A recombinant protein and its application in detecting anti-acetylcholine receptor cluster antibodies using live cell immunofluorescence method

Abstract

The present invention discloses a recombinant protein and its application in detecting anti-acetylcholine receptor cluster antibodies by using a live cell immunofluorescence method, belonging to the field of biomedical engineering technology. The present invention intercepts partial sequences from AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit (or AChRγ subunit) for redesign and fusion expression, so that the fusion-expressed protein can be stably overexpressed on the cell membrane of eukaryotic cells, and can meet clinical detection requirements when using a live cell immunofluorescence method (LCBA method) to detect anti-acetylcholine receptor cluster (AChR) antibodies.

Description

Technical Field

[0001] The present invention relates to a recombinant protein and its application in detecting anti-acetylcholine receptor cluster (AChR cluster) antibodies by live cell immunofluorescence method, and specifically relates to a recombinant protein in which partial sequences of AChRα1 subunit, AChRβ subunit, AChRδ subunit, AChRε subunit / AChRγ subunit are fused and expressed on the cell membrane of eukaryotic cells, and a process of detecting anti-AChR cluster antibodies in live cell immunofluorescence method (Live Cell Based Assay, LCBA method) by overexpressing the recombinant protein on the cell membrane, and belongs to the field of biomedical engineering technology. Background Art

[0002] Myasthenia Gravis (MG) is a disease affecting the motor system caused by autoimmune production of antibodies that attack the neuromuscular junction (NMJ), resulting in decreased neuromuscular transmission in voluntary muscles. Its pathophysiology is well understood; most autoantibodies have been identified and can be detected, and are usually directly pathogenic. Characteristic symptoms associated with this disease process include muscle weakness on exercise or repetitive contractions.

[0003] The acetylcholine receptor (AChR) is a sodium ion channel located at the postsynaptic terminal of the neuromuscular junction. Under normal circumstances, acetylcholine (Ach) plays an important role in nerve signal transmission. After being released from nerve endings, it binds to AChR on muscle cells, causing the sodium ion channel to open, thereby causing muscle contraction. However, when antibodies against AChR are present, they bind to the receptor and accelerate the flipping of the receptor channel. These antibodies also prevent acetylcholine from binding to the receptor. If these antibodies bind complement, they attract phagocytes (such as macrophages) and cause the destruction of the neuromuscular junction. This destruction leads to the loss of the folded structure of the junction, which originally provided a spatial advantage that made acetylcholine more effective when binding to the receptor.

[0004] AChR is a glycoprotein with a molecular weight of about 250 kDa. It is composed of five subunits arranged in a barrel-shaped rod around a central channel through the sarcolemmal protrusions. In the intracellular part, it binds to the synaptic receptor associated protein (Rapsyn) to form a stable and correct protein structure. Rapsyn is a 43-kDa peripheral membrane protein of skeletal muscle, which is essential for clustering nicotinic acetylcholine receptors (nAChR) on the postsynaptic membrane. AChR is divided into several subtypes, among which the adult AChR is composed of two α subunits and one each of β, δ, and ε subunits, and the ε subunit of the fetal type is replaced by the γ subunit. The α subunit of AChR (AChRα) contains both the acetylcholine binding site and the main epitopes recognized by self-reactive T cells and autoantibodies in MG patients. In addition, the remaining subunits also contain some antibody recognition epitopes.

[0005] At present, the detection of anti-AChR cluster antibodies mainly adopts radioimmunoprecipitation (RIPA) and enzyme-linked immunosorbent assay (ELISA). 125 I-α-snake toxin is mixed and incubated with the test serum, and then the second antibody is added for precipitation. 125 I-α-snake toxin-AChR-Abs complex, the precipitate is counted and compared with the precipitate using healthy control serum. The sensitivity of this test is high in systemic MG (over 85%) and low in ocular MG. RIPA has become the reference for measuring anti-AChR antibodies, and to ensure that a single test can cover both anti-adult and anti-infant antibodies, both antigens should be included in the antigen preparation. Due to the complexity of the RIPA method and the laboratory configuration required, which is not available in many clinical testing institutions, ELISA is often chosen to replace some AChR antibody detection.

[0006] The ELISA method is to add the serum to be tested to the wells coated with AChR antigen, then add the secondary antibody labeled with alkaline phosphatase or horseradish peroxidase, and after washing, add the colorimetric agent. The advantage of this method over the RIPA method is that it does not require the handling of radioactive isotopes. However, the use of ELISA technology and RIPA technology does not always produce the same results. It was found in previously published literature that compared with the RIPA method, the ELISA method has a certain proportion of false negative rate and false positive rate.

