CN117031014A - Application of MATR3 autoantibody resisting reagent in preparation of products for detecting nervous system autoimmune diseases and reagent kit - Google Patents

Abstract

The invention discloses application of a reagent for resisting MATR3 autoantibodies in preparation of a product for detecting nervous system autoimmune diseases and a kit, and belongs to the technical field of biological medicines. Experiments prove that MATR3 can be used as one of recognition antigens of autoimmune disease related autoantibodies of the central nervous system for the first time, especially MS related diseases, and the reagent for resisting MATR3 autoantibodies can realize auxiliary diagnosis of autoimmune disease of the central nervous system.

Description

Application of MATR3 autoantibody resisting reagent in preparation of products for detecting nervous system autoimmune diseases and reagent kit

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for resisting MATR3 autoantibodies in preparation of a product for detecting autoimmune diseases of a nervous system and a kit.

Background

Autoimmune diseases of the central nervous system (central nervous system, CNS) are autoimmune diseases which take autoimmune cells, autoantibodies and other immune molecules to directly or indirectly attack the nervous system (including neurons, glial cells and myelin) as main pathogenic mechanisms, and comprise inflammatory demyelinating diseases of the central nervous system, autoimmune encephalitis (autoimmune encephalitis, AE), CNS vasculitis and the like, and have complex pathogenesis, high disability rate and various disease types, and the autoantibodies play a very important role in diagnosis and differential diagnosis of the autoimmune encephalitis and CNS inflammatory demyelinating diseases.

Common clinical inflammatory demyelinating diseases of the central nervous system include diseases of the neuromyelitis spectrum (neuromyelitis optic spectrum disorder, NMOSD), myelin oligodendrocyte glycoprotein antibody-related diseases (myelin oligodendrocyte glycoprotein-IgG associated disorders, (MOGAD), multiple sclerosis (multiple sclerosis, MS) and acute disseminated encephalomyelitis (acute disseminated encephalomyelitis, ADEM). Wherein multiple sclerosis (multiple sclerosis, MS) is caused by immune system attacks on the peripheral myelin of neurons in the brain and spinal cord, most vulnerable to the brain stem, cerebellum and periventricular white matter, which can lead to poor communication between neurons and weakening of axons, various parts of the central nervous system can be affected, common symptoms of MS including limb dyskinesia, limb dysesthesia, fatigue, ataxia, vision decline, multiple vision, bladder or rectal dysfunction, etc. clinical typing includes relapsing MS, secondary progressive MS and other types, and the pathogenesis of MS is currently unclear, and may be related to autoimmune reactions, viral infections, genetic factors or genetic factors.

MATR3 (Martin-3), also known as ALS21 or VCPDM, encodes a nuclear matrix protein that is ubiquitously expressed in the brain, thyroid and muscle, has two RNA recognition domains and two zinc finger domains, and can bind RNA and/or DNA. MATR3 has a variety of different RNA-related regulatory functions including transcriptional regulation, mRNA stabilization, alternative splicing, nuclear retention of super-editing RNA, and gene silencing. MATR3 is also involved in DNA damage repair, cell survival, maintenance of neural stem cells, and muscle formation. Matrin 3 plays a role in a variety of tissues including the nervous system and muscles.

The present study found that MATR3 gene mutation was associated not only with distal myopathy, but also with amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) and frontotemporal dementia (FTD). However, no report has been found on MATR3 autoantibodies in autoimmune diseases of the central nervous system, in particular in MS.

Disclosure of Invention

The invention aims to provide an application of an anti-MATR 3 autoantibody reagent in preparing a product for detecting nervous system autoimmune diseases.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses application of an autoimmune disease marker in preparation of a detection reagent or a detection kit for diagnosis of central nervous system autoimmune diseases, wherein the autoimmune disease marker is an autoantibody combined with MATR3 protein, and the autoimmune disease marker is an autoantibody combined with MATR3 protein of a detection sample during detection.

Preferably, the amino acid sequence of the MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and has the function of recognizing MATR3 autoantibodies;

c) The amino acid sequence of a) or b) is modified or mutated and has the function of recognizing MATR3 autoantibodies.

Further preferably, the nucleotide sequence encoding the MATR3 protein comprises any one of I) to III):

i) the nucleotide sequence shown in SEQ ID NO. 2;

II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.2, and encodes an amino acid sequence recognizing MATR3 autoantibodies;

III) after mutation of the nucleotide sequence in I) or II) and encoding an amino acid sequence recognizing MATR3 autoantibodies.

