CN111272998A - Method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP - Google Patents

Abstract

The invention discloses a method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP, which comprises the following steps: respectively introducing AQP4, MOG and MBP genes into HEK293T cells, respectively incubating with a sample to be detected, detecting by an immunofluorescence method with HEK293T cells transfected with empty vectors as a control, and determining that the sample to be detected is positive if a fluorescence signal is stronger than the control; if the fluorescence signal is equal to or less than the control signal, it is negative. The method for simultaneously detecting the central demyelinating autoantibodies AQP4, MOG and MBP in the same system provided by the invention adopts a fluorescence immunocyte staining technology with higher sensitivity and specificity to be used for clinically and qualitatively detecting the AQP4, MOG and MBP antibodies, so that the detection result is more stable, and the labor and time cost is greatly saved.

Description

Method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP

(I) technical field

The invention relates to detection of central demyelinating autoantibodies, in particular to a method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP.

(II) background of the invention

The demyelinating disease of the central nervous system is an autoimmune system disease mainly based on multifocal and inflammatory demyelination of the central nervous system, and the clinical characteristics of the demyelinating disease mainly comprise repeated attack, repeated remission and relapse. The most common clinical demyelinating diseases of the central nervous system include Multiple Sclerosis (MS), neuromyelitis optica (NMO), acute disseminated encephalomyelitis, and the like. In recent years, the incidence of demyelinating diseases of the central nervous system tends to rise year by year, which has serious influence on the life quality of patients and poses serious threat to the physical and psychological health of patients.

The diagnosis of multiple sclerosis and neuromyelitis optica are the most common demyelinating diseases of the central nervous system, and become one of the primary diseases caused by non-traumatic disability in young and middle-aged patients, both diseases usually involve Optic nerves and spinal cords, so that clinical differentiation is difficult, particularly early onset, the research considers that the MS is autoimmune diseases mediated by CD4 helper T cells (including Th1 and Th17 subgroups), so that the treatment mainly aims at immune regulation, such as β -interferon and the like, while the NMO mainly aims at humoral immunity and mainly treats immunosuppression, so that early differential diagnosis is very important for guiding treatment and evaluation prognosis of the two diseases, the detection of Lennon and the like by an indirect immunofluorescence staining method in 2004 discovers that a specific autoantibody NMO-IgG exists in blood serum of neuromyelitis optica (Optic neuronitis, ON), the diagnosis of long-segment transverse myelitis (a pathological serodiagnosis of neuroleptic travernitis), the diagnosis of the AQO-leukoneuritis is proved by the indirect immunofluorescence staining method, the detection of the existence of a specific autoantibody in 2004, the NMO-CD-293, the serum of the monoclonal antibody, the protein.

Myelin Basic Protein (MBP), one of the major components that make up the myelin sheath of nerves, accounts for about 30% of the total myelin protein, is the most resistant protein in myelin sheath, and is synthesized and secreted by oligodendrocytes and schwann cells in the central and peripheral nervous systems, respectively. The autoimmune inflammatory response induced by anti-MBP antibodies is considered to be an important cause of a variety of neurological diseases including Multiple Sclerosis (MS). MS patients have a variety of myelin-specific antibodies, one of the important antibodies being anti-MBP antibodies. Research reports that patients with positive MBP antibodies have short remission periods and high recurrence rate compared with patients with negative MBP antibodies, and can be used as a powerful adjuvant for diagnosing MS of patients with a single demyelinating lesion; the time for a clinically confirmed isolated syndrome patient positive for an anti-MBP antibody to develop MS is shorter than that for an antibody negative patient; therefore, anti-MBP antibodies can be used as important indicators for the diagnosis of demyelinating diseases. However, the results of detection of anti-MBP antibodies are highly controversial at home and abroad. Research shows that the positive rate of the anti-MBP antibody in the MS acute phase serum is 77 percent; research also indicates that the anti-MBP antibody is related to the clinical type and course of MS, and the positive rate is only 28% in the first attack and can be increased by 60% along with the progress of the disease. The research shows that the MBP plays an important role in the differential diagnosis and the prognosis analysis of the central demyelinating disease.

The role of myelin oligodendrocyte glycoprotein immunoglobulin G antibodies (MOG-IgG) in the serum of patients with Central Nervous System (CNS) inflammatory demyelinating diseases has gradually been re-established over the past few years. While MOG antibodies have been considered to be involved in Multiple Sclerosis (MS) as a result of enzyme-linked immunosorbent assay using linearized or denatured MOG peptides as antigens, a recent new generation of cell-based assays has demonstrated that full-length, conformationally intact human MOG antibodies are closely associated with Optic Neuritis (ON), which is mostly recurrent, myelitis, brainstem encephalitis, and Acute Disseminated Encephalomyelitis (ADEM) -like manifestations, but not with classical MS. Immunological studies show that MOG-IgG has direct pathogenic effect, and serological detection shows that almost all patients positive to MOG-IgG have negative aquaporin-4 (AQP4) -IgG; the cohort study results show that its clinical presentation, adjuvant testing, treatment response and prognosis differ from that of classical MS and AQP4-IgG positive neuromyelitis optica lineage disease (NMOSD), which is considered a new disease entity known as MOG-IgG associated encephalomyelitis, abbreviated as MOG encephalomyelitis (MOG-EM). MOG-EM and MS share some similarities and their clinical and imaging manifestations often overlap: as with MOG-EM, most patients relapse, and in addition, 33% and 15% of adult MOG-EM patients meet the McDonald and Barkhof diagnostic criteria for MS at least once during the course of the disease, and thus many MOG-EM patients have been misdiagnosed as MS in the past, whereas serological tests that bind the AQP4 antibody and the MOG antibody will aid in the differential diagnosis of MS.

Currently, the international technologies for detecting AQP-4 antibody mainly include indirect immunofluorescence Assay (IIF), Fluoroimmunoprecipitation Assay (FIPA), Radioimmunoprecipitation Assay (RIPA), Cell-based Assay (CBA) (Clin Immunol.2011Mar; 138(3):239-46, Neurology.2012Feb 28; 78(9):665-71) and Enzyme-Linked ImmunoSorbent Assay (ELISA). The traditional method for detecting AQP-4, such as indirect immunofluorescence method, enzyme linked immunosorbent assay and the like, has low sensitivity and specificity, and the radioimmunoprecipitation method relates to the problem of radioactive source pollution. The plasmids used in the traditional CBA method were the EGFP-tagged human AQP4 cDNA plasmid and a control EGFPcDNA plasmid, and transient transfection was performed using Polyethyleneimine (PEI) or liposome methods. The time consumption is long, the manpower and material resources are greatly consumed, the fluorescence efficiency is unstable, and a more stable transfection method is needed to obtain more sensitive and stable results. Most of MOG and MBP autoantibody detection uses a nerve tissue section with high expression of MBP antigen for staining detection, and the method has a plurality of problems, such as large difference among batches due to the difficulty in controlling the tissue section; most of the tissue slices are animal sources (such as monkey nerve tissue slices used by European Union), and the antigens have species differences, so that the sensitivity and specificity of detection are reduced; the presence of antigens other than MBP in the tissue, a positive result of the test may not be positive for MBP antibody. 2. The Western method or the Elisa method has the advantages that although the two techniques are mature, the spatial conformation of the MBP antigen is difficult to maintain, so that the specificity is low, the cost is high, and the popularization of the detection means is greatly limited.

Disclosure of the invention

The invention aims to provide a method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP by applying an immunofluorescence technique, wherein a cell line stably transfected with AQP-4, MOG and MBP is coated on the same glass slide, and the AQP-4, MOG and MBP antibodies are simultaneously and qualitatively detected by adopting a fluorescence immunocyte staining method (CBA) detection technique with higher sensitivity and specificity, so that the detection result is more stable, the labor and time cost is greatly saved, and the clinical diagnosis and treatment level of neuromyelitis optica and multiple sclerosis is favorably improved.

The technical scheme adopted by the invention is as follows:

the invention provides a method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP, which comprises the following steps: respectively introducing AQP4, MOG and MBP genes into HEK293T cells (namely an HEK293T cell line containing AQP-4 genes, an HEK293T cell line containing MBP genes and an HEK293T cell line containing MOG genes), respectively incubating with a sample to be detected, detecting by adopting an immunofluorescence method by taking HEK293T cells transfected with empty carriers as a control, and if the fluorescence signal of the sample to be detected is stronger than that of the control, determining that the sample to be detected is positive; if the fluorescence signal is equal to or less than the control signal, it is negative.