[0007] With further research on AChR antibodies, it was found that in 80%-85% of patients with systemic MG (GMG), antibodies directly target acetylcholine receptors (AChR), and a small number of patients (5%-8%) have antibodies against muscle-specific kinase (MuSK). The remaining patients (about 10%) are called "seronegative" (SN) MG. These antibodies cannot be detected by classic RIPA and ELISA techniques, but can be detected by cell-based immunofluorescence (CBA) on cells transfected with AChR subunits and Rapsyn expression vectors, which is called AChR CBA detection. The CBA method requires that an antigen protein with specific binding ability to the antibody to be tested be overexpressed in cells to construct a specific cell line expressing the recombinant protein.

[0008] For CBA detection of AChR, fixed cell based assay (FCBA) or live cell based assay (LCBA) can be used. The LCBA method has higher sensitivity and can detect AChR antibodies in some SNMG patients. However, the difficulty lies in that this method currently requires the co-transfection of the five subunits of AChR (α, β, δ, ε / γ) and Rapsyn. It is extremely difficult to obtain a cell line that stably overexpresses the co-transgenic gene. If plasmid-mediated transient transfection is used, the probability of six plasmids being simultaneously transfected and highly expressed in the same cell is low, and there may be large differences in their expression levels. There may be a phenomenon that one subunit is expressed at a high level while another subunit is expressed at a low level. In addition, the LCBA method has high requirements for the cell state. Transiently transfected cells are often unhealthy or dead due to the toxicity of the transfection reagent and the transiently highly expressed protein. The effect is not good when the LCBA method is used. When testing clinical MG samples, the diversity of patient antibodies reduces the detection sensitivity, resulting in a low detection rate.

[0009] Prior art literature:

[0010] Chinese patent CN113651881A;

[0011] Chinese patent CN113687067A.

[0012] In summary, the existing technology cannot achieve the expression of the five subunits of AChR (α, β, δ, ε / γ) on the cell membrane at the same time in a structure and copolymer form including the correct antigenic domain part, and can be used for detecting the level of anti-AChR cluster antibodies in human biological samples with high sensitivity and accuracy. Summary of the invention

[0013] The purpose of the present invention is to provide a recombinant protein and its application in the detection of anti-acetylcholine receptor cluster antibodies by live cell immunofluorescence method (LCBA method). Partial sequences are intercepted from AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit (or AChRγ subunit) for redesign and fusion expression, and there is no need to co-transfect with Rapsyn. The fusion expressed protein can be stably overexpressed on the cell membrane of eukaryotic cells, and can meet clinical detection requirements when anti-acetylcholine receptor cluster (AChR) antibodies are detected by LCBA method.

[0014] The present invention is achieved through the following technical solutions:

[0015] A recombinant protein, wherein two recombinant proteins with different structures are designed for the two isomers of adult type (AChRε) and infant type (AChRγ), namely, adult type AChR recombinant protein and infant type AChR recombinant protein, which are as follows:

[0016] The partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit are fused to obtain an adult AChR recombinant protein as shown in SEQ ID NO: 1.

[0017] Alternatively, partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRγ subunit are fused to construct an infant AChR recombinant protein with a sequence number as shown in SEQ ID NO: 2.

[0018] The partial sequences of the above-mentioned AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit (or AChRγ subunit) are respectively extracted from the amino acid sequences of their specific domains, as follows:

[0019] The partial sequence of the AChRα1 subunit includes the partial sequence of the AChRα1 subunit containing a signal peptide as shown in SEQ ID NO: 3, and the partial sequence of the AChRα1 subunit without a signal peptide as shown in SEQ ID NO: 4;

[0020] The partial sequence of the AChRβ1 subunit is shown in SEQ ID NO: 5;

[0021] The partial sequence of the AChRδ subunit is shown in SEQ ID NO: 6;

[0022] The partial sequence of the AChRε subunit is shown in SEQ ID NO: 7;

[0023] Or the partial sequence of AChRγ subunit is shown in SEQ ID NO:8.

[0024] Furthermore, the connecting peptide can be used to sequentially connect and fuse the partial sequence of the AChRα1 subunit containing the signal peptide, the partial sequence of the AChRβ1 subunit, the partial sequence of the AChRδ subunit, the partial sequence of the AChRα1 subunit without the signal peptide, the partial sequence of the AChRε subunit, the transmembrane sequence and the fluorescent tag to construct an adult AChR recombinant protein.

