Fusion protein for detecting aquaporin 4 antibody as well as preparation method and application of fusion protein

Abstract

The invention provides a fusion protein for detecting an aquaporin 4 antibody as well as a preparation method and application of the fusion protein, and belongs to the technical field of aquaporin 4 antibody detection. The nucleotide sequence of the fusion protein is shown as SEQ ID NO.5. The fusion protein is used as a coating antigen, M1 type and M23 type aquaporin 4 can be detected at the same time, and the sensitivity and the accuracy of detecting an aquaporin 4 antibody are high, and the repeatability is good. When the kit is used for the aquaporin 4 antibody, the detection method is simple and rapid, and the kit is suitable for clinical and scientific research large-scale screening of the aquaporin 4 antibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaporin 4 antibody detection, and in particular relates to a fusion protein for detecting aquaporin 4 antibody, and a preparation method and application thereof. Background Art

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease of the central nervous system that mainly affects the optic nerve and spinal cord. The disease is more common in young and middle-aged people, mainly women, with high recurrence and disability rates, and is easily misdiagnosed as other eye diseases such as primary open-angle glaucoma and macular degeneration. If not diagnosed and treated in time, most patients will be left with severe visual impairment, limb dysfunction, and urinary and bowel disorders.

[0003] Aquaporin 4 antibodies (AQP4-IgG) can be detected in the blood of the vast majority of NMOSD patients. This antibody mistakenly attacks a specific protein in the central nervous system, namely aquaporin-4 (AQP4). AQP4 was isolated using homologous cloning of the aquaporin family. It is widely distributed in the central nervous system and is the most important aquaporin in the mammalian brain. At present, there are 13 subtypes of aquaporins found in humans, namely AQP0 to AQP12. AQP4 is one of them. Its gene is located at the junction of human chromosomes 18q11.2 and q12.1. It contains 4 exons responsible for the encoding of amino acid sequences at positions 127, 55, 27, and 92, and 3 introns are located in between. Structurally, AQP4 located on the cell membrane is composed of 6 transmembrane helical structures, forming 5 ring structures, of which rings A, C and E are located outside the cell, and rings B, D and the carboxyl and amino termini are located inside the cell, mostly expressed in astrocytes. AQP4 has two major subtypes, M1 (32kDa) and M23 (30kDa), both of which have the same extracellular domain, but M1 has an extra 22 amino acid sequence at the intracellular N-terminus. AQP4 usually exists in the form of a tetramer, with the ratio of M23 to M1 being 3:1.

[0004] As a specific biomarker for diagnosing NMOSD, the discovery of AQP4-IgG laid the foundation for the immune pathogenesis of neuromyelitis optica. Currently, serum AQP4-IgG positivity has been included in one of the diagnostic criteria for NMOSD. Therefore, it is of great significance to obtain high-quality target proteins as raw materials for detection reagents for the detection of AQP4-IgG, which helps to improve the accuracy of early diagnosis.

[0005] In addition, expressing M1 or M23 proteins alone for use in detection reagents can easily lead to missed detection. Therefore, how to improve the accuracy of AQP4-IgG detection is an urgent problem to be solved. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a fusion protein for detecting aquaporin 4 antibodies and a preparation method and application thereof. The fusion protein can simultaneously detect M1 and M23 aquaporin 4, and has high sensitivity, accuracy and good repeatability in detecting aquaporin 4 antibodies.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a fusion protein for detecting aquaporin 4 antibodies, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.5.

[0009] The present invention provides a method for preparing the above fusion protein, comprising the following steps:

[0010] The coding gene sequences of M1-type AQP4 and M23-type AQP4 were double-digested and then connected to the backbone vector to obtain a recombinant vector;

[0011] transforming the recombinant vector into Escherichia coli, amplifying the recombinant vector, and obtaining the amplified recombinant vector;

[0012] The amplified recombinant vector is transfected into human embryonic kidney cells HEK293, and cultured to obtain transfected cells;

[0013] Finally, the fusion protein in the transfected cells was extracted.

[0014] Preferably, the coding gene sequence of the M1 type AQP4 is shown as SEQ ID NO.1, and the coding gene sequence of the M23 type AQP4 is shown as SEQ ID NO.3; the dual enzymes are EcoRI enzyme and XhoI enzyme; and the backbone vector is pET-28a(+) vector.