Further preferably, the central nervous system autoimmune disease comprises a central nervous system demyelinating disease.

Still more preferably, the central nervous system demyelinating disease includes multiple sclerosis.

Preferably, the symptom of the central nervous system autoimmune disease is one or more of limb distal paresthesia, limb weakness, limb fatigue, vision disorder, limb stiffness, gait disorder, dizziness, affective disorder, bladder control difficulty, cognitive disorder, affective disorder, depression, and epilepsy.

Preferably, the test sample is one or more of whole blood, serum and cerebrospinal fluid.

The invention also discloses application of the protein for detecting autoimmune diseases in preparing detection reagents or detection kits for diagnosing the autoimmune diseases of the central nervous system, wherein the protein for detecting autoimmune diseases is one or more epitopes derived from MATR3 protein or fusion protein fused with other amino acids; wherein the amino acid sequence of MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and recognizing the amino acid sequence of MATR3 autoantibody;

c) After modification or mutation of the amino acid sequence in a) or b), the amino acid sequence of MATR3 autoantibodies is recognized.

The invention also discloses a kit for detecting the autoimmune disease of the central nervous system, which comprises a reagent for detecting the anti-MATR 3 antibody and a labeled antibody; wherein:

the reagent for detecting the anti-MATR 3 antibody comprises one or more of MATR3 protein, cells expressing MATR3 protein, tissues expressing MATR3 protein and lysates containing MATR3 protein;

the labeled antibody is an antibody capable of binding to the Fc fragment of human IgG.

Preferably, the amino acid sequence of the MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and recognizing the amino acid sequence of MATR3 autoantibody;

c) After modification or mutation of the amino acid sequence in a) or b), the amino acid sequence of MATR3 autoantibodies is recognized.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides application of an anti-MATR 3 autoantibody reagent in preparing a product for detecting the autoimmune disease of a nervous system, experiments prove that MATR3 can be used as one of recognition antigens of the autoimmune disease related to the central nervous system, especially MS related diseases, and the anti-MATR 3 autoantibody reagent can realize auxiliary diagnosis of the autoimmune disease of the central nervous system.

Drawings

FIG. 1 shows the staining results of patient 1 and normal control serum on frozen sections of rat brain tissue;

FIG. 2 shows the co-staining results of patient 1 serum with neuronal marker NeuN antibodies;

FIG. 3 shows the results of staining the patient 1 and normal control serum on MATR3 overexpressing cell slide;

FIG. 4 shows the co-staining of patient 1 serum with MATR3 antibodies on overexpressing cells;

FIG. 5 shows the co-staining of patient 1 serum with MATR3 antibody on rat brain tissue sections;

FIG. 6 shows the results of MATR3 antibodies validating over-expressed protein on WB;

FIG. 7 is a graph showing the signals detected by serum neutralization experiments to verify patient serum on overexpressing cells;

FIG. 8 is a graph showing the staining of serum recovery samples on overexpressing cell slide;

FIG. 9 is the result of staining of serum recovered samples on rat brain tissue sections;

FIG. 10 shows the staining results of 3 patients screened positive.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention. It should be noted that the terms "comprises" and "comprising," along with any variations thereof, in the description and claims of the present invention are intended to cover a non-exclusive inclusion, such as a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus.

Term interpretation:

according to the invention, a "cell" is any prokaryotic or eukaryotic host cell capable of being transformed into a vector. For example, the cells may be bacterial cells (e.g., E.coli cells) or eukaryotic cells (e.g., immortalized human cells, insect cells, yeast). Such cells include HEK293 cells, hela cells, CHO, pichia, saccharomyces cerevisiae, sf9, BL21, rosetta, etc. cells commonly used by those skilled in the art.

"immobilized" within the scope of the present invention refers to molecules which are bound to a solid support which is insoluble in aqueous solution, more preferably by means of covalent bonds, electrostatic interactions, encapsulation or entrapment or by means of hydrophobic interactions, most preferably by means of one or more covalent bonds. Such as glass slides, polystyrene plates, glass plates, membranes (nylon membranes, nitrocellulose membranes, or PVDF membranes), magnetic beads, column chromatography media, biochips, polyacrylamide gels, and the like.

