RyR1 antigen, antigen expression gene, antigen expression vector, cell line and uses thereof

Abstract

The present invention relates to RyR1 antigens, antigen expression genes, antigen expression vectors, cell lines and uses. The present invention provides two effective antigens for detecting anti-RyR1 autoantibodies: MGTR1 and MGTR2, which have the characteristics of small molecule size and good expression. Expression plasmids carrying the MGTR1 and MGTR2 genes are transfected into cell lines to prepare cell-based detection materials. The prepared detection materials have good sensitivity, specificity and stability for RyR1 antibodies. The indirect immunofluorescence method using cells is used to screen for anti-RyR1 autoantibodies in MG patients and MG patients with thymoma. Based on the RyRl cell detection matrix material, a two-color fluorescence system is adopted, and its co-localization effect is good. Therefore, it has good sensitivity, specificity and stability. The RyR1 cell detection matrix of the present invention can meet the needs of auxiliary clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of bioanalysis and detection, and particularly to RyR1 antigen, antigen expression gene, antigen expression vector, cell line and uses thereof. Background Art

[0002] Myasthenia gravis (MG) is a disease of neuromuscular junction transmission disorder, mainly an autoimmune disease mediated by autoantibodies such as acetylcholine receptors antibody (AChR-Ab), assisted by cellular immunity and involving complement. Detection methods for myasthenia gravis include: ① thymus CT and MRI; ② single fiber electromyogram; ③ detection of acetylcholine receptor antibody titer, etc.

[0003] In recent years, studies have found that in addition to AChR-Ab, there are some commonly used detection antibodies in myasthenia gravis clinically, such as anti-muscle specific receptor tyrosine kinase (MuSK) antibody, anti-low density lipoprotein receptor related protein 4 (LRP4) antibody, anti-titin antibody, anti-ryanodine receptor (RyR) antibody, etc.

[0004] There are also various autoantibodies in the serum of MG patients, and anti-ryanodine receptor (RyR1) antibody can be detected in patients with SNMG, mostly in elderly MG patients, and has a relatively high directivity for MG patients with thymoma (AChR antibody positive).

[0005] RyR1 is a large protein containing 5038 amino acids and is the largest ion channel protein known so far. In 1992, Ase Mygland et al. discovered anti-RyR1 antibody in myasthenia gravis patients with thymoma, establishing the connection between RyR1 and MG patients. The antigen initially used was to extract sarcoplasmic reticulum protein from muscle tissue, and this crude extract carried many other miscellaneous proteins, which would increase the risk of false positive test results; RyR1 is a membrane protein, and the purification of membrane proteins is a time-consuming and laborious task, and the main antigenic determinant region of RyR1 protein has not been clearly defined.

[0006] There are three types of RyR proteins in mammals, among which RyR1 is mainly distributed on the sarcoplasmic reticulum in skeletal muscle cells. The commonly used detection methods for anti-RyR1 antibodies are Radioimmunoassay (RIA) and Enzyme-Linked Immunosorbent Assay (ELISA). Currently, it has been reported that the antigenic epitopes bound by anti-RyR1 antibodies are dispersed inside RyR1 (Geir Olve Skeie, Ase Mygland, Susan Treves, et al. Ryanodine receptor antibodies in myasthenia gravis: epitope mapping and effect on calcium release in vitro, Muscle Nerve. 2003 Jan;27(1):81-9.), but the exact binding site is still unclear. Some people have tried to use some fragments of RyR1 to prepare antigens, and detect them by Western-blot using recombinantly expressed truncated proteins such as peptide chain 2 as antigens (Yu Hong, Hai-Feng Li, Geir Olve Skeie, et al. Autoantibody profile and clinical characteristics in a cohort of Chinese adult myasthenia gravis patients, J Neuroimmunol. 2016 Sep 15;298:51-7). Some people also choose some peptide segments to detect anti-RyR1 autoantibodies by ELISA (M Takamori, M Motomura, N Kawaguchi, et al. Anti-ryanodine receptor antibodies and FK506 in myasthenia gravis, Neurology. 2004 May 25;62(10):1894-6.). Initially, RIA was mostly used, and later it was replaced by ELISA and Western-blot. The antigen proteins have also changed from muscle extracts in the past to recombinantly expressed RyR1 peptide segments or large antigen fragments. For the ELISA detection method, since short peptides are used and the antigenicity represented by short peptides is not highly recognized, it is not widely used at present.Western-blot is a commonly used method for current detection. The antigen used in its detection is a large fragment, and there is also more experimental evidence proving better effects. However, the problem with Western-blot is that the process is cumbersome, the operation is relatively complex, and the stability is slightly poor.