[0015] Preferably, the Escherichia coli includes Escherichia coli DH5α.

[0016] Preferably, the transfection method comprises: adding the amplified recombinant vector and PEI transfection reagent to human embryonic kidney cells HEK293, mixing, standing, and culturing to obtain transfected cells.

[0017] Preferably, the extraction method comprises: mixing the transfected cells with a cell lysate, repeatedly freezing and thawing, centrifuging at 2-6°C and 800-1200g for 5-15 min to obtain a supernatant, centrifuging the supernatant at 2-6°C and 12000-16000g for 25-35 min to obtain a precipitate, dissolving the precipitate with a membrane protein extract to obtain a cell membrane protein solution; mixing a His purification resin with the cell membrane protein solution to obtain a mixed solution, centrifuging the mixed solution at 2-6°C and 800-1200g for 5-15 min, obtaining a first precipitation resin, washing the first precipitation resin to obtain a second precipitation resin, and then eluting with an elution solution containing imidazole, centrifuging to obtain a supernatant to obtain a fusion protein.

[0018] The present invention also provides an application of the above fusion protein or the fusion protein obtained by the preparation method in the preparation of an antibody product for detecting aquaporin 4.

[0019] The present invention also provides a kit for detecting aquaporin 4 antibodies, the kit comprising the above fusion protein or the fusion protein obtained by the preparation method.

[0020] Preferably, the kit comprises an ELISA kit.

[0021] Preferably, the kit further comprises a horseradish peroxidase-labeled secondary antibody, a standard substance, a quality control substance, an enzyme substrate, a washing solution and a stop solution.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a fusion protein for detecting aquaporin 4 antibodies, and a preparation method and application thereof. The fusion protein for detecting aquaporin 4 antibodies is used as a coating antigen, and M1-type and M23-type aquaporin 4 can be detected simultaneously, and the sensitivity and accuracy of detecting aquaporin 4 antibodies are high and the repeatability is good. The present invention uses the fusion protein as a coating antigen to prepare an enzyme-linked immunosorbent assay (ELISA) kit, and the detection method is simple and rapid, and is suitable for large-scale screening of aquaporin 4 antibodies in clinical and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an electrophoresis diagram of SDS-PAGE detection of fusion protein expression, with the left lane being the fusion protein and the right lane being the marker. DETAILED DESCRIPTION

[0025] The present invention provides a fusion protein for detecting aquaporin 4 antibodies, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.5.

[0026] The present invention provides a method for preparing the above fusion protein, comprising the following steps:

[0027] The coding gene sequences of M1-type AQP4 and M23-type AQP4 were double-digested and then connected to the backbone vector to obtain a recombinant vector;

[0028] transforming the recombinant vector into Escherichia coli, amplifying the recombinant vector, and obtaining the amplified recombinant vector;

[0029] The amplified recombinant vector is transfected into human embryonic kidney cells HEK293, and cultured to obtain transfected cells;

[0030] Finally, the fusion protein in the transfected cells was extracted.

[0031] In the preparation method of the above fusion protein, the coding gene sequence of M1 type AQP4 and the coding gene sequence of M23 type AQP4 are respectively subjected to double enzyme digestion and connected to the backbone vector to obtain a recombinant vector. The coding gene sequence of M1 type AQP4 is shown in SEQ ID NO.1, and the coding gene sequence of M23 type AQP4 is shown in SEQ ID NO.3; the double enzymes are EcoRI enzyme and XhoI enzyme; and the backbone vector is pET-28a(+) vector.

[0032] After obtaining the recombinant vector, the recombinant vector is transformed into Escherichia coli, and the recombinant vector is amplified to obtain the amplified recombinant vector. The Escherichia coli includes Escherichia coli DH5α. The present invention has no particular limitation on the transformation method, and a conventional transformation method in the art can be used to achieve the purpose of amplifying the recombinant vector.