Further, the detection method comprises the following steps: (1) mixing the cell adherence reagent and PBS according to the volume ratio of 1:10, adding the mixture into a 6-well plate containing a cover glass, and keeping the temperature at 37 ℃ and 5% CO₂Incubating for 1 hour; then adding a stable HEK293T cell line which is respectively transferred with AQP4, MOG and MBP genes, wherein the cell inoculation amount is 1 multiplied by 10⁶/ml，37℃，5％CO₂Culturing to 80% fusion degree;

(2) the cover glass (preferably cutting the cover glass to a small square with the side length of 5 mm) with the cell surface facing upwards is pasted on the glass slide (preferably shadowless glue is pasted, and ultraviolet irradiation is carried out for 30 seconds for pasting), and the cover glass is a reaction area; PBST wash (preferably 50 ul/reaction zone, 5 minutes wash); blocking with 3% BSA (preferably 50 ul/reaction zone for 30 min) and discarding; adding test serum (preferably 50 μ l per well) diluted by PBST at a volume ratio of 1:10, and adding 5% CO at 37 deg.C₂Incubating for 1 hour under the condition; PBST wash (preferably 3 times for 5 minutes each); goat anti-human (Biotin-SP-conjugated Affinipure goat coat anti-human IgG) (preferably 50. mu.l per well) with Biotin conjugated to streptomycin diluted in PBST at a volume ratio of 1:700 was added at 37 ℃ with 5% CO₂Incubating for 30 minutes under the condition; PBST wash (preferably 3 times for 5 minutes each);add the fluorescein 488 monthly streptomycin conjugate (Alex fluor 488-conjugated streptavidin) diluted by PBST at a volume ratio of 1:700 (preferably 50. mu.l per well), PBST wash (preferably 3 times, 5 minutes each); sealing by using a sealing agent; HEK293T cells transfected with the empty vector were used as controls under the same conditions; then, carrying out fluorescence microscope detection, and if the fluorescence signal is stronger than the contrast, determining that the sample to be detected is positive; if the fluorescence signal is equal to or less than the control signal, it is negative.

Further, the AQP4, MOG and MBP genes are respectively introduced into HEK293T cells in a form of lentiviral plasmid vectors, and the nucleotide sequence of the lentiviral plasmid vector containing the AQP4 gene is shown in SEQ ID NO. 1; the nucleotide sequence of the slow virus plasmid vector containing the MBP gene is shown in SEQ ID NO. 2; the nucleotide sequence of the lentiviral plasmid vector containing the MOG gene is shown in SEQ ID NO. 3.

Compared with the prior art, the invention has the following beneficial effects: the method for simultaneously detecting the central demyelinating autoantibodies AQP4, MOG and MBP in the same system provided by the invention adopts a fluorescence immunocyte staining technology with higher sensitivity and specificity to be used for clinically and qualitatively detecting the AQP4, MOG and MBP antibodies, so that the detection result is more stable, and the labor and time cost is greatly saved. The establishment of a platform of the central demyelination autoantibody detection technology is beneficial to improving the clinical diagnosis and treatment level of neuromyelitis optica and multiple sclerosis. The antibody detection is an important diagnosis index of autoimmune diseases, and the establishment of the item can greatly improve the disease spectrum of neuromyelitis optica and the diagnosis and treatment level of multiple sclerosis, improve the labor capacity and the life quality of patients and generate great social and economic benefits.

(IV) description of the drawings

FIG. 1 is a schematic representation of the construction of the AQP4 viral vector.

FIG. 2 is a schematic diagram of the construction of MBP viral vectors.

FIG. 3 is a schematic diagram of the construction of MOG viral vectors. .

FIG. 4 is an AQP-4 protein expression electrophoresis diagram after the AQP-4 virus expression vector is infected by HEK293 cells.

FIG. 5 is an electrophoresis diagram of MOG protein expression after HEK293 cells are infected with MOG virus expression vectors.

FIG. 6 shows the MBP protein level expression electrophoretogram (A) and fluorometric chart (B) in MBP plasmid-transfected cells.

FIG. 7 is a fluorescence diagram of AQP-4/MOG/MBP antibody expression level of serum to be detected by cell immunofluorescence assay.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1 construction of Stable HEK293T cells transfected with AQP4, MOG, MBP genes

1. Vector construction:

(1) construction of overexpression vector fusing and expressing human AQP-4 gene and fluorescence labeling gene EGFP

Using pEGFP-N1 vector as template, adopting primers EGFP and EGFP to amplify EGFP sequence, wherein the primers are as follows: 5'-CTA GGATCC ATGGTGAGCAAGGGCGAGGAG-3' and 5'-CAGGTCGACGAATTCGCGGCCGCCTCGAGCTGCAGTTACTTGTACAGCTCGTCCATGCCG-3' of EGPF, cutting the amplified fragment into gel, recovering the gel, connecting the gel with a T vector, performing double enzyme digestion by BamHI and SalI after the sequence is aligned to be correct, connecting the gel with a pCDH-CMV-MCS-EF1-CopGFP vector fragment subjected to double enzyme digestion by the same enzyme, and replacing an EF1-copGFP sequence in the original vector by EGFP to obtain a plasmid vector pCDH-CMV-MCS-EGFP.

The AQP-4(M1+ M23) sequence is amplified by PCR by using human optic nerve cDNA as a template and AQP4F and AQP4R as primers to obtain an AQP-4 amplified fragment.

AQP4F:5’-ACTTCTAGAATGAGTGACAGACCCACAGCAAG-3’；

AQP4R：5’-CCAAGATCTTGAACCGCCTCCACCTACTGAAGACAATACCTCT CCAGATTG-3’。

Cutting gel of the AQP-4 amplified fragment, recovering the gel, connecting the gel with a T vector, performing sequencing comparison to obtain a correct sequence, performing double enzyme digestion on the gel by EcoRI and XhoI, and connecting the gel with a vector pLV-Puro subjected to double enzyme digestion by EcoRI and XhoI to obtain a vector pLV-Puro-AQP-4; then XbaI and BglII are used for double enzyme digestion and then are connected with the obtained plasmid vector pCDH-CMV-MCS-EGFP to obtain the lentiviral plasmid vector pLenti-CMV-AQP-4-EGFP, the nucleotide sequence is shown in SEQ ID NO.1, and the plasmid is shown in FIG. 1.

(2) Construction of overexpression vector fusing and expressing human MBP gene and fluorescence labeling gene EGFP

Using human oligodendrocyte as template (

CRL-3216^TM) And carrying out PCR amplification on the MBP sequence by taking MBP F and MBP R as primers.

MBP F:5’-TTTCTAGAAATGTACAAGGAATTCGCCACCATGGCGTCACAGAAGA-3’；

MBP R:5’-TCTAGGGATCCGGGCCCGGGTTCGAACTCGAGTCAGCGTCTAGCCATGGGT-3’。

And (2) cutting the MBP amplified fragment into gel, recovering the gel, connecting the gel with a T vector, performing sequencing comparison to obtain a correct sequence, performing double enzyme digestion on the gel by using EcoRI and XhoI, connecting the gel with a vector pLV-Puro subjected to double enzyme digestion by using EcoRI and XhoI to obtain a vector pLV-Puro-MBP, performing double enzyme digestion on the gel by using XbaI and BglII, and connecting the gel with the plasmid vector pCDH-CMV-MCS-EGFP obtained in the step (1) to obtain the lentiviral plasmid vector pLV-EGFP (2) Puro-MBP-V1, wherein the nucleotide sequence is shown in SEQ ID NO.2, and the plasmid is shown in FIG. 2.

(3) Construction of overexpression vector fusing and expressing human MOG gene and fluorescence labeling gene EGFP

Using human oligodendrocyte as template (

CRL-3216^TM) And carrying out PCR amplification on the MOG sequence by taking MOG F and MOG R as primers.

MOG F：5’-GGGTAGTTTAGAGTGATAGGATTAAGATAT-3’；

MOG R：5’-TAAAAATAAACCACCCTAAAAAAAA-3’

And (3) carrying out electrophoresis on the amplified fragment, cutting and recovering the gel, connecting the recovered gel with a T vector, carrying out sequencing comparison, confirming the correct sequence, carrying out double enzyme digestion on the obtained product by EcoRI and XhoI, and connecting the obtained product with a pCDH-CMV-MCS-EGFP particle vector to obtain a lentiviral plasmid vector pLenti-CMV-MOG-EGFP for over-expressing the human MOG full-length gene, wherein the nucleotide sequence is shown as SEQ ID NO.3, and the plasmid is shown as figure 3.

2. Viral packaging and acquisition of HEK293T stably transfected cell line

By using CaCl₂Packaging lentivirus expressing target gene fusion protein, collecting virus suspension, infecting HEK293 cells in the presence of Polybrene (Polybrene) of 6 μ g/ml, and after 1 week, sorting positive stable transfected cells. After continuous subculture, secondary screening is carried out to obtain a 100% transfected HEK293 cell line stably expressing the fusion protein, which specifically comprises the following steps:

1) cell culture: inoculating HEK293T cell line of human embryonic kidney into low-sugar DMEM medium containing 10% inactivated fetal calf serum, and placing at 37 deg.C and 5% CO₂Culturing in an incubator, taking HEK293T cells in logarithmic growth phase, resuspending the cells in a low-sugar DMEM medium containing 10% of inactivated Fetal Bovine Serum (FBS) and 1% of double antibodies (penicillin and streptomycin with final concentration of 100 mu g/mL), and preserving in liquid nitrogen in a freezing tube for experiment;

2) recovery and passage of cells: taking out the frozen tube obtained in the step 1), quickly putting the frozen tube into a water bath at 37 ℃ and continuously shaking the frozen tube to thaw the frozen tube in the shortest time, sterilizing the outer surface of the frozen tube by 70% alcohol, transferring the cell sap in the frozen tube into a 50ml centrifuge tube filled with 20ml of low-sugar DMEM medium, centrifuging the cell sap for 5 minutes at 1000rmp, removing the supernatant, depositing the cell sap in the low-sugar DMEM medium containing 10% fetal calf serum, and removing 5% CO at 37 ℃ to obtain a precipitate₂Culturing under saturated humidity until the confluence reaches 80%;

3) digestion of cells: adding 1ml of pancreatin into step 2), digesting HEK293T cells for 1-2 min, collecting HEK293T cells, resuspending with low sugar DMEM medium containing 10% FBS, placing in a culture dish, and adding 5% CO₂Incubation is carried out in an incubator at 37 ℃ for 24 hours, and transfection can be started when the cells are completely attached. The medium was changed 2 hours before transfection (1.5 ml of complete medium was used to replace the old medium).

