Fusion protein, fusion protein gene, recombinant cell, recombinant antigen, reagent for detecting anti-myelin-associated glycoprotein antibody, and preparation method, detection method and application of reagent

Abstract

The invention discloses a fusion protein, a fusion protein gene, a recombinant cell, a recombinant antigen, a reagent for detecting an anti-myelin sheath related glycoprotein antibody and a preparation method, a detection method and application of the reagent, the fusion protein is MAG, GlcAT-P and HNK-1ST fusion protein, and the MAG, GlcAT-P and HNK-1ST fusion protein is formed by sequentially connecting MAG, a GGGGS sequence, EGFP, P2A, GlcAT-P, T2A and HNK-1ST; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is as shown in SEQ ID NO. 1; the kit can be used for detecting the MAG autoantibody, has excellent detection sensitivity and specificity, and can ensure the detection accuracy especially for the detection in a low titer range.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay technology, and in particular relates to a fusion protein, a fusion protein gene, a recombinant cell, a recombinant antigen, and a reagent for detecting anti-myelin-associated glycoprotein antibodies, as well as preparation methods, detection methods, and applications thereof. Background Art

[0002] Anti-myelin-associated glycoprotein (MAG) antibody-associated peripheral neuropathy is a refractory autoimmune polyneuropathy. This disease is caused by the deposition of MAG-IgM in the myelin sheath and is an immune-mediated peripheral neuropathy seen in IgM monoclonal gammopathy-associated peripheral neuropathy. Typical clinical manifestations include a slowly progressive neuropathy with prominent deep sensory disturbances in middle-aged or elderly patients, or a sensorimotor neuropathy with predominant sensory disturbances. Limb weakness and sensory disturbances are symmetrically distributed and length-dependent. Electrophysiological examinations suggest a demyelinating peripheral neuropathy. Typical pathological changes are the deposition of monoclonal IgM in the myelin sheath and widening of the myelin interstitial spaces under electron microscopy.

[0003] Myelin-associated glycoprotein (MAG) is a transmembrane glycoprotein with a relative molecular mass of 100 kDa. It is present in the Schwann cell membrane, the Schwann-Lan notch, and the inner and outer axonal membranes that form the myelin sheath around the axon. It is essential for maintaining the structure and function of the myelin sheath and axons. MAG protein contains five extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic domain. Due to mRNA splicing, the cytoplasmic domain is expressed in two developmentally regulated isoforms, MAG-L and MAG-S. MAG contains approximately 30% carbohydrate by weight, composed of a heterogeneous set of N-linked oligosaccharides at eight extracellular sites. The expression of the HNK-1 carbohydrate epitope at multiple glycosylation sites on MAG makes it highly immunoreactive. The antigenic epitope of MAG is located in the carbohydrate portion of the molecule, and its reactive determinant is a sulfated trisaccharide, also known as the HNK-1 epitope. Anti-MAG autoantibodies target the HNK-1 sulfated trisaccharide epitope, which is primarily expressed on MAG. The HNK-1 carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO 3 -3GlcAβ1-3Galβ1-4GlcNAc-) and is biosynthesized sequentially by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST).

[0004] There are many existing tests for anti-MAG antibodies. Braun and Steck first used purified myelin and purified MAG as antigens and measured serum anti-MAG antibodies by Western blot. This technique can verify antibodies against the typical 100 kDa MAG protein, rather than antibodies against the purified myelin fraction. Caudie and Kuijfet used ELISA to detect anti-MAG antibodies in patient serum based on MAG purified from human brain as an antigen. Because anti-MAG-reactive serum can also recognize SGPG glycolipids, SGPG can also be used as an antigen instead of purified human MAG. However, using MAG as a target antigen is better than SGPG because the binding strength of anti-MAG antibodies to MAG antigens is 10-100 times that of SGPG antigens. Therefore, if SGPG is used as an antigen in the test, low levels of anti-MAG antibodies may be missed.

[0005] Existing studies have shown that ELISA is more sensitive than Western blotting for detecting anti-MAG antibodies. Anti-MAG antibody cell immunofluorescence detection typically involves co-transfection of MAG and galactosyl-xylosyl protein-3-β-glucuronyltransferase 1 to prepare cell slides, but the sensitivity and specificity of this method are incompletely evaluated.

[0006] An article used a cell-based immunofluorescence assay (CBA) to diagnose anti-MAG antibody-associated peripheral neuropathy. Testing in 95 patients and 55 controls, CBA demonstrated near-99% sensitivity and 100% specificity at a 1:100 dilution, significantly outperforming the complexity of traditional ELISA. However, the article only presented the test results and did not describe the specific CBA reagent preparation method.

[0007] Although most studies have shown that ELISA can be used as a sensitive and reliable screening method for detecting anti-MAG antibodies, the ideal cutoff value for positive anti-MAG autoantibody ELISA has been controversial, and a gray area of ​​false positives exists in the low titer range. Therefore, it is important to perform careful clinical and electrophysiological evaluation of patients with low titers to distinguish CIDP from anti-MAG neuropathy. Studies have shown that CBA is an efficient and standardized detection tool and has the potential to become a new clinical standard.

[0008] Currently, there is no specific method described for detecting anti-MAG antibodies by cellular immunofluorescence. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a fusion protein that can be used for the detection of MAG antibodies, has excellent detection sensitivity, specificity and accuracy, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0010] To solve the above technical problems, the technical solution adopted by the present invention is: a fusion protein, which is a MAG, GlcAT-P and HNK-1ST fusion protein, and the MAG, GlcAT-P and HNK-1ST fusion protein is composed of MAG, GGGGS sequence, EGFP, P2A, GlcAT-P, T2A and HNK-1ST connected in sequence; the GGGGS sequence is a connecting peptide between MAG and EGFP, the P2A is a connecting peptide between EGFP and GlcAT-P, and the T2A is a connecting peptide between GlcAT-P and HNK-1ST; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.1.

