CN114487448B

CN114487448B - Composition and kit for detecting myasthenia gravis-related antibody and application of composition and kit

Info

Publication number: CN114487448B
Application number: CN202210072137.2A
Authority: CN
Inventors: 施福东; 李敏淑; 金维娜; 李治国; 郑培; 魏常娟; 陈京山; 章雷; 李�浩
Original assignee: Tianjin Tianhai Xinyu Biotechnology Co ltd
Current assignee: Tianjin Tianhai Xinyu Biotechnology Co ltd
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-10-18
Anticipated expiration: 2042-01-21
Also published as: CN114487448A

Abstract

The invention belongs to the field of immunodetection, and particularly relates to a composition for detecting an antibody related to myasthenia gravis, a kit and application thereof, and a method for detecting the antibody related to the myasthenia gravis. The composition for detecting the antibody related to the myasthenia gravis provided by the invention can construct a fluorescent detection system of 'primary anti-HRP secondary antibody-TSA' by using cells expressing the antigen related to the myasthenia gravis, a secondary antibody and a chromogenic substance, realizes high-efficiency detection of an anti-AChR antibody, an anti-MuSK antibody and an anti-LRP 4 antibody, has high sensitivity and specificity, provides effective diagnostic information for early diagnosis and prognosis evaluation of MG patients, and has important clinical application value.

Description

Composition and kit for detecting myasthenia gravis-related antibody and application

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a composition for detecting an antibody related to myasthenia gravis, a kit and application thereof, and a method for detecting the antibody related to the myasthenia gravis.

Background

Myasthenia Gravis (MG) is an autoimmune disease with acquired impairment of neuro-muscular junction transmission mediated by autoantibodies. The global prevalence rate is (150-250)/million, and the estimated annual incidence rate is (4-10)/million. The MG incidence rate of China is about 0.68/10 ten thousand, the female incidence rate is slightly high, the diseases can occur in all age stages, and the age group of 70-74 years is a high incidence population. Skeletal muscles of the whole body are involved, which are manifested as weakness and fatigability, with symptoms of "light morning but heavy afternoon", aggravated after activity and relieved after rest. Early involvement of the cranial nerves was common, with extraocular muscle involvement most commonly manifested as drooping eyelids and diplopia. Currently, MG therapy is based on cholinesterase inhibitors, glucocorticoids, immunosuppressive agents, intravenous immunoglobulin, plasmapheresis, and thymectomy. The disease condition of some patients can rapidly progress in a short period, and the patients are in danger of myasthenia. The hospitalization mortality rate is 14.69 per thousand, and the main death reasons comprise respiratory failure, lung infection and the like.

A variety of autoantibodies can be detected in the serum of MG patients, with acetylcholine receptor (AChR) antibodies being the most common pathogenic antibodies, about 80% MG patient antibody positivity, with the same patient antibody titer positively correlated with disease severity. Late onset, high titers of AChR antibody, and combined thymoma may increase the risk of secondary generalization of MG. The use of immunosuppressive drugs at an early stage is thought to reduce the risk of secondary generalization. Therefore, the early discovery and early diagnosis are of great significance for the treatment and prognosis of MG patients.

MG is also an MG pathogenic antibody, together with a muscle-specific tyrosine kinase (MuSK) antibody, a low-density lipoprotein 4 (anti-low-density lipoprotein receptor-related protein 4, lrp 4) antibody, and the like. In AChR antibody negative patients, about 5% -8% of MG patients are positive in MuSK antibodies, which are mostly seen in young women, eyeball and neck muscles are most seriously affected, the incidence rate of muscle atrophy is higher, the disease course is usually rapid, and life-threatening symptoms such as respiratory failure can appear after a plurality of weeks of disease attack, the clinical types of MuSK-Ab positive MG patients are all full-body types, the titer level of the antibodies is related to the severity of diseases, and the treatment effect of cholinesterase inhibitors and hormones is poor. The low density lipoprotein receptor-related protein 4 antibody (LRP 4-Ab) is a novel MG autoantibody discovered in recent years, and can exist with the two autoantibodies or cause diseases independently. LRP4 is a transmembrane protein present on the postsynaptic membrane at the neuromuscular junction, and the receptor as an aggremin plays an important role in activation of MuSK, aggregation of AChR, and maintenance of the structure of the neuromuscular junction.

Clinically common methods for detecting serum antibodies (AChR, muSK, LRP 4) of patients with myasthenia gravis include Radioimmunoprecipitation (RIPA), enzyme-linked immunosorbent Assay (The enzyme-linked immunosorbant Assay Elisa) and Cell-Based fluorescent staining (CBA). Antibody titer quantification is important for MG disease monitoring. Although RIPA is a good quantitative detection method by quantitatively calculating the actual concentration of antibodies in the serum of MG patients by detecting radioactive markers, the use of radioactivity has certain technical limitations, the effective period of the reagent is short, about 1 month, and radioactive contamination exists. Diagnostic sensitivity of RIPA is higher in adult-onset systemic MGs (80% -85%), but lower in ocular MGs (about 50%), with about 10% of MG patients still having antibodies undetectable by RIPA. Although the enzyme-linked immunosorbent assay (ELISA) has long validity period and high sensitivity, the enzyme-linked immunosorbent assay has low specificity and is easy to generate false positive because the spatial conformation of the antigen is difficult to maintain, and a detection reagent of the enzyme-linked immunosorbent assay pollutes the environment because of strong acidity. The kit based on the two methods depends on import, is expensive and has higher detection cost, and the popularization of the detection means is greatly limited.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art, for example, the method for detecting myasthenia gravis has low sensitivity and specificity, and effective detection for MG patients cannot be achieved. Therefore, the composition for detecting the antibody related to myasthenia gravis can construct a fluorescent detection system of 'primary anti-HRP secondary antibody-TSA' by using cells expressing the antigen related to myasthenia gravis, a secondary antibody and a chromogenic substance, realizes high-efficiency detection of at least one of an anti-AChR antibody, an anti-MuSK antibody and an anti-LRP 4 antibody, has high sensitivity and specificity, and provides effective diagnostic information for early diagnosis and prognosis evaluation of MG patients.

Means for solving the problems

In a first aspect, the present invention provides a composition for detecting an antibody associated with myasthenia gravis, wherein the composition comprises: cells expressing myasthenia gravis-associated antigen, a second antibody, a chromogenic; wherein,

the myasthenia gravis-related antigen comprises one or a combination of more than two of acetylcholine receptor, muscle-specific tyrosine kinase and low-density lipoprotein 4;

the second antibody is conjugated with a label; and when the second antibody is combined with the myasthenia gravis-related antibody, the marker catalyzes the color substance to generate fluorescence or generate fluorescence quenching.

In some embodiments, the composition according to the present invention, wherein the cells expressing an antigen associated with myasthenia gravis comprise one or a combination of two or more of the following groups (i) to (iii):

(i) Comprises a first polynucleotide group encoding each subunit forming the acetylcholine receptor;

(ii) A second polynucleotide encoding a muscle specific tyrosine kinase;

(iii) A third polynucleotide encoding low density lipoprotein 4;

preferably, the first set of polynucleotides comprises the nucleotide sequence set forth as SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: 7; or, comprising the amino acid sequence as set forth in SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: 8;

preferably, the second polynucleotide is as set forth in SEQ ID NO: 1;

preferably, the third polynucleotide is as set forth in SEQ ID NO:2, or a polynucleotide having the sequence shown in figure 2.

In some embodiments, the composition of the invention, wherein the cells expressing an antigen associated with myasthenia gravis comprise: a first cell expressing an acetylcholine receptor, a second cell expressing a muscle specific tyrosine kinase, and a third cell expressing low density lipoprotein 4;

preferably, the first cell is selected from at least one of: comprises a nucleotide sequence as set forth in SEQ ID NO: 3. the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:7 comprising a polynucleotide having a sequence as set forth in SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: 8;

the second cell comprises the nucleotide sequence set forth as SEQ ID NO: 1; the third cell comprises the nucleotide sequence set forth as SEQ ID NO:2, or a polynucleotide having the sequence shown in figure 2.

In some embodiments, the composition of the invention, wherein the label is horseradish peroxidase and the color former is tyramide fluorescein; optionally, the fluorescein is selected from any one of: fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, texas Red, phycoerythrin, propidium iodide, alexa Fluor series, dylight series, iFluor series.

In some embodiments, the composition of the invention, wherein the method for preparing the cells expressing the antigen associated with myasthenia gravis comprises:

and (3) vector construction: constructing a first vector group for expressing each subunit forming the acetylcholine receptor, a second vector for expressing muscle-specific tyrosine kinase and a third vector for expressing low-density lipoprotein 4;

and (3) transfection step: transfecting a cell with at least one of the first vector group, the second vector, and the third vector;

a screening step: screening cells expressing acetylcholine receptor, muscle specific tyrosine kinase and/or low density lipoprotein 4 to obtain cells expressing myasthenia gravis-associated antigen.

In some embodiments, the composition of the invention, wherein the transfection step comprises: transfecting cells by using the first vector group, the second vector and the third vector respectively to obtain a first transfected cell transferred with the first vector group, a second transfected cell transferred with the second vector and a third transfected cell transferred with the third vector;

the screening step comprises the following steps: screening the first transfected cell, the second transfected cell and the third transfected cell to obtain a first cell for expressing an acetylcholine receptor, a second cell for expressing muscle specific tyrosine kinase and a third cell for expressing low density lipoprotein 4;

preferably, the mass ratio of the first vector combination, the second vector and the third vector transfected cells is 1.

In some embodiments, the composition of the present invention, wherein the first vector set comprises the nucleic acid sequences as set forth in SEQ ID NOs: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:7, the mass ratio of the recombinant expression vector transfected cells is 1; or,

the first vector group comprises the sequences shown as SEQ ID NO: 3. SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:8 in the sequence shown in the specification, the mass ratio of the transfected cells of the recombinant expression vector is 1.