4) Plasmid transfection: a sterile 1.5ml EP tube was put in 400. mu.l of serum-free low-sugar DMEM medium, 6. mu.l of mixed plasmid (target plasmid: packaged plasmid VSVG: packaged plasmid REV: 5:3:2, volume ratio) and 6. mu.l of transfection reagent (Lipofectamine 2000) were added, and the mixture was mixed well and allowed to stand for 15 minutes to obtain 412. mu.l of plasmid mixture. Adding 412 μ l of plasmid mixed solution dropwise into the culture dish with complete adherence in the step 3)Gently shaking the culture dish, mixing well, and placing in a container containing 5% CO₂At 37 ℃ in an incubator. Wherein the target plasmids are respectively a lentivirus plasmid vector pLenti-CMV-AQP-4-EGFP, a lentivirus plasmid vector pLV-EGFP (2) Puro-MBP-V1 and a lentivirus plasmid vector pLenti-CMV-MOG-EGFP.

5) And (3) virus collection: step 4) after 8 hours incubation, the medium was aspirated, 2.5ml of low sugar DMEM medium pre-heated at 37 ℃ was added, and the incubation continued with 5% CO₂Incubating at 37 ℃ for 40 hours in an incubator, collecting the culture solution into a 1.5ml centrifuge tube, centrifuging at 3000rpm for 20 minutes, filtering the supernatant by a 0.45 mu m filter head, and collecting the filtrate, namely the virus solution, to respectively obtain AQP-4 virus solution, MBP virus solution and MOG virus solution. Subpackaging and storing at-80 deg.C for use.

6) Virus titer determination (dilution count method):

titer unit: TU/ml, refers to the number of biologically active viral particles contained per ml. "TU" is an abbreviation for "transducing units" and in Chinese "transducing units" and represents the number of viral genomes that can infect and enter target cells.

First day cell preparation: digesting and counting HEK293T cells with good growth state in the step 2), and diluting to 1 × 10 by using low-sugar DMEM medium⁵Adding into 96-well plate (100 ul/well), dividing into 3 groups (AQP-4 virus solution, MBP virus solution, and MOG virus solution), preparing 10 wells, placing at 37 deg.C and 5% CO₂Culturing in an incubator.

The next day, virus addition: 10-fold gradient dilutions were made in EP tubes, 10 dilutions in series, as follows: preparing 10 EP tubes of 1.5ml for each group of viruses, adding 90ul of DMEM culture solution containing 10% inactivated fetal calf serum and 1% double antibody into each tube, adding 10ul of the virus solution prepared in the step 5) into the first tube, uniformly mixing, sucking 10ul of the virus solution, adding the virus solution into the second tube, and uniformly mixing. Repeating the above steps until ten dilutions (10-10)^-8). The original medium in the 96-well plate was aspirated, and an equal volume of diluted virus solution was added and labeled.

And (3) adding culture solution on the third day: 100ul of low-sugar DMEM medium was added to each well and cultured for 48 hours to the fifth day.

Results were observed and titers calculated on day five: the results were observed under a fluorescent microscope and the number of the last two fluorescent cell clones were counted, assuming X and Y, the titer (TU/ml) was (X + Y × 10) × 1000/2/X well virus content (ul).

7) Virus infection and stable cell line screening: 8ul of virus liquid in the step 5) and PEG8000 according to the volume ratio of 4: 1, every 20 minutes, 4 times, 4 ℃ overnight. The next day, centrifugation was carried out at 4000g and 4 ℃ for 20 minutes, the supernatant was discarded, and 100ul of virus concentrate was obtained by resuspending the pellet in 100ul of DMEM medium containing 10% inactivated fetal calf serum and 1% double antibody. 10ul of virus concentrate containing 6. mu.g/ml polyamine were added to HEK293T cells in logarithmic growth phase from step 1), after 48 hours a final concentration of 15ug/ml puromycin was added, fresh medium was replaced approximately every 2 days at 37 ℃ with 5% CO₂Culturing for about 10 days, sorting the positive stable high expression transfected cells of the protein to be detected by using single cell clone, respectively obtaining HEK293T cells of a transfected gene AQP4, HEK293T cells of a transfected gene MOG and HEK293T cells of a transfected gene MBP, and measuring the relative expression amount and protein level expression of the target antibody transcript in the screened cells and the untransfected 293T cells by using quantitative PCR and western blot technology, as shown in figure 4, figure 5 and figure 6. The results show that the transcripts of the target antibodies (AQP4, MOG, MBP) were significantly increased in the stably transfected cell lines compared to the untransfected 293T cells.

Example 2 measurement of serum AQP4, MOG, MBP antibodies by immunofluorescence staining method

1) Inoculating cells: mixing cell adherence reagent (purchased from Beijing Fine engineering Biotech Co., Ltd., product number: 1027) and PBS at volume ratio of 1:10, adding into 6-well plate containing cover glass, and heating at 37 deg.C with 5% CO₂And incubated for 1 hour. Then adding stable HEK293T cell lines (namely HEK293T cell line containing AQP-4 gene, HEK293T cell line containing MBP gene and HEK293T cell line containing MOG gene) respectively transferred with AQP4, MOG and MBP genes, wherein the cell inoculation amount is 1 × 10⁶/ml，37℃，5％CO₂Cultured to 80% confluence for subsequent testing. HEK293T cells transfected with the empty vector were used as controls under the same conditions.

2) Cellular immunofluorescence: cutting the cover glass with a diamond lettering pen to a small square with the side length of 5mm, enabling the cell surface to face upwards, adhering the shadowless glue on the glass slide, and firmly adhering the cover glass after ultraviolet irradiation for 30 seconds, wherein the cover glass is the reaction area. PBST washed cells 50 ul/reaction zone for 5 min. Blocked with 3% BSA at 50 ul/reaction area for 30 min and discarded. 50 mul of serum to be tested diluted by PBST at a volume ratio of 1:10 is respectively added into corresponding holes, and 5 percent CO is added at 37 DEG C₂Incubate under conditions for 1 hour. PBST was washed 3 times for 5 minutes each. 50 μ l of goat anti-human (Biotin-SP-conjugated Affinipure goat anti-human IgG) diluted with PBST at a volume ratio of 1:700 was added to each dilution, and 5% CO was added at 37 ℃₂Incubate under conditions for 30 minutes. PBST was washed 3 times for 5 minutes each. 50. mu.l of fluorescein 488 monthly streptomycin conjugate (Alex fluor 488-conjugated streptavidin) diluted with PBST at a volume ratio of 1:700 was added, respectively. PBST was washed 3 times for 5 minutes each. The sealing agent seals the sheet.

3) Fluorescence microscopy analysis: the relative amount of antibody concentration was indicated by the intensity of red fluorescence signal in a 20-fold field of the objective lens with the control of cells transfected with empty vector. The results show that the method of the present invention can distinguish the relative amounts of autoantibodies in AQP4, MOG, MBP positive patients based on fluorescence intensity, as shown in figure 7.

Example 3 specific detection

In view of the low positive rate of the MOG antibody detection, in order to further promote the clinical application of the method, 120 sera of patients with nervous system diseases are selected by multiple centers and are respectively sent to European Union third-party companies for detection, and meanwhile, the detection method is used for blind detection. The detection rate of the MOG positive antibody of the method is analyzed by comparing with the EU detection method.

120 sera of patients with neurological diseases (30 patients with demyelinating diseases) were selected and compared with the detection rate of the method of the present invention (example 2) using the detection method of Europe (Europe medical diagnostics, China, Inc.). The results showed that in 4 out of 30 demyelinated patients, omron detected MOG antibody positive sera, 6 positive sera were detected by the present detection method, and the patients with the detected MOG positive sera were central demyelinating diseases and were in accordance with clinical symptoms. In the other 90 cases of non-demyelinated patients, the results of the omron test and the test by the method were negative.