[0011] The structure of the MAG, GlcAT-P and HNK-1ST fusion protein is MAG-GGGGS sequence-EGFP-P2A-GlcAT-P-T2A-HNK-1ST, wherein MAG is fused with EGFP, EGFP is a green fluorescent protein, and the fusion expression product of MAG and EGFP is fluorescently labeled. The GGGGS sequence is added in the middle to reduce the mutual influence between EGFP and MAG protein; the P2A and T2A peptides are added between MAG, GlcAT-P and HNK-1ST proteins respectively. This design enables the three genes of MAG, GlcAT-P and HNK-1ST to be expressed simultaneously in the same ratio, but can be assembled separately without affecting each other. The invention relates to a novel fusion protein comprising a plurality of fusion proteins, a plurality of fusion proteins and a plurality of fusion proteins, and a plurality of fusion proteins, wherein the fusion protein comprises ..., a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of fusion proteins, a plurality of

[0012] Furthermore, the MAG in the MAG, GlcAT-P and HNK-1ST fusion protein is a MAG-S isomer; and the GlcAT-P is a 1GlcAT-P isomer.

[0013] Another technical problem to be solved by the present invention is to provide a fusion protein gene, which encodes the MAG, GlcAT-P and HNK-1ST fusion protein, and the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] By encoding the MAG, GlcAT-P and HNK-1ST fusion protein, the fusion protein gene can express the MAG-EGFP protein of the HNK-1 epitope in the host cell. The MAG-EGFP protein expressing the HNK-1 epitope is used as the target antigen, and the CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids. Antibodies targeting MAG protein and HNK-1 can be detected simultaneously. Therefore, the fusion protein gene of the present invention can be used for the detection of MAG antibodies, has excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0015] The third problem to be solved by the present invention is to provide a recombinant expression vector comprising the aforementioned fusion protein gene.

[0016] The recombinant expression vector can express the MAG-EGFP protein of the HNK-1 epitope in host cells by encoding the MAG, GlcAT-P and HNK-1ST fusion protein. The MAG-EGFP protein expressing the HNK-1 epitope is used as the target antigen, and the CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids. Antibodies targeting MAG protein and HNK-1 can be detected simultaneously. Therefore, the recombinant expression vector of the present invention can be used for the detection of MAG antibodies, has excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0017] The fourth technical problem to be solved by the present invention is to provide a recombinant cell comprising the aforementioned recombinant expression vector.

[0018] The recombinant cell, i.e., the host cell of the recombinant expression vector, expresses the MAG-EGFP protein of the HNK-1 epitope in the host cell. The MAG-EGFP protein expressing the HNK-1 epitope is used as the target antigen, and the CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids. Antibodies targeting MAG protein and HNK-1 can be detected simultaneously. Therefore, the recombinant cell of the present invention can be used for the detection of MAG antibodies, has excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0019] The fifth technical problem to be solved by the present invention is to provide a recombinant antigen, which is a MAG-EGFP protein expressing the HNK-1 epitope. The MAG-EGFP protein expressing the HNK-1 epitope is the expression and biosynthesis product of MAG, GlcAT-P and HNK-1ST fusion protein in host cells.

[0020] The recombinant antigen, i.e., the MAG-EGFP protein expressing the HNK-1 epitope, is used as a target antigen, and the CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids. Antibodies targeting MAG protein and HNK-1 can be detected simultaneously. Therefore, the recombinant antigen of the present invention can be used for the detection of MAG antibodies, has excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0021] The sixth technical problem to be solved by the present invention is to provide a reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0022] In order to solve the above technical problems, the adopted technical solution is: a reagent for detecting anti-myelin-associated glycoprotein antibodies, which contains the recombinant antigen.

[0023] The reagent for detecting anti-myelin-associated glycoprotein antibodies uses a recombinant antigen, namely a MAG-EGFP protein expressing an HNK-1 epitope, as a target antigen, and utilizes a CBA double fluorescence staining analysis method to detect anti-MAG antibodies in human body fluids. It can simultaneously detect antibodies targeting MAG protein and HNK-1. Therefore, the reagent for detecting anti-myelin-associated glycoprotein antibodies of the present invention can be used for the detection of MAG antibodies, has excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0024] The seventh technical problem to be solved by the present invention is to provide an application of a reagent for detecting anti-myelin-associated glycoprotein antibodies in detecting MAG antibodies, and to detect MAG antibodies using the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0025] In order to solve the above technical problems, the adopted technical solution is: the reagent for detecting anti-myelin-associated glycoprotein antibodies is used in detecting MAG antibodies.

[0026] The eighth technical problem to be solved by the present invention is to provide a method for preparing a reagent for detecting anti-myelin-associated glycoprotein antibodies. The reagent for detecting anti-myelin-associated glycoprotein antibodies prepared by this preparation method can detect MAG antibodies.

[0027] To solve the above technical problems, the technical solution adopted is: a method for preparing a reagent for detecting anti-myelin-associated glycoprotein antibodies, comprising the following steps:

[0028] Step S1: constructing a recombinant expression vector of the MAG, GlcAT-P and HNK-1ST fusion protein, and obtaining a recombinant plasmid through the recombinant expression vector; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.2;

[0029] Step S2: transfecting the recombinant plasmid obtained in step S1 into cells to obtain recombinant cells; overexpressing the MAG-EGFP protein target antigen with HNK-1 epitope in the cells;

[0030] Step S3: preparing the recombinant cells in step S2 into a recombinant cell matrix plate, which is the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0031] By designing a fusion protein expression sequence, a recombinant expression vector is constructed; the recombinant plasmid is transfected into cells to overexpress the target antigen in the cells; the recombinant cells carrying the recombinant plasmid are prepared into a recombinant cell matrix plate, which is the reagent for detecting anti-myelin-associated glycoprotein antibodies. The recombinant cell matrix plate can be used to detect MAG antibodies with excellent detection sensitivity and specificity, especially for detection in the low titer range, and can ensure the accuracy of detection.

[0032] Furthermore, the step S1 includes the following steps:

[0033] Step S11: designing PCR amplification primers according to the gene sequence of the fusion protein, and obtaining an amplified product by PCR amplification; the nucleotide sequence of the primer F is shown in SEQ ID NO.6, and the nucleotide sequence of the primer R is shown in SEQ ID NO.7; SEQ ID NO.6: 5'-cccAAGCTTAtgatattcctcacggcactgc-3', SEQ ID NO.7: 5'-ccgGAATTCttagtttagcaaaaagtctgg-3'; Step S12: using a pcDNA3.1(+) vector; using restriction endonucleases Hind III and EcoR I to digest the amplified product and the pcDNA3.1(+) vector, respectively; Step S13: using DNA ligase to connect the digestion product of the amplified product and the digestion product of the pcDNA3.1(+) vector to obtain an overexpression vector; Step S14: transforming competent cells using the overexpression vector; Step S15: using the antibiotic corresponding to the resistance contained in the overexpression vector to screen the transformed competent cells, performing PCR amplification on the screened single colonies, amplifying the positive colonies and extracting the plasmids, and sequencing and identifying the extracted plasmids.