[0025] Alternatively, using a connecting peptide, the partial sequence of the AChRα1 subunit containing a signal peptide, the partial sequence of the AChRβ1 subunit, the partial sequence of the AChRδ subunit, the partial sequence of the AChRα1 subunit without a signal peptide, the partial sequence of the AChRγ subunit, the transmembrane sequence and the fluorescent tag are sequentially connected and fused to construct an infant AChR recombinant protein.

[0026] The connecting peptides used in constructing the above-mentioned adult AChR recombinant protein or infant AChR recombinant protein specifically include:

[0027] Connecting peptide A: used to connect the partial sequence of the AChRα1 subunit containing a signal peptide and the partial sequence of the AChRβ1 subunit, connect the partial sequence of the AChRβ1 subunit and the partial sequence of the AChRδ subunit, connect the partial sequence of the AChRδ subunit and the partial sequence of the AChRα1 subunit without a signal peptide, connect the partial sequence of the AChRα1 subunit without a signal peptide and the partial sequence of the AChRε subunit, and connect the partial sequence of the AChRα1 subunit without a signal peptide and the partial sequence of the AChRγ subunit;

[0028] Connecting peptide B: used to connect the partial sequence and transmembrane sequence of the AChRε subunit, and to connect the partial sequence and transmembrane sequence of the AChRγ subunit;

[0029] Connector peptide C: used to connect the transmembrane sequence and the fluorescent tag.

[0030] Furthermore, the sequence of connecting peptide A is shown in SEQ ID NO:9; the sequence of connecting peptide B is shown in SEQ ID NO:10; and the sequence of connecting peptide C is shown in SEQ ID NO:11.

[0031] Optionally, the transmembrane sequence is CD8a hinge.

[0032] Optionally, the fluorescent label is mCherry.

[0033] The present invention also provides an application of the LCBA method based on the above-mentioned recombinant protein in the detection of anti-acetylcholine receptor cluster antibodies. The LCBA method is used to transfect host cells with nucleic acid encoding the recombinant protein for expression, and the co-transfected cells obtained are used as detection products to detect anti-acetylcholine receptor cluster antibodies.

[0034] Optionally, the host cell includes CHO cells or HEK293 cells.

[0035] Optionally, the detection product includes a detection reagent or a detection kit.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] (1) The present invention constructs a recombinant protein by intercepting a specific part of the sequence of the five subunits of AChR (α, β, δ, ε / γ), redesigning it using a connecting peptide, and fusing it with a transmembrane sequence and a fluorescent tag, thereby achieving stable overexpression on the cell membrane of eukaryotic cells.

[0038] (2) The recombinant protein constructed by fusion of the present invention is anchored on the cell membrane, and does not require co-transfection of multiple subunits or co-transfection using Rapsyn. Only one transfection is required, and a stable cell line that stably expresses the antigen conformation can be easily obtained. It can be used in the process of detecting anti-AChR antibodies by the FCBA method and the LCBA method, and plays an important role in the detection of the antibody and the detection of SNMG patients.

[0039] (3) The present invention designs two fusion-expressed recombinant proteins for the adult type (AChRε) and infant type (AChRγ) isoforms, namely, the adult type AChR recombinant protein and the infant type AChR recombinant protein, which are suitable for the detection of anti-AChR antibodies in two different patient populations, adults and infants, and have a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the three-dimensional conformation of the natural protein cluster (full length) of the five subunits of the adult AChR (top view and side view).

[0041] Figure 2 Schematic diagram of the three-dimensional conformation of the adult AChR recombinant protein (without fluorescent tag) of Example 1 (top view and side view).

[0042] Figure 3 The natural protein cluster of the five subunits of AChR (full length) and Figure 2 Schematic representation of the three-dimensional conformational superposition of the adult-type AChR recombinant protein shown (top and side views).

[0043] Figure 4 This is a diagram of the lentiviral vector of the adult AChR recombinant protein in Example 1.

[0044] Figure 5 This is a diagram of the lentiviral vector of the infant AChR recombinant protein of Example 1.

[0045] Figure 6This is the live cell immunofluorescence image of the AChR antibody positive sample in Example 2.

[0046] Figure 7 This is the live cell immunofluorescence image of the AChR antibody negative sample in Example 2.

[0047] Figure 8 Schematic diagram of the three-dimensional conformation of the adult AChR recombinant protein (without fluorescent label) in Comparative Example 1 (top view and side view).

[0048] Fig. 9 The natural protein cluster (full length) of the five subunits of the adult AChR Figure 8 Schematic representation of the three-dimensional conformational superposition of the adult-type AChR recombinant protein shown (top and side views).