In the present invention, a "vector" includes an insert (e.g., an original or mutant nucleic acid sequence encoding a desired protein) and other features (promoters, multiple cloning sites, selectable markers, replicons, etc.) of circular or linear nucleic acid sequences. Such vectors include vectors of pTriEx vector family, pCDNA3 family, pET series, pBac series and other commercial eukaryotic expression systems or prokaryotic expression systems.

The invention is described in further detail below with reference to the attached drawing figures:

the invention provides application of a reagent for detecting an anti-MATR 3 autoantibody in preparation of a product for detecting central nervous system autoimmune diseases.

In the present invention, the product preferably comprises a kit.

In the present invention, the symptoms of the autoimmune disease of the central nervous system include one or more of limb distal paresthesia, limb weakness, limb fatigue, vision disorder, limb stiffness, gait disorder, dizziness, affective disorder, bladder control difficulty, cognitive disorder, affective disorder, depression, and epilepsy;

the central nervous system autoimmune disease preferably comprises a central nervous system inflammatory demyelinating disease; the central nervous system inflammatory demyelinating disease preferably includes multiple sclerosis.

In the present invention, the reagent for detecting an anti-MATR 3 autoantibody preferably comprises a MATR3 protein, or a homolog of the MATR3 protein, or a derivative of the MATR3 protein, or a vector or a cell capable of expressing the MATR3 protein, or a tissue containing the MATR3 protein, and more preferably a MATR3 protein.

In the present invention, the amino acid sequence of the MATR3 protein preferably comprises any one of a) to c):

a) An amino acid sequence shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and has the function of recognizing MATR3 autoantibodies;

c) The amino acid sequence in a) or b) is modified or mutated and has the function of recognizing MATR3 autoantibodies.

In the present invention, the nucleotide sequence encoding the MATR3 protein preferably comprises any one of I) to III):

i) the nucleotide sequence shown in SEQ ID NO. 2;

II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.2, and can code an amino acid sequence for recognizing MATR3 autoantibodies;

III) after mutation of the nucleotide sequence in I) or II) and can code for an amino acid sequence recognizing MATR3 autoantibodies.

The patient serum in the embodiment of the invention is given by a hospital, and the serum of a healthy subject comes from the serum of a healthy physical examination person given by a physical examination center of the hospital after the consent of the patient, and the patient information is as follows:

patient 1: sex women, age 31 years, no past medical history, no obvious cause of weakness of bilateral limbs, starting from the left side, numbness, difficulty in walking, unstable gait, holding things, no headache, dizziness, choking by drinking water, dysphagia, urine and feces disorder, acute left eye staring sickness, and no pain and compound vision. Checking: the vital sign is stable, consciousness is clear, speech is fluent, blood is normal, blood biochemistry is normal, various indexes of rheumatism immunity are normal, cerebrospinal fluid IgG is increased, and serum AQP4-IgG is negative. The doctor suspects that they have autoimmune diseases of the central nervous system, and sends a sample to a clinical laboratory, which detects that the inflammatory demyelinating lesions of the central nervous system are negative for 6 (AQP 4, MBP, MOG, GFAP, AQP1, flotillin 1/2) antibodies, 6 (including NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABABR) antibodies, and 14 (including Ri, hu, yo, CV2, ma2, amphiphysin, titin, ma1, SOX1, tr, zic4, PKC gamma, recoverin, GAD) antibodies. The case was then diagnosed with multiple sclerosis via comprehensive examination.

Example 1 Indirect immunofluorescence of rat brain tissue sections patient serum was screened out

(1) Preparation of rat brain tissue frozen section

Adult rats are selected for anesthesia, the abdominal cavity is opened after the four limbs of the rats harden, the apex of the heart is exposed, and PBS is infused from the left apex of the heart so as to be convenient for systemic circulation; taking out brain tissue, fixing with methanol for 10-30 min, transferring the sample into 30% sucrose solution for dehydration, and placing at 4 ℃ until the tissue blocks are settled; dripping a small amount of embedding medium OCT onto a sample table, placing into a freezing table of a frozen microtome (manufacturer: LEICA model: CM 1950) at-20deg.C, coating a thin layer on the surface of the sample with OCT when the tissue is slightly whitened, and continuously freezing for 20min for slicing.

(2) Serum incubation

Patient 1 serum and normal control serum were mixed with PBS at 1:10, respectively incubating the diluted PBST onto the rat brain tissue frozen slices prepared in the step (1), incubating the diluted PBST for 1h at room temperature, and washing the PBST for 3 times, wherein each time is 5min; adding 1:200 dilution of FITC-labeled goat anti-human IgG secondary antibody (manufacturer: jackson cat# 109-095-170), 30min incubation at room temperature, 3 PBST washes, 5min each; the results are shown in FIG. 1.