[0034] The fusion sequence of MAG, GlcAT-P and HNK-1ST was ligated to the vector pcDNA3.1(+) using a double enzyme digestion method. Primer F contained AAGCTT, corresponding to the Hind Ⅲ restriction site, and primer R contained GAATTC, corresponding to the EcoR I restriction site. This pair of primers had strong specificity and could efficiently amplify the target fragment.

[0035] Furthermore, in step S2, the cells include any one of HEK293T cells, Sox1 cells, CHO cells and Hela cells; and the transfection reagent used for transfection is any one or any combination of Lipofectamine 3000, Lipofectamine 2000 and PEI.

[0036] The ninth technical problem to be solved by the present invention is to provide a kit for detecting anti-myelin-associated glycoprotein antibodies, by which MAG antibodies can be detected.

[0037] In order to solve the above technical problems, the adopted technical solution is: the kit for detecting anti-myelin-associated glycoprotein antibodies includes the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0038] The kit also includes a fluorescent secondary antibody and a cleaning solution.

[0039] The tenth technical problem to be solved by the present invention is to provide a CBA detection method using the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0040] In order to solve the above technical problems, the technical solution adopted is: the CBA detection method includes the following steps: adding the sample to the reagent for detecting anti-myelin-associated glycoprotein antibodies and performing the first incubation; then adding the fluorescent secondary antibody for the second incubation, washing and microscopic examination, and interpreting the results.

[0041] The eleventh technical problem to be solved by the present invention is to provide another recombinant cell.

[0042] To solve the above technical problems, the technical solution adopted is: a recombinant cell, which includes a recombinant plasmid expressing MAG, a recombinant plasmid expressing GlcAT-P and a recombinant plasmid expressing HNK-1ST, and the recombinant cell is used for the detection of anti-myelin-associated glycoprotein antibodies; the nucleotide sequence of the MAG is shown in SEQ ID NO.3, the nucleotide sequence of the GlcAT-P is shown in SEQ ID NO.4, and the nucleotide sequence of the HNK-1ST is shown in SEQ ID NO.5.

[0043] Recombinant vectors of the three proteins MAG, GlcAT-P and HNK-1ST are separately constructed and then co-transfected into the same host cell, i.e., the recombinant cell. In the host cell, the three proteins are separately expressed. Moreover, similar to the aforementioned MAG, GlcAT-P and HNK-1ST fusion proteins, in the host cell, MAG provides the basic protein, GlcAT-P and HNK-1ST modify the MAG protein, and the MAG-EGFP protein expressing the HNK-1 epitope is obtained as the target antigen. The CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids. It can simultaneously detect antibodies targeting MAG protein and HNK-1, and has excellent detection sensitivity and specificity, especially for detection in the low titer range, and can ensure the accuracy of detection.

[0044] Compared with the prior art, the present invention has the following beneficial effects: (1) A fusion protein expression vector for co-expression of MAG, GlcAT-P, and HNK-1ST was designed, which consists of the following sequences: MAG, GGGGS sequence, EGFP, P2A, GlcAT-P, T2A, and HNK-1ST sequence. P2A and T2A amino acid sequences were used, respectively, so that the three genes of MAG, GlcAT-P, and HNK-1ST were expressed simultaneously in the same ratio, but they could be assembled separately without affecting each other's conformation and function. (2) MAG, GlcAT-P, and HNK-1ST were co-expressed to obtain MAG-EGFP protein expressing the HNK-1 epitope, which is a better antigen material; (3) For the first time, a CBA method for detecting MAG autoantibodies was disclosed. Cells expressing MAG-EGFP protein with HNK-1 epitope were used as the detection matrix to detect anti-MAG antibodies in human body fluids. Antibodies targeting MAG protein and HNK-1 can be detected simultaneously, which improves the specificity and sensitivity of the detection to 100%; (4) The EGFP gene and the MAG target gene were constructed into a chimeric gene so that the recombinant protein expressed by them was fluorescently labeled. The double fluorescence staining analysis method was used in the subsequent CBA detection. Compared with the single fluorescence staining method, the double fluorescence staining analysis method further improved the specificity of the detection, thereby improving the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a schematic diagram of the sequence structure of MAG, GlcAT-P, and HNK-1ST fusion proteins; Figure 2 Schematic diagram of the MAG-EGFP protein structure expressing the HNK-1 epitope and the biosynthesis of the HNK-1 epitope; MAG protein contains five extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic domain; the HNK-1 carbohydrate epitope is expressed at multiple glycosylation sites on MAG and is synthesized by glucuronyltransferase (GlcAT-P) and sulfotransferase (HNK-1ST) in sequence; Figure 3 Figures 1 and 2 are the test results of the same MAG-positive sample; Figures AC are the test results of the positive sample of Comparative Example 1, Figures DF are the test results of the positive sample of Comparative Example 2, and Figure GI is the test results of the positive sample of Example 1; Figure 4 Figures AC and DF show the negative sample detection results of comparative example 1, and Figure GI shows the negative sample detection results of comparative example 2, respectively. DETAILED DESCRIPTION

[0046] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0047] It should be noted that, in the present invention, MAG is the abbreviation of human myelin-associated glycoprotein, GlcAT-P is the abbreviation of galactosylgalactosylxylosyl protein 3-beta-glucuronosyl transferase 1, and HNK-1ST is the abbreviation of HNK-1 sulfotransferase.

[0048] The fusion protein in the embodiment of the present invention is a fusion protein of MAG, GlcAT-P and HNK-1ST, wherein the fusion protein of MAG, GlcAT-P and HNK-1ST is sequentially connected by MAG, GGGGS sequence, EGFP, P2A, GlcAT-P, T2A, and HNK-1ST; the GGGGS sequence is a connecting peptide between MAG and EGFP, the P2A is a connecting peptide between EGFP and GlcAT-P, and the T2A is a connecting peptide between GlcAT-P and HNK-1ST. Figure 1 As shown, MAG is fused with EGFP and a GGGGS sequence is added in the middle to reduce the mutual influence between EGFP and MAG proteins; P2A and T2A peptides can make the three proteins of MAG-EGFP, GlcAT-P and HNK-1ST expressed simultaneously in the same ratio, but can be assembled separately without affecting each other's conformation and function; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.1.