[0049] Fig.10 Schematic diagram of the three-dimensional conformation of the adult AChR recombinant protein (without fluorescent label) in Comparative Example 2 (top view and side view).

[0050] Fig.11 The natural protein cluster (full length) of the five subunits of the adult AChR Fig.10 Schematic representation of the three-dimensional conformational superposition of the adult-type AChR recombinant protein shown (top and side views).

[0051] Fig.12 This is the live cell immunofluorescence image of the AChR antibody positive sample in Comparative Example 3. DETAILED DESCRIPTION

[0052] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0053] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0054] The present invention aims to provide a recombinant protein fused with partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit (or AChRγ subunit), which can be stably overexpressed on the cell membrane of eukaryotic cells and is very easy to obtain a stable cell line that stably expresses the antigen conformation.

[0055] To prove the stable expression of the recombinant protein antigen conformation, the present invention uses Alpha Fold 3 software to predict the three-dimensional structure of the natural protein cluster (full length) of the five subunits of AChR and the recombinant protein, see Figures 1 to 3 As shown, Figure 1 In the middle, five different subunits of AChR are represented respectively; Figure 2 The recombinant protein constructed by the present invention; Figure 3 It can be seen that the three-dimensional structure of the extracellular domain of the recombinant protein overlaps well with the three-dimensional structure of the extracellular domain of the natural protein cluster, and both can normally expose the antigen binding site. The PyMOL software was used for comparison, and the root mean square deviation value RMSD =0.357.

[0056] Therefore, the recombinant protein can be used to establish a live cell immunofluorescence method for detecting anti-AChR antibodies, and is not limited to the FCBA method and the LCBA method.

[0057] The recombinant proteins involved in the present invention are as follows:

[0058] Adult AChR recombinant protein: obtained by fusing partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit with a connecting peptide, a transmembrane sequence and a fluorescent tag, the sequence of which is shown in SEQ ID NO: 1 as follows:

[0059]

[0060] The infantile AChR recombinant protein is obtained by fusing partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRγ subunit with a connecting peptide, a transmembrane sequence and a fluorescent tag, and its sequence is shown in SEQ ID NO: 2 as follows:

[0061]

[0062] Specifically, the partial sequence of the AChRα1 subunit includes a partial sequence of the AChRα1 subunit containing a signal peptide and a partial sequence of the AChRα1 subunit without a signal peptide, wherein:

[0063] The partial sequence of the AChRα1 subunit containing the signal peptide is derived from the 1-230 amino acid sequence of the N-terminal domain of the CHRNA1 transcript isoform NM_000079.4, and its sequence is shown in SEQ ID NO: 3 as follows:

[0064] MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNN ADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRL;

[0065] The partial sequence of the AChRα1 subunit without the signal peptide is derived from the amino acid sequence 21-230 on the N-terminal domain of the CHRNA1 transcript isoform NM_000079.4, and its sequence is shown in SEQ ID NO: 4 as follows:

[0066] SEHETRLVAKLFKDYSSVVRPVEDHRQVVVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKF TKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRL.

[0067] The partial sequence of the AChRβ1 subunit is derived from the amino acid sequence 24-244 on the N-terminal domain of the CHRNB1 transcript isoform NM_000747.3, and its sequence is shown in SEQ ID NO: 5 as follows:

[0068] SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVS SDGSVRWQPPGIYRSSCSIQVTYFPFDWQNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRK.

[0069] The partial sequence of the AChRδ subunit is derived from the amino acid sequence 22-245 on the N-terminal domain of the CHRND transcript isoform NM_000751.3, and its sequence is shown in SEQ ID NO: 6 as follows:

[0070] LNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLV YHYGFVYWLPPAIFRSSCPISVTYFPFDWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSSPRQDITFYLIIRRK.

[0071] The partial sequence of the AChRε subunit is derived from the amino acid sequence 21-239 on the N-terminal domain of the CHRNE transcript isoform NM_000080.4, and its sequence is shown in SEQ ID NO: 7 as follows:

[0072] KNEELRLYHHLFNNYDPGSRPVREPEDTVTISLKVTLTNLISLNEKEETLTTSVWIGIDWQDYRLNYSKDDFGGIETLRVPSELVWLPEIVLENNIDGQFGVAYDANVLV YEGGSVTWLPPAIYRSVCAVEVTYFPFDWQNCSLIFRSQTYNAEEVEFTFAVDNDGKTINKIDIDTEAYTENGEWAIDFCPGVIRRHHGGATDGPGETDVIYSLIIRRK.

