A recombinant protein for detecting anti-myelin associated glycoprotein MAG antibody and a detection reagent thereof

Abstract

The present invention discloses a recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody and a detection reagent thereof, belonging to the technical field of neuro-immunodiagnosis. By performing specific amino acid point mutation substitutions at five amino acid positions in the MAG protein, a MAG protein mutant sequence is obtained, and then it is fused with the full-length sequences of GlcATPase and HNK-1ST enzyme to construct a lentiviral expression vector, which is stably transfected into host cells, enabling the simultaneous stable expression of the three genes in one cell. The constructed recombinant protein has a similar anti-MAG IgM antibody binding domain to the wild-type MAG protein, and without using small molecule agonists of GlcATPase and HNK-1ST enzyme, it improves the sensitivity and specificity of detecting MAG autoantibodies by the CBA method, and is suitable for preparing related detection reagents for in vitro detection of IgM-type anti-MAG antibodies.

Description

Technical Field

[0001] The present invention relates to a recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody and a detection reagent thereof, and particularly relates to a recombinant protein of MAG antigen epitope used in detecting anti-myelin associated glycoprotein (MAG) antibody by cell immunofluorescence assay (Cell Based Assay, CBA method), and the application of the recombinant protein in preparing related detection reagents, belonging to the technical field of neuroimmune diagnosis. Background Art

[0002] Myelin associated glycoprotein is a highly glycosylated transmembrane protein and belongs to the members of the immunoglobulin superfamily (IgSF), with a molecular weight of about 100 kDa. Its structure consists of three parts: an extracellular region, a transmembrane region and an intracellular region. The extracellular region contains 5 immunoglobulin-like domains (Ig-like domains) and 1 type III fibronectin domain (FN3). These domains endow MAG with the ability to specifically bind to the surface receptors of nerve cells, such as gangliosides and sialic acid-binding receptors, thereby mediating the adhesion and signal transmission between myelin sheath and axon. MAG is mainly distributed in the innermost layer (periaxonal loop region) of the myelin sheath formed by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS), directly contacting the axonal membrane, and is a key molecule for maintaining the structural integrity of myelin sheath and axon-glial cell interaction. Under physiological conditions, MAG not only participates in the spiral wrapping and maturation of myelin sheath, but also regulates the metabolic activities of glial cells by activating downstream signaling pathways (such as Fyn kinase pathway); while under pathological conditions, the abnormal glycosylation modification of MAG may become the target of autoimmune attack, leading to demyelinating diseases.

[0003] Anti-MAG antibody syndrome is a chronic demyelinating peripheral neuropathy caused by IgM monoclonal gammopathy (MGUS). Its core pathological mechanism is closely related to the HNK-1 glycoepitope of MAG. The HNK-1 epitope is a product of post-translational modification of MAG protein, which is composed of sulfated glucuronic acid (HSO3-3GlcAβ1–3Galβ1–4GlcNAc-), and this unique structure becomes the main target of autoantibodies. When anti-MAG IgM antibody binds to the HNK-1 epitope, it causes nerve damage through two pathways: First, it activates the classical complement pathway (C1q-C9) to form a membrane attack complex (MAC), directly destroying the Schwann cell membrane and causing demyelination; Second, it interferes with the adhesion function of MAG, weakens the interface stability between the myelin sheath and the axon, and leads to axonal degeneration. Clinically, patients present with distal symmetric sensorimotor disturbances, such as numbness in hands and feet, unsteady gait, and weakened tendon reflexes. Electrophysiological examination shows a significant slowdown in motor nerve conduction velocity, and about 50% of cases are accompanied by monoclonal IgM gammopathy. However, existing diagnostic methods such as Western Blot and ELISA have insufficient sensitivity at low antibody concentrations, and due to the cross-reactivity of MAG with other nerve antigens, the false positive rate is relatively high, which limits the accuracy of early diagnosis.

[0004] The cell-based assay (CBA) can accurately identify the HNK-1 conformational epitope by expressing the naturally folded MAG antigen in mammalian cells, significantly improving the sensitivity and specificity of anti-MAG IgM antibody detection. However, the application of CBA faces two major technical bottlenecks: First, in addition to maintaining a specific protein structure, the HNK-1 epitope requires partial residues of the epitope to undergo N -glycosylation modification to have the correct epitope active site, and these N -glycosylation modifications are highly dependent on the co-expression of MAG with two modifying enzymes (GlcATPase and HNK-1ST enzyme); Second, as rate-limiting enzymes for sugar chain modification, the activities of GlcATPase and HNK-1ST enzyme are easily interfered by environmental factors such as intracellular ATP concentration, pH value, and metabolic by-products. Even if the expression levels of GlcATPase and HNK-1ST enzyme are increased significantly, the synthesis efficiency of the HNK-1 epitope cannot be doubled, but the synthesis efficiency of the HNK-1 epitope can be increased by activating the activities of GlcATPase and HNK-1ST enzyme with small molecules.