[0049] The SEQ ID NO.1 is:

[0050] The MAG in the MAG, GlcAT-P and HNK-1ST fusion protein is a MAG-S isomer, and MAG has two isomers, namely MAG-S and MAG-L, preferably the MAG-S isomer, and the nucleotide sequence of the MAG-S is shown in SEQ ID NO.3; the GlcAT-P in the MAG, GlcAT-P and HNK-1ST fusion protein is a lGlcAT-P isomer, and GlcAT-P has two isomeric proteins, namely lGlcAT-P and sGlcAT-P, preferably the lGlcAT-P isomer, and the nucleotide sequence of the lGlcAT-P is shown in SEQ ID NO.4; the nucleotide sequence of the HNK-1ST is shown in SEQ ID NO.5.

[0051] The SEQ ID NO.3 is: The SEQ ID NO.4 is: The SEQ ID NO.5 is:

[0052] The fusion protein gene in this example encodes the MAG, GlcAT-P and HNK-1ST fusion protein, and its nucleotide sequence is shown in SEQ ID NO.2.

[0053] The recombinant expression vector in this embodiment contains the fusion protein gene.

[0054] The recombinant cell in this embodiment comprises the recombinant expression vector.

[0055] The recombinant antigen in this example is a MAG-EGFP protein expressing the HNK-1 epitope. The MAG-EGFP protein expressing the HNK-1 epitope is the expression product of the MAG, GlcAT-P and HNK-1ST fusion protein in the host cell, i.e., the recombinant cell.

[0056] The reagent for detecting anti-myelin-associated glycoprotein antibodies in this embodiment contains the recombinant antigen.

[0057] The reagent for detecting anti-myelin-associated glycoprotein antibodies in this example is used to detect MAG antibodies.

[0058] The kit for detecting anti-myelin-associated glycoprotein antibodies in this embodiment includes the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0059] The method for preparing a reagent for detecting anti-myelin-associated glycoprotein antibodies in this embodiment comprises the following steps: Step S1: constructing a recombinant expression vector of the MAG, GlcAT-P and HNK-1ST fusion protein, and obtaining a recombinant plasmid through the recombinant expression vector; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.2; Step S2: transfecting the recombinant plasmid obtained in step S1 into cells to obtain recombinant cells; overexpressing the MAG-EGFP protein target antigen with HNK-1 epitope in the cells; Step S3: preparing the recombinant cells in step S2 into a recombinant cell matrix plate, wherein the recombinant cell matrix plate is the reagent for detecting anti-myelin-associated glycoprotein antibodies.

[0060] The step S1 comprises the following steps: Step S11: Design the gene sequence for fusion protein expression, artificially synthesize the above gene sequence, and then use the double enzyme digestion method to connect the fusion sequence of MAG, glucuronyl transferase GlcAT-P, and sulfotransferase HNK-1ST to the vector pcDNA3.1(+).

[0061] Step S111: First, design the gene sequence for fusion protein expression and obtain the fusion protein gene sequence by artificially synthesizing the gene sequence. The sequence structure diagram of MAG, GlcAT-P, HNK-1ST fusion protein (MAG-EGFP-P2A-GlcAT-P-T2A-HNK-1ST) is shown in the figure below. Figure 1 As shown; in order: MAG, GGGGS sequence, EGFP, P2A, GlcAT-P, T2A, HNK-1ST sequence, among which MAG and EGFP are fused and expressed, and the GGGGS sequence is added in the middle to reduce the mutual influence between EGFP and MAG proteins.

[0062] In this embodiment, the P2A peptide sequence is contained between the MAG-EGFP fusion sequence and the GlcAT-P sequence. P2A is a small peptide that can be understood as containing 19 amino acid residues (ATNFSLLKQAGDVEENPGP), but the actual process does not occur by self-cleavage. Instead, it causes the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of 2A elements such as P2A, ultimately leading to the separation of the 2A sequence end and the downstream product. The C-terminus of the upstream target gene expression protein will add some additional P2A residues (GSGATNFSLLKQAGDVEENPGP), while the N-terminus of the downstream protein will have an additional proline. Adding a GSG sequence to the N-terminus of the P2A peptide can improve the cleavage efficiency.

[0063] In this embodiment, the T2A peptide sequence is contained between the GlcAT-P sequence and the HNK-1ST sequence. T2A and P2A have similar functions, with slightly different amino acid residue sequences (EGRGSLLTCGDVEENPGP). The C-terminus of the upstream target gene expression protein will have some additional T2A residues (GSGEGRGSLLTCGDVEENPGP), while the N-terminus of the downstream protein will have additional proline. Similarly, adding the GSG sequence to the N-terminus of the T2A peptide can improve shearing efficiency.

[0064] The three genes (ORFs) are linked by a 2A polypeptide chain into an open reading frame (ORF). The 2A peptide allows the peptide chain translated from this ORF to be separated into several independent peptide chains. The theoretical molar ratio of these three proteins is 1:1. This design allows the MAG, GlcAT-P, and HNK-1ST genes to be expressed simultaneously in the same ratio, yet they can be assembled independently without affecting each other's conformation and function.

[0065] Among them, the nucleotide sequence of the MAG, GlcAT-P, and HNK-1ST recombinant fusion protein is shown in SEQ ID NO.2, and the amino acid sequence of the MAG, GlcAT-P, and HNK-1ST recombinant fusion protein is shown in SEQ ID NO.1.

[0066] Step S112: using PCR amplification primers designed according to the fusion gene sequence to obtain an amplified product through PCR amplification.

[0067] Based on the base sequence shown in SEQ ID NO.1, a PCR amplification primer pair was designed using primer design software in order to efficiently amplify the MAG sequence. Upstream and downstream primers and enzyme cutting sites for PCR amplification were designed according to the MAG gene sequence.

[0068] The nucleotide sequence of the primer F is shown in SEQ ID NO.6, and the nucleotide sequence of the primer R is shown in SEQ ID NO.7; SEQ ID NO.6: 5'-cccAAGCTTAtgatattcctcacggcactgc-3', SEQ ID NO.7: 5'-ccgGAATTCttagtttagcaaaaagtctgg-3'; Primer F contains AAGCTT, corresponding to the restriction site of Hind III, and primer R contains GAATTC, corresponding to the restriction site of EcoR I. This pair of primers has strong specificity and can efficiently amplify the target fragment.