[0073] The partial sequence of the AChRγ subunit is derived from the amino acid sequence 25-241 on the N-terminal domain of the CHRNG transcript isoform NM_005190.4, and its sequence is shown in SEQ ID NO: 8 as follows:

[0074] QEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEALTTNVWIEMQWCDYRLRWDPRDYEGLWVLRVPSTMVWRPDIVLENNVDGVFEVALYCNVLVS PDGCIYWLPPAIFRSACSISVTYFPFDWQNCSLIFQSQTYSTNEIDLQLSQEDGQTIEWIFIDPEAFTENGEWAIQHRPAKMLLDPAAPAQEAGHQKVVFYLLIQRKP.

[0075] The connecting peptides include connecting peptide A, connecting peptide B and connecting peptide C, wherein:

[0076] The sequence of connecting peptide A is shown in SEQ ID NO: 9 as GSTGGGGSGGGGSGGGGSGAASR;

[0077] The sequence of connecting peptide B is shown in SEQ ID NO: 10 as ASGGGGSGGGGSSG;

[0078] The sequence of connecting peptide C is shown in SEQ ID NO: 11 as AEAAAKEAAAKA.

[0079] The transmembrane sequence is CD8a hinge, and its sequence is shown in SEQ ID NO: 12 as follows:

[0080] IYIWAPLAGTCGVLLLSLVITLYC.

[0081] The fluorescent tag is mCherry, and its sequence is shown in SEQ ID NO: 13 as follows:

[0082] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0083] Since the connecting peptide A is used to connect the partial sequence of the AChRα1 subunit containing the signal peptide and the partial sequence of the AChRβ1 subunit, connect the partial sequence of the AChRβ1 subunit and the partial sequence of the AChRδ subunit, connect the partial sequence of the AChRδ subunit and the partial sequence of the AChRα1 subunit without the signal peptide, connect the partial sequence of the AChRα1 subunit without the signal peptide and the partial sequence of the AChRε subunit, and connect the partial sequence of the AChRα1 subunit without the signal peptide and the partial sequence of the AChRγ subunit. Connecting peptide B is used to connect the partial sequence of the AChRε subunit and the transmembrane sequence, and connect the partial sequence of the AChRγ subunit and the transmembrane sequence. Connecting peptide C is used to connect the transmembrane sequence and the fluorescent label.

[0084] Therefore, two recombinant proteins of different configurations were constructed by fusing the transmembrane sequence and the fluorescent tag with the partial sequences of the above-mentioned five subunits of AChR (α, β, δ, ε / γ) through the connecting peptide A, connecting peptide B and connecting peptide C as follows:

[0085] Adult AChR recombinant protein:

[0086] Partial sequence of CHRNA1 (including signal peptide)—connecting peptide A—partial sequence of CHRNB1—connecting peptide A—partial sequence of CHRND—connecting peptide A—partial sequence of CHRNA1 (excluding signal peptide)—connecting peptide A—partial sequence of CHRNE—connecting peptide B—CD8a hinge—connecting peptide C—mCherry.

[0087] Infant AChR recombinant protein:

[0088] Partial sequence of CHRNA1 (including signal peptide)—connecting peptide A—partial sequence of CHRNB1—connecting peptide A—partial sequence of CHRND—connecting peptide A—partial sequence of CHRNA1 (excluding signal peptide)—connecting peptide A—partial sequence of CHRNG—connecting peptide B—CD8a hinge—connecting peptide C—mCherry.

[0089] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0090] Example 1: Construction of lentiviral vector

[0091] According to the above method of the present invention, lentiviral vectors capable of fusion-expressing adult-type AChR recombinant protein and infant-type AChR recombinant protein are constructed respectively. The map of the constructed lentiviral vectors is shown in Figure 4 and Figure 5 shown.

[0092] The details are as follows:

[0093] Adult type: CHRNA1 (1-230aa) (including signal peptide)—CHRNB1 (24-244aa)—CHRND (22-245aa)—CHRNA1 (21-230aa)—CHRNE (21-239aa)—CD8a hinge—mCherry.

[0094] Infant type: CHRNA1 (1-230aa) (including signal peptide)—CHRNB1 (24-244aa)—CHRND (22-245aa)—CHRNA1 (21-230aa)—CHRNG (25-241aa)—CD8a hinge—mCherry.

