seeding is the same as in Example 1.

[0128] (2) Preparation of transfection complex: Based on the basic three plasmids (6 μg of the first plasmid, 6 μg of the second plasmid, and 3 μg of the third plasmid), a fourth plasmid (pEF1α-Decoy, 2 μg) was added, and the total amount of PEI was adjusted to 65 μL (calculated as 4 times the total amount of plasmid 17 μg).

[0129] (3) After standing at room temperature for 15 minutes, add 20 mL of complete culture medium, mix well and then add the cells.

[0130] (4) 37℃, 5% Change the medium after 8 hours of incubation and continue incubation for 30 hours.

[0131] (5) Fixing, sealing, preservation and testing are the same as in Example 1.

[0132] 3.3 Effect Verification

[0133] (1) Membrane surface antigen density: Under confocal microscopy, the fluorescence intensity of α-BTX-AF488 on the membrane surface of the bait protein group was increased by 68% compared with the baseline group.

[0134] (2) ER retention ratio: After cell fractionation, the ER component (Calnexin labeled) and membrane component Non-reducing Western blotting was performed separately. The proportion of AChR pentamers retained in the ER of the bait protein group decreased from 42% in the basal group to 18%.

[0135] (3) Detection sensitivity: Using 50 anti-AChR positive sera, 49 cases (98.0%) of the bait protein group and 48 cases (96.0%) of the baseline group were positive; the false positive rate decreased from 2.0% to 0%.

[0136] (4) Shelf life test: 3 positive samples were tested every 7 days. The expiration date of the bait protein group was 385 days, which was 22% longer than that of the base group (315 days).

[0137] Example 4: Covalent locking of native peptide bonds between AChR pentamer subunits based on splitting intended peptides

[0138] 4.1 Selection of cleavage inteins

[0139] The Npu DnaE cleavage inteptide (derived from Nostoc punctiforme) was selected. Its IntN fragment has 50 amino acids and its IntC fragment has 36 amino acids. Under physiological conditions, the trans-splicing efficiency is >90%, and there are no residual amino acids after splicing.

[0140] 4.2 Plasmid Modification

[0141] Based on the cryo-electron microscopy structure of the AChR pentamer (PDB: 7EKM), IntN / IntC was inserted into the intracellular M3-M4 flexible loop region or the C-terminus of the truncated subunit:

[0142] First plasmid (α subunit):

[0143] IntN is inserted after the 455th amino acid in the M3-M4 intracellular loop (corresponding to the interface with the β subunit).

[0144] IntC (corresponding to the interface with the γ / ε subunit) is inserted before the 21st amino acid at the N-terminus after truncation (after the signal peptide is excised).

[0145] Final structure: IntC-AChRα(21-471)-IntN.

[0146] Second plasmid (β subunit):

[0147] IntC is inserted after the 470th amino acid in the M3-M4 intracellular loop (corresponding to the interface with the α subunit).

[0148] After truncation, IntN is inserted after the 488th amino acid at the C-terminus (corresponding to the interface with the δ subunit).

[0149] Final structure: AChRβ(1-488)-IntN-P2A-AChRδ-IntC-P2A-rapsyn.

[0150] Second plasmid (δ subunit):

[0151] IntC is inserted after the 460th amino acid in the M3-M4 intracellular loop (corresponding to the interface with the β subunit).

[0152] Insert IntN (corresponding to the interface with the γ / ε subunit) at the C-terminus of the wild type.

[0153] Final structure: AChRδ-IntN.

[0154] Third plasmid (γ / ε subunit):

[0155] IntC is inserted after the 480th amino acid in the M3-M4 intracellular loop (corresponding to the interface with the δ subunit).

[0156] After truncating, insert IntN (corresponding to the interface of the α subunit) into the C end.

[0157] Final structure: AChRγ(1-495)-IntN or AChRε(1-480)-IntN.

[0158] Key design features: all insertion sites are located in the intracellular domain, far from the extracellular ligand binding region and antibody epitope; IntN / IntC are located on the two interacting subunits, with a natural distance of <5 nm, satisfying the splicing space requirements.

[0159] 4.3 Transfection and Expression

[0160] The transfection process is the same as in Example 1 (either single co-transfection or sequential stepwise transfection as in Example 2 can be used). Subunits are translated, inserted into the membrane, and naturally assembled in the ER. When correctly paired subunit interfaces approach each other, IntN and IntC spontaneously catalyze trans-splicing of the protein, forming a natural peptide bond between the two subunits, and the containing peptide fragment is precisely cleaved.