[0069] In this example, a eukaryotic expression vector containing a complete open reading frame of the MAG target antigen tagged with EGFP was constructed using the pcDNA3.1(+) vector. Two restriction endonucleases, Hind III (Brand: NEB, Catalog Number: R3104T, Lot Number: 10041452) and EcoR I (Brand: NEB, Catalog Number: R3101T, Lot Number: 10035422), were used to achieve specific enzyme cleavage.

[0070] Preferably, in the PCR amplification reaction system, the primer concentration is 200 nM, the amount of DNA polymerase is 1.0 U, the dNTP concentration is 200 μM, the amount of template DNA is 100 ng; and the amplification reaction system volume is 50 μL.

[0071] Preferably, the PCR amplification reaction procedure includes: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 60 s, annealing at 62°C for 60 s, and extension at 72°C for 120 s, for a total of 25 cycles; and final extension at 72°C for 5 min.

[0072] Step S12: using restriction endonucleases corresponding to the two restriction sites included in the PCR amplification primers, respectively digest the amplified product and the pcDNA3.1(+) vector (brand: Invitrogen, catalog number: V79020).

[0073] Preferably, in the reaction system of the enzyme digestion reaction, the amount of Hind III is 5-20 U, the amount of EcoR I is 5-20 U, the amount of gene amplification product is 0.5-3 μg, and the amount of vector is 0.5-3 μg; the volume of the enzyme digestion reaction system is 10-50 μL; and the reaction procedure of the enzyme digestion reaction includes continuing at 37°C for 5 hours.

[0074] Step S13: using DNA ligase to connect the enzyme-digested product of the gene amplification product and the enzyme-digested product of the vector to obtain an overexpression vector.

[0075] Preferably, in the reaction system of the ligation reaction, the amount of DNA ligase used is 200 U, the amount of the enzyme digestion product of the gene amplification product used is 100 ng, and the amount of the enzyme digestion product of the vector used is 100 ng; the volume of the ligation reaction system is 10 pcDNA3.1(+); and the reaction procedure of the ligation reaction includes overnight at 16°C.

[0076] Step S14: transforming competent cells using the overexpression vector.

[0077] The transformation reaction was carried out as follows: the competent cells were taken out of the -80°C freezer and dissolved on ice, 10 μL of the overexpression vector after ligation was added to 100 μL of the competent cells, the cells were gently mixed, and the cells were allowed to stand on ice for 30 min. The competent cells were then heat-shocked in a 42°C water bath for 60 s, and then quickly transferred to ice and allowed to stand for 5 min. 0.5 mL of antibiotic-free LB medium was added in proportion, and the cells were then cultured in a constant temperature shaker at 37°C for 45-60 min.

[0078] Step S15: The transformed competent cells are screened with the antibiotic corresponding to the resistance contained in the overexpression vector, and the screened single colonies are amplified by PCR. The positive colonies are amplified by shaking and the plasmid of the overexpression vector is extracted. The extracted plasmid is sent for sequencing for identification.

[0079] Based on this, the specific steps for the identification of transformed cells and plasmid extraction operations include: Step S151: Spread the transformed competent cells onto an LB agar plate with ampicillin resistance and culture them upside down in a 37°C incubator overnight; Step S152: On the second day, select some colonies for PCR amplification. Colonies that successfully connect to the MAG overexpression vector and can amplify the target fragment are considered positive colonies. Positive colonies are selected and shaken at 37°C for 12-18 hours. The plasmids of the overexpression vector are extracted and sent for sequencing for identification. Step S153: After the constructed recombinant expression vector is sequenced correctly, it is extracted using a plasmid extraction kit and set aside.

[0080] Step S1531: Bacterial expansion culture: 15 μL of Amp / Kan antibiotics was added to 15 mL of LB medium. Positive colonies were picked and added to the LB medium. The culture was shaken at 180 rpm and 37°C overnight for about 12-16 hours. Step S1532: Use a plasmid extraction kit (brand: OMEGA, product number: D6950-02) to extract the plasmid.

[0081] Furthermore, in step S2, the cells include any one of HEK293T cells, Sox1 cells, CHO cells and Hela cells, preferably immortalized human cells. In this embodiment, the preferred cell line is HEK293T cells; the transfection reagent used for transfection is any one or any combination of Lipofectamine 3000, Lipofectamine 2000 and PEI.

[0082] The step S2 comprises the following steps: Step S21 Cell culture: (1) Take out the DMEM medium containing 10% Fetal Bovine Serum and 1% P / S (penicillin-streptomycin double antibody), the DMEM medium containing 10% Fetal Bovine Serum, Trypsin-EDTA and PBS from the 2-8 ℃ refrigerator and equilibrate at room temperature for 30 min; (2) Take out the 100 mm cell culture dish and observe under a microscope. Start subculturing when the cell confluence reaches about 90%; (3) Trypsin digestion: Use an electric aspirator to discard the supernatant, slowly add 1.5 mL of Trypsin-EDTA to each dish along the wall, gently shake the culture dish crosswise to make the Trypsin-EDTA evenly cover the bottom of the dish, and incubate in a 37°C incubator for 1 minute. Remove the culture dish after incubation and use an electric pipette controller to quickly add 3 mL of DMEM medium containing 10% Fetal Bovine Serum to terminate the digestion; (4) Use a 10 mL pipette to gently blow off the cells and transfer them all to a centrifuge tube. Add 2 mL of PBS to wash the culture dish and transfer the washed solution to a centrifuge tube; (5) Place the above centrifuge tube in a centrifuge, set the speed to 1000 RPM, and the time to 3 minutes.

[0083] (6) Remove the centrifuge tube after centrifugation, discard the supernatant, add 5 mL of DMEM culture medium containing 10% Fetal Bovine Serum and 1% P / S, and mix well by pipetting; (7) Prepare a cell counting plate and Trypan Blue stain solution. Take 10 μL of cell suspension and 10 μL of Trypan Blue stain solution and mix them by pipetting 3-5 times. Then count the cells using a cell counter. Take a 100 mm cell culture dish and ensure that the number of cells in each dish is (1.3-1.5) × 10 6 Place the cells in a CO2 cell incubator and shake them crosswise before culturing. (8) After the culture is completed, remove the cells from the cell culture incubator and observe the cell growth status and whether the cell solution is contaminated under an inverted microscope. When the cell density reaches 80%-90% under the microscope, the second passage of HEK293T cells can be performed.