As can be seen from fig. 1, patient 1 serum showed positive signals at the hippocampal and cortical sites on frozen sections of rat brain tissue, whereas normal control serum showed no positive signals at this site, suggesting that antibodies recognizing neuronal cell antigens may be present in patient serum.

(3) Antibody co-staining

Use 1: incubation of 200 diluted neuron-specific Marker NeuN antibody (manufacturer: wuhan Sanying goods number: 26975-1-AP) frozen sections of rat brain tissue incubated in step (2), incubation at room temperature for 30min, and PBST washing 3 times for 5min each; adding 1:200 dilutions of Alexa Fluor 594-labeled goat anti-rabbit IgG (manufacturer: jackson cat. Number: 115-585-144) were incubated for 30min at room temperature, washed 3 times with PBST for 5min each; the results are shown in FIG. 2.

As can be seen from fig. 2, the signals of patient 1 serum appear overlapping with the neuron-specific Marker NeuN antibody signals at the hippocampal and cortical sites, indicating that the antigen recognized by the antibodies in patient 1 serum is located on the neuronal cells.

Example 2 screening and identification of target antigens

Searching reported target antigens related to the autoantibodies of the nervous system, preparing cell climbing sheets which overexpress the target antigens, assembling a plurality of cell climbing sheets which overexpress the target antigens into a biological detection chip material, and performing immunofluorescence staining on a patient sample, wherein the specific method is as follows:

(1) Searching 60 reported target antigens of autoantibodies of the nervous system (including DPPX, igLON5, glyR, GABAAR alpha 1, GABAAR gamma 2, GABAAR beta 3, mGluR5, D2R, neurexin alpha, AQP4, MBP, MOG, GFAP, AQP1, flotillin1/2, home 3, ITPR1, MGLUR5, ca/ARHGAP26, kcna4/kv1.4, ATP1A3, NCDN, septin5, nrCAM, gliomedin, KLHL11, gephyrin, ca8/CA-VIII, TGM2, TGM6, trib2, tpo, ak5, GRIA3/GLUR3, MUNC18-1, KCTD16, GRM1/MGLUR1, CACNNA 2D1/CaV alpha 2 delta, RY 1, PLP1, PDE10A, adam, ROCK2, mGluR2/GRM2, rab 6/3875, NF1, CNLUR 1, CNR 78, searching gene sequences encoding the 60 proteins from NCBI and sending the gene sequences to a sequencing company for gene synthesis to obtain recombinant vectors of the corresponding genes, then transfecting the recombinant vectors into 293T cells to obtain recombinant cells, preparing a biological detection chip material for over-expressing target antigens, detecting whether serum/cerebrospinal fluid of a patient can generate immune reaction after incubation with the biological detection chip by an immunofluorescence method, and searching whether the serum/cerebrospinal fluid of the patient contains specific autoantibodies of the series of genes or not, wherein the specific steps are as follows:

a. recombinant vector construction: connecting the 60 genes to pCDNA3.1 through a molecular cloning method by a PCR or artificial synthesis method to obtain 60 recombinant vectors, and carrying out large extraction on the constructed recombinant vectors for later use after the sequencing is correct;

b. transfection of the target Gene: high sugar culture using 10% FBS-DMEM based on 37℃and 5% CO ₂ 293T cells with 6cm multiplied by 6cm climbing sheets are paved at the bottom of a culture dish in a cell culture box, 61 dishes in total, when the cell density reaches 30% -40%, a PEI transfection reagent (manufacturer: thermo, goods number: BMS 1003) is used for respectively transfecting recombinant vectors of 60 corresponding genes and empty pCDNA3.1 into the 293T cells, and marking is carried out;

c. cell climbing sheet fixation: washing 48h of cells after transfection with PBS for 2 times, adding acetone for fixing for 5min, washing with PBS for 2 times, drying at 45 ℃ for 30min, cutting the climbing slices into 2.5mm multiplied by 2.5mm, and pasting 61 cell climbing slices with the size of 2.5mm multiplied by 2.5mm on a glass slide to prepare a biological detection chip for screening target antigens for later use;