Sequence listing

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agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140

aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200

tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260

aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320

gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380

cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440

gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500

ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560

actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620

gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680

aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740

ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800

tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860

actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920

cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980

cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040

gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100

aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160

acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220

atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280

ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460

ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520

agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580

ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640

acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700

gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760

tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgaa 2820

tttctagaaa tgtacaagga attcgccacc atggcgtcac agaagagacc ctcccagagg 2880

cacggatcca agtacctggc cacagcaagt accatggacc atgccaggca tggcttcctc 2940

ccaaggcaca gagacacggg catccttgac tccatcgggc gcttctttgg cggtgacagg 3000

ggtgcgccca agcggggctc tggcaaggta ccctggctaa agccgggccg gagccctctg 3060

ccctctcatg cccgcagcca gcctgggctg tgcaacatgt acaaggactc acaccacccg 3120

gcaagaactg ctcactacgg ctccctgccc cagaagtcac acggccggac ccaagatgaa 3180

aaccccgtag tccacttctt caagaacatt gtgacgcctc gcacaccacc cccgtcgcag 3240

ggaaagggga gaggactgtc cctgagcaga tttagctggg gggccgaagg ccagagacca 3300

ggatttggct acggaggcag agcgtccgac tataaatcgg ctcacaaggg attcaaggga 3360

gtcgatgccc agggcacgct ttccaaaatt tttaagctgg gaggaagaga tagtcgctct 3420

ggatcaccca tggctagacg ctgactcgag ttcgaacccg ggcccggatc cctagataat 3480

tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 3540

cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg 3600

gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc 3660

tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag 3720

tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag 3780

gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg 3840

gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa 3900

ggtcctccgg aggcccggca ttctgcacgc ttcgaagcgc acgtctgccg cgctgttctc 3960

ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccgccac catggtgagc 4020

aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 4080

aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 4140

accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 4200

accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 4260

ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 4320

gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 4380

atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 4440

tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 4500

gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 4560

cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 4620

acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 4680

ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga atttggcagc 4740

ggccagtgca ccaactatgc cctgctgaaa ctggctggcg atgtggaaag caaccccgga 4800

cccatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc 4860

gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg 4920

gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 4980

gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 5040

gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 5100

ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag 5160

gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 5220

ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc 5280

ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 5340

gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 5400

tgaccgcgtc tggaacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 5460

tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 5520

tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 5580

tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 5640

tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 5700

cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 5760

gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc 5820

ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 5880

cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 5940

gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 6000

cccgcctgga attaattctg cagtcgagac ctagaaaaac atggagcaat cacaagtagc 6060

aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg 6120

ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat 6180

cttagccact ttttaaaaga aaagagggga ctggaagggc taattcactc ccaacgaaga 6240

caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac 6300

tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta 6360

ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac 6420

cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac 6480

agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc 6540

tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg 6600

ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg 6660

tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 6720

cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 6780

gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 6840

tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat 6900

atcagagagt gagaggcctt gacattgcta gcgttttacc gtcgacctct agctagagct 6960

tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 7020

acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 7080

tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 7140

tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 7200

cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 7260

actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 7320

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 7380

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 7440

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 7500

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 7560

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 7620

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 7680

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 7740

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 7800

acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 7860

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 7920

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 7980

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 8040

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 8100

tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 8160

ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 8220

taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 8280

cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 8340

gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 8400

gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 8460

tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 8520

gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 8580

ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 8640

ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 8700

cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 8760

ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 8820

gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 8880

ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 8940

ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 9000

tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 9060

ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 9120

cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa tggttacaaa 9180

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 9240

ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat aactcaagct 9300

aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa ttacctgtgg 9360

tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag aaattgtatt 9420

tgttaaatat gtactacaaa cttagtagtt tttaaagaaa ttgtatttgt taaatatgta 9480

ctacaaactt agtagt 9496

<210>3

<211>22392

<212>DNA

<213> Unknown (Unknown)