In a second aspect, the present invention provides a kit for detecting an antibody associated with myasthenia gravis, wherein the kit comprises the composition according to the first aspect.

In a third aspect, the present invention provides a composition according to the first aspect or a kit according to the second aspect for use in at least one of the following (a) - (b):

(a) Detecting the myasthenia gravis related antibody, or preparing a kit for detecting the myasthenia gravis related antibody;

(b) Preparing a kit for diagnosis or prognosis of myasthenia gravis.

In a fourth aspect, the present invention provides a method for detecting an antibody associated with myasthenia gravis, wherein the method comprises the following steps:

contacting a sample to be tested with a composition according to the first aspect or a kit according to the second aspect;

and acquiring a fluorescent signal, and judging whether the sample to be detected contains an anti-acetylcholine receptor antibody, an anti-muscle specific tyrosine kinase antibody and/or an anti-low density lipoprotein 4 antibody or not according to the fluorescent signal.

ADVANTAGEOUS EFFECTS OF INVENTION

In some embodiments, the composition for detecting myasthenia gravis-related antibodies provided by the invention can construct a fluorescent detection system of 'primary anti-HRP secondary antibody-TSA' by using cells expressing myasthenia gravis-related antigens, secondary antibodies and chromogenic substances, realizes efficient detection of at least one of an anti-acetylcholine receptor (AChR) antibody, an anti-muscle specific tyrosine kinase (MuSK) antibody and an anti-low density lipoprotein 4 (LRP 4) antibody, has high sensitivity and specificity, and provides effective diagnostic information for early diagnosis and prognosis evaluation of myasthenia gravis patients.

The present invention utilizes cells to express myasthenia gravis-related antigens and utilizes the CBA method to diagnose MG patients. The CBA method can maintain AChR antigen conformation, has high specificity similar to the RIPA method in the aspect of detecting antibody, and simultaneously improves the detection rate of the clustered AChR antibody by 21-56 percent compared with an radioimmunoassay. Therefore, the CBA method has the advantages of better specificity and sensitivity in the aspect of detection of AChR and MuSK antibodies.

In addition, because the proportion of patients with positive MuSK antibodies and LRP4 antibodies in MG patients is lower (less than or equal to 10%), the invention develops a composition for detecting myasthenia gravis-related antibodies (containing three pathogenic antibodies of AChR, muSK and LRP 4), thereby maximally improving the detection rate of MG antibodies and providing more comprehensive antibody detection support for MG clinical diagnosis and treatment. In addition, the kit utilizes a novel fluorescent detection system of 'primary anti-HRP secondary antibody-TSA', can realize high-efficiency detection of low-abundance AChR antibody, muSK antibody and anti-LRP 4 antibody in serum, has high sensitivity and specificity, provides effective diagnostic information for early diagnosis and prognosis evaluation of MG patients, and has important clinical application value.

In some preferred embodiments, the composition provided by the invention can simultaneously realize combined detection of an anti-AChR antibody, an anti-MuSK antibody and an anti-LRP 4 antibody, remarkably improve the diagnosis accuracy of myasthenia gravis, reduce the occurrence of missed detection and false detection, and has important clinical application value.

In some embodiments, the present invention detects MG directly in cells expressing an antigen associated with myasthenia gravis, and by expressing the antigen in the cells, the native three-dimensional conformation of the antigen can be maintained, and the specificity and affinity of the antigen binding to the antibody can be increased, thereby increasing the accuracy of detecting myasthenia gravis.

In some preferred embodiments, the polynucleotide or recombinant expression vector with a specific nucleic acid sequence used in the present invention expresses the target antigen, which can improve the detection rate of patients with negative clinical antibody detection, and has important diagnostic significance.

In some embodiments, the compositions provided herein are capable of achieving effective detection of embryonic and adult AChR antibodies, with higher detection rates of anti-AChR antibodies in different MG patients.

Drawings

FIG. 1 shows a vector map of the vector PEGFP-N1 in example 1;

FIG. 2 shows the observation result of cell fluorescence of the recombinant plasmid transfected AChR in example 1;

FIG. 3 shows fluorescence observation results of detection of AChR, muSK, and LRP4 antibodies in the kit of example 2, wherein A is a first cell positive for AChR, B is a second cell positive for MuSK, C is a blank control, and D is a third cell positive for LRP 4;

FIG. 4 shows the results of comparing the detection rates of four subunits of MuSK for AChR antibody-negative MG patients in example 3;

FIG. 5 shows the results of comparing the specificity and sensitivity of diagnosing MG patients with CBA-TSA, elisa and RIPA in example 4.

Detailed Description

Definition of

The words "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but may also mean "one or more", "at least one", and "one or more than one".

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this application, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative and "and/or," the term "or" in the claims means "and/or" unless it is explicitly stated that only alternatives or mutual exclusions between alternatives are mutually exclusive.

The term "HRP" is Horseradish Peroxidase (HRP), which catalyzes "Donor + H ₂ O ₂ →Oxidized donor+2H ₂ And O' in the reaction process.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component). The polypeptides may be isolated from natural sources, may be produced by recombinant techniques from eukaryotic or prokaryotic hosts, and may be the product of synthetic methods.

The term "polynucleotide" or "nucleic acid molecule" refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments, or may be a component of a larger nucleotide sequence structure, derived from nucleotide sequences that have been isolated at least once in quantity or concentration, and which are capable of being recognized, manipulated, and recovered in sequence, and their component nucleotide sequences, by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individually or as a whole) or may be an integral part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "recombinant polynucleotide" and "recombinant nucleic acid molecule" belong to "polynucleotide".

The term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences for the expression of a gene of interest in a suitable host. The term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors can include, for example, a collection comprising i) genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons.

As used in this disclosure, the terms "expression", "encoding" and "encoding" include any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "sample to be tested" relates to any kind of sample in which it is desired to determine whether or not antibodies associated with myasthenia gravis are contained. For example, the sample to be tested may be any product produced by the subject, or any product derived from a product produced by the subject. The sample may be taken from any tissue or body fluid, such as a blood sample (including samples derived from blood), a serum sample, a cerebrospinal fluid sample, a lymph sample, a saliva sample, a joint synovial fluid, and the like.

Technical scheme

In the technical scheme of the invention, the meanings represented by the numbers of the nucleotide and amino acid sequence table in the specification are as follows:

the amino acid sequence of SEQ ID NO:1 shows a nucleotide sequence encoding a muscle-specific tyrosine kinase (MUSK);

SEQ ID NO:2 shows a nucleotide sequence encoding low density lipoprotein 4 (LRP 4);

SEQ ID NO:3 shows a nucleotide sequence encoding a rapn subunit forming an acetylcholine receptor (AChR);

SEQ ID NO:4 shows a nucleotide sequence encoding the alpha subunit forming acetylcholine receptor (AChR);

the amino acid sequence of SEQ ID NO:5 shows a nucleotide sequence encoding the β subunit forming the acetylcholine receptor (AChR);

SEQ ID NO:6 shows a nucleotide sequence encoding the delta subunit forming the acetylcholine receptor (AChR);

the amino acid sequence of SEQ ID NO:7 shows a nucleotide sequence encoding the gamma subunit forming the acetylcholine receptor (AChR);

SEQ ID NO: the nucleotide sequence encoding the epsilon subunit forming the acetylcholine receptor (AChR) is shown at 8.

Composition for detecting myasthenia gravis-related antibodies

The invention provides a composition for detecting an antibody related to myasthenia gravis, which comprises the following components: cells expressing myasthenia gravis-associated antigen, a second antibody, a chromogenic; wherein,

The composition provided by the invention utilizes cells to express myasthenia gravis-related antigens, can be combined with at least one myasthenia gravis-related antibody, the second antibody is further combined with the myasthenia gravis-related antibody, and when a chromogenic substance exists, the marker coupled with the second antibody catalyzes the chromogenic substance to generate fluorescence or generate fluorescence quenching, so that immunofluorescence detection of at least one of an anti-AChR antibody, an anti-MuSK antibody and an anti-LRP 4 antibody is realized, and effective diagnostic information is provided for clinical diagnosis of myasthenia gravis patients.

The invention successfully constructs a tyramide signal amplification system of 'primary anti-HRP secondary antibody-TSA' by using cells expressing myasthenia gravis related antigens, a secondary antibody and a chromogenic substance, has high sensitivity and specificity, and solves the problems of low detection rate, false positive and the like existing in the conventional diagnosis of myasthenia gravis.

< cells expressing an antigen associated with myasthenia gravis >

In the present invention, the cell expressing one or a combination of two or more of acetylcholine receptor, muscle-specific tyrosine kinase, and low-density lipoprotein 4 can be used to bind at least one of anti-AChR antibody, anti-MuSK antibody, and anti-LRP 4 antibody in a sample to be tested, thereby realizing immunodetection of MG patients.

In some embodiments, the cells expressing an antigen associated with myasthenia gravis include a first cell that expresses an acetylcholine receptor (AChR). The AChR is expressed by the first cell, so that the natural spatial conformation of the AChR can be maintained, the binding affinity of the AChR and the AChR antibody is improved, and the detection sensitivity and specificity of the AChR antibody are further improved.

Further, the first cell comprises a first polynucleotide set comprising polynucleotides encoding polypeptides that form acetylcholine receptors. The acetylcholine receptor is a pentameric transmembrane glycoprotein, and in some embodiments, the first set of polynucleotides comprises polynucleotides that: polynucleotides encoding the alpha subunit of AChR, polynucleotides encoding the beta subunit of AChR, polynucleotides encoding the delta subunit of AChR, polynucleotides encoding the gamma subunit of AChR, and polynucleotides encoding the synaptic receptor associated protein Rapsn. The first cell can stably express acetylcholine receptor by using the polypeptide encoded by the first polynucleotide group, and realizes specific binding and detection of the anti-AChR antibody.