[0007] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, when the applicant made this invention, a large number of documents and patents were studied, but due to space limitations, all details and contents were not listed in detail. However, this by no means means that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. Summary of the Invention

[0008] Myasthenia gravis (MG) is a neuromuscular junction transmission disorder disease, mainly an autoimmune disease mediated by autoantibodies such as acetylcholine receptors antibody (AChR-Ab), assisted by cellular immunity and participated by complement. In addition to identifying this disease by detecting the titer of acetylcholine receptor antibody, there are also some commonly used detection antibodies in myasthenia gravis clinically, such as anti-muscle specific receptor tyrosine kinase (MuSK) antibody, anti-low density lipoprotein receptor related protein 4 (LRP4) antibody, anti-titin (Titin) antibody, anti-ryanodine receptor (RyR) antibody, etc. In the detection of autoantibodies of RyR1, the key problem is how to obtain its antigen substrate. In the past, the antigen used was to extract sarcoplasmic reticulum protein from muscle tissue. This crude extract carried many other miscellaneous proteins, which would increase the risk of false positive detection results; and RyR1 is a membrane protein, and its purification process is difficult. Currently, recombinant expressed RyR1 peptide segments or large antigen fragments are used for detection, but the commonly used detection methods are radioimmunoassay and enzyme-linked immunosorbent assay. The short peptide is used in ELISA detection, and the antigenicity recognition represented by the short peptide is not high, so it is not widely used currently. Western-blot is a commonly used method for current detection. The antigen used in its detection is a large fragment, and there is also more experimental evidence proving better effects. However, the problem with Western-blot is that the process is cumbersome, the operation is relatively complex, and the stability is slightly poor.

[0009] In view of the deficiencies of the prior art, the present invention provides a RyR1 antigen, and the amino acid sequence of the antigen is shown in SEQ ID NO.1. The antigen is a fusion protein composed of the N-terminal of RyR1 and the middle part sequence spliced together. The antigen is linked with a fluorescent tag. Preferably, the fluorescent tag is an mCherry fluorescent tag.

[0010] The present invention also provides another RyR1 antigen, and the amino acid sequence of the antigen is as shown in SEQ ID NO.2. The antigen is a fusion protein formed by splicing partial sequences of the N-terminal, middle, and C-terminal of RyR1. The antigen is linked with a fluorescent label. Preferably, the fluorescent label is an mCherry fluorescent label.

[0011] The two RyR1 antigens provided by the present invention contain different fragments of the extracellular region of RyR1, can efficiently detect anti-RyR1 antibodies in MG patients, and have the advantages of high sensitivity, good specificity and stability.

[0012] The present invention also provides a nucleic acid molecule, and the nucleic acid molecule encodes the antigen with the amino acid sequence as SEQ ID NO.1.

[0013] The present invention also provides another nucleic acid molecule, and the nucleic acid molecule encodes the antigen with the amino acid sequence as SEQ ID NO.2.

[0014] The present invention also provides a RyR1 antigen expression vector, and the expression vector includes the nucleic acid molecule encoding the antigen with the amino acid sequence as SEQID NO.1 and a vector. According to a preferred embodiment, the vector is pmCherry-N1, and the nucleotide sequence of the vector is as shown in SEQ ID NO.3.

[0015] The present invention also provides another RyR1 antigen expression vector, and the expression vector includes the nucleic acid molecule encoding the antigen with the amino acid sequence as SEQ ID NO.2 and a vector. According to a preferred embodiment, the vector is pmCherry-N1, and the nucleotide sequence of the vector is as shown in SEQ ID NO.3.

[0016] The present invention also provides an expression cell line of RyR1 antigen, and the expression cell line contains the expression vector.

[0017] The present invention also provides a preparation method of the expression cell line, including the following steps: transferring the RyRl antigen expression vector into a cell line, and obtaining the cell line after screening. Preferably, the expression cell line is a Hep2 cell.

[0018] The present invention provides the uses of RyR1 antigen, nucleic acid molecule, expression vector, and expression cell line in detecting RyR1 antibody.

[0019] The present invention also provides the uses of the RyR1 antigen with the nucleotide sequence as SEQ ID NO.1, the nucleic acid molecule encoding the antigen, the expression vector, and the expression cell line in detecting RyR1 antibody.

[0020] The present invention also provides the use of the RyR1 antigen with the nucleotide sequence as set forth in SEQ ID NO.2, the nucleic acid molecule encoding the antigen, the expression vector and the expression cell line in detecting the RyR1 antibody.

[0021] The present invention also provides the application of the RyR1 antigen with the nucleotide sequence as set forth in SEQ ID NO.1, the nucleic acid molecule encoding the antigen, the expression vector and the expression cell line in the preparation of a kit for detecting the RyR1 antibody.

[0022] The present invention also provides the application of the RyR1 antigen with the nucleotide sequence as set forth in SEQ ID NO.2, the nucleic acid molecule encoding the antigen, the expression vector and the expression cell line in the preparation of a kit for detecting the RyR1 antibody.