<400>3

gtacagtgaa gtatacaact ttgtgcaggg caatttatca atatttattg aaattaccaa 60

aaaacatgct ctctgaacaa actattctac cagtgtagaa agcagagtaa acttcatggg 120

tgagtgacca gggcagtcac acaagggccc catgcttaga agggatactg tgtttgggtt 180

ctaaagctct gtggttcctg tcttgaaatt cttaataatt ttatctttca atttgtgtct 240

tataatgaag tccgatgaga aagcagaaca tgggctagag acttttggag cctggctcaa 300

gcgaggtcct gctccccatg cctcccagcc tccccaggac tggttttcag ctgccggctc 360

caccaccttc tgtgcaggct cgctcccagc aggggcctgg gaacagtgga aaggagggga 420

gcggtcaggc atacacacct cccttgccaa atggaaggca tggccctagg cacttgtgaa 480

gatctgcact tccccctagg tactcctgtg cctggagtgt gacattaaat taaaaaaaaa 540

aaggccgggc gcggtggctc acgcctgtaa tcccaccatt atgggaggcc aaggcaggcg 600

gatcacgagg tcaggcgatc gagaccatcc tggctaacac ggtgaaaccc cgtctccact 660

aaaaatacaa aaaaattatc tgggcatggt ggcgagcgcc tgtagtccca gctacttggg 720

aggctgagac aggagaatgg cttgaacccg ggaggcggag gttgcagtga accgagattg 780

cgccactgca ctccagcctg ggcgacagag cgagactcca tctcaaaaaa aaaaaaaaaa 840

aagaaaagaa aaaaacccca cataataggt tgacagtgga accacagaaa aaaggaaaag 900

gttgggtttt ttttctgctt tttattttct attttattat tttttaatag atttatttaa 960

ctagagatgg ggtctcacta tgttgtaaag gctggactcg agaccctggg cccgagcgat 1020

cctccaacct ggtcctccca aagtgatggg attacaggcg tgagccactg cacctggtct 1080

tttcctgcta ttaaacaagg agctccatag tttcattttg cccctcaaaa tatgtagctg 1140

gccttagtag actgatattc attgccaaat tatatgtaag agcaaaaagg ttgaaaatga 1200

tggcctgaca ttgatcaatt tgtgccttta ggtaacatat aactgtaata taactgcaat 1260

acaactagaa tataactcat aaaggcaaga atcttgtctg ccttgctgaa agttttataa 1320

tcagggccta atataaagta tgacacatag cacttgcttt taaatatgta ttgatttaaa 1380

ttaattgagt acatttttgc ttcatcctag taaaaatagg tatttaaaaa actgaaacag 1440

tctaaatgtc ttgggatgct acttaaataa ctatattata ttcatccaat aaaatattgt 1500

aagctgttta aaaataacaa ggatgttctt taggtactga taaggaaaga gcttcaagat 1560

aaattgttac catttatgta aaacaggtgg gagaagggag agggagggat gtgtgagcgc 1620

tacttgcagt actcacaggc agtgactttc gtggagcgcc ctctagtggt atatatatac 1680

aaacggaagg atttagagaa aatacagatc ggctttagct ggctgagatt tattttcaaa 1740

gcatgttact ttataagaat caatttttat ttaaaaaatt tttttgagat agggtctcac 1800

tctgtcgcac aggttggagt gcagcagcac gatcagtgct cactgcagcc tctctctctt 1860

gggctcaaca ggtgcatgtc accacgtcca gctaacaatc aattttcaaa agtacaaaaa 1920

agccatatta tgtattaatg tggaattatg aattaagtag acaacaagaa tcaaaacagg 1980

gtgtctatta tcacttctga taacataaat aatgtaaaga tacatatttt acagattatc 2040

tgtaaaagct tatacagtac tgttgctggg tatttatgta ggaaagctac catttattga 2100

atgcttacta tttcacatat ggacagcata gagcatgtta aaaaattacc acacacattt 2160

actgtattca atgtgtcact ctgaatatat tactgtgtac atggtctgtc attggacatg 2220

gtgagagatg cagattaagc tgaaattact gaggacagca acactggaag aaaattgagc 2280

tgggtgtagt ggctcagcct gtaattccaa catttcagaa ggctgaggca ggaggatcac 2340

ttgagtccag gagtttgaga ccaagggaaa gaaaagaaaa gaagctttca tttagccagg 2400

catgctggca catacctgta gtttcagcta ctcaggaggt tgaggcataa ggttcactta 2460

aacttgagag gtaaaggctg cagtgagccc tgatcacgcc actgctctcc agcctgtgac 2520

agagagagac cctgtctcaa aaacgagaaa gaaagaaaaa aagaggcaac tcaagaactc 2580

aggaatactt gcaggatctc ataacatatg ctatacaaaa tcaattaaaa taatatttaa 2640

atgctgaaag aaatgagcag ctcccagggt gatacagggt ggtttcactt cttggacaca 2700

tctacactga gctctattcc tggcaatacc tgatgttccc ataccccaga tttctttatt 2760

ttattttgag acggagtttt gctcttcttg cccaggctgg agtgcaatgg cgggatcttg 2820

gctcaccgca atctccgcct cctgggttca agggattacc ctgcctcagc ctcccgagta 2880

gctgggatta caggcgcacg ccaccatgcc cagctaattt ttgtattttt agtagaggca 2940

gggtttctcc atgttggtca ggctggtctt gaactcccaa cctcaggtaa tctgcccgct 3000

tcggcctccc aaagtgctgg gattacaggc gtgagcgggc ccagccccta taccccagat 3060

ttctgcaagt ggcaacacca ctggcttcat tttgctggtg gcccctctgg ccttcccttg 3120

tatatatcac ctttgcccaa agaccatgtc agccaaggga ctgctctcac agctccagga 3180

atcctcccct ttcaggaaat ttgaggcagt tgagggcatg aaagtaaata agctgagctc 3240

atcagaggcc ttgtattgtg gtggttaaaa gagccagttc taggactaga aagcctggct 3300

tgaaatccca gctctgccac tccctagtgg tgtgacttta gcaagttcct ttacctcttt 3360

tgtacctccc ttttctcacc tgtaagatat gggtgataat agtttaatat ttgttttgtt 3420

gttgtgagga ttaaaggtgt taatgcaagt aaaccactta gaaccacagc acatagaata 3480

tctcagtaag gtggttagtt ttttttattg ttgttttcag agatgctgtg atttctccaa 3540

agtggctgtg atggctcggc aggctcctga ccctctctgc tcccacatgc ctccaccctc 3600

atcctgatct cccatccagc ttttgacagc ttgcttggtg ctggacaatt gcacacatct 3660

taccaccccc aaatcctgcc cagaagcatc ttgtgcataa ctctcctacc tgaatatgca 3720

acagggagaa agagcgtccc aggacatttt aggtttttga agaaaaaaaa acccctttgg 3780

taaaaagcca gagatccaca gcggccactt tttccatggg attgacccct gcaatcttga 3840

ctttcaacca cacagcacca gagtagccaa acattgcttg tgtccaaacg ctggctgcct 3900

tgaagggtga aagaataagc agttcccaaa ctcagctgac cttaatgtcc ttctagctcc 3960

ttacgcccat ctcggacaaa agcagaaatg tatgtctcag ttgtgtttct accccttgct 4020

gcccaatata aatttttgtg ttgcccaata taattttttg tgacgatgga aatgttctgt 4080

atttgtgttg tctgatgaga taaccactaa ctgtagtgct attgagcatt tgaaacatgg 4140

ctagtgtaat caatgaacca aatttttaat tttatttaat tgtaattaat tttaagtggc 4200

cacatgcagg gagtgactgc tgcattggac agcacggctc taaattgagc cttttttcct 4260

tatttggtga ggcatacttg ccttaagatt gggaagtcta tttttggaac ctgctaccaa 4320

tgctggtctc acacttgcaa ttctcagctg agccaagagg tgagagaaag gtcattttcc 4380

attccagatc tcactctccc ctgtgacact gaggaaactg gcaagtgatg tgaaggctgg 4440

agagcgtgtc ctgtatgctg gctctgtccc ttctgcctgt gttgactgac atagttagtt 4500

gctgcccttg ctggtctccc ttcctccaac cttgcctctc tgagcacacc tgacattcat 4560

ctcatgactt ccctaaaaac attctttggg aacaagaaac taacaaatcc caagtgacct 4620

atcacatata caaacataca gggcagagtt tggattcgcg gtagaagaaa gggaggttag 4680

acattaagaa gaatggtctg gtgatgacag ttgtgagata atagaaacag gaaaaagaaa 4740

tctaagtttt ctttcttttt ttaagaacca ataataattt ctctcttttg actagtcagt 4800

agggctgggg tggattggag gaagcttaca tattccatga acaagcctct tcctaaggtc 4860

ctgtaagtga tcctgcccca ctgattagcc cctagaagac ccttcaaagg ttggatctcc 4920

aggagggagt gggggaggaa agccctgtac caggcagcct ctgctccatt gctctggggg 4980

ggtggggaag gcaaaccctg gtcatcccct cagtctgtag cccttttgtg tgagtgcctg 5040

gcaagggtga cgtggggctg tttctgcggg cacagctgca gcaattaccg gagtggaggc 5100

agggcccagg cagcactgcc ctccaagatc ttcccttggg cttttcagca gtaaggggac 5160

atgcacccca agggcctcca cttggcctga ccttgctgcg ggggctctct gtccccagga 5220

acagtagaga tggcaagctt atcaagaccc tctctgccca gctgcctctg ctccttcctc 5280

ctcctcctcc tcctccaagt gtcttccagc tatgcaggta agacatgttt tttttcctgc 5340

cctggggaga ccctgaaaac agaaaggcta gtttcctggg gcttagctcc ttcaaacatc 5400

ctcaagttgc tatattatct ttctaaaaca tagacctact gacatgcctc ccttcctcag 5460

aaaccttccg tgggtggttc ttacagcctt caagatggag tccagactct tttttttttt 5520

tgagacagag tctccctctg ttgctcaggc tggagtgcag tggcatgatc tcggctcact 5580

gcaacctcag cctccctggt tcaagcgatt ctcctgactt ggcctcccaa gtagcggaga 5640

ctacaggcgc ctgccaccac acccagctaa attttttctt ttcttttttt tttttttttt 5700

ttttgtattt tagtacagac ggggtttcac atgttggcca ggatggtctc gatctcttga 5760

cctgctgatc cgcccgcctc agcttcccaa agtactggga ttatgggcgt gagccactgc 5820

actaggccta atttttttat ttttagtaga gatggggttt caccatgttg gccaggctgg 5880

tctggaaccc ctgacctcaa gtggtctgcc ctcctcagcc tcccaaagtg ctgagattac 5940

aggcatgagc cattgcgtct gacccagact ccttaatgtg actaactcaa ggctttcctt 6000

gaactacttc ttacttgtct ttccagcttt gtcttttcac ctctcaaatt gagataaaat 6060

aataacaacc tcttggagtt ctcatcagga ttacatgaaa tgagatatgt aacatgctta 6120

gcagtgcctg tccatagtaa atctcaataa atgtttgtgg aattataata tcttgtcatg 6180

tttgagactt tgctctgcat aatcaggcac cagtaggttt ttataaagga acccggctgt 6240

cacgtgcaga ggagaaataa acagaaagtt tcccatcctc agggagccac ctgactgaca 6300

gaggcacagt gcatccactc tccaggtcta ggggagaaag cagccttatt tcttagtagc 6360

tcagaatctg acttgagaaa cacatccaca tagaaaaaaa caaggaactt tttcgggtca 6420

gggtccggga gccacagtga ggtggaagat acaggggaag gaagagggaa atagagccat 6480

ccccagggtg gaagatctca gaagagaatt tgggaaacaa ggtatgaaca aggactgaat 6540

agtgagaagt gatggagaga cagttaaagt agatggagtg acaaaagcaa aacctctaag 6600

ggtagaatag gcagcaattt ggccaagtcc taacagggag gcccatagga ggattcaacc 6660

tcaagatgct gtgccacatt ccaagaggga acctaaaggc tgggctgaag agtcagagat 6720

ggctacagct ggcaaaaaga tgggcagatg ctgagaggag atgattgcta aaatgttctg 6780

tccaggacat tcacagtatc tctataacca gagtcttttt tgtcgttgtt gttctcaaga 6840

aggaaacttg aggccgggtg tggtggttta tgcccataat cccagcgctt tggggccaag 6900

gcaggcggat cacctgaggt caggagttcg agaccagcct ggccaacagt gtgaaacctc 6960

atctttacta aaaatacaaa aattagctgg atgcggcggt aggtgcctgt aatgccagct 7020

actcgggagg ctgaggcagg agaatcactt gaacctggga ggcggaggtt gcagggaggc 7080

ggaggttgca gtgagccaag attgcaccac tgcactccag cctgggcgac agagagtaag 7140

actgtctcaa aaaataaatg aataaataaa aaggaagaag aagaagaaga acaattgcaa 7200

tcctccctgg ctctagaatg tcatttaaaa gtcgagtgtc ttcttccttc cctgttttga 7260

agcagccctt ctcatgacag gcttgcttgc caaggttccc tctgacctta aatctcttcc 7320

ttttggtgtc ttggacaggg cagttcagag tgataggacc aagacaccct atccgggctc 7380

tggtcgggga tgaagtggaa ttgccatgtc gcatatctcc tgggaagaac gctacaggca 7440

tggaggtggg gtggtaccgc ccccccttct ctagggtggt tcatctctac agaaatggca 7500

aggaccaaga tggagaccag gcacctgaat atcggggccg gacagagctg ctgaaagatg 7560

ctattggtga gggaaaggtg actctcagga tccggaatgt aaggttctca gatgaaggag 7620

gtttcacctg cttcttccga gatcattctt accaagagga ggcagcaatg gaattgaaag 7680

tagaaggtga gtagtgccat ataatattag gtattaactg ttgggtggcc aagaacaatt 7740

attctctcaa ctgagatgag atccctcaac ccaaacatct cagtcctggg aatgatttcc 7800

ataaaaatgt acacatcaat aaacagaaac tcatgcttag ggatgtctgt tgcatcatta 7860

ttcagagtag caaggaaatt gggatcaaaa tcaatgcctt tgagtaggta agtgacagaa 7920

tgaacaatgg tagccatact gtgaatatta tgcaggcatt aaaaagatta ttttagcact 7980

aggccagatg gtttggaggc cttctataag gtattattga gtgataagag caagctgctg 8040

taggatacaa aaacaaaaac aaaaccctag ggcatggtgg tttgcctcgc agctactcag 8100

gaggctgaga cgggaggctg gcttgagccc aggggtttgc agttacagtg agctatgatt 8160

gcaccactgc actccaaccc gggtgacaga gcaaagacct tcacccccac tccctacccg 8220

tctctaaaaa aaacaaaaac aaaaacaaaa aaacccttgg gcccagcgcc gtggctcacg 8280

cctgtaatcc cagcactgtg ggaggccgag gtgggcagat cacaaggtca ggagatcgag 8340

accatcctgg ctaaaacggt gaaaccccgt ctctactaaa aatacaaaaa aaaaaaaaaa 8400

attagccagg catggtagca ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga 8460