[0005] In response to these problems, the optimization strategies of the prior art focus on genetic engineering and cell metabolism regulation. For example, as reported in "Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: Possible clinical relevance", neures, 2014.09.10 ("Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: Possible clinical relevance", Journal of Neurology), and "Expression of glycosylated recombinant human myelin-associated glycoprotein on a neuroblastoma cell line and its reactivity with HNK-1 but not human anti-MAG antibodies", Neuroscience letters, 1998, 00246-3 ("Expression of glycosylated recombinant human myelin-associated glycoprotein on a neuroblastoma cell line and its reactivity with HNK-1 but not human anti-MAG antibodies", Journal of Neuroscience), the efficient co-expression of MAG, β-1,3-glucuronosyltransferase (GlcATP), and carbohydrate sulfotransferase 10 (HNK-1ST) is achieved using IRES or 2A peptide chain; adding forskolin in cell culture to activate adenylate cyclase to increase cAMP level, or using retinoic acid to enhance glycosyltransferase activity, thereby promoting the synthesis of HNK-1 epitope. However, the prior art does not perform structural re-design on the MAG protein structure and HNK-1 site, and regulates the increase of the synthesis efficiency of the HNK-1 epitope of MAG by means of gene co-expression and promoting protein glycosylsulfonation modification. Summary of the Invention

[0006] The present invention aims to provide a recombinant protein for detecting anti-myelin-associated glycoprotein MAG antibody and its detection reagent. By performing specific amino acid point mutation substitution on five amino acid positions in the MAG protein to obtain a MAG protein mutant sequence, and then fusing it with the full-length sequences of GlcATP enzyme and HNK-1ST enzyme to construct a lentiviral expression vector, and stably transfecting it into host cells, the three genes are stably expressed simultaneously in one cell, so that the constructed recombinant protein has a similar anti-MAG IgM antibody binding domain to the wild-type MAG protein. Without using small molecule agonists of GlcATP enzyme and HNK-1ST enzyme, the sensitivity and specificity of detecting MAG autoantibodies by the CBA method are improved, and it is suitable for preparing related detection reagents for in vitro detection of IgM-type anti-MAG antibodies.

[0007] The present invention is achieved through the following technical solutions: A recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody, wherein the recombinant protein is obtained by mutating five amino acid sites of the MAG protein gene, then fusing the mutated MAG protein gene with the full-length sequences of GlcATPase and HNK-1ST enzyme and constructing them into a lentiviral expression vector, and then expressing and purifying in a host cell.

[0008] The five amino acid mutation sites involved in the present invention are respectively: A263N, L366N, V434N, I487N and A496N. The sequence of the mutated MAG protein gene is as shown in SEQ ID NO: 1, specifically as follows:

[0009] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVNKSNPEPSVAFELPSRNVTVNESEREFVYSERSGLVLTSILTLRGQAQAPPRVNCTARNLYGNKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK.

[0010] The full-length sequence of GlcATPase is as shown in SEQ ID NO: 2, specifically as follows:

[0011] MPKRRDILAIVLIVLPWTLLITVWHQSTLAPLLAVHKDEGSDPRRETPPGADPREYCTSDRDIVEVVRTEYVYTRPPPWSDTLPTIHVVTPTYSRPVQKAELTRMANTLLHVPNLHWLVVEDAPRRTPLTARLLRDTGLNYTHLHVETPRNYKLRGDARDPRIPRGTMQRNLALRWLRETFPRNSSQPGVVYFADDDNTYSLELFEEMRSTRRVSVWPVAFVGGLRYEAPRVNGAGKVVGWKTVFDPHRPFAIDMAGFAVNLRLILQRSQAYFKLRGVKGGYQESSLLRELVTLNDLEPKAANCTKILVWHTRTEKPVLVNEGKKGFTDPSVEI。

[0012] The full-length sequence of the HNK-1ST enzyme is shown in SEQ ID NO: 3 and is as follows:

[0013] MHHQWLLLAACFWVIFMFMVASKFITLTFKDPDVYSAKQEFLFLTTMPEVRKLPEEKHIPEELKPTGKELPDSQLVQPLVYMERLELIRNVCRDDALKNLSHTPVSKFVLDRIFVCDKHKILFCQTPKVGNTQWKKVLIVLNGAFSSIEEIPENVVHDHEKNGLPRLSSFSDAEIQKRLKTYFKFFIVRDPFERLISAFKDKFVHNPRFEPWYRHEIAPGIIRKYRRNRTETRGIQFEDFVRYLGDPNHRWLDLQFGDHIIHWVTYVELCAPCEIMYSVIGHHETLEDDAPYILKEAGIDHLVSYPTIPPGITVYNRTKVEHYFLGISKRDIRRLYARFEGDFKLFGYQKPDFLLN。

[0014] During the fusion construction, a fluorescent tag and a linker peptide are also included. The linker peptide includes a first linker peptide for connecting the mutated MAG protein gene and the fluorescent tag, a second linker peptide for connecting the fluorescent tag and the full-length sequence of the GlcATP enzyme, and a third linker peptide for connecting the full-length sequence of the GlcATP enzyme and the full-length sequence of the HNK-1ST enzyme.

[0015] The sequence of the first linker peptide is shown in SEQ ID NO: 4 and is specifically: SGGGGSGGGGSGGGGS.

[0016] The sequence of the second linker peptide is as shown in SEQ ID NO: 5, specifically: GSGEGRGSLLTCGDVEENPGP.

[0017] The sequence of the third linker peptide is as shown in SEQ ID NO: 6, specifically: RAKRSGSGAT NFSLLKQAGDVEENPGP.

[0018] The fluorescent label is EGFP, and its sequence is as shown in SEQ ID NO: 7, specifically as follows:

[0019] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0020] The host cell is a CHO cell.

[0021] Specifically, in the present invention, the mutated MAG protein gene, EGFP label, GlcATP enzyme and HNK-1ST enzyme are respectively connected in series through linker peptides (the first linker peptide, the second linker peptide, the third linker peptide), constructed into a lentiviral expression vector, and then co-transfected into CHO cells, so as to obtain a cell line stably co-expressing MAG—EGFP—GlcATP—HNK-1ST. Therefore, it can be used to prepare a detection reagent for detecting anti-MAG antibodies.

[0022] For this reason, the present invention also provides a detection reagent, that is, a reagent for detecting anti-myelin associated glycoprotein MAG antibodies is prepared by using the above recombinant protein, and the detection method is immunofluorescence assay.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) After the five specific amino acid sites of the MAG protein gene are subjected to site-directed mutagenesis, and then recombined with GlcATPase and HNK-1ST enzyme, fused with a fluorescent tag using a linker peptide, a recombinant protein is constructed, which can achieve stable overexpression on the cell membrane of eukaryotic cells. In the process of detecting anti-MAG antibodies by the CBA method, it can improve the detection rate of weak antibodies and plays an important role in the detection of patients.

[0025] (2) The five specific amino acid sites of the present invention are selected at A 263 N, L 366 N, V 434 N, I 487 N, and A 496 N of the MAG protein gene. Through site-directed mutagenesis at these sites, the recombinant protein constructed with GlcATPase and HNK-1ST enzyme can exhibit an anti-MAG IgM antibody binding domain similar to that of the wild-type MAG protein, thereby realizing the sensitivity and specificity of IgM-type anti-MAG antibody detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a map of the lentiviral expression vector.

[0027] Figure 2 It is an immunofluorescence image of positive samples in Example 4.

[0028] Figure 3 It is an immunofluorescence image of negative samples in Example 4.

[0029] Figure 4 It is a comparison of the three-dimensional protein conformations between Example 4 and Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.

[0031] Example 1: MAG Mutation Sequence

[0032] This example relates to the process of mutating specific amino acid sites of the MAG protein gene to obtain a MAG mutation sequence.

[0033] Using the MAG protein gene (NM_002361.4), mutations are made at five amino acid sites of the MAG protein gene: A 263 N, L 366 N, V 434 N, I 487 N, and A 496 N. By means of amino acid site-directed mutagenesis, a MAG mutation sequence is obtained, and its sequence is shown in SEQ ID NO: 1.