d. immunofluorescent staining: patient 1 serum and healthy subject serum (control) were each in accordance with 1:10, incubating the diluted solution on a biological detection chip at room temperature for 1h, and washing the solution with PBST for 3 times, each time for 5min; use 1:200 diluted FITC-labeled goat anti-human secondary antibodies are incubated for 30min at room temperature, and PBST is washed 3 times for 5min each time; the fluorescence microscope showed that the signals of the serum of the patient 1 and 60 biological detection chips were not significantly stronger than the signals of the serum (control) of the healthy subjects, but the staining results of the rat brain tissue sections in example 1 showed that the staining results of the serum of the patient 1 were obvious at the hippocampal and cortical regions of neurons, so that it was suspected that there might be a new autoantibody different from the previously reported identifiable neuronal cells in the serum of the patient 1.

(2) 100 proteins with relatively high expression levels in the hippocampus and cortex of the human brain (which are not reported in the prior art to be recognized by autoantibodies of autoimmune diseases of the nervous system) are searched from the human protein atlas (https:// www.proteinatlas.org /), a biological detection chip is prepared according to the step (1) of reference example 2, whether the serum or cerebrospinal fluid of the patient 1 can react with the biological detection chip after incubation is detected by immunofluorescence, and whether the serum or cerebrospinal fluid of the patient 1 contains novel autoantibodies which are different from the previously reported identifiable neuron cells is explored, wherein the specific steps are as follows:

a. preparing a biological detection chip: the gene sequences encoding the proteins are searched from NCBI, and sent to sequencing companies to synthesize the genes onto pcDNA3.1, the synthesized recombinant vector is transformed into clone TOP10 for amplification, and then the plasmid is extracted for later use. The recombinant vector is respectively transfected into 293T cells growing on a 6cm multiplied by 6cm climbing plate by using a PEI transfection reagent, the cell climbing plate of 6cm multiplied by 6cm is cut into a size of 2.5mm multiplied by 2.5mm for standby after being washed, fixed and dried after 48 hours of transfection, and the cell climbing plate is stuck to a glass slide after being prepared for sample detection.

b. Immunofluorescent staining: patient 1 serum and healthy subject serum (control) were each in accordance with 1:10, incubating the diluted solution on a biological detection chip at room temperature for 1h, and washing the solution with PBST for 3 times, each time for 5min; use 1:200 diluted FITC-labeled goat anti-human secondary antibodies are incubated for 30min at room temperature, and PBST is washed 3 times for 5min each time; the results are shown in FIG. 3.

As can be seen from fig. 3, the patient 1 serum developed a significantly stronger color reaction with one antigen on the bioassay chip than the healthy subject serum (control), producing a positive signal, whereas none of the antigens on the healthy subject serum (control) and bioassay chip produced a signal. The target antigen is MATR3 (matrine-3, accession number: NM_ 199189.3), the amino acid sequence is shown as SEQ ID NO.1, and the coding sequence is shown as SEQ ID NO. 2.

EXAMPLE 3 commercial antibodies verify patient serum detection signals in overexpressed cells and rat brain tissue

(1) Commercial antibody validation of MATR3 Signal detected by serum on over-expressed cell slide

Commercial MATR3 antibodies (manufacturer: proteontech) were diluted 1:200 with PBST, incubated to MATR3 overexpressing cell slide incubated with patient 1 serum in example 2, incubated 30min at room temperature, washed 3 times with PBST 5min each, added 1:200 diluted Alexa Fluor 594-labeled goat anti-rabbit IgG (manufacturer: jackson, cat# 115-585-144), incubated 30min at room temperature, and washed 2 times with PBST 5min each; the results are shown in FIG. 4.

As can be seen from fig. 4, the staining signal of the patient serum on the overexpressed MATR3 cell slide overlaps with the staining signal of the commercial MATR3 antibody on the overexpressed MATR3 cell slide, indicating that the antibody in the patient 1 serum specifically recognizes the MATR3 protein on the overexpressed MATR3 cell slide.

(2) Commercial antibody validation of MATR3 Signal from serum on rat brain tissue sections

a. Reference example 1 step (1) preparation of rat brain tissue sections;

b. patient serum was diluted 1:10 with PBST, rat brain tissue sections were incubated, incubated for 1h at room temperature, and PBST washed 3 times for 5min each; adding 1:200 dilution of FITC-labeled goat anti-human IgG (manufacturer: jackson), 30min incubation at room temperature, 3 washes with PBST 5min each; co-staining with commercial MATR3 antibody (manufacturer: proteontech) diluted 1:200, incubating for 30min at room temperature, washing with PBST 3 times, each for 5min, adding Alexa Fluor 594-labeled goat anti-rabbit IgG (manufacturer: jackson, cat# 115-585-144) diluted 1:200, incubating for 30min at room temperature, and washing with PBST 2 times, each for 5min; the results are shown in FIG. 5, when observed under a microscope.