In some preferred embodiments, the first set of polynucleotides comprises the sequences set forth as SEQ ID NOs: 3. the amino acid sequence of SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5. the amino acid sequence of SEQ ID NO:6 and SEQ ID NO:7, or a polynucleotide having the sequence shown in seq id no. The polynucleotide can encode and form the embryo type AChR, maintain the natural three-dimensional conformation of the protein and realize the accurate detection of the embryo type AChR antibody.

In some embodiments, the first polynucleotide set comprises polynucleotides that: polynucleotides encoding the alpha subunit of AChR, polynucleotides encoding the beta subunit of AChR, polynucleotides encoding the delta subunit of AChR, polynucleotides encoding the epsilon subunit of AChR, and polynucleotides encoding the synaptic receptor associated protein Rapsn. The first cell can stably express acetylcholine receptor by using the polypeptide encoded by the first polynucleotide group, and realizes specific binding and detection of the anti-AChR antibody.

In some preferred embodiments, the first set of polynucleotides comprises the sequences set forth as SEQ ID NOs: 3. the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:8, or a variant thereof. The polynucleotide can encode and form the adult AChR, maintain the natural three-dimensional conformation of the protein and realize the accurate detection of the adult AChR antibody.

The composition disclosed by the invention can realize detection of an adult type AChR antibody and detection of an embryonic type AChR antibody, is beneficial to improving the detection rate of the anti-AChR antibody of a patient with myasthenia gravis, and has high clinical diagnosis significance.

In some embodiments, the cell expressing an antigen associated with myasthenia gravis comprises a second cell expressing a muscle-specific tyrosine kinase (MuSK). The second cell is used for expressing the MuSK, so that the natural spatial conformation of the MuSK can be maintained, the binding affinity with the anti-MuSK antibody is improved, and the detection sensitivity and specificity of the anti-MuSK antibody are further improved.

Further, the second cell comprises a second polynucleotide encoding a muscle specific tyrosine kinase, and the second polynucleotide is used within the second cell to encode the muscle specific tyrosine kinase in a native three-dimensional conformation.

In some preferred embodiments, the second polynucleotide is a polynucleotide as set forth in SEQ ID NO: 1. The present invention finds use with SEQ ID NO:1, the MuSK coded by the polynucleotide with the sequence shown in the specification has higher detection rate for MG patients with positive anti-MuSK antibodies, and is favorable for improving the accuracy of MG patient diagnosis.

In some embodiments, the cell expressing an antigen associated with myasthenia gravis comprises a third cell expressing low density lipoprotein 4 (LRP 4). Expression of LRP4 by a third cell maintains the native spatial conformation of LRP4, increases the affinity of binding to the anti-LRP 4 antibody, and thus increases the sensitivity and specificity of detection of the anti-LRP 4 antibody.

Further, the third cell comprises a third polynucleotide encoding low density lipoprotein 4, and the third cell encodes low density lipoprotein 4 having a native three-dimensional conformation using the third polynucleotide.

In some preferred embodiments, the third polynucleotide is as set forth in SEQ ID NO:2, or a polynucleotide having the sequence shown in figure 2. The present invention utilizes SEQ ID NO:2, and the anti-LRP 4 antibody has high binding specificity and sensitivity, and is favorable for improving the accuracy of diagnosis of MG patients.

In some embodiments, the cells expressing an antigen associated with myasthenia gravis include a first cell expressing acetylcholine receptor (AChR), a second cell expressing muscle-specific tyrosine kinase (MuSK), and a third cell expressing low density lipoprotein 4 (LRP 4). In other embodiments, the cells expressing the myasthenia gravis-related antigen may also simultaneously express acetylcholine receptor, muscle-specific tyrosine kinase, and low-density lipoprotein 4, can simultaneously detect anti-AChR antibody, anti-MuSK antibody, and anti-LRP 4 antibody, and have high specificity and high sensitivity, thereby achieving accurate detection of MG patients.

< method for producing cells expressing an antigen associated with myasthenia gravis >

The preparation method of the cell for expressing the myasthenia gravis-related antigen provided by the invention comprises the following steps:

and (3) vector construction: constructing a first vector group for expressing polypeptides forming acetylcholine receptors, a second vector for expressing muscle-specific tyrosine kinase, and a third vector for expressing low-density lipoprotein 4;

In some embodiments, the transfection step comprises: transfecting cells by using the first vector group, the second vector and the third vector respectively to obtain a first transfected cell transfected with the first vector group, a second transfected cell transfected with the second vector and a third transfected cell transfected with the third vector;

the screening step comprises: and screening the first transfected cell, the second transfected cell and the third transfected cell to obtain a first cell for expressing an acetylcholine receptor, a second cell for expressing muscle specific tyrosine kinase and a third cell for expressing low-density lipoprotein 4.

In some preferred embodiments, the mass ratio of the first vector combination, the second vector, and the third vector transfected cells is 1.

In some preferred embodiments, the first vector set comprises the sequences set forth as SEQ ID NOs: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:7 in the sequence shown in the specification, the mass ratio of the recombinant expression vector transfected cells is 1.

In some other preferred embodiments, the first vector set comprises the sequences set forth as SEQ ID NOs: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: the mass ratio of the recombinant expression vector with the sequence shown in 8 to transfected cells is 1.

The method provided by the invention can construct cells expressing acetylcholine receptors, muscle specific tyrosine kinases and/or low density lipoprotein 4, and has the advantages of high specificity and accuracy of combination of reformed myasthenia related antigens and antibodies, high stability and low detection cost.

< second antibody >

In the present invention, the anti-AChR antibody, the anti-MuSK antibody and/or the anti-LRP 4 antibody contained in the sample to be tested is a primary antibody capable of antigen-antibody specific binding to a myasthenia gravis-associated antigen expressed by cells. The second antibody can be combined with the first antibody in an antigen-antibody specific manner, and the enzymatic detection of the high-density in-situ labeling of the target protein is realized under the catalytic action of a labeling substance.

In some embodiments, the label is an enzyme, exemplary including, but not limited to, horseradish peroxidase (HRP), alkaline Phosphatase (AP) or derivatives thereof, and the like. The enzyme and the second antibody may be directly linked or indirectly linked. When the second antibody is combined with the antibody related to myasthenia gravis, the enzyme catalyzes the chromogenic substance to enable the chromogenic substance to generate fluorescence or generate fluorescence quenching.

In some preferred embodiments, the label is horseradish peroxidase (HRP), which can be used to label the target protein in situ at high density and can be combined with a high performance dye Alexa

Phase of conventional fluorescent dyes and colorimetric detection systemsAnd (3) compatible construction of a tyramide signal amplification system.

In some preferred embodiments, the HRP is indirectly linked to a secondary antibody, illustratively, a label linked to biotin and a secondary antibody linked to avidin; or HRP linked avidin and secondary antibody linked biotin. HRP is labeled on a secondary antibody by high affinity binding between biotin and avidin, and a multi-stage signal amplification effect is achieved.

In some embodiments, the second antibody is an anti-human antibody. Exemplary, secondary antibodies include, but are not limited to, goat anti-human antibodies, rat anti-human antibodies, mouse anti-human antibodies, pig anti-human antibodies, donkey anti-human antibodies, sheep anti-human antibodies, chicken anti-human antibodies, horse anti-human antibodies, rabbit anti-human antibodies, hamster anti-human antibodies, dog anti-human antibodies, cow anti-human antibodies, and the like, preferably goat anti-human antibodies.

In some embodiments, the type of second antibody includes, but is not limited to, igG, igA, igM, igG (Fc), igG (ab') ² Etc.; igG is preferred.

< color-developing Material >

In the invention, the color developing substance can generate fluorescence or generate fluorescence quenching under the catalysis of the marker, and generate a fluorescence signal for detection.

In some preferred embodiments, the color-developing substance is tyramine fluorescein. Tyramine fluorescein is activated under the action of HRP and hydrogen peroxide and covalently coupled with tyrosine residues of adjacent proteins, so that the protein sample is stably combined with the fluorescein. Further, fluorescein includes, but is not limited to fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, texas Red, phycoerythrin, propidium iodide, alexaFluor series, dylight series, iFluor series. When the second antibody binds to the myasthenia gravis-related antibody, tyramine fluorescein becomes activated and covalently couples to the target protein, producing a fluorescent signal that can be detected.

The cell, the second antibody and the chromogenic substance for expressing the myasthenia gravis-related antigen provided by the invention can construct an anti-HRP (horse radish peroxidase) secondary antibody-TSA (TSA) system, and a method for detecting an anti-AChR antibody, an anti-Musk antibody and/or an anti-LRP 4 antibody in a sample to be detected with high sensitivity and high specificity is established, the detection sensitivity and specificity are obviously higher than those of an ELISA (enzyme-linked immunosorbent assay) detection method, the sensitivity is higher than that of a radioimmunoassay, the method has an important significance for improving the detection rate of MG patients with different antibody types, and reliable clinical diagnosis information is improved for early diagnosis and prognosis evaluation of myasthenia gravis.

Kit for detecting myasthenia gravis-related antibody

The kit for detecting the antibody related to the myasthenia gravis provided by the invention comprises a composition for detecting the antibody related to the myasthenia gravis.