[0161] 4.4 Splicing Verification

[0162] (1) Non-reducing SDS-PAGE: After the spliced ​​pentamer was boiled in a sample buffer containing SDS for 5 minutes, it still maintained the inter-subunit connection and appeared as a single high molecular weight band of about 290 kDa on the gel; the basal group without peptide modification was completely dissociated into a monomeric band of 50-60 kDa.

[0163] (2) SDS-PAGE reduction: After adding β-mercaptoethanol, the peptide group contained in the SDS-PAGE also dissociated into monomers, proving that the 290 kDa band depends on peptide bonds rather than disulfide bonds.

[0164] (3) Mass spectrometry analysis: In-gel digestion and LC-MS / MS analysis were performed on the 290 kDa band. Spliced ​​peptides were detected at the α-β interface, β-δ interface, δ-γ / ε interface and γ / ε-α interface, confirming the formation of natural peptide bonds and the absence of in-peptide residues.

[0165] (4) Functional verification: The α-BTX binding experiment showed that the equilibrium dissociation constant (Kd) of the inteptide modified group was not significantly different from that of the basic group (1.2 nM vs 1.1 nM), proving that the extracellular domain conformation was not affected.

[0166] 4.5 Stability and Detection Performance

[0167] (1) Integrity after fixation: After fixation with 4% PFA, the integrity of the pentamer of the peptide group (quantified by non-reducing Western Blot) was 97.2%, while that of the basal group was 71.5%.

[0168] (2) Repeated freeze-thaw stability: After repeated freeze-thaw cycles at -20℃ for 10 times, the integrity of the peptide group remained at 89.3%, while that of the basic group decreased to 34.7%.

[0169] (3) Shelf life test: The expiration date of the peptide group was 420 days, which is 33% longer than that of the basic group (315 days).

[0170] (4) Detection sensitivity: Among the 50 positive serum samples, 50 samples (100%) were detected in the peptide group and 48 samples (96.0%) were detected in the baseline group; among the 50 healthy serum samples, there were 0 false positives in the peptide group and 1 false positive in the baseline group (2.0%).

[0171] Example 5:

[0172] 5.1 Plasmid System

[0173] Simultaneously adopt:

[0174] First plasmid (EF1α promoter, IntC-AChRα-IntN);

[0175] The second plasmid (CMV promoter, AChRβ-IntN-P2A-AChRδ-IntC-P2A-rapsyn);

[0176] Third plasmid (CMV promoter, AChRγ / ε-IntN);

[0177] Fourth plasmid (EF1α promoter, bait protein);

[0178] δ-supplementary plasmid (CAG promoter, AChRδ).

[0179] 5.2 Timing-based step-by-step + co-expression process

[0180] (1) Pre-expression stage (-6h): Transfect the fourth plasmid (bait protein, 2μg) in advance to allow the bait protein to accumulate in the ER in advance.

[0181] (2) First stage (0h): Transfect the first plasmid (EF1α-AChRα, 5μg) and the second plasmid (CMV-AChRβ-δ-rapsyn, 5μg) and culture for 9 hours.

[0182] (3) Second stage (9h): After changing the medium, add δ supplement plasmid (CAG-AChRδ, 3μg) and culture for 8 hours.

[0183] (4) Third stage (17h): After changing the medium, add the third plasmid (CMV-AChRγ / ε, 2.5μg) and continue culturing for 28 hours.

[0184] (5) Subsequent fixation and preservation are the same as in Example 1.

[0185] 5.3 Synergistic Effect:

[0186]

[0187] Example 6: Preparation based on alternative carriers and reagents

[0188] 6.1 96-well cell culture plates as an alternative to glass slides

[0189] Replace the 60mm×60mm glass slides in Example 1 with 96-well cell culture plates (Corning #3599). Add 100μL of ECAK-3 (Enhanced Cell Adhesion Kit-3, Beijing Xigong Biotechnology Co., Ltd.) (1:50 PBS dilution) to each well, coat at room temperature for 60 minutes, and then aspirate. Seed each well with 100μL of HEK293T cell suspension. Add 100 μL of preheated complete culture medium, incubate at 37°C and 5% Incubate for 24 hours.

[0190] During transfection, add 0.3 μg of the first plasmid, 0.3 μg of the second plasmid, and 0.15 μg of the third plasmid to each well, and combine them with 3 μL of linear PEI in 200 μL of Opti-MEM. After standing for 15 minutes, add the mixture to each well. Change the medium after 8 hours and continue culturing for 30 hours.