[0023] The present invention designs a variety of truncated forms of RyR1 by combining the antigen fragment that has been reported to be used for detecting anti-RyR1 autoantibodies with the protein structure (PDB) of the protein binding to RyR1 and the hydrophilicity of the protein, constructs them into eukaryotic expression plasmids respectively, transfects the cell line, fixes the cells after 24 hours of expression, uses them as cell detection materials, and performs immunofluorescence tests with the sera of MG patients with thymoma. After screening, two new antigens with excellent performance are obtained. They are conjugated by fragments at different positions in the extracellular region of RyR1. And the present invention adopts a cell-based immunofluorescence method. The new antigen plus the CBA method can efficiently detect anti-RyR1 antibodies in the blood of MG patients with thymoma, and the positive detection rate reaches 50%, which is basically consistent with the reported ratio (Yu Hong, Hai-Feng Li, Geir Olve Skeie, et al. Autoantibody profile and clinical characteristics in a cohort of Chinese adult myasthenia gravis patients, J Neuroimmunol. 2016 Sep 15; 298: 51-7). This fully demonstrates the effectiveness of the antigen selection of the present invention. The above experimental results prove that the MGTR1 and MGTR2 antigens can be effectively used for the detection of anti-RyR1 antibodies. Moreover, the indirect immunofluorescence method for cells is more reliable than ELISA, and is more convenient and stable compared with Western-blot. The present invention adopts a two-color fluorescence system. The antigen protein itself has red light, and the detection signal of the patient sample is green light. If there is a RyR1-specific antibody in the sample, green light will be detected to co-localize with the red light spontaneously emitted by the protein, which is beneficial to the specificity of the detection. Therefore, the present invention is innovative in the antigen material, and the detection technology used is also more reliable and convenient. Description of the Drawings

[0024] Figure 1 This is the plasmid map of the eukaryotic expression plasmid of MGTR1 / MGTR2 provided by the present invention;

[0025] Figure 2 This is the expression of MGTR1 / MGTR2 conjugated with mCherry in Hep2 provided by the present invention;

[0026] Figure 3 This is the MGTR1 for detecting RyR1-positive serum provided by the present invention;

[0027] Figure 4 This is the MGTR2 for detecting RyR1-positive serum provided by the present invention;

[0028] Figure 5 These are the detection results of using MGTR2 for positive patients with different concentrations of RyR1 antibodies. Specific Embodiments

[0029] The following will be described in detail with reference to the accompanying drawings.

[0030] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention will be given. The present invention is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] The main reagents, instruments and their sources used in the present invention: PCR enzymes: Max DNAPolymerase (Takara, catalog number: R045A), KOD DNA Polymerase (brand Toyobo, product number KOD-201); T4 DNA ligase (NEB catalog number: M0202T); cell lines: HEK293T and Hep2 cells; gel extraction kit: Omega GelExtraction Kit; plasmid: pEGFP-N1 (purchased from Clontech, catalog number 6085-1); plasmid extraction kit: Omega plasmid mini kit; ultrapure water instrument: Millipore 5UV Water PurificationSystem; pipette: eppendorf; PCR instrument: Bio-Rad T100.

[0032] The present invention provides two effective antigens (MGTR1 and MGTR2) for detecting anti-RyR1 autoantibodies, which are characterized by small molecules and good expression. The expression plasmids carrying MGTR1 and MGTR2 genes are transfected into cell lines to prepare cell-based detection materials, and the indirect immunofluorescence assay can be used for screening anti-RyR1 autoantibodies in MG patients and MG patients with thymoma.

[0033] Example 1

[0034] Construction of MGTR1 expression vector and localization of MGTR1 expression, the preparation steps are as follows:

[0035] I. Gene synthesis

[0036] The RyR1 gene sequence was retrieved from the GenBank sequence database, and a partial RyR1 gene sequence was synthesized. MGTR1 is a fusion protein composed of the N-terminal and middle sequences of RyR1. The specific sequence of MGTR1 is shown in SEQ ID NO.1.

[0037] II. Vector construction

[0038] 1. Amplify the MGTR1 gene by PCR;

[0039] PCR amplification system (50 μl): 50 ng of template, 1.5 μl of MGTR1-NheI-F (10 μM), 1.5 μl of MGTR1-XbaI-R (10 μM), 25 μl of Primestar (2×), and the remaining Nuclease-free Water.

[0040] PCR amplification program: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 20 s, annealing at 58 °C for 10 s, extension at 72 °C for 30 s, 35 cycles from denaturation to extension; extension at 72 °C for 1 min again; store at 4 °C.

[0041] 2. Perform agarose gel electrophoresis on the PCR product, successfully amplify the band, and cut and recover the target band (the gel recovery kit used was purchased from OMEGA).

[0042] 3. Double-digest the gel recovery product and the pmCherry-N1 vector with NheI and XhoI respectively;

[0043] Digestion system (50 μl): about 1 μg of gel recovery product, about 1 μg of pmCherry-N1, 1 μl of NheI-HF, 1 μl of XhoI, 1O×rCutSmart, and the remaining Nuclease-free Water.