The Tyramide Signal Amplification (TSA) technique is also called as Catalytic Signal Amplification (CSA), and the TSA technique is an enzymatic detection method for high-density in-situ labeling of target protein or nucleic acid by using Horseradish Peroxidase (HRP). The application is wide at present, and comprises Immunofluorescence (IF) and immunohistochemistry. The technology can be compatible with a high-performance dye Alexa Fluor, a traditional fluorescent dye and a colorimetric detection system. The principle of the TSA technology is that tyramine-fluorescent compounds are combined with tyrosine residues in situ and nearby of a target through covalent bonds under the catalysis of HRP to generate stable fluorescent compounds. Tyramine-hapten compounds (such as tyramine-biotin) can also be combined on tyrosine residues in situ and nearby of a target through HRP catalysis, and then combined with Streptavidin-HRP/fluorescent group added later, and after several rounds of cyclic amplification, a large number of enzyme molecules or fluorescent groups can be combined, so that the detection signal is amplified by 100-1000 times. Low abundance targets that cannot be detected by traditional methods can be detected by using this technique. The method has the advantages that the amplified fluorescent signal has ideal light, heat and pH stability, the sample can be preserved for a long time, and the multicolor fluorescent labeling by the TSA is not limited by an antibody species.

The kit provided by the invention can construct a 'primary anti-HRP secondary antibody-TSA' system, realizes high-sensitivity and high-specificity detection on anti-AChR antibody, anti-Musk antibody and/or anti-LRP 4 antibody in a sample to be detected, and has important significance for improving the detection rate and diagnosis accuracy of MG patients.

Examples

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

All reagents used in the examples were commercially available unless otherwise noted.

Example 1: preparation of kit for detecting AChR/Musk/LRP4 antibody

1. Amplification and extraction of plasmids

And synthesizing each subunit of AChR, the MuSK full-length gene and the LRP4 full-length gene by using a full-gene synthesis mode. The target genes shown as SEQ ID NO. 1-8 are connected with the vectors respectively by using endonuclease, DNA ligase and the like to obtain the recombinant expression vectors respectively containing the target genes shown as SEQ ID NO. 1-8. In this example, a PEGFP-N1 vector is used to construct (GenBank Accession: U55762.1) a recombinant expression vector, and the target genes shown in SEQ ID NOS: 1-8 are linked to the multiple cloning site region (MSC) of the PEGFP-N1 vector. The constructed plasmid was introduced into competent E.coli. After being coated, the escherichia coli is cultured in an incubator, and a monoclonal strain is picked out for sequencing and conservation. The clone bacteria which are identified to be sequenced correctly are smeared on a solid culture medium. Culturing in 37 deg.C incubator for 1-2 days, placing the grown colony in liquid culture medium, and shake culturing overnight. And (4) centrifugally amplifying the bacterial liquid, and extracting plasmids by using a plasmid mass extraction kit according to the specification requirements. The plasmid concentration was measured by nanodrop and stored at-20 ℃ after split charging.

2. Culture of 293T cells

(1) Cell recovery: a15 mL centrifuge tube was added with 2-4mL of medium and preheated to 37 ℃. Frozen cells were removed from liquid nitrogen, gently swirled in a 37 ℃ water bath for <1min until thawed, quickly transferred to a 15mL centrifuge tube after sterilization with 75% alcohol, centrifuged at 1000rmp for 5min, the supernatant discarded, and the resuspended cells placed in DMEM medium containing 10% fetal bovine serum, incubated at 37 ℃ 5% CO 2, saturated humidity.

(2) Cell passage: observing the cells under a mirror, and carrying out passage when the growth density reaches more than 90%; discarding the culture medium, adding 1.5-3 mL of sterile PBS buffer solution along the cell-free side of the bottle wall, shaking for one circle slightly, immediately discarding the PBS, adding 500 mu LPBS and 1mL of pancreatin digestive cells again, shaking in a cross manner until a white membrane falls off, wherein the digestion time is about 1min, and adding 3mL of DMEM complete culture medium to stop digestion; transferring the liquid into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5min; discarding the supernatant, adding 1mL of complete culture medium for resuspension, transferring to a 96-well plate added with the culture medium, wherein each 96-well plate needs 100-150 mu L of cells and 9.6mL of culture medium; and (3) uniformly mixing the cell suspension, adding 100 mu L of cell suspension into each hole, uniformly mixing by cross shaking, and transferring to an incubator for continuous culture.

3. Cell transfection

(1) Plasmid preparation:

mu.g of plasmid was added to prepare Musk plasmid and LRP4 plasmid transfection reagents.

AChR plasmid combination: for a total of 6 subunits, in 2 combinations, a total of 5 μ g of each combination was transfected:

TABLE 1

	Ratio of	Amount of the use		Ratio of	Amount of the composition used
						α	2	1.66	α	2	1.66
β	1	0.83	β	1	0.83
						Rapsn	1	0.83	Rapsn	1	0.83
δ	1	0.83	δ	1	0.83
						ε	1	0.83	ε	1	0.83

(2) Cell transfection: cells were observed under the mirror and transfection was initiated at cell densities around 60% -70%. Taking a sterile 1.5ml EP tube, marking A, respectively and sequentially adding 125 mu L of OPTI-MEM, 5 mu g of plasmid and 10 mu L of P3000 mu L, and gently blowing uniformly for 2-3 times to avoid bubbles; another sterile 1.5ml EP tube is marked with B, 125 mu L of OPTI-MEM and 3000.8 mu L of Lipofectamine are respectively added in sequence, and the mixture is evenly blown for 2-3 times to avoid bubbles. And adding the solution A into the solution B, blowing the solution A evenly for 5 to 7 times to avoid bubbles, and standing the solution for 13 minutes. Adding the AB mixed solution into a 96-well plate dropwise, shaking gently, culturing overnight in an incubator at 37 ℃, changing into a new DMEM complete culture medium at 6 hours, and continuing to CO ₂ Culturing in incubator for 1-2 days until the cells are full. The three plasmids are separately transfected into cells to obtain a first cell which is respectively transferred into the AChR plasmid combination, a second cell which is transferred into the Musk plasmid combination and a third cell which is transferred into the AChR plasmid combination.

The transfection effect was observed under a fluorescence microscope: 293T cells transfected with the AChR recombinant plasmid were able to express Green Fluorescent Protein (GFP) and acetylcholine receptor protein (AChR protein). As can be seen in FIG. 2, 293T cells were successfully transfected with AChR recombinant plasmid and expressed green fluorescent protein.

(3) And (3) fixing and preserving transfected cells: after transfection, cells were washed 2 times with Phosphate Buffered Saline (PBS), 5min each time; adding 4% paraformaldehyde into each well, and fixing for 30min; PBS wash for 2 times, 5min each time; adding protein preserving solution, and preserving at 4 deg.C.

Example 2: application of AChR/Musk/LRP4 antibody kit in detection of sample to be detected

(1) Rewarming: the well plate and related reagents (including washing solution and sealing solution) are taken out of a 4-degree refrigerator, and dyeing is carried out after room temperature rewarming for 10-15 minutes.

(2) Washing: the storage solution was discarded, and 150. Mu.l of PBS solution was added to each well, followed by washing 2 times.

(3) Membrane rupture: adding 50 μ l of 0.5% Triton X-100 (PBS diluted) solution per well, and standing at room temperature for 20 minutes.

(4) Washing the plate: add 150. Mu.l PBS solution to each well and wash on a shaker 3 times X3 min.

(5) And (3) peroxidase treatment: mu.l of peroxidase solution was added to each well, and the mixture was allowed to stand at room temperature for 20 minutes.

(6) Washing the plate: add 150. Mu.l PBST solution to each well and wash on a shaker 3 times X3 min.

(7) Preparing a PBST solution: 500ml PBS + 800. Mu.l Tween20+ 300. Mu.l Triton-100, shaken to complete dissolution, and the reagents are stored at 4 ℃.

(8) Serum blocking: mu.l of serum blocking solution was added to each well and allowed to stand at room temperature for 30 minutes.

(9) Primary antibody incubation: mu.l of the suspension was added to each well and allowed to stand in a thermostat at 37 ℃ for 40min.

(10) Serum preparation: 1: dilution 40 (serum blocking dilution).

(11) Washing the plate: add 150. Mu.l PBST solution to each well and wash on a shaker 3 times X7 min.

(12) And (3) secondary antibody incubation: mu.l of the secondary antibody working solution was added to each well and allowed to stand at room temperature for 30 minutes.

(13) Preparing a secondary antibody working solution: goat anti-human IgG,1: dilution 2000 (PBST dilution).

(14) Washing the plate: add 150. Mu.l PBST solution to each well and wash on a shaker 3 times X7 min.

(15) TSA color development: 50. Mu.l of TSA color developing solution was added to each well, and the mixture was allowed to stand at room temperature for 1 minute (note: 1min for each row separately).

(16) Washing the plate: add 150. Mu.l PBST solution to each well and wash on a shaker for 2X 4min.

(17) And (4) observation: 50-100. Mu.l PBST was added to each well and observed under a mirror (care was taken to avoid air bubbles).

And observing the experimental result under a fluorescence microscope. The relative amount of antibody concentration was indicated by the intensity of the signal of fluorescence excitation (excitation wavelength 555 nm) in a 20-fold field of the objective lens with cell control transfected with empty vector. As shown in fig. 3, where a is a first cell positive for AChR, B is a second cell positive, C is a blank control, and D is a third cell positive for LRP 4.

Example 3: comparison of M negative to AChR antibody by different MuSK subunitsDetection Rate of G patients

In this example, 4 Musk overexpression plasmids were constructed, where subunit 1 is the Musk subunit of the sequence shown in (NCBI: NM-005592.4), subunit 2 is the Musk subunit of the sequence shown in (NCBI: NM-001166280.2), subunit 3 is the Musk subunit of the sequence shown in (NM-001166281.2), and subunit 4 is the Musk subunit of the sequence shown in (NM-001369398.1).