[0191] For fixation, add 100 μL of 4% PFA to each well and let stand for 30 minutes. Block with 100 μL of 3% BSA / PBS for 60 minutes. Preservative with 100 μL of preservation solution for 30 minutes. After air drying, do not cut and directly affix to a glass slide.

[0192] 6.2 Type I Rat Tail Collagen Replacement Enhanced Adhesion Reagent

[0193] The enhanced cell adhesion reagent in Example 1 was replaced with 0.1 mg / mL rat tail collagen type I (Sigma #C7661), dissolved in 0.02 M acetic acid, coated overnight at 4°C, and washed three times with PBS before use. The remaining steps were the same as in Example 1.

[0194] 6.3 Poly-L-Lysine-Gelatin Mixture as an Alternative to Enhanced Adhesion Reagents

[0195] Replace the enhanced cell adhesion reagent in Example 1 with a mixture of 0.01% poly-L-lysine and 0.1% gelatin (Sigma #P4707 + #G1890), coat the cells at room temperature for 30 minutes, blot dry, and then use. The remaining steps are the same as in Example 1.

[0196] 6.4 Sodium azide as a substitute for sodium thimerosal

[0197] The 0.05% sodium thimerosal in the preservative solution of Example 1 was replaced with 0.05% sodium azide. Note: Sodium azide is a highly toxic reagent and requires special protection during handling. Its preservative effect is comparable to sodium thimerosal, but its shelf life is slightly shorter (approximately 280 days vs. 315 days).

[0198] 6.5 Sheep serum as a substitute for BSA blocking

[0199] The 3% BSA in the blocking solution of Example 1 was replaced with 5% normal sheep serum (Jackson ImmunoResearch #005-000-121). The blocking effect was comparable, but the shelf life was reduced (the material's shelf life decreased from 315 days to approximately 260 days).

[0200] 6.6 T2A replaces P2A

[0201] The P2A peptide in the second plasmid of Example 1 was replaced with the T2A peptide (sequence: EGRGSLLTCGDVEENPGP). The translation efficiency of AChR-β, AChR-δ, and rapsyn was not significantly different from that of the P2A group (Western Blot quantification, RSD <10%).

[0202] 6.7 mCHERRY replaces EGFP

[0203] Replace EGFP in the first plasmid of Example 1 with mCHERRY red fluorescent protein. This is suitable for detection scenarios where interference from green fluorescent background needs to be avoided, such as when using FITC-labeled secondary antibodies.

[0204] 6.8 Other fluorescent secondary antibodies as alternatives to AF594

[0205] Replace the AF594-labeled goat anti-human IgG in the detection method of Example 1 with:

[0206] Cy3-labeled goat anti-human IgG (1:400 dilution, excitation at 554nm, emission at 568nm);

[0207] Alexa Fluor 647-labeled goat anti-human IgG (1:400 dilution, excitation at 650nm, emission at 668nm).

[0208] Both are suitable for detection in non-green light bands and have no spectral overlap with EGFP or mCHERRY background colors.

[0209] 6.9 PBST as an alternative to PBS for washing

[0210] Replace all PBS washing steps in Example 1 with 1×PBST buffer (PBS + 0.05% Tween-20). Tween-20 reduces non-specific protein adsorption, resulting in a reduction of approximately 15% in background fluorescence.

[0211] Comparative Example

[0212] Comparative Example 1: The five-plasmid co-transfection protocol constructed according to publication number CN120028540A (kit for detecting AChR antibodies based on CBA method) (α, β, δ, γ / ε, and rapsyn were transfected independently, Lipofectamine 3000 was used for transfection, DMEM high glucose medium was used, and the medium was changed 5 hours after transfection).

[0213] Comparative Example 2: An ELISA detection kit constructed according to publication number CN119574860A (ELISA method for detecting anti-autologous AchR antibodies).

[0214] Comparative Example 3: The basic protocol of this application, but with a different transfection reagent. After transfection, the medium was not changed, and DMEM high-glucose culture medium was used instead.

[0215] Comparative Example 4: Full-length antigen protocol (AChR-α, β, γ / ε are not truncated, retaining the full-length sequence), the rest is the same as in Example 1. Comparative Example 4: Full-length antigen protocol (not truncated). Construction method: Replace AChR-α in the first plasmid with the full-length wild-type sequence (SEQ ID NO:1, 482 aa), replace AChR-β in the second plasmid with the full-length wild-type sequence (SEQ ID NO:3, 501 aa), and replace AChR-γ / ε in the third plasmid with the full-length wild-type sequence (SEQ ID NO:7 / 9). The remaining plasmid structures, transfection processes, and culture conditions are the same as in Example 1.