[0044] Digestion conditions: 37 °C, 2 h.

[0045] Perform agarose gel electrophoresis on the above digestion products, and cut and recover the target bands.

[0046] 4. Ligate the recovered digested target fragment and vector with T4 DNA ligase;

[0047] Ligation conditions: 25 °C, 1 h.

[0048] 5. Transform the ligation product into DH5α competent cells (purchased from TIANGEN BIOTECH). After heat shock, add 500 μl of LB medium, place it on a shaker at 37 °C for 30 min, evenly spread it on an LB plate containing kanamycin antibiotic, and culture it overnight at 37 °C.

[0049] 6. Pick monoclonal colonies on the plate into an LB liquid medium containing ampicillin antibiotic, shake the bacteria, extract the plasmid (the plasmid extraction kit is purchased from OMEGA). After digestion and identification, sequence the positive plasmid. The sequencing results are completely correct, the plasmid construction is successful, and the plasmid map is as Figure 1 shown.

[0050] III. Cell Transfection

[0051] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, discard the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0052] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with polylysine (PDL). After the glass slides in the dish are dried, add the prepared cell suspension to the culture dish, gently mix, and place it in an incubator at 37 °C and 5% CO₂ overnight. Observe the cells the next day, and transfection can be carried out when the density reaches 30 - 40%.

[0053] 3. Mix the prepared RyR1 expression plasmid (MGTR1, 6 μg) and pmCherry-N1 (empty vector control) plasmid with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortex, and let stand for 10 min. Then transfect them into the prepared cells respectively, and culture at 37 °C and 5% CO₂ for 48 h.

[0054] The transfection methods also include lipofectamin2000, lipofectamin3000, other liposome transfection reagents, electroporation, or other transfection methods.

[0055] IV. Observe fluorescence expression under the microscope and take pictures

[0056] After 24 - 48 h of cell transfection, the expression of MGTR1 was observed through the coupled mCherry tag, and the results were observed and photographed under a microscope.

[0057] MGTR1 is the extracellular domain fragment of RyR1 without a transmembrane domain. Theoretically, MGTR1 should show a uniform cytoplasmic distribution. The experimental results are as Figure 2 shown. MGTR1 coupled with the mCherry tag showed red fluorescence in the results. MGTR1 coupled with the mCherry tag was normally expressed in cells. The cells transfected with MGTR1 grew normally. The coupled mCherry tag indicated that the protein indeed behaved as a cytoplasmic protein, and MGTR1 was evenly distributed within the cells.

[0058] Example 2

[0059] Construction of the MGTR2 expression vector and localization of MGTR2 expression. The preparation steps are as follows:

[0060] I. Gene synthesis

[0061] The RyR1 gene sequence was retrieved from the GenBank sequence database, and a partial RyR1 gene sequence was synthesized. MGTR2 is a fusion protein composed of partial sequences of the N - terminal, middle, and C - terminal of RyR1. The specific sequence of MGTR2 is shown in SEQ ID NO.2.

[0062] II. Vector construction

[0063] 1. The MGTR2 gene was amplified by PCR;

[0064] PCR amplification system (50 μl): 50 ng of template, 1.5 μl of MGTR2 - NheI - F (10 μM), 1.5 μl of MGTR2 - XbaI - R (10 μM), 25 μl of Primestar (2×), and the remaining Nuclease - free Water.

[0065] PCR amplification program: Pre - denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing at 58°C for 10 s, extension at 72°C for 30 s, 35 cycles from denaturation to extension; extension at 72°C for 1 min again; preservation at 4°C.

[0066] 2. The PCR product was subjected to agarose gel electrophoresis, and the target band was successfully amplified. The target band was excised and recovered (the gel recovery kit used was purchased from OMEGA).

[0067] 3. The gel - recovered product and the pmCherry - N1 vector were double - digested with NheI and XhoI respectively;

[0068] Restriction Enzyme Digestion System (50 μl): Approximately 1 μg of gel-extracted product, approximately 1 μg of pmCherry-N1, 1 μl of NheI-HF, 1 μl of XhoI, 10× rCutSmart, and the remaining volume of Nuclease-free Water.

[0069] Restriction Enzyme Digestion Conditions: 37 °C, 2 h.

[0070] Perform agarose gel electrophoresis on the above restriction enzyme digestion products, and excise and recover the target band.

[0071] 4. Ligate the recovered digested target fragment and vector with T4 DNA ligase;

[0072] Ligation Conditions: 25 °C, 1 h.

[0073] 5. Transform the ligation product into DH5α competent cells (purchased from TIANGEN BIOTECH). After heat shock, add 500 ml of LB medium, place it on a shaker at 37 °C for 30 min, evenly spread it on an LB plate containing kanamycin antibiotic, and culture it overnight at 37 °C.