[0034] Example 2: Construction of Lentiviral Expression Vector

[0035] This example relates to the process of constructing a lentiviral expression vector for the fusion protein MAG-EGFP-GlcATP-HNK-1ST.

[0036] The mutant sequence of the MAG protein gene, the fluorescent tag EGFP, the full-length sequence of GlcATP enzyme, and the full-length sequence of HNK-1ST enzyme are successively connected by three linker peptides to form a lentiviral expression vector. Among them, the mutant sequence of the MAG protein gene is the MAG mutant sequence of Example 1; the sequence of the fluorescent tag EGFP is as shown in SEQ ID NO: 7; the full-length sequence of GlcATP enzyme is as shown in SEQ ID NO: 2; the full-length sequence of HNK-1ST enzyme is as shown in SEQ ID NO: 3.

[0037] The three linker peptides include the first linker peptide, the second linker peptide, and the third linker peptide. The first linker peptide is used to connect the mutant sequence of the MAG protein gene and the fluorescent tag EGFP, and its sequence is as shown in SEQ ID NO: 4; the second linker peptide is used to connect the fluorescent tag EGFP and the full-length sequence of GlcATP enzyme, and its sequence is as shown in SEQ ID NO: 5; the third linker peptide is used to connect the full-length sequence of GlcATP enzyme and the full-length sequence of HNK-1ST enzyme, and its sequence is as shown in SEQ ID NO: 6.

[0038] The map of the lentiviral expression vector constructed thereby is as Figure 1 shown.

[0039] Example 3: Preparation of detection reagent

[0040] This example relates to the preparation process of a detection reagent for detecting anti-MAG antibody by the CBA method.

[0041] The lentiviral expression vector prepared in Example 2 is transfected into CHO cells, and then the transfected cells are screened by the limiting dilution method to obtain monoclonal cells expressing the MAG-EGFP-GlcATP-HNK-1ST fusion protein. Then, take the culture dish filled with MAG-EGFP-GlcATP-HNK-1ST transfected cells, digest it into a single cell suspension with trypsin (trypsin-EDTA) solution, calculate the cell density with a cell counting chamber, dilute the cells with complete medium, and dilute the cells to 4 cells / 100 μL, 2 cells / 100 μL, 1 cell / 100 μL by the limiting dilution method. Then, inoculate 100 μL / well into a 96-well plate respectively, screen the wells with single cell populations, and select the wells with strong green fluorescence for cryopreservation and primary antibody immunofluorescence detection. The qualified cells can be used to prepare the detection reagent for subsequent immunofluorescence experiments.

[0042] Inoculation of cell sheets: Digest the MAG-EGFP-GlcATP-HNK-1ST monoclonal cells and control empty vector cells, mix them evenly at a ratio of monoclonal cells: control cells = 1:2, and inoculate them into 96-well plates or cell slides at a total density of 1×2×10 5 / mL, and culture them with F12K + 10% FBS + NEAA. When culturing the cells, add 6 μM Forskolin and 3 μM Retinoic acid and grow for about 48 hours. Stop culturing when the confluence reaches 90 - 100%.

[0043] The cell sheets can be fixed with paraformaldehyde and stored for later use.

[0044] Example 4:

[0045] According to the methods of Examples 1 to 3, fuse the constructed MAG mutant sequence (mutation sites: A 263 N, L 366N, V 434 N, I 487 N, A 496 N) + fluorescent tag EGFP + the full-length sequence of GlcATP enzyme + the full-length sequence of HNK-1ST enzyme to express and detect anti-MAG antibodies. Use the fixed CBA method to screen the stable transfected cells and wild-type CHO cells and spread them onto a 96-well cell culture plate. After adding the reagents 6 μM Forskolin and 3 μM Retinoic acid and culturing for 48 hours, fix them with paraformaldehyde.

[0046] Comparative Example 1:

[0047] This comparative example is a process of expressing and detecting anti-MAG antibodies using the MAG protein gene (NM_002361.4), the full-length sequence of GlcATP enzyme, and the full-length sequence of HNK-1ST enzyme.

[0048] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfect CHO cells with Lipofectamine™ 3000 transfection reagent. After culturing for 48 hours, fix them with paraformaldehyde.

[0049] Comparative Example 2:

[0050] This comparative example is a process of expressing and detecting anti-MAG antibodies using the MAG mutant sequence with two-site mutations (mutation sites: A 263 N; L 366 N), the full-length sequence of GlcATP enzyme, and the full-length sequence of HNK-1ST enzyme.