gaatggcgtg aacccggaag cggaggttgc agtgagccaa aatccttcca ctgcactcca 8520

gcatggggga cacagcgaga ctccgtctca aaaaaaaaaa aaaaaccctg tatttgtgag 8580

cgcacacaca cacacacaca cacacacacc tgtgcttggt cctagtgaat aagcaagtaa 8640

atcaaatgtc taaatataat tatagaaagg agatgtcact ttttggctgt acctccacta 8700

tttcattctg cagaattgca gaatttcttt ttttttttcc tttctttctt ttcttttttt 8760

ttttgacaca gagtctcgct ctgtcaccca ggctggagtg caatggcgcc ctccgcctcc 8820

tgggttcaag tgattctcct gcctcagcct cccgagtagc tgagattaca ggtgcccacc 8880

accacaccca gctaattttt gtatttttag tagagacagg gtttcaccag gttgtcaagg 8940

ttggtctcaa actcctgacc tcaggtgatc cactcgcctc agcctcccaa agtgctggga 9000

ttacaggcat gagccatggt gcccggcctc agaatttcat tttcaacatg ttttgcatga 9060

tgggtgattt tggagaatat tttttgctct atcgcaggat gattaagatg tggacaaggt 9120

gaagccgatg gagggggagc tttgaaagtt acttgctatt taattgagga actaaactgc 9180

tttgagagcc tgggggtcag atcctctgcc ttttcctcct ccccacctgc agtgcaaaca 9240

tcagacaatt gatcactatt gtatcttgga ggtgggagtg accattgcag tgctgggacc 9300

agaagatggc attgtatgtg gaacaacaaa gcactatttc tagagactgc ctgcagggat 9360

atggaaatag ctttatgtgt ctcagaatgt tcttcataca gctgttttta ttggggaaat 9420

tctacttgcc gaaaagtttg atagtgagac cctctccagt ttgcagattt ttctccttcc 9480

tgctcaacaa cttcctagct cagtaactgc ctctcccaac aaactccctc agtttcacca 9540

caccaaaaaa ggaagacaag ccggttgcgg tggctcacac ctataatccc aaaactttgg 9600

gaggccgagg cgggtggatc acctgaggtc gggagttcga gactagcctg accaacatgg 9660

agaaaccctg tctctactaa aaacacaaaa ttagcctggc gtggtggcgc attcctgtaa 9720

tcccagctgg gaggctgagg caggagaatc gcttgaaccc cggaggcgga ggttgcagtg 9780

agccaagatc gtgccattac actccagtct gggcaagaaa agtggaactc catctcaaaa 9840

aaaaaaaaaa aaaaaaaaca aggaagacaa aaagaaaagc agctaaagac tttgcctcag 9900

gggagaaagt tctcttttgg gttgctatcc acattccaac ctcctgttcc cacctcttcg 9960

tctgcatgcc taagaaactg ttttacaagt aaataaggga cgctttgtct aggctttgga 10020

gccaggaagt tgagacaaat ttaggaatga gatgaagtaa tggtattatt gcaagtctca 10080

ggtgtaacta cctctgctct ttctctgaag agtttctaat ttctcttgtt tacttatttt 10140

tttcttgtca tttttgtgat tttattacta gttgtctcta atcctttctt taaattcttc 10200

attatgaaac ataaaaacaa atgccaggcg cggcagctca cgcctgtaat cccagcactt 10260

tgggaggccg aagcgggcag atcacccgag gtcaggagtt cgagaccagc ctgatcaaca 10320

tggagaaacc ccgtctctac taaaaaatac aaaattagct aggcgtggtg gcacatgcca 10380

gtaatcccag ctacttgaga gactgaggca ggagaatcgc ttgaaccggg aggcagaggt 10440

tgcggtgagc caagatcgcg ccattgcact ccagcctggg caacaagagc aaaactctgt 10500

ctcaaaaaaa aaaaaccaca tacaaaccag agataatatt ataatgagcc tccaagtgcc 10560

taccaccttg ctgcagcact tgtcaatcca gggaccaccc acctcaccgg ctccccactc 10620

attaccaccc tcccctactc aattactgag gtaaatccta ggcagcatga tcatttcttt 10680

tttttctttt tatttatttt gagacaggat ctgtctctgt cacccaggct ggagtgtagt 10740

ggcatatctc tgctcactgc agcctctgcc tcccgggcag aagccatcct cccacctcag 10800

cctacatagt agctgggacc acaggcacac accaccacac actgctaatg ttttgtattt 10860

tttgtagaga ctgggtttta ccatgttgat caggctggtc tcaaactcct aggctcaagc 10920

aatcctccca cctcggcctc ccaaagtgct agaattacag gcgcgagcca ctgcacccag 10980

cgaagaacac tttttaaaaa ataaataggc cgggcgcggt ggctcacacc tgtaatccca 11040

gtactttggg agcccaagga gggcgaatca tgaggtcaag agattgagac catcctagct 11100

aacatggtga aaccccattt ctactacaaa tacaaaaaca aaattagcct ggcgtggtgg 11160

caggcgcctg tagtcccagc tacttgggag ctgaggcagg agaatggagt gaacccggga 11220

ggcggagctt gcagtgagct gagatcatgc cactgcactc cagcctgggg caacagagtg 11280

agactcaaaa aaaaaaaaaa aaaagccccc cctccccaca cacaataata taaataaata 11340

aataaccaca atactattat cacatcttac aaactcaaca aaaatttctt aatatcatca 11400

aatacccagt ttgtgttcaa attttcctga ttgtttcata aatatactct tacagttggt 11460

ttcttttagc gagattcaaa tgagacccac ctgttgacct ttgcccttag ggtttcccag 11520

ggtctgaatt ttgttgacga cattcccatg ttgctatgta atacggtcct ccatgccctg 11580

tgtttttctg taaactgata gatgtggagg tgcaatgaca tttgtgtttg atttactttg 11640

gcaaatatag ttcatcagtg atactctata cttcttgttg ctttacatcc ggaggctgat 11700

aatgtctgct tttctctctt ttctaattat ttgtgaaagg aaaaatgtgg ggggttggga 11760

gaaaaaaacc cttaagtaca tactcgctaa atcacattgc tacaggtaac ttccattaag 11820

aacttgaaag taaaggtagc tgcattttcc cctagggaac acaatgatag acaggagcct 11880

tagtctacag cttgaaggat tgtaattata cctaagcaac cctcctggac cagtttaatg 11940

ttattagctg tgatgtatcc ctacctttga tgtcattatc cttactcagc tcccttaaag 12000

cagagatcaa gatgaaaagg gcttcagctg cagcatggca catggagatt agagtggggc 12060

ttttggatgc tgaggagcag acctagaatg ggaaatagat gggagccaca gaagtgaagg 12120

tccccctccc tcattgctca acctactcca catctccagg tctgcacatc tgttcagtta 12180

ctgaatcctg tgtaagctac cttctttttc ttttttcttt tatttattta tttatttttt 12240

ttttgagatg gagttttgct cttgttaccc aggctggagt gcaatggtgc aatctcggct 12300

cactgcaccc tccaactccc aggttcatgc aattctcctc cctcagcctt ccaagtagct 12360

gggattacag gctgcaccac catgtctggc taatttttgt attatcagta gagagagggt 12420

ttcaccatgt tggccaagcc ggtctcgaac tcctgacctc aagtgatcca cccaccttgg 12480

cctcccaaaa tgctgggatt acaggtgtga gccaccatgc ccgctgtaaa ctaccttctt 12540

aaaagctcta gaagagggct cttaaccttt tgttgtgtgt catgcacctt ccgcaagctg 12600

atgaagttga tagacccatc tcagaatttt tttttttttt ttgagacagt gtctcactct 12660

gtcacccagg attggttgca gtggcacgat catggctcat tgcagcctcc acctcccagg 12720

ctcaagtgat cctcctgact cagcctcttg aatagctgag accacaggct tgtgtcacca 12780

tgcccaggta atttttaatt ttttttcgta gaggcagggt ctcacattat gttgcccagt 12840

ctggcctcga gaactcctgg gctcaagcaa tcttcctgcc ttggcctccc aaagtggtgg 12900

gattacaggg gagagccacc acacctagcc agaagaatgt tttaaataca ccaaataaaa 12960

catttatacc aaaatacagt tatcaaaata ttaaattaac aagagttagg gtgaccctat 13020

taattagtgt aatttcaaaa tagtaatgaa cataagtgat agtttgagat ttctgtgact 13080

tttctaatgt gacgtgaaaa tatttgtgat ttttcttttt cttttttttt tttgagatgg 13140

agtttcgctc ttgttgccca ggctggagtg caatggcaag atctcggctc acctcaacct 13200

ccgcctcctg ggttcaagcg attctcctgc ctcagcctct tgagtagctg ggattacagg 13260

actgtgccac cacgtccagc taattttgta tttttagtag aaacagggtt tctccatgtt 13320

ggtcaggctg gtcttgaact cccaacctca ggcgatccgc ccgcctcggc ctcccaaagt 13380

gctgggatta caggtgtgag ccaccgcacc tggccaatat ttgtgatttt tattgacgac 13440

aaagtcaaag gttctcttca tattattgtg gtgtatcgcc tacaagcata attaaaataa 13500

acactaaatt tcagtttaaa gtttactgaa aataaatatg tattttttat tccctattta 13560

agctttgaat cccctgactt cctataccat taccactgtc ctagttcagg ttcatgttgt 13620

tttttacttt aattgttatc acagtctctt aacatttctc cctatgttct ccagtcctgt 13680

aggtgctaaa tctgacgtgg tcacttctca gcttggaatc cttcagtgca ccaccacagc 13740

cttgaactac atatttgaaa tacatattta ttttcagtaa actttaaact gaaatttagt 13800

gtttatttta attatgcttg taggcgatac accacaataa tatgaagaga acctttgact 13860

ttgtcgtcaa taaaaagtcc cttgagggac ttcagatgta agtcccttag ctgctcgtta 13920

aaactccccc agcctgaccc aatacacaat cttgacttta aaccacttgt cattctaaat 13980

cactagcatt tcctggaaaa aaaagccatt tttccttcag ggctaagctc agggaccaat 14040

tctgtgtcac cttctttgaa tcctgatgat attcacttct ttatttgacc tgatttattg 14100