As can be seen from fig. 5, the staining signal of patient 1 serum on the rat brain tissue section overlapped with the staining signal of the commercial MATR3 antibody on the rat brain tissue section, indicating that the antibody in patient 1 serum specifically recognizes with MATR3 protein on the rat brain tissue section.

EXAMPLE 4 serum neutralization experiments verify the signals detected by the patient's serum

(1) Preparation of neutralizing proteins

293T cells overexpressing MATR3 and empty pCDNA3.1 cells were collected from 1 dish according to example 2 step (2), centrifuged at 800rpm at room temperature to remove the supernatant, 200. Mu.L of PBS was added, and sonicated (disruption conditions: 10% power, disruption for 3s, disruption for 6s, co-sonication for 1 min) as MATR 3-neutralized proteins; preparing a control protein by using cells transfected with empty pCDNA3.1, wherein the preparation conditions and the preparation method are the same as those of MATR3 neutralizing protein;

(2) Identification of neutralizing proteins

Taking the harvested MATR3 neutralization protein and the control protein as sample loading samples, measuring the concentration, respectively taking 40 mug protein to carry out SDS-PAGE gel electrophoresis, and carrying out wet transfer membrane under the condition of 300mA and 90min after the electrophoresis is finished; sealing 5% of skimmed milk powder for 1h at room temperature; commercial MATR3 antibodies were diluted 1:1000 using TBST and incubated overnight at 4 ℃; the next day, TBST is washed 3 times for 5min each time; adding HRP-labeled goat anti-rabbit secondary antibody (manufacturer: jackson), and incubating for 1h at room temperature; TBST is washed for 3 times, each time for 5min; the chemiluminescent solution was added for color development and photographing, and the result is shown in FIG. 6.

(3) Serum neutralization assay signals detected by patient serum were validated on an overexpressing MATR3 cell slide

3 parts of patient 1 serum diluted 1:10 are prepared by PBST, each 100 mu L of serum is added with 20 mu LPBST, 20 mu L of MATR3 neutralizing protein and 20 mu L of control neutralizing protein respectively, the serum is incubated for 30min at room temperature, the prepared cell climbing tablets over-express MATR3 are incubated respectively, the serum is incubated for 1h at room temperature, and the serum is washed 3 times by PBST for 5min each time; FITC-labeled goat anti-human secondary antibody (manufacturer: jackson cat# 109-095-170) diluted 1:200 was added, incubated at room temperature for 30min, PBST washed 3 times for 5min each, and observed under a fluorescence microscope, and the results are shown in FIG. 7.

As can be seen from fig. 6, the color development of the overexpressed MATR3 protein was significantly stronger than that of the control protein, and thus it was considered that the MATR3 protein was successfully overexpressed on 293T cells; as can be seen from fig. 7, on the overexpressed MATR3 cell slide, the patient serum signal was blocked by MATR3 neutralizing protein, whereas the control protein did not block the signal present on the overexpressed MATR3 cell slide, indicating that this signal is a signal specifically recognizing MATR3 antigen.

EXAMPLE 5 recovery of autoantibodies from patient serum to verify patient serum detection Signal

(1) Patient serum recovery experiment

a. Transfection of the target Gene: 293F suspension cells were cultured in 20mL in 100mL flasks using CD05 medium (manufacturer: olprimex), 2 flasks total, and placed at 37℃in 5% CO ₂ In a cell culture shaker. When the cell density reaches 3X 10 ⁶ at/mL, the pCDNA3.1-MATR3 recombinant vector and empty pCDNA3.1 were transfected into 293F cells, respectively, using PEI transfection reagent (manufacturer: thermo, cat. BMS 1003), and labeled, and centrifuged the next day for liquid exchange;

b. cell fixation: respectively centrifuging two bottles of suspension cells grown for 96 hours after transfection, washing with PBS for 2 times, re-suspending with 5mLPBS, adding absolute ethanol for fixing for 10min, centrifuging to remove ethanol, washing with PBS for 2 times, placing in a 2mL EP tube, and adding 1mLPBS for re-suspending;