An MG patient diagnosed as MG clinically but negative with AChR antibody is selected, 4 Musk overexpression plasmids are used for detecting anti-Musk antibody in the serum of the patient, the result is shown in figure 4, in the serum of the MG patient, 10 cases of antibody positive patients of MuSK subunit 1, 8 cases of antibody positive patients of MuSK subunit 2 and 3 and 7 cases of antibody positive patients of MuSK subunit 4 are shown, and the fact that the cells constructed based on the recombinant expression vector (the sequence shown in SEQ ID NO: 2) expressing the MuSK subunit 1 in the disclosure can be combined with the anti-MuSK antibody in high specificity and the detection rate of the MG patient negative with the AChR antibody is improved.

Example 4: detection rate of detection kit for anti-AChR antibody of MG patient

In this example, 335 clinical diagnosis patients were collected as MG patients, and AChR antibody subtypes, including embryonic type and adult type, were detected using the kit constructed in example 1. The results are shown in Table 1, and in 335 patients, 179 patients with positive embryonal type and human type AChR antibodies, 50 patients with positive single human type AChR antibody, 35 patients with positive single embryonal type AChR antibody, and 70 patients with negative single embryonal type and human type AChR antibody.

The above results demonstrate that the composition for detecting an antibody associated with myasthenia gravis constructed in the present invention can be used for detecting an antibody against AchR (including a, β, δ, e, rapsn) of an adult type as well as an antibody against AchR (including a, β, δ, γ, rapsn) of an embryonic type, thereby improving the detection rate of AchR in myasthenia gravis.

Table 2 tests of embryonal and adult AChR antibodies in 335 MG patients

	Embryo type AChR positive	Embryo type AChR negative
			Adult type AChR positive	179	50
Adult type AChR negative	36	70

Example 5: sensitivity and specificity of the detection kit

To further verify the sensitivity and specificity of the CBA-TSA method (using the kit constructed in example 1) for detection of AChR antibody, this example compared the sera of 34 patients (29 MG patients; 5 non-MG patients) collected at Total Hospital of Tianjin medical university from month 2 to month 5 of 2021. These sera were used to detect AChR antibodies using radioimmunoassay, elisa and CBA-TSA blinded methods, respectively. The results show that: 26 MG patients with positive AChR antibody are detected by using three methods in 34 selected samples, wherein 3 non-MG patients (1 nervous system paraneoplastic syndrome; 1 limb weakness to be examined; 1 paraneoplastic syndrome-related myasthenia syndrome) are all negative AChR antibody by using three methods. Of note, 2 non-MG patients (1 immune-related pancytopenia; 1 thymocyst) were positive for AChR antibody using the Elisa method, while the RIPA and CBA-TSA tests were negative; in 2 cases of patients with myasthenia gravis, the antibody to AChR was positive by CBA-TSA, while the antibody to RIPA and Elisa was negative. These results suggest that the CBA-TSA method detects AChR antibody with specificity consistent with RIPA and sensitivity higher than RIPA and Elisa (FIG. 5).

The present invention is not intended to be limited in scope by the specifically disclosed embodiments, which are provided, for example, to illustrate aspects of the present disclosure. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

SEQUENCE LISTING

<110> Tianjin Tianhai New zone Biotechnology Co., ltd

<120> composition and kit for detecting myasthenia gravis-related antibody and application