[0051] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfect CHO cells with Lipofectamine™ 3000 transfection reagent. After culturing for 48 hours, fix them with paraformaldehyde.

[0052] For the MAG mutant sequence with two-site mutations (mutation sites: A263N; L366N), its sequence is as shown in SEQ ID NO: 8, specifically as follows:

[0053] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVVKSNPEPSVAFELPSRNVTVNESEREFVYSERSGLVLTSILTLRGQAQAPPRVICTARNLYGAKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK.

[0054] Comparative Example 3:

[0055] This comparative example is a process of expressing and detecting anti-MAG antibodies using a MAG mutant sequence with four-site mutations (mutation sites: A263N; L366N; V434N; I487N), the full-length sequence of GlcATPase, and the full-length sequence of HNK-1ST enzyme.

[0056] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfecting CHO cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0057] For the MAG mutant sequence with four-site mutations (mutation sites: A 263 N; L 366 N; V 434 N; I 487 N), its sequence is as shown in SEQ ID NO: 9, specifically as follows:

[0058] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVNKSNPEPSVAFELPSRNVTVNESEREFVYSERSGLVLTSILTLRGQAQAPPRVNCTARNLYGAKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK。

[0059] Comparative Example 4:

[0060] In this comparative example, the process of expressing and detecting anti-MAG antibodies was carried out using the MAG mutant sequence with five-site mutations (mutation sites: A 263 N; L 366 N; V461N; V 434 N; I 487 N), the full-length sequence of GlcATPase, and the full-length sequence of HNK-1ST enzyme.

[0061] By constructing a eukaryotic transient expression vector containing the above sequences and transiently transfecting CHO cells with Lipofectamine™ 3000 transfection reagent, after culturing for 48 hours, it was fixed with paraformaldehyde.

[0062] For the MAG mutant sequence with five-site mutations (mutation sites: A263N; L366N; V461N; V434N; I487N), its sequence is as shown in SEQ ID NO: 10, specifically as follows:

[0063] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVNKSNPEPSVAFELPSRNVTVNESEREFNYSERSGLVLTSILTLRGQAQAPPRVNCTARNLYGAKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK。

[0064] Comparative Example 5:

[0065] This comparative example is a process of expressing and detecting anti-MAG antibodies using a MAG mutant sequence with six-site mutations (mutation sites: A263N; L366N; V434N; V461N; I487N; A496N), the full-length sequence of GlcATPase, and the full-length sequence of HNK-1ST enzyme.

[0066] By constructing a eukaryotic transient expression vector containing the above sequences and transiently transfecting CHO cells with Lipofectamine™ 3000 transfection reagent, after culturing for 48 hours, the cells were fixed with paraformaldehyde.

[0067] For the MAG mutant sequence with six-site mutations (mutation sites: A 263 N; L 366 N; V 434 N; V 461N; I 487 N; A 496 N), its sequence is shown in SEQ ID NO: 11 as follows:

[0068] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVNKSNPEPSVAFELPSRNVTVNESEREFNYSERSGLVLTSILTLRGQAQAPPRVNCTARNLYGNKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK.

[0069] Comparative Example 6:

[0070] This comparative example is a process of expressing and detecting anti-MAG antibodies using the MAG mutant sequence with eight-site mutations (mutation sites: E131N; A263N; A285N; L366N; V434N; V461N; I487N; A496N), the full-length sequence of GlcATPase, and the full-length sequence of HNK-1ST enzyme.

[0071] By constructing a eukaryotic transient expression vector containing the above sequences, transiently transfecting CHO cells with Lipofectamine™ 3000 transfection reagent, and fixing with paraformaldehyde after culturing for 48 hours.

[0072] For the MAG mutant sequence with eight-site mutations (mutation sites: E131N; A263N; A285N; L366N; V434N; V461N; I487N; A496N), its sequence is shown in SEQ ID NO: 12 as follows:

[0073] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSNHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGNDSNPPPLLTWMRDGTVLREAVNESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQINSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVNKSNPEPSVAFELPSRNVTVNESEREFNYSERSGLVLTSILTLRGQAQAPPRVNCTARNLYGNKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK。

[0074] Add the fixed cells prepared in Example 4 and Comparative Examples 1 to 6 above to 110 positive samples clinically diagnosed with anti-MAG antibody disease, respectively, and perform immunofluorescence detection.