[0074] 6. Pick monoclonal colonies on the plate into an LB liquid medium containing ampicillin antibiotic, shake the bacteria, extract the plasmid (the plasmid extraction kit is purchased from OMEGA). After restriction enzyme digestion identification, sequence the positive plasmid. After the sequencing results are completely correct, the plasmid construction is successful, and the plasmid map is as Figure 1 .

[0075] III. Cell Transfection

[0076] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, discard the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0077] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with polylysine (PDL). After the glass slides in the dish are dried, add the prepared cell suspension to the culture dish, gently mix, and place it in an incubator at 37 °C and 5% CO2 overnight. Observe the cells the next day, and transfection can be performed when the density reaches 30 - 40%.

[0078] 3. Mix the prepared RyR1 expression plasmid (MGTR2, 6 μg) and pmCherry-N1 (empty vector control) plasmid with the transfection reagent PEI at a mass-to-volume ratio of 1:2 respectively, vortex, let it stand for 10 min, and then transfect them into the prepared cells, and culture at 37 °C and 5% CO2 for 48 h.

[0079] The transfection methods also include Lipofectamin 2000, Lipofectamin 3000, other liposome transfection reagents, electroporation or other transfection methods.

[0080] IV. Observe the fluorescence expression under a microscope and take pictures

[0081] At 24 - 48 h after cell transfection, observe the expression of MGTR2 through the coupled mCherry tag, observe the results under a microscope and take pictures.

[0082] MGTR2 is obtained from the extracellular region fragment of RyR1 and does not contain a transmembrane domain. Theoretically, MGTR2 should show a uniform cytoplasmic distribution. The experimental results are as Figure 2 shown. MGTR2 coupled with the mCherry tag shows red fluorescence in the results. MGTR1 coupled with the mCherry tag is normally expressed in cells. The cells transfected with MGTR2 grow normally. The coupled mCherry tag indicates that the MGTR2 protein indeed behaves as a cytoplasmic protein and MGTR2 is evenly distributed in the cells.

[0083] Example 3

[0084] MGTR1 antigen is used for the detection of anti - RyR1 positive serum

[0085] I. Cell transfection:

[0086] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum. Aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, pour out the supernatant, add DMEM complete medium containing 10% serum, and gently pipette to mix evenly to prepare a cell suspension.

[0087] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with poly - L - lysine (PDL). After the glass slides in the dish are dried, add the prepared cell suspension to the dish, gently mix evenly, and place it in an incubator at 37°C and 5% CO2 overnight. Observe the cells the next day. When the density reaches 30 - 40%, transfection can be carried out.

[0088] 3. Mix the prepared RyR1 expression plasmid (MGTR1, 6 μg) and pmCherry - N1 (empty vector control) plasmid with the transfection reagent PEI at a mass - to - volume ratio of 1:2 respectively, vortex, and let stand for 10 min. Then transfect them into the prepared cells and culture at 37°C and 5% CO2 for 48 h.

[0089] The transfection methods also include Lipofectamin 2000, Lipofectamin 3000, other liposome transfection reagents, electroporation or other transfection methods.

[0090] 4. Fixed-immunofluorescence staining;

[0091] (1) Fix the cells transfected for 48 h with acetone / cold methanol to prepare cell smears, dilute the serum of RyR1-positive patients 1:10, and incubate the cell smears for 1 h;

[0092] (2) Wash 3 times with PBS, 5 min each time;

[0093] (3) Incubate with the fluorescently labeled secondary antibody Alexa Fluor 488 (purchased from Thermo fisher) for 30 min;

[0094] (4) Wash 3 times with PBS, 5 min each time;

[0095] (5) Observe the results and take pictures under a microscope. The picture results are as Figure 3 shown.

[0096] It can be seen from Figure 3 that after cell fixation, MGTR 1-mCherry shows obvious red fluorescence signals at 546, and MGTR 1-mCherry shows obvious green fluorescence signals at 488. Compared with the control mCherry (Ctr), the RyR1 antibody-positive serum can detect obvious positive signals on the cells transfected with the MGTR1 antigen, and the two fluorescences can be well co-localized.

[0097] Example 4

[0098] Detection of MGTR2 antigen for anti-RyR 1 positive serum

[0099] I. Cell transfection:

[0100] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, pour out the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0101] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with polylysine (PDL). After the glass slides in the dish are dried, add the prepared cell suspension to the culture dish, gently mix, and place it in an incubator at 37°C and 5% CO2 overnight. Observe the cells the next day, and transfection can be carried out when the density reaches 30 - 40%.

[0102] 3. Mix the prepared RyR1 expression plasmid (MGTR2, 6 μg) and pmCherry-N1 (empty vector control) plasmid with the transfection reagent PEI at a mass-to-volume ratio of 1:2, vortex, and let stand for 10 min. Then, transfect them into the prepared cells and culture at 37°C and 5% CO2 for 48 h.

[0103] The transfection methods described above also include lipofectamin2000, lipofectamin3000, other liposome transfection reagents, electroporation, or other transfection methods.