<130> 6886-2143600I

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 2610

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of MUSK

<400> 1

atgagagagc tcgtcaacat tccactggta catattctta ctctggttgc cttcagcgga 60

actgagaaac ttccaaaagc tcctgtcatc accactcctc ttgaaacagt ggatgcctta 120

gttgaagaag tggctacttt catgtgtgca gtggaatcct acccccagcc tgagatttcc 180

tggactagaa ataaaattct cattaaactc tttgacaccc ggtacagcat ccgggagaat 240

gggcagctcc tcaccatcct gagtgtggaa gacagtgatg atggcattta ctgctgcacg 300

gccaacaatg gtgtgggagg agctgtggag agttgtggag ccctgcaagt gaagatgaaa 360

cctaaaataa ctcgtcctcc cataaatgtg aaaataatag agggattaaa agcagtccta 420

ccatgtacta caatgggtaa tcccaaacca tcagtgtctt ggataaaggg agacagccct 480

ctcagggaaa attcccgaat tgcagttctt gaatctggga gcttgaggat tcataacgta 540

caaaaggaag atgcaggaca gtatcgatgt gtggcaaaaa acagcctcgg gacagcatat 600

tccaaagtgg tgaagctgga agttgaggtt tttgccagga tcctgcgggc tcctgaatcc 660

cacaatgtca cctttggctc ctttgtgacc ctgcactgta cagcaacagg cattcctgtc 720

cccaccatca cctggattga aaacggaaat gctgtttctt ctgggtccat tcaagagagt 780

gtgaaagacc gagtgattga ctcaagactg cagctgttta tcaccaagcc aggactctac 840

acatgcatag ctaccaataa gcatggggag aagttcagta ctgccaaggc tgcagccacc 900

atcagcatag cagaatggag taaaccacag aaagataaca aaggctactg cgcccagtac 960

agaggggagg tgtgtaatgc agtcctggca aaagatgctc ttgtttttct caacacctcc 1020

tatgcggacc ctgaggaggc ccaagagcta ctggtccaca cggcctggaa tgaactgaaa 1080

gtagtgagcc cagtctgccg gccagctgct gaggctttgt tgtgtaacca catcttccag 1140

gagtgcagtc ctggagtagt gcctactcct attcccattt gcagagagta ctgcttggca 1200

gtaaaggagc tcttctgcgc aaaagaatgg ctggtaatgg aagagaagac ccacagagga 1260

ctctacagat ccgagatgca tttgctgtcc gtgccagaat gcagcaagct tcccagcatg 1320

cattgggacc ccacggcctg tgccagactg ccacatctag attataacaa agaaaaccta 1380

aaaacattcc caccaatgac gtcctcaaag ccaagtgtgg acattccaaa tctgccttcc 1440

tcctcctctt cttccttctc tgtctcacct acatactcca tgactgtaat aatctccatc 1500

atgtccagct ttgcaatatt tgtgcttctt accataacta ctctctattg ctgccgaaga 1560

agaaaacaat ggaaaaataa gaaaagagaa tcagcagcag taaccctcac cacactgcct 1620

tctgagctct tactagatag acttcatccc aaccccatgt accagaggat gccgctcctt 1680

ctgaacccca aattgctcag cctggagtat ccaaggaata acattgaata tgtgagagac 1740

atcggagagg gagcgtttgg aagggtgttt caagcaaggg caccaggctt acttccctat 1800

gaacctttca ctatggtggc agtaaagatg ctcaaagaag aagcctcggc agatatgcaa 1860

gcggactttc agagggaggc agccctcatg gcagaatttg acaaccctaa cattgtgaag 1920

ctattaggag tgtgtgctgt cgggaagcca atgtgcctgc tctttgaata catggcctat 1980

ggtgacctca atgagttcct ccgcagcatg tcccctcaca ccgtgtgcag cctcagtcac 2040

agtgacttgt ctatgagggc tcaggtctcc agccctgggc ccccacccct ctcctgtgct 2100

gagcagcttt gcattgccag gcaggtggca gctggcatgg cttacctctc agaacgtaag 2160

tttgttcacc gagatttagc caccaggaac tgcctggtgg gcgagaacat ggtggtgaaa 2220

attgccgact ttggcctctc caggaacatc tactcagcag actactacaa agctaatgaa 2280

aacgacgcta tccctatccg ttggatgcca ccagagtcca ttttttataa ccgctacact 2340

acagagtctg atgtgtgggc ctatggcgtg gtcctctggg agatcttctc ctatggcctg 2400

cagccctact atgggatggc ccatgaggag gtcatttact acgtgcgaga tggcaacatc 2460

ctctcctgcc ctgagaactg ccccgtggag ctgtacaatc tcatgcgtct atgttggagc 2520

aagctgcctg cagacagacc cagtttcacc agtattcacc gaattctgga acgcatgtgt 2580

gagagggcag agggaactgt gagtgtctaa 2610

<210> 2

<211> 5718

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of LRP4

<400> 2

atgaggcggc agtggggcgc gctgctgctt ggcgccctgc tctgcgcaca cggcctggcc 60

agcagccccg agtgtgcttg tggtcggagc cacttcacat gtgcagtgag tgctcttgga 120

gagtgtacct gcatccctgc ccagtggcag tgtgatggag acaatgactg cggggaccac 180

agcgatgagg atggatgtat actacctacc tgttcccctc ttgactttca ctgtgacaat 240

ggcaagtgca tccgccgctc ctgggtgtgt gacggggaca acgactgtga ggatgactcg 300

gatgagcagg actgtccccc ccgggagtgt gaggaggacg agtttccctg ccagaatggc 360

tactgcatcc ggagtctgtg gcactgcgat ggtgacaatg actgtggcga caacagcgat 420

gagcagtgtg acatgcgcaa gtgctccgac aaggagttcc gctgtagtga cggaagctgc 480

attgctgagc attggtactg cgacggtgac accgactgca aagatggctc cgatgaggag 540

aactgtccct cagcagtgcc agcgcccccc tgcaacctgg aggagttcca gtgtgcctat 600

ggacgctgca tcctcgacat ctaccactgc gatggcgacg atgactgtgg agactggtca 660

gacgagtctg actgctcctc ccaccagccc tgccgctctg gggagttcat gtgtgacagt 720

ggcctgtgca tcaatgcagg ctggcgctgc gatggtgacg cggactgtga tgaccagtct 780

gatgagcgca actgcaccac ctccatgtgt acggcagaac agttccgctg tcactcaggc 840

cgctgtgtcc gcctgtcctg gcgctgtgat ggggaggacg actgtgcaga caacagcgat 900

gaagagaact gtgagaatac aggaagcccc caatgtgcct tggaccagtt cctgtgttgg 960

aatgggcgct gcattgggca gaggaagctg tgcaacgggg tcaacgactg tggtgacaac 1020

agcgacgaaa gcccacagca gaattgccgg ccccggacgg gtgaggagaa ctgcaatgtt 1080

aacaacggtg gctgtgccca gaagtgccag atggtgcggg gggcagtgca gtgtacctgc 1140

cacacaggct accggctcac agaggatggg cacacgtgcc aagatgtgaa tgaatgtgcc 1200

gaggaggggt attgcagcca gggctgcacc aacagcgaag gggctttcca atgctggtgt 1260

gaaacaggct atgaactacg gcccgaccgg cgcagctgca aggctctggg gccagagcct 1320

gtgctgctgt tcgccaatcg catcgacatc cggcaggtgc tgccacaccg ctctgagtac 1380

acactgctgc ttaacaacct ggagaatgcc attgcccttg atttccacca ccgccgcgag 1440

cttgtcttct ggtcagatgt caccctggac cggatcctcc gtgccaacct caacggcagc 1500

aacgtggagg aggttgtgtc tactgggctg gagagcccag ggggcctggc tgtggattgg 1560

gtccatgaca aactctactg gaccgactca ggcacctcga ggattgaggt ggccaatctg 1620

gatggggccc accggaaagt gttgctgtgg cagaacctgg agaagccccg ggccattgcc 1680

ttgcatccca tggagggtac catttactgg acagactggg gcaacacccc ccgtattgag 1740

gcctccagca tggatggctc tggacgccgc atcattgccg atacccatct cttctggccc 1800

aatggcctca ccatcgacta tgccgggcgc cgtatgtact gggtggatgc taagcaccat 1860

gtcatcgaga gggccaatct ggatgggagt caccgtaagg ctgtcattag ccagggcctc 1920

ccgcatccct tcgccatcac agtgtttgaa gacagcctgt actggacaga ctggcacacc 1980

aagagcatca atagcgctaa caaatttacg gggaagaacc aggaaatcat tcgcaacaaa 2040

ctccacttcc ctatggacat ccacaccttg cacccccagc gccaacctgc agggaaaaac 2100

cgctgtgggg acaacaacgg aggctgcacg cacctgtgtc tgcccagtgg ccagaactac 2160

acctgtgcct gccccactgg cttccgcaag atcagcagcc acgcctgtgc ccagagtctt 2220

gacaagttcc tgctttttgc ccgaaggatg gacatccgtc gaatcagctt tgacacagag 2280

gacctgtctg atgatgtcat cccactggct gacgtgcgca gtgctgtggc ccttgactgg 2340

gactcccggg atgaccacgt gtactggaca gatgtcagca ctgataccat cagcagggcc 2400

aagtgggatg gaacaggaca ggaggtggta gtggatacca gtttggagag cccagctggc 2460

ctggccattg attgggtcac caacaaactg tactggacag atgcaggtac agaccggatt 2520

gaagtagcca acacagatgg cagcatgaga acagtactca tctgggagaa ccttgatcgt 2580

cctcgggaca tcgtggtgga acccatgggc gggtacatgt attggactga ctggggtgcg 2640

agccccaaga ttgaacgagc tggcatggat gcctcaggcc gccaagtcat tatctcttct 2700

aatctgacct ggcctaatgg gttagctatt gattatgggt cccagcgtct atactgggct 2760

gacgccggca tgaagacaat tgaatttgct ggactggatg gcagtaagag gaaggtgctg 2820

attggaagcc agctccccca cccatttggg ctgaccctct atggagagcg catctattgg 2880

actgactggc agaccaagag catacagagc gctgaccggc tgacagggct ggaccgggag 2940

actctgcagg agaacctgga aaacctaatg gacatccatg tcttccaccg ccgccggccc 3000

ccagtgtcta caccatgtgc tatggagaat ggcggctgta gccacctgtg tcttaggtcc 3060

ccaaatccaa gcggattcag ctgtacctgc cccacaggca tcaacctgct gtctgatggc 3120

aagacctgct caccaggcat gaacagtttc ctcatcttcg ccaggaggat agacattcgc 3180

atggtctccc tggacatccc ttattttgct gatgtggtgg taccaatcaa cattaccatg 3240

aagaacacca ttgccattgg agtagacccc caggaaggaa aggtgtactg gtctgacagc 3300

acactgcaca ggatcagtcg tgccaatctg gatggctcac agcatgagga catcatcacc 3360

acagggctac agaccacaga tgggctcgcg gttgatgcca ttggccggaa agtatactgg 3420

acagacacgg gaacaaaccg gattgaagtg ggcaacctgg acgggtccat gcggaaagtg 3480

ttggtgtggc agaaccttga cagtccccgg gccatcgtac tgtaccatga gatggggttt 3540

atgtactgga cagactgggg ggagaatgcc aagttagagc ggtccggaat ggatggctca 3600

gaccgcgcgg tgctcatcaa caacaaccta ggatggccca atggactgac tgtggacaag 3660

gccagctccc aactgctatg ggccgatgcc cacaccgagc gaattgaggc tgctgacctg 3720

aatggtgcca atcggcatac attggtgtca ccggtgcagc acccatatgg cctcaccctg 3780

ctcgactcct atatctactg gactgactgg cagactcgga gcatccaccg tgctgacaag 3840

ggtactggca gcaatgtcat cctcgtgagg tccaacctgc caggcctcat ggacatgcag 3900

gctgtggacc gggcacagcc actaggtttt aacaagtgcg gctcgagaaa tggcggctgc 3960

tcccacctct gcttgcctcg gccttctggc ttctcctgtg cctgccccac tggcatccag 4020

ctgaagggag atgggaagac ctgtgatccc tctcctgaga cctacctgct cttctccagc 4080

cgtggctcca tccggcgtat ctcactggac accagtgacc acaccgatgt gcatgtccct 4140

gttcctgagc tcaacaatgt catctccctg gactatgaca gcgtggatgg aaaggtctat 4200

tacacagatg tgttcctgga tgttatcagg cgagcagacc tgaacggcag caacatggag 4260

acagtgatcg ggcgagggct gaagaccact gacgggctgg cagtggactg ggtggccagg 4320

aacctgtact ggacagacac aggtcgaaat accattgagg cgtccaggct ggatggttcc 4380