[0095] Example 2: Establishment of LCBA method for anti-AChR antibodies

[0096] The lentiviral vector prepared in Example 1 was transfected into CHO or HEK293 cells, and then screened by limiting dilution method to obtain monoclonal cells stably expressing recombinant protein, treated with trypsin (trypsin-EDTA) solution to form a single cell suspension, and after cell counting, the cells were diluted to 1 cell / 100μL by limiting dilution method, and then inoculated into 96-well plates at 100μL / well, respectively, and the wells of single cell groups were screened out, and the wells with strong red fluorescence were selected for freezing and primary antibody immunofluorescence detection. The qualified cells were used to prepare the detection reagent or detection kit for subsequent immunofluorescence experiments.

[0097] Cell sheet inoculation: The recombinant protein-depleted monoclonal cells and control empty cells were mixed evenly at a ratio of monoclonal cells to control cells = 1:2, and the total density was 2×10 5 / mL was inoculated into 96-well plates or cell slides, cultured with F12K+10% FBS+NEAA, grown for about 48 hours, and cultured when the confluence reached 90-100%.

[0098] The LCBA method was used. The specific operation was as follows: the screened stably transfected monoclonal cells and empty control cells were mixed and plated into a 96-well cell culture plate. After culturing for 48 hours, they were directly used for live cell immunofluorescence detection of anti-AChR antibodies.

[0099] Live cell immunofluorescence detection includes the following steps:

[0100] Serum from patients with myasthenia gravis who are clinically confirmed to be positive for ACHR antibodies or normal human serum is prepared as a sample. The sample diluent contains the following components:

[0101] DMEM medium, BSA (1%);

[0102] The samples were diluted with sample diluent for the initial assay, with the serum dilution ratio being 1:10;

[0103] Sample incubation: incubate in a carbon dioxide incubator at 37 degrees Celsius for 1 hour, wash 3 times with PBS;

[0104] Dilute the secondary antibody with sample diluent: use goat anti-human IgG (H+L) at a dilution ratio of 1:500, incubate at room temperature for 45 minutes, and wash 3 times with PBS;

[0105] Interpretation: Interpret the positive and negative of the sample under a microscope, see Figure 6 and Figure 7 shown.

[0106] Figure 6 This is the LCBA fluorescence image of the AChR antibody positive sample. Figure 6 A is the fluorescence of the recombinant protein itself, B is the fluorescence of the secondary antibody after incubation of the positive sample with the secondary antibody, and C is the in situ overlap of A and B, which can indicate that the sample is detected as positive.

[0107] Figure 7 This is the LCBA fluorescence image of the AChR antibody negative sample. Figure 7 A is the fluorescence of the recombinant protein itself, and B is the fluorescence of the secondary antibody after incubation of the negative sample with the secondary antibody, which indicates that the sample is negative.

[0108] In general, when the recombinant protein of this example is used for LCBA detection of anti-AChR antibodies, the immunofluorescence staining image is clear and the interpretation accuracy is high.

[0109] Comparative Example 1:

[0110] According to the method of Example 1 and Example 2, construct:

[0111] The adult recombinant protein vector of CHRNA1 (1-300aa) (including signal peptide) - CHRNB1 (24-244aa) - CHRND (22-245aa) - CHRNA1 (21-300aa) - CHRNE (21-239aa) - CD8a hinge - mCherry was expressed by fusion, and the anti-AChR antibody was detected by LCBA method.

[0112] In addition, the infant-type recombinant protein vector of CHRNA1 (1-300aa) (including signal peptide) - CHRNB1 (24-244aa) - CHRND (22-245aa) - CHRNA1 (21-300aa) - CHRNG (25-241aa) - CD8a hinge - mCherry was expressed by fusion, and the anti-AChR antibody was detected by LCBA method.

[0113] The sequence of CHRNA1 (1-300aa) (including signal peptide) is shown in SEQ ID NO: 14 as follows:

[0114] MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEI IVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGKYMLF.

[0115] The sequence of CHRNA1 (21-300aa) is shown in SEQ ID NO: 15 as follows:

[0116] SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQ NCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGKYMLF.

[0117] Comparative Example 2:

[0118] According to the method of Example 1 and Example 2, construct:

[0119] The adult recombinant protein vector of CHRNA1 (1-230aa) (including signal peptide) - CHRNB1 (24-300aa) - CHRND (21-300aa) - CHRNE (21-239aa) - CD8a hinge - mCherry was expressed by fusion, and the anti-AChR antibody was detected by LCBA method.

[0120] In addition, the infant-type recombinant protein vector of CHRNA1 (1-230aa) (including signal peptide) - CHRNB1 (24-300aa) - CHRND (21-300aa) - CHRNG (25-241aa) - CD8a hinge - mCherry was expressed by fusion, and the anti-AChR antibody was detected by LCBA method.