[0104] 4. Fixation-immunofluorescence staining;

[0105] (1) Fix the cells transfected for 48 h with acetone / cold methanol to prepare cell smears. Dilute the RyR1-positive patient serum 1:10 and incubate the cell smears for 1 h.

[0106] (2) Wash with PBS 3 times, 5 min each time.

[0107] (3) Incubate with the fluorescently labeled secondary antibody Alexa Fluor 488 (purchased from Thermo fisher) for 30 min.

[0108] (4) Wash with PBS 3 times, 5 min each time.

[0109] (5) Observe and take pictures of the results under a microscope. The pictures are as shown in Figure 4 shown.

[0110] It can be seen from Figure 4 that MGTR2-mCherry shows obvious red fluorescence signals in 546, and MGTR2-mCherry shows obvious green fluorescence signals in 488. Compared with the control mCherry (Ctr), the RyR1 antibody-positive serum can detect obvious positive signals on the cells transfected with the MGTR2 antigen, and the two fluorescences can be well co-localized.

[0111] Figure 5 The following are the fluorescence results of using the cell matrix expressing the MGTR2 antigen to detect samples with different concentrations of RyR1 antibodies: Different RyR1 antibody titers (the titer represents the maximum dilution ratio at which a positive signal can be successfully detected. For example, a titer of 1:100 means diluting the serum sample 100 times) result in different detected fluorescence intensities and numbers of positive cells. Figure 5 It can be seen that the cell matrix expressing the MGTR2 antigen can effectively detect and distinguish RyR1 antibody signals at various different concentrations, indicating that the MGTR2 antigen has good sensitivity, specificity, and stability for the detection of anti-RyR1 positive serum.

[0112] Example 5

[0113] Analysis of the results of using the MGTR1 antigen to detect MG patients with thymoma.

[0114] The RyR1 antibody has a strong correlation with MG patients with thymoma. Past reports have shown that 40 - 60% of MG patients with thymoma can detect a positive RyR1 signal. In this example, 55 MG patients with thymoma and 75 MG patients without thymoma were selected, and cell materials prepared with the MGTR1 antigen were used for screening. 30 cases of positive RyR1 antibody were detected, indicating that the MGTR1 antigen designed in this example has good sensitivity and stability for the RyR1 antibody.

[0115] I. Cell transfection

[0116] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, pour off the supernatant, add DMEM complete medium containing 10% serum, and gently pipette to mix evenly to prepare a cell suspension.

[0117] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with polylysine (PDL). After the glass slides in the dish are air-dried, add the prepared cell suspension to the culture dish, gently mix evenly, and place it in an incubator at 37°C and 5% CO2 overnight. Observe the cells the next day, and transfection can be carried out when the density reaches 30 - 40%.

[0118] 3. Mix the prepared RyR1 expression plasmid (MGTR1, 6 μg) and pmCherry-N1 (empty vector control) plasmid with the transfection reagent PEI at a mass-volume ratio of 1:2 respectively, vortex, and let stand for 10 min. Then transfect them into the prepared cells and culture at 37°C and 5% CO2 for 48 h.

[0119] The transfection method described above also includes lipofectamin2000, lipofectamin3000, other liposome transfection reagents, electroporation, or other transfection methods.

[0120] 4. Fixation - immunofluorescence staining

[0121] (1) Fix the cells transfected for 48 h with acetone / PFA / cold methanol to prepare cell smears. Dilute 55 MG patients with thymoma and 75 MG patients without thymoma at a ratio of 1:10 and incubate the cell smears for 1 h;

[0122] (2) Wash with PBS 3 times, 5 min each time;

[0123] (3) Incubate with the fluorescently labeled secondary antibody Alexa Fluor 488 (purchased from Thermo Fisher) for 30 min;

[0124] (4) Wash 3 times with PBS, 5 min each time;

[0125] (5) Observe the results under a microscope and take pictures.

[0126] 5. Result analysis

[0127] The sera of 55 MG patients with thymoma and 75 MG patients without thymoma were analyzed by immunofluorescence using cells transfected with the MGTR 1 antigen. Taking the MGTR1 antigen as an example, 30 patients with positive anti-RyR1 antibodies were detected from 55 MG patients with thymoma, and 3 patients with positive anti-RyR1 antibodies were detected from 75 MG patients without thymoma. As shown in Table 1, the results of this example indicate that the MGTR1 antigen has good sensitivity and stability for the RyR1 antibody, and the MGTR1 antigen can be used for the detection and diagnosis of patients with positive RyR1 antibodies.

[0128] Example 6

[0129] Result analysis of using the MGTR2 antigen to detect MG patients with thymoma

[0130] I. Cell transfection

[0131] 1. Digest the cultured Hep2 cells with trypsin, terminate the digestion with DMEM complete medium containing 10% serum, aspirate the digested cells into a centrifuge tube, centrifuge at 800 - 1000 rpm for 3 min, pour out the supernatant, add DMEM complete medium containing 10% serum, and gently pipette and mix to make a cell suspension.