ggccccagac accatgctga gtgttgggga ttcagctctg gacaatgtca aatgtcagtc 14160

ctgcctttca gatcctttct actgggtgag ccctggagtg ctggttctcc tcgcggtgct 14220

gcctgtgctc ctcctgcaga tcactgttgg cctcatcttc ctctgcctgc agtacagact 14280

gagaggtaca gggcagaggg tgggtggatc aggatccttt ctttaaatga gctggcttct 14340

tggagctaca ccacttaaca tgtatttgtg agtgacttct gggttcagaa gttcttctca 14400

ctattgagtg ataaagaaaa aaaataactc catgatgaaa gagttttaca tcttacggaa 14460

tgctttcata tgaataatcg gacctagcat ttccctatga gctaactatg ccatatagta 14520

accccatttt acagaggata caactgaggc caggagtagt tcagtgactt actcaaaccg 14580

atataactta taagtggtag agctgaggcc tctgtatcat acctagcagc tccatgcaac 14640

ttgggagagt gtgagcttcg aagtcagaca ggtctaggct attaggagtt ttgaataaag 14700

atactgaagt gaaagtctct accacacagt aggcgttcga aaattgtttc ctctttctcc 14760

attcaacact gaggactcag gttcagctgc tgatgaagct cctctttttt gcctagagct 14820

ttcattctga gccttctcct cctaccaagt gtctccccaa tgccagagca ggaagagtct 14880

tcactcctcc ccatgcccca cctcccattt gttactaaga ggagaggaga aagtagcaag 14940

gagggtatgg ggaatgttct gggggaatgg gtgttggtgc gatcaacaac aaagtccttt 15000

ctctcacctt gaattcatcc cagatgcctg cttgtttact tcttccacac aaaaaaaggc 15060

cttcagccct catggctgag cagaaagaat ctgaatgtta gagtcaggca gcctgggttt 15120

gaattccatc tcaggtactg aactctatag caaaattctt agattctcca agcttcagtt 15180

gccttgtctg tcaaatagag aaaacatcct tcgtcctaaa ttgtagggag gattaaagtc 15240

atgcaaagtg cctactacaa atccagtcac aaagtagcta gctactcact aaatgttcag 15300

ctcctccctc ctcattcaga tgggaagtgg ctttagataa acaaagtggc aacgcagtgg 15360

gctggagcag ctctgtgaac tgagaatcca agaaaagggg cgaagagcag ctgggatgta 15420

ttggatgctt gtgctggctt ggagcattgc tcacattctt tattcgctat tgtatctaga 15480

ctatagctag agaaagagcc gcaaccattg gctttaaatc cagtgctctt cctactctcc 15540

tgaggttgtt tccaggctgc agagaaatag cctgcacaag gggcccaggc gctgggtgtg 15600

ggagggtccc caccgagagc cagaacatgc aggaactaaa atgttgcctt tttctatttt 15660

aggaaaactt cgagcagaga taggtgagtt ccagtcatcg tttctcccaa ttcttgcctt 15720

ttggtttttt ggcataacgg aaatggtccc gttcttggac cgtctctccc tctcaatacc 15780

ctgttttccc ctcagtttcc ctttctctac agtgggtgtg tcgtgcctag aacaagtttt 15840

aagtaattaa ataacaaaga ctcaggataa aagatccttt ttgagtgccc tactaaatcc 15900

atttccattt gtttctcttt cagagaatct ccaccggact tttggtaagt tccggcatgt 15960

ctaggccctc ccaggtcaac ttggtatttc actctagttc cagtcacctg ggggaacaag 16020

gacccctggc tcctggttga gtcccttcct ctcttctctt ttctttcttt aaataagaag 16080

tcatttgcat ttaggattgg taaaatcata ataaaaatac tcatgtactg tttttatgtg 16140

ccaggcacta ttctaactac tttacaaaaa tgttatctta ttctgtttaa ctccttatgc 16200

acatgatctc tcttttcagg aatggcaaaa cagaggtaaa tagatcgttt acacgtaaac 16260

ctgatgtctg gttggggagg tgaaacaaac agaaacaaga cacaactgta tcacctgtac 16320

ttatatttct gctttacaaa ctcaggatgt ttccatgagt acagaacatg actaatcaga 16380

gaagacctca tagaggaata gaaaagccac caagccccac taggaattga cccctcaagg 16440

acatggtttc tagccttttt gttcactgca gattgcccaa tgcctaaaga taatggcaac 16500

agaagagcac ccaaatattt gttagataaa tgttgcagac actagaaggt gtcattaggg 16560

cacagatggt accttctctg agcaaacttc cttcacagct cctcctcccg aggctgtagg 16620

tgactctact cttgtcacct ggcacacaga gttctatcgt acgatttagg aaattagacc 16680

agtgtgtgga ccacacacac acacatcttt acacacccaa agaggaggaa tagtatcttt 16740

gttttggagg acttgactat gaaaggtctt aactcctttt tgtaccatga atctctctgg 16800

cactccagtg aagtctaaag gacccctttg cagaatgttt ttaaatatac acataaaata 16860

gaacacatag gattgcaaaa acaatcattg tactaaaata cagttatcaa ccgataatca 16920

catttgtgat atagtaacat aaatgtttct tttttttttt ttttgaggca gagttttgct 16980

cttgtcaccc aggctggagt gcaatggcgc gatctaggct cactgaaacc tctgcctccc 17040

gggttcaagc gattctcagc ctcctgagta gctgggatta caggtgcccg ccaccacacc 17100

cagctaattt ttgtattttt agtagagact aggtttcacc aggttggcca ggctggcctc 17160

gaactcctga cctcaggtga tccacctgcc ttggcctccc aaagtgctgg gattacgggc 17220

atgagccacc gtgcccggcc ataaatattt ctttagccaa agtaatacat taagtaatgt 17280

agcagcaagt ctaataacct gtaatttctt tctttctttc tttctttctt tttttttgag 17340

atgaagtttt tttgagatgg agtgcaatgg cacaatctcg gctcactgca acctccacct 17400

cctgggttca agcgattctc ctgcctcagc ctcccaagtt gctggaacta caggcgcatg 17460

ccaccatgcc cagctaattt ttgtattttt agtagagacg gggtttcacc atgttggcca 17520

ggctggtctt gaacccctga cctcaggtga tctgcctgcc ttggccttcc aaagtgctgg 17580

gattacaggc atgagccacc aggcccagcc caataacctt taatttcaac atactaataa 17640

acataaacag tatttcaaga tttctgcaat aactctaatg ggaatgaaaa catctgtggc 17700

ttccattggt aattaagtca caggtactgc tcatattgtg gttagttgta aaatgttttg 17760

gtttgttttg ttttttccaa gacttggggg aatgggtgtt ggtgggatca acaagagtct 17820

tgctctgtgg cccaggctgg agtgcagggg caggatcttg gctcactgca acctccgcct 17880

cccaggttca agcgattctc ctgcctcagc ctcctgagta gctggcatta caggcatgtg 17940

ccaccacgcc cacctaattt ttacattttt agtagagatg gggtttcacc atgttggcct 18000

ggctggtctt gaactcttgg cctcatgatc cacccgtctc ggactcccag agtgttggga 18060

ttacaggcat gagccaccac acctggcagt tgttacattt ttaatgaaag aaaatgttaa 18120

atccagttat tgaaaataag gaggcagtac ttttctcatc caagttcatg gactttctga 18180

attttgtccc cagagtcctt tggtgttcta ggaccccagg ttaaggaacc aaaaaagaca 18240

ggtgggtggg gcatgagggg gaacacatgt taaccctgtt tgttctggtg aacaattcag 18300

atccccactt tctgagggtg ccctgctgga agataaccct gtttgtaatt gtgccggttc 18360

ttggaccctt ggttgccttg atcatctgct acaactggct acatcgaaga ctagcaggtg 18420

cagtggctgg gcagcaggca agaccaccaa atagtggggg accaagtcag ctctgaatgg 18480

gaagccaaaa gagaatagaa ccaggactca agattagggg agctgggatt tccttattcc 18540

tctgtcccca tgcccaaccc caggctcttc tgagaaactg tgaagagaac cacttactgg 18600

atctgtggga tcccccagtg gaaagggcag tgtgggtcac tccaaatgtc catagggagg 18660

atgtggggaa ggtgctattc atcttccact aatcacatat ttgtttcttt ttgttttcag 18720

ggcaattcct tgaagagcta cgtaagttct cttctctctg ttataagcag agaataaaaa 18780

gccaggaaag ggagacagaa gcaacaagag gaagaggcgg gctattgagg gatcacattc 18840

ccagaggaaa ggaggagctg gagagcctgg gtggagggaa gactcctcct gggaggtaga 18900

gggcaaagaa gccagctgtt agagacacat ttacaggtgg cagagaagct ggaggcactc 18960

ctatctgcca cctgatccat tcctccttca ctgcccctaa gcaggaatcc aaccctagct 19020

ggtctcattg cccattccac agcaactgcc cagtgcctca cctctcagat caaccattga 19080

ggcaggaatg gagacaagat gaccccaagg gcttttcttc tccctagttc aatggtttta 19140

tgatacaaac tactgacata cgtttttcaa gttattttct ccttcttcta ggaaatccct 19200

tctgagtgat gtcacatctt ggcaggggtg gaggagagcc tggttgccca gggatttgtc 19260

cttggggaca tctcatccat caagttgcac actcactggc atctttgcta tggggacatt 19320

ccaatttgca ctttcaggaa cactctgaat tccaagtaga attgatttcc cttcttctgt 19380

catctacctt ttctcttcat tttcccattt ttattaccct tctttccatt tctctctcca 19440

gtcttccacc tggaagccct ctctggctaa ggacaggcag gtgcccctct ctccatcaga 19500

ggacacctgt actggagagc aacacaggat ggtctctgcc atgaactgga ggccaggaat 19560

ctcctcactg aaaattacag tatggtaact ttgcaaatgg tggttgtttc ttccaagact 19620

ccagccctga ttgcgcaaaa ctgaaaggca tgtgaaggga aggaagagga agagtgcaaa 19680