[0084] Step S22 Cell transfection: (1) After the cells have been passaged for more than three times, when the cell confluence reaches 80% to 90%, transfection is performed using the plasmid and reagent combinations according to Table 1 below; (2) First, add Opti-MEM culture medium and P3000 reagent (brand: Invitrogen, catalog number: L3000150) to one of the centrifuge tubes, then add the plasmid to be transfected to prepare the plasmid premix, and pipette to mix it thoroughly. Then, add Opti-MEM culture medium to another centrifuge tube, and then add Lipofectamine 3000 reagent (brand: Invitrogen, catalog number: L3000150) to prepare the liposome premix, and pipette to mix it thoroughly. The amount of material added to each culture dish of plasmid premix and liposome premix is ​​shown in Table 1 below: Table 1 Plasmid-liposome complex preparation table (I)

[0085] (3) Add the diluted plasmid premix to the liposome premix, mix well, and let it stand at room temperature for 10 minutes to obtain a DNA-liposome complex; (4) Take out the cell culture dish to be transfected, discard the original culture medium, and then use an electric pipette controller to slowly add 10 mL of antibiotic-free DMEM culture medium containing 10% Fetal Bovine Serum along the wall of the dish. Then, add the plasmid-liposome complex all the way along the wall of the dish into the culture dish, and shake the culture dish crosswise to evenly distribute the complex in the culture medium. (5) After transfection, the cells were cultured in a 37°C cell culture incubator for 16-26 h. The transfected recombinant expression vectors were transcribed in the cells and expressed the corresponding proteins, namely MAG, GlcAT-P, and HNK-1ST. The schematic diagram of the MAG-EGFP protein structure expressing the HNK-1 epitope and the schematic diagram of the biosynthesis of the HNK-1 epitope are shown in Figure 2. Figure 2 As shown, the MAG protein contains five extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic domain. HNK-1 carbohydrate epitopes are expressed at multiple glycosylation sites on MAG, and their synthesis is catalyzed sequentially by glucuronyltransferase (GlcAT-P) and sulfotransferase (HNK-1ST).

[0086] The step S3 comprises the following steps: Using the recombinant cells in step S2, a recombinant cell matrix plate is prepared according to the following method; (1) Coating a 96-well plate: Dilute polylysine to 100 μg / mL with sterile water in a biosafety cabinet to obtain a polylysine solution. Add 50 μL of polylysine solution to each well of the 96-well plate and place it in a 37°C incubator for coating for 1 h. After coating, remove the 96-well plate and add 100 μL of sterile water to each well for cleaning. Then, aspirate and discard the sterile water, place the 96-well plate in a biosafety cabinet to air dry, and then use it later. (2) On the second day after transfection, observe the cell transfection status under a fluorescence microscope. Take photos of the same area using white light and green excitation light respectively. Repeat the observation under three fields of view. The transfection efficiency is considered qualified if it exceeds 50%; (3) Take the successfully transfected cell culture dish and collect the cells after trypsin digestion; (4) Use trypan blue to mark dead cells and then count them using a cell counter. Based on the counting results, dilute the cells to a density of 3~5×10 using DMEM medium containing 10% Fetal Bovine Serum and 1% P / S. 5 cells / mL; (5) Add 100 μL of the above cell suspension to the coated 96-well plate, and then transfer to a cell culture incubator and culture for 20-24 h; (6) After the culture is completed, remove the 96-well cell culture plate with cells from the incubator, remove the culture medium in the 96-well plate, and wash twice with PBS; (7) Take out the 4% paraformaldehyde from the refrigerator in advance and melt it, then equilibrate it to room temperature. Add 100 μL of the prepared 4% paraformaldehyde fixative to each well and fix it at room temperature for 10 min. It should be noted that cell fixation is performed using a fixative when the confluency of HEK293T cells carrying the recombinant plasmid is 80% to 90%. The cell fixation time can also be any time between 10 and 20 minutes. The purpose of fixation is to preserve the original morphological structure of cells and tissues. The fixative can prevent endogenous lysosomal enzymes from autolyzing their own tissues and cells and inhibit the growth of bacteria and molds. Fixatives are divided into aldehyde fixatives, mercury fixatives, alcohol fixatives, oxidant fixatives, picrate fixatives, etc. The fixative can be selected from one or more of paraformaldehyde, formalin, ethanol, glutaraldehyde, methanol, and acetone. (8) Washing: Aspirate the fixative solution in the 96-well plate. Wash the plate with 100 μL of PBS, then aspirate the PBS again. Repeat this operation twice. (9) Prepare the blocking solution in advance. The blocking solution used is 1% Bovine Serum Albumin, 3% goat serum, and 0.05 M EDTA, prepared in PBS. Add 100 μL of the blocking solution prepared in advance to each well and block at room temperature for 0.5 h-2 h. The blocking solution is used to block nonspecific sites to reduce nonspecific binding and improve the specificity and sensitivity of the test. It should be noted that the blocking solution usually contains a high molecular weight protein, such as bovine serum albumin (BSA), serum or casein, which can occupy and bind to unknown or non-specific sites on the surface of the experimental sample, thereby preventing them from non-specifically binding to the detection reagent. The blocking step can reduce the background signal generated by the non-specific binding of the primary and secondary antibodies to non-target sites. It can not only reduce the background, but also weaken the cause of the stray signal and eliminate the non-specific binding of the antibody. Ideally, the blocking agent will occupy the "sticky" sites in the sample, such as exposed charged or hydrophobic protein surfaces, while not interfering with the recognition of the target epitope by the primary / secondary antibody, thereby maximizing the signal-to-noise ratio; (10) Washing: Aspirate the blocking solution in the 96-well plate. Wash the plate with 100 μL PBS, then aspirate the PBS again and repeat this operation once more. (11) Storage: Aspirate the blocking solution in the detection well; if the cell matrix culture plate is not used immediately, add 200 μL of PBS containing 0.05% Proclin 300; after all operations are completed, stick a sealing film on each recombinant cell matrix plate, seal it and store it in the dark at 2-8°C; the protective solution is a mixed liquid containing preservatives, anti-fluorescence quenchers and stabilizers, which is used to protect samples or reagents, prevent microbial contamination, extend the shelf life of the product, and maintain the stability of the sample and the fluorescence signal.