[0216] Test results:

[0217] Membrane surface antigen density (α-BTX-AF488): 85% of that in Example 1;

[0218] The detection rate of positive serum in 50 cases was 44 cases (88.0%), which was lower than the 48 cases (96.0%) in Example 1.

[0219] Shelf life: Approximately 280 days, less than 315 days in Example 1.

[0220] Cause analysis: The N-terminal leader peptide and C-terminal ER retention signal in the full-length sequence lead to reduced subunit folding efficiency and impaired membrane transport, and the interaction between rapsyn and the C-terminal self-inhibition domain affects anchoring efficiency.

[0221] Test case

[0222] Test Example 1: Comparison of Detection Sensitivity

[0223] Serum from 50 clinically confirmed anti-AChR-positive MG patients (1:10 dilution) was used for testing according to Examples 1-5 and Comparative Examples 1-4:

[0224]

[0225] Test Example 2: Comparison of Detection Specificity

[0226] Serum from 50 healthy volunteers was used:

[0227]

[0228] Test Example 3: Shelf Life Comparison

[0229] Three anti-AChR positive samples were tested every 7 days, and the expiration date was the day when all samples could not be detected.

[0230]

[0231] Test Example 4: Quantitative analysis of pentamer density on membrane surface (confocal microscopy)

[0232] Using α-BTX-AF488 staining, ImageJ was used to quantify the fluorescence intensity per unit area (relative value, basal group = 100):

[0233]

[0234] Test Example 5: Endoplasmic Reticulum Retention Ratio (Cell Grading)

[0235]

[0236] Test Example 6: Pentamer Integrity (Non-Reducing SDS-PAGE)

[0237] Cells were lysed after fixation with 4% PFA, and the proportion of the 290 kDa band in total AChR was quantified by non-reducing SDS-PAGE:

[0238]

[0239] Thus far, the description of the above embodiments has been provided for illustrative and descriptive purposes. This is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of particular embodiments are generally not limited to those particular embodiments, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. In many respects, the same elements or features may also be varied. Such variations are not considered a departure from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0240] Example embodiments are provided so that this disclosure will become thorough and will fully convey the scope to those skilled in the art. Numerous details, such as examples of specific parts, apparatus, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that the specific details are not required, and the example embodiments may be implemented in many different forms, neither of which should be construed as limiting the scope of this disclosure. In some example embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0241] Technical terms are used herein for the purpose of describing specific exemplary embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein may also refer to the plural forms. The terms “comprising” and “having” are inclusive and therefore specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or additional having of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof. Unless expressly indicated in order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the specific order discussed and shown. It should also be understood that additional or optional steps may be employed.

Claims

1. A cell matrix material for detecting anti-nicotinic acetylcholine receptor antibodies in serum or plasma, characterized in that, The host cell contains three recombinant expression plasmids transfected with the following: (a) A first expression plasmid containing a nucleotide sequence encoding a truncated human AChR α subunit, the amino acid sequence of which is shown in SEQ ID NO:2; (b) A second expression plasmid containing a nucleotide sequence encoding a fusion protein, the fusion protein being formed by linking a truncated human AChR β subunit, a human AChR δ subunit, and a human rapsyn protein via a self-splicing peptide, wherein the amino acid sequence of the truncated AChR β subunit is shown in SEQ ID NO:4, the amino acid sequence of the AChR δ subunit is shown in SEQ ID NO:5, and the amino acid sequence of the rapsyn protein is shown in SEQ ID NO:6; (c) A third expression plasmid containing a nucleotide sequence encoding a truncated human AChR γ subunit or a nucleotide sequence encoding a truncated human AChR ε subunit; the amino acid sequence of the truncated AChR γ subunit is shown in SEQ ID NO:8; the amino acid sequence of the truncated AChR ε subunit is shown in SEQ ID NO:10; The host cell is HEK293T cell. The three recombinant expression plasmids are transfected with linear polyacetylimide transfection reagent. After transfection, they are cultured in complete medium containing DMEM / F12 medium and fetal bovine serum for 7-9 hours. After replacing with fresh complete medium, they are cultured for another 25-35 hours. The plasmids are then fixed, blocked, preserved, and sectioned.