c. antibody elution: adding 12 mu L of patient 1 serum into the two tubes of fixed cells, incubating at 4 ℃ for the next day, re-suspending with PBS after centrifugation, and repeatedly washing for 5min each time; after washing, adding 500 mu L of 0.1M glycine eluent with pH=3 into each tube, eluting for 15min by a rotary table at room temperature, centrifugally collecting eluent after eluting, adding 10 mu L of 1M Tris into the eluent to neutralize until the pH of the eluent is 7.0-8.0, and adding 1/10 volume of PBS to obtain 2 parts of 500 mu L serum recovered samples, wherein one part is the recovered sample after the combination of patient 1 serum and over-expressed MATR 3; the other is a recovered sample of patient 1 serum after binding to control pcdna 3.1.

(2) Verification of serum recovery samples on overexpressed cell slide

Referring to example 2, an overexpressing MATR3 protein cell slide and an empty pcdna3.1 control cell slide were prepared, and the cell slide was incubated with 2 sample eluents from step (1), incubated for 1h at room temperature, and pbst washed 3 times for 5min each; incubation with FITC-labeled goat anti-human IgG at room temperature for 30min, and PBST washing 3 times for 5min each with 1:200 dilution; the results of observation under the fluorescence microscope are shown in FIG. 8.

As can be seen from fig. 8, the serum recovered sample combined with the overexpressed MATR3 suspension cells had a positive signal on the overexpressed MATR3 cell slide, whereas the control serum recovered sample did not, indicating that the serum recovered sample combined with the overexpressed MATR3 suspension cells specifically recognized the MATR3 antigen on the overexpressed cell slide, which can be used as an autoantibody eluent (i.e., as a positive sample), and the recovered sample combined with pcdna3.1 can be used as a control eluent (i.e., as a negative sample).

(3) Serum recovery samples verify target antigens on rat brain tissue sections

Preparing frozen sections of rat brain tissue according to the step (1) of the reference example 1, respectively incubating the sections of rat brain tissue with the autoantibody eluent obtained in the step (2) of the example 5 and the control eluent, incubating for 1h at room temperature, and washing with PBST for 3 times each for 5min; incubation with FITC-labeled goat anti-human IgG at room temperature for 30min, and PBST washing 3 times for 5min each with 1:200 dilution; the results of observation under the fluorescence microscope are shown in FIG. 9.

As can be seen from fig. 9, the autoantibody eluate had a positive signal on the rat brain tissue section, whereas the control eluate did not, indicating that the autoantibody eluate can specifically recognize MATR3 antigen expressed on the rat brain tissue section.

Example 6 detection Rate of anti-MATR 3 autoantibodies in samples suspected of autoimmune neurological disease

2758 patients with neurological disease were sampled and exhibited symptoms of: suspected encephalitis, paraneoplastic syndrome, myasthenia gravis, neuromyelitis optica. The test was performed using the MATR 3-overexpressing cell slide of example 2, MATR3 autoantibody positive serum samples were screened, 8 MATR3 autoantibody positive samples were screened out (partial patient results are shown in FIG. 10), the detection rate of anti-MATR 3 antibodies was 0.29%, and the patients detected for anti-MATR 3 antibodies had multiple sclerosis by the diagnosis by the clinician, one of the MS patients had ALS disease in combination. The invention provides a new antigen combined with an autoantibody to be tested for diagnosing the multiple sclerosis, which shows that the antibody has an auxiliary effect on diagnosing the multiple sclerosis.

Example 7 cell-based immunofluorescence assay to verify specificity of anti-MATR 3 autoantibodies

Serum from 100 patients with neurological autoimmune disease and 50 healthy controls was selected for immunofluorescence assays using the MATR3 overexpressing cell slide prepared in example 2, for specific procedures with reference to example 2, wherein at least one of AQP4, MBP, MOG, GFAP, AQP1, PLP1, flotillin-1/2, NF155, NF186, CNTN1, CNTN2, CASPR1, hu, yo, CV2, ma2, amphiphysin, ma1, SOX1, NMDAR, AMPAR1, AMPAR2, LGI1, CASPR2, GABABR, DPPX, igLON5, D2R, neurexin3, KCNA4, GABARy 2, ATP1A3, homer3, ARHGAP26, ITPR1/2, mGluR1, CARP VIII, AP3B2, septin5, GM1, GD1B, GQ1B, sulfatides, GT B, GT1a, GD3, GD2, GD1a, GM4, GM3 and GM2 autoantibodies were present in the serum from 100 patients.