[0121] The sequence of CHRNB1 (24-300aa) is shown in SEQ ID NO: 16 as follows:

[0122] SEAEGRLREKLFSGYDSSVRPAREVGDRVRVSVGLILAQLISLNEKDEEMSTKVYLDLEWTDYRLSWDPAEHDGIDSLRITAESVWLPDVVLLNNNDGNFDVALDISVVVSSDGSVRWQPPGIYRSSCSIQVTYFPFDW QNCTMVFSSYSYDSSEVSLQTGLGPDGQGHQEIHIHEGTFIENGQWEIIHKPSRLIQPPGDPRGGREGQRQEVIFYLIIRRKPLFYLVNVIAPCILITLLAIFVFYLPPDAGEKMGLSIFALLTLTVFLLLLADKVPE.

[0123] The sequence of CHRND (21-300aa) is shown in SEQ ID NO: 17 as follows:

[0124] GLNEEERLIRHLFQEKGYNKELRPVAHKEESVDVALALTLSNLISLKEVEETLTTNVWIEHGWTDNRLKWNAEEFGNISVLRLPPDMVWLPEIVLENNNDGSFQISYSCNVLVYHYGFVYWLPPAIFRSSCPISVTYFPF DWQNCSLKFSSLKYTAKEITLSLKQDAKENRTYPVEWIIIDPEGFTENGEWEIVHRPARVNVDPRAPLDSSPRQDITFYLIIRRKPLFYIINILVPCVLISFMVNLVFYLPADSGEKTSVAISVLLAQSVFLLLISKRLP.

[0125] The Alpha Fold 3 software was used to predict the three-dimensional structures of the recombinant proteins of Comparative Example 1 and Comparative Example 2, respectively.

[0126] See also Figure 8 and Fig. 9 Shown is the three-dimensional structure prediction of Comparative Example 1, wherein, Figure 8 The front view and side view of the adult AChR recombinant protein in Comparative Example 1 are shown. Fig. 9 The top view and side view of the adult AChR recombinant protein of Comparative Example 1 were overlapped with the natural protein cluster (full length) of the five subunits of the adult AChR, and the PyMOL software was used for comparison, and the RMSD was 15.475.

[0127] See also Fig.10 and Fig.11 Shown is the three-dimensional structure prediction of Comparative Example 2, wherein, Fig.10 The front view and side view of the adult AChR recombinant protein in Comparative Example 2 are shown in FIG. Fig.11 The top view and side view of the adult AChR recombinant protein of Comparative Example 2 were overlapped with the natural protein cluster (full length) of the five subunits of the adult AChR, and the PyMOL software was used for comparison, and the RMSD was 16.771.

[0128] Compared with the present invention Figure 2 and Figure 3 The recombinant protein and the natural protein cluster extracellular domain were compared. Figures 8 to 11 There are large differences in the conformational comparison of the extracellular domains of the adult-type AChR recombinant proteins of representative comparative examples 1 and 2 with the natural protein clusters. At the same time, the RMSD values ​​of the comparison of the recombinant proteins of comparative examples 1 and 2 with the natural protein clusters are also greater than the comparison values ​​of the present invention, indicating that there are greater differences in the structures of the recombinant proteins of comparative examples 1 and 2 with the natural protein clusters.

[0129] Comparative Example 3:

[0130] The conventional LCBA method in the current literature was used to construct eukaryotic expression vectors for the five subunits of AChR (α, β, δ, ε / γ) and Rapsyn. The pcDNA3.1 vector framework was used to construct six eukaryotic expression vectors for the five subunits of AChR (α, β, δ, ε, γ) and Rapsyn according to the following transcript full-length sequences (see NCBI for detailed sequences):

[0131] CHRNA1 (NM_000079.4) expression vector;

[0132] CHRNB1 (NM_000747.3) expression vector;

[0133] CHRND (NM_000751.3) expression vector;

[0134] CHRNE (NM_000080.4) expression vector;

[0135] CHRNG (NM_005190.4) expression vector;

[0136] RAPSN (NM_005055.5) - Connector peptide C - mCherry expression vector;

[0137] Use an endotoxin-free plasmid extraction kit to extract the plasmids of the above six vectors. After testing the concentration and purity, follow the following steps:

[0138] The adult type: CHRNA1∶CHRNB1∶CHRND∶CHRNE∶RAPSN=2∶1∶1∶1∶1 and the infant type: CHRNA1∶CHRNB1∶CHRND∶CHRNG∶RAPSN=2∶1∶1∶1∶1 were used to transiently transfect CHO cells using the lipofectamine3000 kit. Two days after transfection, the adult and infant type transiently transfected cells were used to detect anti-AChR antibodies using the LCBA method.