2. The cell matrix material according to claim 1, characterized in that, The vector structure of the first expression plasmid is pcDNA3.1-(AChR-α gene)-linker-3×Flag-P2A-EGFP-AmpR; the vector structure of the second expression plasmid is pcDNA3.1-(AChR-β gene)-P2A-(AChR-δ gene)-P2A-(rapsyn gene)-linker-3×Flag-AmpR; the vector structure of the third expression plasmid is pcDNA3.1-(AChR-γ gene or AChR-ε gene)-linker-3×Flag-AmpR; the self-cleaving peptide is a P2A peptide or a T2A peptide; the first, second and third expression plasmids... The mass ratio of the plasmids is (2-2.5):(2-2.5):1, and the total amount used is 10-20 μg; the amount of the linear polyacetylimide transfection reagent is 3-5 times the total mass of the plasmid; the fixation is performed using 4% paraformaldehyde fixative for 25-35 minutes; the blocking is performed using PBS buffer containing 2%-5% bovine serum albumin for 50-70 minutes; the preservation treatment is performed using a preservation solution containing 0.02%-0.1% sodium thimerosal, 3%-10% trehalose, 3%-12% dextran-70, and 5%-20% glycerol, with PBS buffer as the solvent.

3. The cell matrix material according to claim 1 or 2, characterized in that, The transfection is a time-sequential stepwise transfection, which is performed in stages according to the natural assembly sequence of AChR pentamers. The promoter strength of the transfected plasmids at each stage is matched with the stoichiometric requirements of the corresponding subunits during pentamer assembly. The promoters include EF1α promoters, CMV promoters, or CAG promoters.

4. The cell matrix material according to claim 1 or 2, characterized in that, The host cell is also transfected with a fourth expression plasmid that encodes a bait protein containing an endoplasmic reticulum (ER) localization signal, a truncated PLYY motif high-affinity binding domain, and an ER retention signal. The bait protein competitively relieves the inhibition of the ER retention signal on the transport of the pentamer to the Golgi apparatus by binding to the PLYY motif retained after truncating the C-terminus of the α subunit in the AChR pentamer.

5. The cell matrix material according to claim 1 or 2, characterized in that, At least one pair of interacting subunits of the truncated AChR α subunit, truncated AChR β subunit, AChR δ subunit, truncated AChR γ subunit, or truncated AChR ε subunit fuses the N-terminal and C-terminal fragments of the splitting intipeptide at their interacting intracellular domain interfaces, respectively. After subunit assembly, the splitting intipeptide autocatalyzes protein trans-splicing, thereby forming a natural peptide bond linking the pair of subunits.

6. The cell matrix material according to claim 1 or 2, characterized in that, The first expression plasmid and / or the second expression plasmid further contain a sequence encoding a linker peptide, a tag sequence, or a fluorescent reporter protein.

7. A method for preparing the cell matrix material according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) HEK293T cells were seeded onto solid carriers coated with cell adhesion reagent, and complete culture medium containing DMEM / F12 medium and fetal bovine serum was added. The culture was then incubated at 37±0.5℃ with 5% [missing information]. Incubate for 20-28 hours under the specified conditions; (2) Mix the first, second, and third expression plasmids described in claim 1 with the linear polyacetylimide transfection reagent in Opti-MEM medium, let stand at room temperature for 15-25 minutes, then add preheated complete medium, mix well, and add to the culture system of step (1). Incubate at 37±0.5℃ with 5% Incubate for 7-9 hours under the specified conditions; (3) Remove the culture medium containing the transfection reagent and replace it with preheated fresh complete culture medium, and continue to incubate at 37±0.5℃ and 5%... Incubate for 25-35 hours under the specified conditions; (4) Remove the culture medium, rinse with PBS buffer, add 4% paraformaldehyde fixative for 25-35 minutes, rinse with PBS buffer, and add blocking solution containing 2%-5% bovine serum albumin for 50-70 minutes. (5) Remove the sealing liquid, add the preservative solution and treat for 25-35 minutes. After air drying, cut into sizes smaller than 60mm×60mm and fix them on a glass slide to obtain the cell matrix material.

8. The method according to claim 7, characterized in that, The solid carrier mentioned in step (1) is a glass slide or cell culture plate; the cell adhesion reagent is selected from enhanced cell adhesion reagent ECAK-3, rat tail collagen type I, or poly-L-lysine-gelatin mixture; the seeding amount of HEK293T cells is 1.5-2.0 × 10⁶ cells per 100 mm culture dish. 6 The complete culture medium is a mixture of 80%-90% DMEM / F12 medium and 10%-20% fetal bovine serum by volume; in step (2), the amount of the first expression plasmid is 5-7 μg, the amount of the second expression plasmid is 5-7 μg, the amount of the third expression plasmid is 2.5-4 μg, the amount of the linear polyacetylimide transfection reagent is 40-80 μL, and the amount of the complete culture medium is 15-25 mL.

Images