tgccgcaaag tactgatcaa caatagcctg gatgagcccc gggccattgc tgttttcccc 4440

aggaaggggt acctcttctg gacagactgg ggccacattg ccaagatcga acgggcaaac 4500

ttggatggtt ctgagcggaa ggtcctcatc aacacagacc tgggttggcc caatggcctt 4560

accctggact atgatacccg caggatctac tgggtggatg cgcatctgga ccggatcgag 4620

agtgctgacc tcaatgggaa actgcggcag gtcttggtca gccatgtgtc ccaccccttt 4680

gccctcacac agcaagacag gtggatctac tggacagact ggcagaccaa gtcaatccag 4740

cgtgttgaca aatactcagg ccggaacaag gagacagtgc tggcaaatgt ggaaggactc 4800

atggatatca tcgtggtttc ccctcagcgg cagacaggga ccaatgcctg tggtgtgaac 4860

aatggtggct gcacccacct ctgctttgcc agagcctcgg acttcgtatg tgcctgtcct 4920

gacgaacctg atagccggcc ctgctccctt gtgcctggcc tggtaccacc agctcctagg 4980

gctactggca tgagtgaaaa gagcccagtg ctacccaaca caccacctac caccttgtat 5040

tcttcaacca cccggacccg cacgtctctg gaggaggtgg aaggaagatg ctctgaaagg 5100

gatgccaggc tgggcctctg tgcacgttcc aatgacgctg ttcctgctgc tccaggggaa 5160

ggacttcata tcagctacgc cattggtgga ctcctcagta ttctgctgat tttggtggtg 5220

attgcagctt tgatgctgta cagacacaaa aaatccaagt tcactgatcc tggaatgggg 5280

aacctcacct acagcaaccc ctcctaccga acatccacac aggaagtgaa gattgaagca 5340

atccccaaac cagccatgta caaccagctg tgctataaga aagagggagg gcctgaccat 5400

aactacacca aggagaagat caagatcgta gagggaatct gcctcctgtc tggggatgat 5460

gctgagtggg atgacctcaa gcaactgcga agctcacggg ggggcctcct ccgggatcat 5520

gtatgcatga agacagacac ggtgtccatc caggccagct ctggctccct ggatgacaca 5580

gagacggagc agctgttaca ggaagagcag tctgagtgta gcagcgtcca tactgcagcc 5640

actccagaaa gacgaggctc tctgccagac acgggctgga aacatgaacg caagctctcc 5700

tcagagagcc aggtctaa 5718

<210> 3

<211> 1239

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of RAPSN

<400> 3

atggggcagg accagaccaa gcagcagatc gagaaggggc tccagctgta ccagtccaac 60

cagacagaga aggcattgca ggtgtggaca aaggtgctgg agaagagctc ggacctcatg 120

gggcgcttcc gcgtgctggg ctgcctggtc acagcccact cggagatggg ccgctacaag 180

gagatgctga agttcgctgt ggtccagatc gacacggccc gggagctgga ggatgccgac 240

ttcctcctgg agagctacct gaacctggca cgcagcaacg agaagctgtg cgagtttcac 300

aagaccatct cctactgcaa gacctgcctt gggctgcctg gtaccagggc aggtgcccag 360

ctcggaggcc aggtcagcct gagcatgggc aatgccttcc tgggcctcag cgtcttccag 420

aaggccctgg agagcttcga gaaggccctg cgctatgccc acaacaatga tgacgccatg 480

ctcgagtgcc gcgtgtgctg cagcctgggc agcttctatg cccaggtcaa ggactacgag 540

aaagccctgt tcttcccctg caaggcggca gagcttgtca acaactatgg caaaggctgg 600

agcctgaagt accgggccat gagccagtac cacatggccg tggcctatcg cctgctgggc 660

cgcctgggca gtgccatgga gtgttgtgag gagtctatga agatcgcgct gcagcacggg 720

gaccggccac tgcaggcgct ctgcctgctc tgcttcgctg acatccaccg gagccgtggg 780

gacctggaga cagccttccc caggtacgac tccgccatga gcatcatgac cgagatcgga 840

aaccgcctgg ggcaggtgca ggcgctgctg ggtgtggcca agtgctgggt ggccaggaag 900

gcgctggaca aggctctgga tgccatcgag agagcccagg atctggccga ggaggtgggg 960

aacaagctga gccagctcaa gctgcactgt ctgagcgaga gcatttaccg cagcaaaggg 1020

ctgcagcggg aactgcgggc gcacgttgtg aggttccacg agtgcgtgga ggagacggag 1080

ctctactgcg gcctgtgcgg cgagtccata ggcgagaaga acagccggct gcaggcccta 1140

ccttgctccc acatcttcca cctcaggtgc ctgcagaaca acgggacccg gagctgtccc 1200

aactgccgcc gctcatccat gaagcctggc tttgtatga 1239

<210> 4

<211> 1449

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of α subunit

<400> 4

atggagccct ggcctctcct cctgctcttt agcctttgct cagctggcct cgtcctgggc 60

tccgaacatg agacccgtct ggtggcaaag ctatttaaag actacagcag cgtggtgcgg 120

ccagtggaag accaccgcca ggtcgtggag gtcaccgtgg gcctgcagct gatacagctc 180

atcaatgtgg atgaagtaaa tcagatcgtg acaaccaatg tgcgtctgaa acagggtgac 240

atggtagatc tgccacgccc cagctgcgtg actttgggag ttcctttgtt ttctcatctg 300

cagaatgagc aatgggtgga ttacaaccta aaatggaatc cagatgacta tggcggtgtg 360

aaaaaaattc acattccttc agaaaagatc tggcgcccag accttgttct ctataacaat 420

gcagatggtg actttgctat tgtcaagttc accaaagtgc tcctgcagta cactggccac 480

atcacgtgga cacctccagc catctttaaa agctactgtg agatcatcgt cacccacttt 540

ccctttgatg aacagaactg cagcatgaag ctgggcacct ggacctacga cggctctgtc 600

gtggccatca acccggaaag cgaccagcca gacctgagca acttcatgga gagcggggag 660

tgggtgatca aggagtcccg gggctggaag cactccgtga cctattcctg ctgccccgac 720

accccctacc tggacatcac ctaccacttc gtcatgcagc gcctgcccct ctacttcatc 780

gtcaacgtca tcatcccctg cctgctcttc tccttcttaa ctggcctggt attctacctg 840

cccacagact caggggagaa gatgactctg agcatctctg tcttactgtc tttgactgtg 900

ttccttctgg tcatcgtgga gctgatcccc tccacgtcca gtgctgtgcc cttgattgga 960

aaatacatgc tgttcaccat ggtgttcgtc attgcctcca tcatcatcac tgtcatcgtc 1020

atcaacacac accaccgctc acccagcacc catgtcatgc ccaactgggt gcggaaggtt 1080

tttatcgaca ctatcccaaa tatcatgttt ttctccacaa tgaaaagacc atccagagaa 1140

aagcaagaca aaaagatttt tacagaagac attgatatct ctgacatttc tggaaagcca 1200

gggcctccac ccatgggctt ccactctccc ctgatcaaac accccgaggt gaaaagtgcc 1260

atcgagggca tcaagtacat cgcagagacc atgaagtcag accaggagtc taacaatgcg 1320

gcggcagagt ggaagtacgt tgcaatggtg atggaccaca tactcctcgg agtcttcatg 1380

cttgtttgca tcatcggaac cctagccgtg tttgcaggtc gactcattga attaaatcag 1440

caaggatga 1449

<210> 5

<211> 1506

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of β subunit

<400> 5

atgaccccag gggctctgct gatgctgctg ggggcgctgg gggcgccgct cgccccaggc 60

gtccgcggct cggaggcgga gggtcgactc cgggagaaac ttttctctgg ctatgatagc 120

tccgtgcggc cagcgcggga ggtgggagac cgtgtcaggg tcagcgttgg tctcatcctg 180

gcgcaactca tcagcctgaa cgagaaggat gaagagatga gcacaaaggt gtacttagac 240

ctggagtgga ctgactacag gctgagctgg gaccctgcgg agcacgacgg catcgattcg 300

ctccgcatca cggcggaatc cgtgtggctc cctgacgtgg tgctactgaa caacaatgat 360

gggaattttg acgtggctct ggacattagc gtcgtggtgt cctccgacgg ctccgtgcgt 420

tggcaacccc cgggcatcta tcgcagcagc tgcagcatcc aggtcaccta cttccccttc 480

gactggcaga attgcactat ggtgttcagc tcctacagct acgacagctc ggaggtcagc 540

ctgcagacag gcctgggtcc tgacgggcaa gggcatcagg aaatccacat tcatgaaggg 600

actttcattg agaatggcca gtgggagatt atccacaagc cctctcggct aatccagcct 660

ccaggcgatc ctaggggagg gagggaagga cagcgccagg aagtcatctt ctacctcatc 720

atccgccgca agcctctctt ctacctggtc aacgtcattg ccccatgcat cctcatcact 780

cttctggcca tcttcgtctt ctacctgcca ccagatgcag gagagaagat ggggctctca 840

atctttgccc tgctgaccct tactgtgttc ctgctgctgc tggctgacaa agtacctgag 900

acctcactat cagtacccat tattatcaag tacctcatgt ttaccatggt cctcgtcacc 960

ttctcagtca tccttagtgt cgtggttctc aacctgcacc accgctcacc ccacacccac 1020

caaatgcccc tttgggtccg tcagatcttc attcacaaac ttccgctgta cctgcgtcta 1080

aaaaggccca aacccgagag agacctgatg ccggagcccc ctcactgttc ttctccagga 1140

agtggctggg gtcggggaac agatgaatat ttcatccgga agccgccaag tgattttctc 1200

ttccccaaac ccaataggtt ccagcctgaa ctgtctgccc ctgatctgcg gcgatttatc 1260

gatggtccaa accgggctgt ggccctgctt ccggagctac gggaggtcgt ctcctctatc 1320

agctacatcg ctcgacagct gcaggaacag gaggaccacg atgcgctgaa ggaggactgg 1380

cagtttgtgg ccatggtagt ggaccgcctc ttcctgtgga ctttcatcat cttcaccagc 1440

gttgggaccc tagtcatctt cctggacgcc acgtaccact tgccccctcc agaccccttt 1500

ccttga 1506

<210> 6

<211> 1554

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of δ subunit

<400> 6

atggaggggc cagtgctgac actggggctg ctggctgccc tggcggtgtg tggcagctgg 60

gggctgaacg aggaggagcg gctgatccgg cacctgtttc aagagaaggg ctacaacaag 120

gagctccggc ccgtggcaca caaagaggag agtgtggacg ttgccctggc cctcacactc 180

tccaacctca tctccctgaa agaagttgag gagaccctca ctaccaatgt gtggatagag 240

cacggctgga cagacaaccg gctgaagtgg aatgctgaag aatttggaaa catcagtgtc 300

ctgcgcctcc ccccggacat ggtgtggctc ccagagattg tgctggagaa caacaatgac 360

ggctccttcc agatctccta ctcctgcaac gtgcttgtct accactacgg cttcgtgtac 420

tggctgccac ctgccatctt ccgctcctcc tgccccatct ctgtcaccta tttccccttc 480

gactggcaga actgctccct caagttcagt tccctcaagt atacggccaa agagatcacc 540

ctgagcctga aacaggatgc caaggagaac cgcacctacc ccgtggagtg gatcatcatt 600

gatcctgaag gcttcacaga gaacggggag tgggagatag tccaccggcc ggccagggtc 660

aacgtggacc ccagagcccc tctggacagc cccagccgcc aggacatcac cttctacctc 720

atcatccgcc gcaagcccct cttctacatc atcaacatcc tggtgccctg cgtgctcatc 780

tccttcatgg tcaacctggt cttctaccta ccggctgaca gtggtgagaa gacatcagtg 840

gccatctcgg tgctcctggc tcagtctgtc ttcctgctgc tcatctccaa gcgtctgcct 900

gccacatcca tggccatccc ccttatcggc aagttcctgc tcttcggcat ggtgctggtc 960

accatggttg tggtgatctg tgtcatcgtg ctcaacatcc acttccgaac acccagcacc 1020

catgtgctgt ctgagggggt caagaagctc ttcctggaga ccctgccgga gctcctgcac 1080

atgtcccgcc cagcagagga tggacccagc cctggggccc tggtgcggag gagcagctcc 1140

ctgggataca tctccaaggc cgaggagtac ttcctgctca agtcccgcag tgacctcatg 1200

ttcgagaagc agtcagagcg gcatgggctg gccaggcgcc tcaccactgc acgccggccc 1260

ccagcaagct ctgagcaggc ccagcaggaa ctcttcaatg agctgaagcc agctgtggat 1320

ggggcaaact tcattgttaa ccacatgagg gaccagaaca attacaatga ggagaaagac 1380