[0132] 2. Place the autoclaved glass slides in a cell culture dish, and then treat them with polylysine (PDL). After the glass slides in the dish are dried, add the prepared cell suspension to the culture dish, gently mix, and incubate overnight in an incubator at 37°C and 5% CO2. Observe the cells the next day, and transfection can be carried out when the density reaches 30 - 40%.

[0133] 3. Mix the prepared RyR1 expression plasmid (MGTR2, 6 μg) and pmCherry-N1 (empty vector control) plasmid with the transfection reagent PEI at a mass-volume ratio of 1:2 respectively, vortex, let stand for 10 min, and then transfect them into the prepared cells, and culture at 37°C and 5% CO2 for 48 h.

[0134] The transfection methods described above also include lipofectamin 2000, lipofectamin 3000, other liposome transfection reagents, electroporation, or other transfection methods.

[0135] 4. Fixation-Immunofluorescence Staining

[0136] (1) Fix the cells transfected for 48 h with acetone / PFA / cold methanol to prepare cell smears. Dilute 55 cases of thymoma-associated MG patients and 75 cases of non-thymoma-associated MG patients at a ratio of 1:10 and incubate the cell smears for 1 h.

[0137] (2) Wash with PBS three times, 5 min each time.

[0138] (3) Incubate with the fluorescently labeled secondary antibody Alexa Fluor 488 (purchased from Thermo fisher) for 30 min.

[0139] (4) Wash with PBS three times, 5 min each time.

[0140] (5) Observe the results and take pictures under a microscope.

[0141] 5. Result Analysis

[0142] Using the MGTR2 antigen, 27 anti-RyR1 antibody positive patients were detected from 55 thymoma-associated MG patients, and 4 anti-RyR1 antibody positive patients were detected from 75 non-thymoma-associated MG patients. As shown in Table 1, the results of this example indicate that the MGTR2 antigen has good sensitivity and stability for the RyR1 antibody, and the MGTR2 antigen can be used for the detection and diagnosis of RyR1 antibody positive patients.

[0143] Table 1 Statistical table of the results of detecting thymoma / non-thymoma MG patients with MGTR1 / MGTR2

[0144]

[0145] Example 7

[0146] This example provides a RyR1 antigen, the antigen is MGTR1, and the amino acid sequence of the antigen is shown in SEQ ID NO.1. The antigen is a fusion protein formed by splicing the RyR1 N-terminal and the middle part sequences. The antigen is linked with a fluorescent tag. Preferably, the fluorescent tag is an mCherry fluorescent tag. The preparation method of the antigen is the method provided in Example 1.

[0147] Example 8

[0148] This example provides a RyR1 antigen, the antigen being MGTR2, and the amino acid sequence of the antigen being as shown in SEQ ID NO.2. The antigen is a fusion protein formed by splicing partial sequences of the RyR1 N-terminus, middle, and C-terminus. The antigen is linked with a fluorescent label. Preferably, the fluorescent label is an mCherry fluorescent label. The preparation method of the antigen is the method provided in Example 2.

[0149] Example 9

[0150] This example provides a nucleic acid molecule that encodes an antigen with the amino acid sequence of SEQ ID NO.1. The preparation method of the nucleic acid molecule is the method provided in Example 1.

[0151] Example 10

[0152] This example provides a nucleic acid molecule that encodes an antigen with the amino acid sequence of SEQ ID NO.2. The preparation method of the nucleic acid molecule is the method provided in Example 2.

[0153] Example 11

[0154] This example provides a RyR1 antigen expression vector, and the antigen expression vector in this example is the MGTR1 expression vector. The expression vector includes a nucleic acid molecule encoding an antigen with the amino acid sequence of SEQ ID NO.1 and a vector. According to a preferred embodiment, the vector is pmCherry-N1, and the nucleotide sequence of the vector is as shown in SEQ ID NO.3. The construction method of the MGTR1 expression vector is the method described in Example 1.

[0155] Example 12

[0156] This example provides a RyR1 antigen expression vector, and the antigen expression vector in this example is the MGTR2 expression vector. The expression vector includes a nucleic acid molecule encoding an antigen with the amino acid sequence of SEQ ID NO.2 and a vector. According to a preferred embodiment, the vector is pmCherry-N1, and the nucleotide sequence of the vector is as shown in SEQ ID NO.3. The construction method of the MGTR2 expression vector is the method described in Example 2.

[0157] Example 13

[0158] This example provides an expression cell line of a RyR1 antigen, and the expression cell line contains a RyR1 antigen expression vector.

[0159] Example 14

[0160] This example provides a preparation method of the expression cell line, and the preparation method is the method provided in Example 1 or 2.