acattgaaga gagagctgag tgagctgaag agtgaggata tgagtagccc caacccaaac 19740

ctggagatgg ggagaaacct acagaatact agccagagct cctccttgtc ttggcagcct 19800

actagggacc tggggaagca aaaacgaaag ctgggcaaca tgcctgcttt agaatgtttt 19860

ccttctactt acacatcttc cacaggtctc agaatctttc cttcctctca tccttttctc 19920

ctatcttcat atctatcaga gtatccactg tttattcaac aactactact tgatggtcag 19980

acacaaacaa acaagctagg tgctaattaa taaagatacg agttttggcc gggtgcggtg 20040

gctcacgcct gtaatcccag cactttggga ggccgaggcg ggcgaatcac gaggtcagga 20100

gttcaagacc agcctggcca acatggtgaa accccatctc tactaaaaat acaaacaatt 20160

aactgagcat agtggtgggc acctataata ccagctactc cggaggctga ggcaggagaa 20220

tcgcttgaac ccaggaggca gaggttgcag tgagctgaga tcgtgccact gcactctagc 20280

cggagtgaca gagtaagact ctgtctcaaa aataaataaa taaataaata aataaataaa 20340

taaataaata aataaaaaat aataatacaa gttttcataa gcacacttct aaccccttgt 20400

cttttatgta tttccttcct tatccacgca cctgtctccc tctactccag cctcattacc 20460

ccagaggtca gtcctcagga aaactaaaca caaagaaaga gctcagtcag aaaggccatt 20520

tatttatgtt tcaagatgct cactgcctcc tttgttttgt ctcctttgca ggccttctct 20580

cttaggcctc ttctcctggg ggtatggatc ctggggggag attgatcacc tccatgcttc 20640

cattcctccc cagccatagt ggggacatca tgagagaagc caagccactg gcccaggatc 20700

acccggcatt tatggtggct gctctggcac aggtccttgc ctttatagcc cctccagtga 20760

tccataaggc cctctttctc cccaaaggag aggtcacaga tagggcaaag gtagctcttc 20820

tgcttccagt gggtctgctg gtgtctgacc agcctggaaa atgagctgaa agacttgctg 20880

caatggaagc agtagttggg cggctctgtg aggtgggcct tctggtgtct ggagagatag 20940

gatttcttgc taaaagtcaa agaacaatgg gggcaacaga agacattgag tcttgagggc 21000

ttcactggat gagagttgga tctggcatcc tgacagaggg ttccagtgat gggtgcctgg 21060

gtcctggtca caggtgcttg gttcttaagt acagatgcct ggttctgggc cataggaccc 21120

tcagttctaa atatgggttc ctgggacctg gccactggtg catggttcac atccaaaagc 21180

ccctggatgg acctctggct tctggcgatg ggtgtctgga attcagcctg ggtgcctgga 21240

atcctcaaag tacactcctg gtttccatcc actggctcct ggttttggtg tatcttctgg 21300

tggcgtttga gctcagactg gtcccggaag ctcttcccac acacagagca tgaatggggc 21360

cggtaaccca gatggacgcg gcggtgacga cttagtccag aagcatcaca gtaggtcttg 21420

tcacagagcg tgcaacagaa gggcctctcc ccaagatgca tgcgtctgtg atagctgagg 21480

gacttggggc tccgaaacaa cttcccacac tgactgcagc tgttagtcag cttgggattg 21540

tgaacaaact ggtggctata gaggtaggag cgcctgctga aacatttgcc acaggtgtag 21600

caaaaaaagg gtggcccagc ctgggatgct tgaagcaccc gggtcctgtc catagtccca 21660

gctggggcag atagggggca ctggccggcc cctctgcatg caaggaagac cttgtcatca 21720

ctagtcccct catctctcag actgggatgt tgttctcgaa gctctttctt cttgccttct 21780

acagtgaatg aggaagaata acacaaaatt cactgtaaga actccaacag aggcttggca 21840

tggtggctca cacctgtaat cccagcactt tgggaggccg aggccagcgg atcacctgag 21900

gttaggagtt cgaaaccagc ctgaccaaca tggtgaaacc ctgtctctac tacaaataca 21960

aaaattagct gggcgtcatg gcatctgcct gtaatctcag ctactaggga gactgaggca 22020

ggacaatcac tcgaacccgg gaggcggagg ttgcagtgag ccaagatggt gccactgcac 22080

tcctgcctgg gcaactagag tgaaactctg tctcaaaaaa aaaaaaagaa agaaagaaaa 22140

agaagaagaa gaaggagaag gagaagaagg agaaggagaa gagaaggaga agaagaagaa 22200

gaaggaagaa gaagaagaag aaaagaaaag aagaagaaga agaagacgaa gacgaagaag 22260

aagaagaaga ggaagaagaa gaactccaac acagcactcc attcagccta acacacttct 22320

tgtctctgcc cttgctctcc cacccaacac attcatcctt acccttgggc ctcataggct 22380

agaaataaga ag 22392

Claims (9)

1. A method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP, the method comprising: respectively introducing AQP4, MOG and MBP genes into HEK293T cells, respectively incubating with a sample to be detected, detecting by an immunofluorescence method with HEK293T cells transfected with empty vectors as a control, and determining that the sample to be detected is positive if a fluorescence signal is stronger than the control; if the fluorescence signal is equal to or less than the control signal, it is negative.

2. The method of claim 1, wherein the detecting is performed by: (1) mixing the cell adherence reagent and PBS according to the volume ratio of 1:10, adding the mixture into a 6-well plate containing a cover glass, and keeping the temperature at 37 ℃ and 5% CO₂Incubating for 1 hour; then adding a stable HEK293T cell line which is respectively transferred with AQP4, MOG and MBP genes, and carrying out cell culture at 37 ℃ and 5% CO₂Culturing to 80% fusion degree;

(2) the cover glass with the cell surface facing upwards is stuck on a glass slide, washed by PBST, sealed by 3 percent BSA and discarded; adding the serum to be tested diluted by PBST at a volume ratio of 1:10, and adding 5% CO at 37 DEG C₂Incubating for 1 hour under the condition; PBST washing; adding 50 μ l of goat anti-human IgG conjugated with biotin and streptomycin diluted by PBST at a volume ratio of 1:700, 37 deg.C, 5% CO₂Incubating for 30 minutes under the condition; PBST washing; adding fluorescein 488 monthly streptomycin conjugate diluted by PBST at the volume ratio of 1:700, and washing by PBST; sealing a sheet; HEK293T cells transfected with the empty vector were used as controls under the same conditions; then, carrying out fluorescence microscope detection, and if the fluorescence signal is stronger than the contrast, determining that the sample to be detected is positive; if the fluorescence signal is equal to or less than the control signal, it is negative.

3. The method according to claim 2, wherein the cells of step (1) are inoculated in an amount of 1X 10⁶/ml。

4. The method of claim 2, wherein in step (2) said coverslip is attached to said slide with a shadowless adhesive and fixed by ultraviolet radiation.

5. The method according to claim 2, wherein 50 μ l of the diluted test serum of step (2) is added to each well; the addition amount of goat anti-human IgG and fluorescein 488 monthly streptomycin conjugate is 50 mul/well.

6. The method according to claim 1, wherein the AQP4, MOG and MBP genes are introduced into HEK293T cells in the form of lentiviral plasmid vectors, respectively, and the nucleotide sequence of the lentiviral plasmid vector containing the AQP4 gene is shown in SEQ ID No. 1; the nucleotide sequence of the slow virus plasmid vector containing the MBP gene is shown as SEQ ID NO. 2; the nucleotide sequence of the lentiviral plasmid vector containing the MOG gene is shown in SEQ ID NO. 3.

7. The method of claim 6, wherein the AQP4 gene-containing lentiviral plasmid vector is constructed by transferring AQP4 gene into a plasmid vector pCDH-CMV-MCS-EGFP.

8. The method of claim 6, wherein the MBP gene-containing lentiviral plasmid vector is constructed by transforming the MBP gene into a plasmid vector pCDH-CMV-MCS-EGFP.

9. The method of claim 6, wherein the lentiviral plasmid vector containing the MOG gene is constructed by transferring the MOG gene into a plasmid vector pCDH-CMV-MCS-EGFP.

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