agctggaacc gagtggcccg cacagtggac cgcctctgcc tgtttgtggt gacgcctgtc 1440

atggtggtgg gcacagcctg gatcttcctg cagggcgttt acaaccagcc accaccccag 1500

ccttttcctg gggaccccta ctcctacaac gtgcaggaca agcgcttcat ctag 1554

<210> 7

<211> 1554

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of γ subunit

<400> 7

atgcatgggg gccaggggcc gctgctcctc ctgctgctgc tggctgtctg cctgggggcc 60

cagggccgga accaggagga gcgcctgctc gcagacctga tgcaaaacta cgaccccaac 120

ctgcggcccg cggaacgaga ctcggatgtg gtcaatgtca gcctgaagct aaccctcacc 180

aacctcatct ccctgaacga gcgagaggaa gccctcacca ccaatgtctg gatagagatg 240

cagtggtgcg actatcgcct gcgctgggat ccgcgagact acgaaggcct gtgggtgctg 300

agggtgccgt ccaccatggt gtggcggccg gatatcgtgc tggagaacaa cgtggacggt 360

gtcttcgagg tggccctcta ctgcaatgtg ctcgtgtccc ctgacggctg tatctactgg 420

ctgccgcctg ccatcttccg ttccgcctgc tctatctcag tcacctactt ccccttcgac 480

tggcagaact gctcccttat cttccagtcc cagacttaca gcaccaatga gattgatctg 540

cagctgagtc aggaagatgg ccagaccatc gagtggattt tcattgaccc tgaggccttc 600

acagagaatg gggagtgggc catccagcac cgaccagcca agatgctcct ggacccagcg 660

gcgccagccc aggaagcagg ccaccagaag gtggtgttct acctgctcat ccagcgcaag 720

cccctcttct acgtcatcaa catcatcgcc ccctgtgtgc tcatctcctc tgtcgccatc 780

ctcatccact tccttcctgc caaggctggg ggccagaagt gtaccgtcgc catcaacgtg 840

ctcctggccc agactgtctt cctcttcctt gtggccaaga aggtgcctga aacctcccag 900

gcggtgccac tcatcagcaa gtacctgacc ttcctcctgg tggtgaccat cctcattgtc 960

gtgaatgctg tggttgtgct caatgtctcc ttgcggtctc cacacacaca ctccatggcc 1020

cgaggggtcc gcaaggtgtt cctgaggctc ttgccccagc tgctgaggat gcacgttcgc 1080

ccgctggccc cggcagctgt gcaggacacc cagtcccggc tacagaatgg ctcctcggga 1140

tggtcgatca caactgggga ggaggtggcc ctctgcctgc ctcgcagtga actcctcttc 1200

cagcagtggc agcggcaagg gctggtggcg gcagcgctgg agaagctaga gaaaggcccg 1260

gagttagggc tgagccagtt ctgtggcagc ctgaagcagg ctgccccagc catccaggcc 1320

tgtgtggaag cctgcaacct cattgcctgt gcccggcacc agcagagtca ctttgacaat 1380

gggaatgagg agtggttcct ggtgggccga gtgctggacc gcgtctgctt cctggccatg 1440

ctctcgctct tcatctgtgg cacagctggc atcttcctca tggcccacta caaccgggtg 1500

ccggccctgc cattccctgg agatccacgc ccctacctgc cctcaccaga ctga 1554

<210> 8

<211> 1482

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence of ε subunit

<400> 8

atggcaaggg ctccgcttgg ggtcctgctc ctcttggggc ttctcggcag gggtgtgggg 60

aagaacgagg aactgcgtct ttatcaccat ctcttcaaca actatgaccc aggaagccgg 120

ccagtgcggg agcctgagga tactgtcacc atcagcctca aggtcaccct gacgaatctc 180

atctcactga atgaaaaaga ggagactctc accactagcg tctggattgg aatcgattgg 240

caggattacc gactcaacta cagcaaggac gactttgggg gtatagaaac cctgcgagtc 300

ccttcagaac tcgtgtggct gccagagatt gtgctggaaa acaatattga tggccagttc 360

ggagtggcct acgacgccaa cgtgctcgtc tacgagggcg gctccgtgac gtggctgcct 420

ccggccatct accgcagcgt ctgcgcagtg gaggtcacct acttcccctt cgattggcag 480

aactgttcgc ttattttccg ctctcagacg tacaatgccg aagaggtgga gttcactttt 540

gccgtagaca acgacggcaa gaccatcaac aagatcgaca tcgacacaga ggcctatact 600

gagaacggcg agtgggccat cgacttctgc ccgggggtga tccgccgcca ccacggtggc 660

gccaccgacg gcccagggga gactgacgtc atctactcgc tcatcatccg ccggaagccg 720

ctcttctacg tcattaacat catcgtgccc tgtgtgctca tctcgggcct ggtgctgctc 780

gcctacttcc tgccggcgca ggccggcggc cagaaatgca cggtctccat caacgtcctg 840

ctcgcccaga ccgtcttctt gttcctcatt gcccagaaaa tcccagagac ttctctgagc 900

gtgccgctcc tgggcaggtt ccttattttc gtcatggtgg tcgccacgct cattgtcatg 960

aattgcgtca tcgtgctcaa cgtgtcccag cggacgccca ccacccacgc catgtccccg 1020

cggctgcgcc acgttctcct ggagctgctg ccgcgcctcc tgggctcccc gccgccgccc 1080

gaggcccccc gggccgcctc gcccccaagg cgggcgtcgt cggtgggctt attgctccgc 1140

gcggaggagc tgatactgaa aaagccacgg agcgagctcg tgtttgaggg gcagaggcac 1200

cggcagggga cctggacggc tgccttctgc cagagcctgg gcgccgccgc ccccgaggtc 1260

cgctgctgtg tggatgccgt gaacttcgtg gccgagagca cgagagatca ggaggccacc 1320

ggcgaggaag tgtccgactg ggtgcgcatg gggaatgccc ttgacaacat ctgcttctgg 1380

gccgctctgg tgctcttcag cgtgggctcc agcctcatct tcctcggggc ctacttcaac 1440

cgagtgcctg atctccccta cgcgccgtgt atccagcctt ag 1482

Claims

1. A composition for detecting an antibody associated with myasthenia gravis, wherein the composition comprises: cells expressing an antigen associated with myasthenia gravis, a second antibody, a chromogenic; wherein,

the myasthenia gravis-related antigen comprises a combination of acetylcholine receptors, muscle-specific tyrosine kinase, and low density lipoprotein 4;

the second antibody is conjugated with a label; and when the second antibody is combined with the myasthenia gravis-related antibody, the marker catalyzes the color development to generate fluorescence or generate fluorescence quenching;

the cells expressing an antigen associated with myasthenia gravis comprise a combination of (i) to (iii) below:

(i) A first set of polynucleotides encoding the various subunits forming the acetylcholine receptor;

(ii) A second polynucleotide encoding a muscle specific tyrosine kinase; and

(iii) A third polynucleotide encoding low density lipoprotein 4;

the first polynucleotide set comprises the nucleotide sequence set forth as SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: 7; alternatively, the first set of polynucleotides comprises the sequences set forth as SEQ ID NOs: 3. SEQ ID NO: 4. SEQ ID NO: 5. the amino acid sequence of SEQ ID NO:6 and SEQ ID NO: 8;

the second polynucleotide is as set forth in SEQ ID NO: 1;

the third polynucleotide is as set forth in SEQ ID NO: 2;

the cells expressing the myasthenia gravis-associated antigen include: a first cell expressing an acetylcholine receptor, a second cell expressing a muscle-specific tyrosine kinase, and a third cell expressing low density lipoprotein 4;

the first cell is selected from at least one of: comprises the amino acid sequence shown as SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:7, or a polynucleotide comprising a sequence as set forth in SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. the amino acid sequence of SEQ ID NO:6 and SEQ ID NO: 8;

2. The composition of claim 1, wherein the label is horseradish peroxidase and the color former is tyramide fluorescein.

3. The composition of claim 2, wherein the fluorescein is selected from any one of: fluorescein isothiocyanate, rhodamine, tetramethylrhodamine isothiocyanate, texas Red, phycoerythrin, propidium iodide, alexa Fluor series, dylight series, iFluor series.

4. The composition of claim 1, wherein the cells expressing an antigen associated with myasthenia gravis are prepared by a method comprising:

and (3) vector construction: constructing a first vector group for expressing and forming each subunit of the acetylcholine receptor, a second vector for expressing muscle-specific tyrosine kinase and a third vector for expressing low-density lipoprotein 4;

a transfection step comprising: transfecting cells by using the first vector group, the second vector and the third vector respectively to obtain a first transfected cell transferred with the first vector group, a second transfected cell transferred with the second vector and a third transfected cell transferred with the third vector;

a screening step comprising: and screening the first transfected cell, the second transfected cell and the third transfected cell to obtain a first cell for expressing an acetylcholine receptor, a second cell for expressing muscle specific tyrosine kinase and a third cell for expressing low density lipoprotein 4.

5. The composition of claim 4, wherein the mass ratio of the first vector group, the second vector and the third vector transfected cells is 1.

6. The composition of claim 4, wherein each of the first vector group comprises the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO:7 in the sequence shown in the specification, the mass ratio of the recombinant expression vector transfected cells is 1; or,

the first vector group respectively comprises the following sequences as shown in SEQ ID NO: 3. SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6 and SEQ ID NO: the mass ratio of the recombinant expression vector with the sequence shown in 8 to transfected cells is 1.

7. A kit for detecting an antibody associated with myasthenia gravis, wherein the kit comprises the composition of any one of claims 1-6.

8. Use of a composition according to any one of claims 1 to 6 for the preparation of a kit for the detection of antibodies associated with myasthenia gravis.

9. Use of a composition according to any one of claims 1 to 6 for the preparation of a kit for the diagnosis or prognosis of myasthenia gravis.