[0087] In this embodiment, the CBA detection method using the reagent for detecting anti-myelin-associated glycoprotein antibodies includes the following steps:

[0088] Based on the recombinant cell matrix plate prepared in step S3 above, sample testing was performed as follows: (1) Take the recombinant cell matrix plate out of the refrigerator and dilute the clinical serum sample with sample diluent. The starting dilution of the sample detection system is 1:10. Further dilution factors can also be made, such as 1:32, 1:100, 1:320, 1:1000, etc. There is no upper limit to the detection range; Preferably, the sample diluent comprises 1% BSA and 0.05 M EDTA; It should be noted that the sample may also be plasma, whole blood or cerebrospinal fluid, containing antibodies from the patient, and the sample is preferably serum; the dilution is performed according to the blocking solution doubling dilution method; (2) Sample (primary antibody) incubation: Take the reagent out of the refrigerator and return it to room temperature; add the prepared sample and incubate in a 37°C constant temperature incubator for 30 min to allow the target antibody in the sample to fully bind to the target antigen; after incubation, use PBS to elute; (3) Secondary antibody incubation: The bound antibody reacts specifically with a goat anti-human IgM secondary antibody labeled with Alexa Fluor® 594 (Brand: Jackson ImmunoResearch, Catalog No.: 109585129, Lot No.: 165946). Incubate at 37°C in the dark for 30 minutes. Excess secondary antibody is washed away during the wash process. After elution, add 100 μL of PBS and photograph and read the slides under a fluorescence microscope. Dilution is performed according to the blocking solution doubling dilution method; (4) Microscopic examination: Microscopic examination is the abbreviation of microscopic examination. It is to observe, analyze and judge the above matrix plate under a microscope to assist in the diagnosis of diseases; (5) Interpretation of results: The evaluation of test results must be based on the evaluation of positive and negative quality controls, so the original results must be photographed and saved for traceability; the positive control must be positive under any circumstances. The positive control ensures that the experiment is performed correctly and that the various components are normal. If the positive control result is not good, the experimental data cannot be read.

[0089] Positive result: If a sample shows overlap of red fluorescence (secondary antibody fluorescence) and green fluorescence (transfected plasmid), the sample is considered positive.

[0090] Negative result: If a sample only shows green fluorescence (transfected plasmid) and no red fluorescence (secondary antibody fluorescence); or if the red fluorescence (secondary antibody fluorescence) and green fluorescence (transfected plasmid) do not overlap, the sample is considered negative.

[0091] Depending on actual needs, when culturing cells, polylysine-treated cell slides can be placed in 96-well cell culture plates, and HEK293T cells carrying the recombinant plasmid can be added for culture. Diagnostally useful carriers are intended for microscopic immunofluorescence analysis and can include cell culture plates, slides, or glass slides.

[0092] In this example, clinical sample testing and sensitivity / specificity evaluation were also performed.

[0093] Two other comparative plasmid vectors were constructed using step S1 of this example. The recombinant cells prepared in step S2 were used to prepare cell matrix plates based on the method in step S3. Clinical samples were tested using the CBA detection method of the above-mentioned reagent for detecting anti-myelin-associated glycoprotein antibodies. The test results of different groups were compared to evaluate the sensitivity and specificity of the reagent detection. The specific steps include: (1) Vector design: First, the gene sequence for fusion expression was designed. Based on the fusion expression plasmid designed and constructed in this example: MAG, GlcAT-P, HNK-1ST fusion protein (MAG-EGFP-P2A-GlcAT-P-T2A-HNK-1ST), the comparative example 1 - MAG, EGFP fusion expression plasmid (MAG-EGFP) and the comparative example 2 - MAG, GlcAT-P fusion expression plasmid (MAG-EGFP-P2A-GlcAT-P) were designed and constructed; wherein the GGGGS sequence was added between MAG and EGFP. The above gene sequences were artificially synthesized, and then the corresponding fusion sequences were ligated to the vector pcDNA3.1(+) using the double enzyme digestion method; (2) HEK293T cell culture and transfection: DMEM high glucose medium and FBS were prepared at a ratio of 9:1 to prepare 10% FBS-DMEM high glucose medium, and 10% penicillin-streptomycin double antibody was added. When the cells were confluent, the cells were passaged at a ratio of 1:5-1:6 and cultured overnight in a cell culture incubator at 37°C and 5% CO2. When the cell density reached 80%-90%, the genes were transfected according to the combination in Table 2 below, and fresh culture medium was replaced after 6 hours. Table 2 Plasmid-liposome complex preparation table (II)

[0094] (3) The successfully transfected cells are used to prepare recombinant cell matrix plates and test clinical samples according to step S3.

[0095] To further promote the clinical application of the method of the present invention, 30 MAG-positive serum samples and 50 MAG-negative serum samples were selected, whose positive and negative characteristics were consistent with the clinical symptoms. The above three groups of reagents were used for testing respectively, and the sensitivity and specificity of different groups of reagents were compared.

[0096] like Figure 3 The following are the test results for the same MAG-positive sample: Figures AC and DF are the test results for the positive sample of Comparative Example 1, Figures DF are the test results for the positive sample of Comparative Example 2, and Figures GI are the test results for the positive sample of this embodiment. The green fluorescent signal is EGFP fused with MAG, indicating the expression of the MAG antigen; the red fluorescent signal is the positive detection signal of the serum sample; Merged is the combined result of red and green fluorescence. The test results show that the addition of GlcAT-P significantly improves the detection sensitivity compared to MAG alone; however, the group expressing MAG, GlcAT-P, and HNK-1ST simultaneously has higher sensitivity.

[0097] like Figure 4 Figures 1 and 2 show the test results for the same MAG-negative sample. Figures AC show the negative sample test results for Comparative Example 1, Figures DF show the negative sample test results for Comparative Example 2, and Figures GI show the negative sample test results for this example. The green fluorescent signal is EGFP fused with MAG, indicating the expression of the MAG antigen. No obvious red fluorescent signal was observed in either the example or the comparative example, indicating a negative test result.