[0161] Example 15

[0162] This example provides the use of the RyR1 antigen with the amino acid sequence SEQ ID NO.1, the nucleic acid molecule encoding the RyR1 antigen, the RyR1 expression vector, and the RyR1 expression cell line in detecting RyR1 antibodies. The RyR1 antigen includes the antigens provided in Examples 1, 3, 5, and 7. The nucleic acid molecule includes the nucleic acid molecules provided in Examples 1, 3, 5, and 9. The eukaryotic expression vector includes the expression vectors provided in Examples 1, 3, 5, and 11. The expression cell line includes the cell lines provided in Examples 1, 3, 5, and 14.

[0163] Example 16

[0164] This example provides the use of the RyR1 antigen with the amino acid sequence SEQ ID NO.2, the nucleic acid molecule encoding the RyR1 antigen, the RyR1 expression vector, and the RyR1 expression cell line in detecting RyR1 antibodies. The antigen includes the antigens provided in Examples 2, 4, 6, and 8. The nucleic acid molecule includes the nucleic acid molecules provided in Examples 2, 4, 6, and 10. The expression vector includes the expression vectors provided in Examples 2, 4, 6, and 12. The expression cell line includes the cell lines provided in Examples 2, 4, 6, and 14.

[0165] Example 17

[0166] This example provides the application of the RyR1 antigen with the amino acid sequence SEQ ID NO.1, the nucleic acid molecule encoding the antigen, the expression vector, and the expression cell line in preparing a kit for detecting RyR1 antibodies. The RyR1 antigen includes the antigens provided in Examples 1, 3, 5, and 7. The nucleic acid molecule includes the nucleic acid molecules provided in Examples 1, 3, 5, and 9. The eukaryotic expression vector includes the expression vectors provided in Examples 1, 3, 5, and 11. The expression cell line includes the cell lines provided in Examples 1, 3, 5, and 14.

[0167] Example 18

[0168] This example provides the application of the RyR1 antigen with the amino acid sequence SEQ)ID NO.2, the nucleic acid molecule encoding the antigen, the expression vector, and the expression cell line in preparing a kit for detecting RyR1 antibodies. The antigen includes the antigens provided in Examples 2, 4, 6, and 8. The nucleic acid molecule includes the nucleic acid molecules provided in Examples 2, 4, 6, and 1O. The expression vector includes the expression vectors provided in Examples 2, 4, 6, and 12. The expression cell line includes the cell lines provided in Examples 2, 4, 6, and 14.

[0169] The present invention uses two different RyR antigens: MGTR1 and MGTR2. The positive rates of anti-RyR1 detected in MG patients with thymoma are 54% and 49% respectively, while the detection rates in MG patients without thymoma are approximately 4% and 5%. This is basically consistent with the reported ratios, indicating that the RyR1 antibody index does have good directivity for MG patients with thymoma. The two RyR1 antigens (MGTRl and MGTR2) provided by the present invention can specifically recognize RyR1 antibody and stably bind to RyR1 antibody; the present invention adopts a two-color fluorescence system. The antigen protein itself has red light, and the detection signal of the patient sample is green light. If there is a RyR1-specific antibody in the sample, the co-localization of green light and the red light spontaneously emitted by the protein will be detected, which is beneficial to the specificity of the detection. The detection material prepared by transfecting cells with plasmids carrying MGTRl and MGTR2 genes has good sensitivity, specificity and stability for RyR1 antibody. Based on the good sensitivity, specificity and stability of the RyR1 cell detection matrix material, the RyR1 cell detection matrix of the present invention can meet the needs of assisting clinical diagnosis.

[0170] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosed content of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation to the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. Expressions such as "preferably", "according to a preferred embodiment" or "optionally" indicate that the corresponding paragraphs disclose an independent concept. The applicant reserves the right to file divisional applications according to each inventive concept. Throughout the text, the features guided by "preferably" are only an optional manner and should not be understood as a must. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A RyR1 antigen, characterized in that, The amino acid sequence of the antigen is as shown in SEQ ID NO.

1.

2. A RyR1 antigen, characterized in that, The amino acid sequence of the antigen is as shown in SEQ ID NO.

2.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antigen according to claim 1 or claim 2.

4. A RyR1 antigen expression vector, characterized in that, The expression vector comprises the nucleic acid molecule according to claim 3 and a vector.

5. The RyR1 antigen expression vector according to claim 4, wherein, The nucleotide sequence of the vector is as shown in SEQ ID NO.

3.

6. An expression cell line of RyR1 antigen, characterized in that, Containing the expression vector according to claim 4 or claim 5.

7. The method for preparing the expression cell line according to claim 6, characterized in that, Comprising the following steps: transferring the RyR1 antigen expression vector according to claim 4 into a cell line and obtaining it after screening.

8. The method for preparing an expression cell line according to claim 7, wherein Comprising the following steps: the expression cell line is a Hep2 cell.

9. Use of the RyR1 antigen according to claim 1 or claim 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 or claim 5, and the expression cell line according to claim 7 in the preparation of a kit for detecting RyR1 antibodies.

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