The immunofluorescence assay showed that none of the selected 100 patients with neurological autoimmunity and 50 healthy controls had similar cell morphology to the 8 patients screened in example 6, as demonstrated by the over-expressed MATR3 cell slide. Patients with the autoimmune diseases of the nervous system described herein have autoantibodies to MATR3 protein, while other patients with autoimmune diseases of the nervous system and healthy subject samples do not have such antibodies. The invention provides a new antigen combined with autoantibodies to be tested for realizing diagnosis of nervous system diseases.

In summary, by incubating rat brain tissue sections with patient serum and healthy human serum suffering from the neurological disease described in the present invention, by amplifying signals with fluorescent secondary antibodies and co-staining with neuron-specific Marker antibodies, it was found that autoantibody MATR3 exists in the patient serum compared with healthy human serum, and by incubating patient serum with overexpressed target antigen suspension cells and recovering serum active antibody components, an antibody-containing eluent and a control eluent were prepared, and incubating the overexpressed cell climbing sheet and rat brain tissue sections verifies the authenticity and specificity of the target antigen, indicating that MATR3 protein is expressed in rat brain tissue and a significant signal appears, MATR3 can be used as one of the recognition antigens of central nervous system autoimmune disease-related autoantibodies, in particular, an MS-related disease, and the reagent against MATR3 autoantibodies can realize the auxiliary diagnosis of central nervous system autoimmune disease.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. The application of an autoimmune disease marker in preparing a detection reagent or a detection kit for diagnosing the autoimmune disease of the central nervous system is characterized in that the autoimmune disease marker is an autoantibody combined with MATR3 protein, and the autoimmune disease marker is an autoantibody combined with MATR3 protein of a detection sample during detection.

2. The use according to claim 1, wherein the amino acid sequence of the MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and has the function of recognizing MATR3 autoantibodies;

c) The amino acid sequence of a) or b) is modified or mutated and has the function of recognizing MATR3 autoantibodies.

3. The use according to claim 2, wherein the nucleotide sequence encoding the MATR3 protein comprises any one of i) to iii):

i) the nucleotide sequence shown in SEQ ID NO. 2;

II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.2, and encodes an amino acid sequence recognizing MATR3 autoantibodies;

III) after mutation of the nucleotide sequence in I) or II) and encoding an amino acid sequence recognizing MATR3 autoantibodies.

4. The use of claim 2, wherein the central nervous system autoimmune disease comprises a central nervous system demyelinating disease.

5. The use of claim 4, wherein the central nervous system demyelinating disease comprises multiple sclerosis.

6. The use of claim 1, wherein the symptom of the central nervous system autoimmune disease is one or more of limb distal paresthesia, limb weakness, limb fatigue, vision disorder, limb stiffness, gait abnormalities, dizziness, affective disorder, bladder control difficulty, cognitive disorder, affective disorder, depression, and epilepsy.

7. The use of claim 1, wherein the test sample is one or more of whole blood, serum, and cerebrospinal fluid.

8. Use of a protein for detecting autoimmune diseases in the preparation of a detection reagent or a detection kit for diagnosis of autoimmune diseases in the central nervous system, characterized in that the protein for detecting autoimmune diseases is one or more epitopes derived from MATR3 protein or is a fusion protein fused with other amino acids; wherein the amino acid sequence of MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and recognizing the amino acid sequence of MATR3 autoantibody;

c) After modification or mutation of the amino acid sequence in a) or b), the amino acid sequence of MATR3 autoantibodies is recognized.

9. A kit for detecting autoimmune diseases of the central nervous system, comprising a reagent for detecting an anti-MATR 3 antibody and a labeled antibody; wherein:

the reagent for detecting the anti-MATR 3 antibody comprises one or more of MATR3 protein, cells expressing MATR3 protein, tissues expressing MATR3 protein and lysates containing MATR3 protein;

the labeled antibody is an antibody capable of binding to the Fc fragment of human IgG.

10. The kit for detecting a neurological related disorder of claim 8 wherein the amino acid sequence of MATR3 protein comprises any one of a) to c):

a) An amino acid sequence as shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown as SEQ ID NO.1, and recognizing the amino acid sequence of MATR3 autoantibody;

c) After modification or mutation of the amino acid sequence in a) or b), the amino acid sequence of MATR3 autoantibodies is recognized.