[0098] As shown in Table 3 below, a summary table of clinical sample test results of the three groups of reagents is shown. The results show that the sample detection sensitivity of Comparative Example 1 is 20% (6 / 30), and the sample detection specificity is 100% (50 / 50); the sample detection sensitivity of Comparative Example 2 is 73.3% (22 / 30), and the sample detection specificity is 100% (50 / 50); the sample detection sensitivity of this embodiment is 100% (30 / 30), and the sample detection specificity is 100% (50 / 50).

[0099] Table 3 Summary of clinical sample test results of three groups of reagents

[0100] The results in Table 3 show that the co-transfection method of MAG, GlcAT-P and HNK-1ST in this example can obtain a more sensitive detection reagent with higher sensitivity and higher diagnostic accuracy, and the specificity of both is 100%.

[0101] Another recombinant cell in this embodiment contains a recombinant plasmid expressing MAG, a recombinant plasmid expressing GlcAT-P, and a recombinant plasmid expressing HNK-1ST. The recombinant cell is used to detect anti-myelin-associated glycoprotein antibodies; the nucleotide sequence of the MAG is shown in SEQ ID NO.3, the nucleotide sequence of the GlcAT-P is shown in SEQ ID NO.4, and the nucleotide sequence of the HNK-1ST is shown in SEQ ID NO.5.

[0102] The three proteins MAG, GlcAT-P and HNK-1ST are separately constructed into recombinant vectors, which are then co-transfected into the same host cell, i.e., the recombinant cell. In the host cell, the three proteins are separately expressed. Moreover, similar to the aforementioned MAG, GlcAT-P and HNK-1ST fusion proteins, in the host cell, MAG provides the basic protein, and GlcAT-P and HNK-1ST perform post-translational modifications on the MAG protein to obtain the MAG protein expressing the HNK-1 carbohydrate epitope as the target antigen. The CBA double fluorescence staining analysis method is used to detect anti-MAG antibodies in human body fluids, and can simultaneously detect antibodies targeting MAG protein and HNK-1, with excellent detection sensitivity and specificity, and can ensure the accuracy of detection, especially for detection in the low titer range.

[0103] The present invention constructs a recombinant expression vector for a fusion protein of MAG, GlcAT-P, and HNK-1ST. The cDNA genes of the three proteins are fused and constructed into a single expression vector, in which MAG is expressed in a fusion with EGFP. The constructed expression vector is then transfected into mammalian cells. The myelin-associated glycoprotein expressed by the cells is catalyzed by the enzymes GlcAT-P and HNK-1ST to produce the HNK-1 carbohydrate epitope. The MAG-EGFP protein expressing the HNK-1 epitope is used as an antigen for detecting anti-myelin-associated glycoprotein antibodies. Cells expressing this antigen are fixed and blocked to produce an immunofluorescence assay reagent for detecting anti-myelin-associated glycoprotein antibodies. This reagent can be used to detect anti-myelin-associated glycoprotein (MAG) antibodies in patient serum or cerebrospinal fluid with high sensitivity and specificity, resulting in excellent detection results.

[0104] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A fusion protein, characterized in that The fusion protein is a fusion protein of MAG, GlcAT-P and HNK-1ST, and the MAG, GlcAT-P and HNK-1ST fusion protein is sequentially connected by MAG, GGGGS sequence, EGFP, P2A, GlcAT-P, T2A and HNK-1ST; the GGGGS sequence is a connecting peptide between MAG and EGFP, the P2A is a connecting peptide between EGFP and GlcAT-P, and the T2A is a connecting peptide between GlcAT-P and HNK-1ST; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.

1.

2. The fusion protein according to claim 1, characterized in that The MAG in the MAG, GlcAT-P and HNK-1ST fusion protein is a MAG-S isomer; the GlcAT-P is a 1GlcAT-P isomer.

3. A fusion protein gene, characterized in that: It encodes the MAG, GlcAT-P and HNK-1ST fusion protein according to claim 1, and its nucleotide sequence is shown in SEQ ID NO.

2.

4. A recombinant antigen, characterized in that The recombinant antigen is a MAG-EGFP protein expressing the HNK-1 epitope, and the MAG-EGFP protein expressing the HNK-1 epitope is the product of the expression and biosynthesis of the MAG, GlcAT-P and HNK-1ST fusion protein according to claim 1 in a host cell.

5. A reagent for detecting anti-myelin-associated glycoprotein antibodies, characterized in that: The reagent for detecting anti-myelin-associated glycoprotein antibodies comprises the recombinant antigen according to claim 4.

6. Use of the reagent for detecting anti-myelin-associated glycoprotein antibodies according to claim 5 in detecting MAG antibodies.

7. A method for preparing a reagent for detecting anti-myelin-associated glycoprotein antibodies according to claim 5, characterized in that: The following steps are involved: Step S1: constructing a recombinant expression vector for the MAG, GlcAT-P and HNK-1ST fusion protein, and obtaining a recombinant plasmid through the recombinant expression vector; the amino acid sequence of the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the MAG, GlcAT-P and HNK-1ST fusion protein is shown in SEQ ID NO.2; Step S2: transfecting the recombinant plasmid obtained in step S1 into cells to obtain recombinant cells; overexpressing the MAG-EGFP target antigen with the HNK-1 epitope in the cells; Step S3: preparing the recombinant cells in step S2 into a recombinant cell matrix plate, which is the reagent for detecting anti-myelin-associated glycoprotein antibodies.

8. A kit for detecting anti-myelin-associated glycoprotein antibodies, characterized in that: The kit comprises the reagent for detecting anti-myelin-associated glycoprotein antibodies according to claim 5.

9. A CBA detection method using the reagent for detecting anti-myelin-associated glycoprotein antibodies according to claim 5, characterized in that: The CBA detection method comprises the following steps: adding a sample to the reagent for detecting anti-myelin-associated glycoprotein antibodies and performing a first incubation; then adding a fluorescent secondary antibody and performing a second incubation, washing, microscopically inspecting, and interpreting the results.

10. A recombinant cell, characterized in that The recombinant cell includes a recombinant plasmid expressing MAG, a recombinant plasmid expressing GlcAT-P and a recombinant plasmid expressing HNK-1ST, and the recombinant cell is used for detecting anti-myelin-associated glycoprotein antibodies; the nucleotide sequence of the MAG is shown in SEQ ID NO.3, the nucleotide sequence of the GlcAT-P is shown in SEQ ID NO.4, and the nucleotide sequence of the HNK-1ST is shown in SEQ ID NO.5.

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