[0075] The immunofluorescence detection method is as follows:

[0076] Dilute the sample (serum) at a ratio of 1:32. Add the diluted sample to the well plate of confluent live cells, place it in a carbon dioxide incubator, incubate at 37 °C for 1 hour, then aspirate the sample solution and wash the cell slides 3 times with DMEM, 5 minutes each time. Next, dilute the secondary antibody with the same sample diluent. The secondary antibody is goat anti-human IgM labeled with Delight 594, and the dilution ratio of the secondary antibody is 1:500. Add the diluted secondary antibody to the sample, incubate at room temperature for 40 minutes, aspirate the secondary antibody, and wash the cell slides 3 times with DMEM, 5 minutes each time. Then add a little phosphate buffer to completely immerse the cell slides and observe under a fluorescence microscope as soon as possible to judge the positivity or negativity of the sample.

[0077] The results of immunofluorescence detection are shown in Figure 2 and Table 1 below: Comparison of the detection rates of positive samples for anti-MAG antibody disease.

[0078] Figure 2 Figure

[0079] Table 1: Comparison of the detection rates of positive samples for anti-MAG antibody disease

[0080]

[0081] Add the fixed cells prepared in Example 4 and Comparative Examples 1 to 6 above to 82 cases of healthy human serum samples and 56 cases of sera from other patients without MAG respectively, and perform immunofluorescence detection (the immunofluorescence detection method is the same as above).

[0082] The results of immunofluorescence detection are shown in Figure 3 and Table 2 below: Comparison of negative samples between healthy people and other patients without MAG.

[0083] Figure 3 Figure

[0084] Table 2: Comparison of negative samples between healthy people and other patients without MAG

[0085]

[0086] In summary, from Figure 2As can be seen from Table 1, positive samples of anti-MAG antibody disease can be well detected by using Example 4 of the present invention. When comparing with the test results of Comparative Examples 1 to 6, the positive detection rate of Example 4 is significantly better than that of Comparative Examples 1 to 6. At the same time, by detecting different dilution ratios of positive samples, Example 4 has the ability to identify antibodies at lower concentrations compared with Comparative Documents 1 to 6 and has sensitivity. From Figure 3 As can be seen from Table 2, negative samples can be well detected by using Example 4 of the present invention, and the specificity is in line.

[0087] Furthermore, to prove that the recombinant protein constructed by the present invention has an anti-MAG IgM antibody binding domain similar to that of the wild-type MAG protein, see Figure 4 as shown. Figure 4 For the comparison of the three-dimensional conformations of the proteins of Example 4 and Comparative Example 1, Figure 4 in which A is a schematic diagram of the three-dimensional conformation of the wild-type MAG protein of Comparative Example 1, B is a schematic diagram of the three-dimensional conformation of the MAG fusion protein in Example 4, and C is the overlapping of the three-dimensional conformation diagrams of the wild-type MAG protein of Comparative Example 1 and the MAG fusion protein of Example 4. It can be seen that the three-dimensional structure of the recombinant protein in Example 4 has good consistency with the wild-type MAG protein in Comparative Example 1.

[0088] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Any simple modification or equivalent change made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody, characterized in that: the recombinant protein is obtained by mutating five amino acid sites of the MAG protein gene, then fusing the mutated MAG protein gene with the full-length sequences of a fluorescent tag, GlcATPase and HNK-1ST enzyme into a lentiviral expression vector, and then expressing and purifying in a host cell; during the fusion construction, the mutated MAG protein gene and the fluorescent tag are connected by a first linker peptide, the fluorescent tag and the full-length sequence of GlcATPase are connected by a second linker peptide, and the full-length sequence of GlcATPase and the full-length sequence of HNK-1ST enzyme are connected by a third linker peptide; the sequence of the mutated MAG protein gene is as shown in SEQ ID NO: 1; the full-length sequence of GlcATPase is as shown in SEQ ID NO: 2; the full-length sequence of HNK-1ST enzyme is as shown in SEQ ID NO: 3; the sequence of the first linker peptide is as shown in SEQ ID NO: 4; the sequence of the second linker peptide is as shown in SEQ ID NO: 5; the sequence of the third linker peptide is as shown in SEQ ID NO: 6; the fluorescent tag is EGFP, and its sequence is as shown in SEQ ID NO:

7.

2. A detection reagent, characterized in that: A reagent for detecting anti-myelin associated glycoprotein (MAG) antibody is prepared by using the recombinant protein according to claim 1, and the detection method is immunofluorescence assay.

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