[0139] Interpretation: The positive and negative of the sample can be determined under a microscope. For positive results, refer to Fig.12 shown.

[0140] Fig.12 The LCBA fluorescence image of the AChR antibody positive sample, where A is the fluorescence of the recombinant protein itself, B is the fluorescence of the secondary antibody after the positive sample and the secondary antibody are incubated, and C is the in situ overlap of A and B, which can indicate that the sample is positive. Figure 6 By comparison, it can be seen that when comparative example 3 is used to perform AChR antibody live cell immunofluorescence detection, the co-transfection rate of the cells themselves is low, and the detection effect is not as good as that of example 2.

[0141] Further, the following tests were performed for the above embodiments and comparative examples:

[0142] (1) Clinical positive sample testing

[0143] Samples and number of samples: 25 adult-type AChR-positive serum samples and 4 infant-type AChR-positive serum samples.

[0144] Sample inclusion criteria: Refer to the "Expert Consensus on Laboratory Diagnosis of Myasthenia Gravis Autoantibodies (2022)" published by the Neuroimmunology Group of the Neurology Branch of the Chinese Medical Association in the "Chinese Journal of Neurology" in March 2023, and select serum samples from adult and infantile AChR-positive myasthenia gravis patients.

[0145] Detection method: Prepare sample diluent, the specific formula is as follows: add 1% bovine serum albumin to DMEM cell culture medium, filter and sterilize after dissolving, dilute human serum sample with sample diluent, the serum sample dilution ratio is 1:10, add the diluted sample to the living cell culture plate that has been overgrown, incubate at 37℃ in a carbon dioxide incubator for 1 hour, aspirate and discard the sample solution, wash the cell sheet with DMEM 3 times, 5 minutes each time. Next, dilute the secondary antibody with the same sample diluent, the secondary antibody is Delight488-labeled goat anti-human IgG (Fc), the dilution ratio of the secondary antibody is 1:500, add the diluted secondary antibody, incubate at room temperature for 40 minutes, aspirate and discard the secondary antibody, wash the cell sheet with DMEM 3 times, 5 minutes each time, add a little phosphate buffer to completely immerse the cell sheet, and observe under a fluorescence microscope as soon as possible.

[0146] The test results are shown in Table 1 below. As can be seen from the results in Table 1, the positive detection rate of Example 2 is significantly better than that of Comparative Example 1, Comparative Example 2 and Comparative Example 3.

[0147] Table 1

[0148]

[0149] (2) Specificity detection

[0150] Samples and sample numbers: 42 clinical specimens were tested, including 27 healthy control sera and 15 sera from myasthenia gravis patients who were MuSK antibody-positive and anti-AChR antibody-negative.

[0151] The sample inclusion criteria and detection methods were the same as above.

[0152] The test results of Example 2 and Comparative Examples 1, 2 and 3 show that no positive results were detected in all 42 AChR negative samples, and the specificity is 100%.

[0153] (3) Sensitivity test

[0154] Samples and number of samples: 14 samples that were positive in Example 2, Comparative Example 1, and Comparative Example 3 were selected from the sample library for positive sample detection and were subjected to gradient dilution sensitivity detection. The serum dilution ratios were 1:10, 1:32, and 1:100.

[0155] The sample inclusion criteria and detection methods were the same as above.

[0156] The test results are shown in Table 2. As can be seen from Table 2, the detection sensitivity of Example 2 is significantly better than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0157] Table 2

[0158]

[0159] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that: The partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRε subunit are fused to obtain an adult AChR recombinant protein as shown in SEQ ID NO:

1. Alternatively, partial sequences of AChRα1 subunit, AChRβ1 subunit, AChRδ subunit and AChRγ subunit are fused to construct an infant AChR recombinant protein with a sequence number as shown in SEQ ID NO:

2.

2. The use of the recombinant protein according to claim 1 in the preparation of a detection product for detecting anti-acetylcholine receptor cluster antibodies using live cell immunofluorescence method, characterized in that: The live cell immunofluorescence method was used to detect the anti-acetylcholine receptor cluster antibodies by transfecting the nucleic acid encoding the recombinant protein into the host cells for expression and obtaining the co-transfected cells as the detection product.

3. The use according to claim 2, characterized in that: The host cells include CHO cells or HEK293 cells.

4. The use according to claim 2, characterized in that: The detection product includes a detection reagent or a detection kit.

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