[0033] After obtaining the amplified recombinant vector, the amplified recombinant vector is transfected into human embryonic kidney cells HEK293, cultured, and transfected cells are obtained. The transfection method comprises: adding the amplified recombinant vector and PEI transfection reagent to human embryonic kidney cells HEK29, mixing, standing, and culturing to obtain transfected cells. The mass volume ratio of the recombinant vector and the PEI transfection reagent is 5-15:15-25 μg / μL; the standing time is preferably 10-20 min, more preferably 12-18 min, and more preferably 13, 14, 15, 16 or 17 min. The complete culture medium refers to a culture medium prepared by adding 10% fetal bovine serum by volume to the basic culture medium and mixing evenly. The basic culture medium includes DMEM or MEM. The standing time again is preferably 10-20 min, more preferably 12-18 min, and more preferably 13, 14, 15, 16 or 17 min. The culture conditions are 37° C., 5% CO2 for 40 to 50 hours, more preferably 37° C., 5% CO2 for 42 to 48 hours, and more preferably 37° C., 5% CO2 for 48 hours. The present invention utilizes HEK293 cells to express fusion proteins, which can efficiently obtain fusion proteins, meet the needs of large-scale production, and help produce more biologically active fusion proteins.

[0034] After obtaining the transfected cells, extract the fusion protein in the transfected cells. The extraction method includes: mixing the transfected cells with the cell lysate, repeatedly freezing and thawing, centrifuging at 2-6°C, 800-1200g for 5-15min to obtain the supernatant, centrifuging the supernatant at 2-6°C, 12000-16000g for 25-35min to obtain the precipitate, dissolving the precipitate with the membrane protein extract to obtain a cell membrane protein solution; mixing the His purification resin with the cell membrane protein solution to obtain a mixed solution, centrifuging the mixed solution at 2-6°C, 800-1200g for 5-15min, taking the first precipitation resin, washing the first precipitation resin, obtaining the second precipitation resin, and then eluting with an elution solution containing imidazole, centrifuging and obtaining the supernatant to obtain the fusion protein. In terms of 5mL, the preparation method of the cell lysate is to mix 4.95mL1×PBS and 50μL 100×protease inhibitor (PI) evenly. The number of repeated freezing and thawing is 3 times to achieve the purpose of destroying the cells. Among them, the preparation method of 1mL cell membrane protein extract is to mix 50μL of 1M Tris-HCl at pH 7.4, 150μL of 1M NaCl, 10μL of NP-40, 5μL of Triton-X-100, 10μL of 100×PI and 775μL of H2O. The His purification resin is preferably a His purification resin rinsed with PBS, and more preferably a His purification resin rinsed with PBS three times. The washing method is to wash the first precipitation resin 2 to 4 times with 1×PBS precooled at 2 to 6°C, and more preferably to wash the first precipitation resin 3 times with 1×PBS precooled at 4°C; each washing method is to centrifuge at 4°C and 1000g for 5 minutes, and retain the precipitation resin.

[0035] The present invention can successfully obtain a fusion protein with a sequence as shown in SEQ ID NO.5 through the above preparation method.

[0036] The present invention also provides an application of the above fusion protein or the fusion protein obtained by the preparation method in the preparation of an antibody product for detecting aquaporin 4.

[0037] In the present invention, the product is preferably a reagent or a kit.

[0038] The present invention also provides a kit for detecting aquaporin 4 antibodies, the kit comprising the above fusion protein or the fusion protein obtained by the preparation method.

[0039] In the present invention, the kit includes an ELISA kit. Furthermore, the kit also includes a secondary antibody labeled with HRP, a standard, a quality control product, an enzyme substrate, a washing solution and a stop solution. Taking the ELISA kit as an example, the ELISA kit contains a microplate coated with a fusion protein, and the preparation method of the microplate coated with the fusion protein includes: using the 8-12 μg / mL fusion protein solution to coat a 96-well microplate, adding 100 μL of 8-12 μg / mL fusion protein solution to each well, overnight at 4°C, adding 200 μL of phosphate buffer containing 1% bovine serum albumin by volume to each well to block, incubating at 37°C for 0.5-1.5h, washing 2-4 times with PBS buffer, and obtaining a microplate coated with a fusion protein. The secondary antibody labeled with HRP is an anti-human IgG secondary antibody labeled with HRP. The preparation method of the HRP-labeled anti-human IgG secondary antibody comprises: labeling the anti-human IgG secondary antibody with horseradish peroxidase (HRP), diluting it with a PBS buffer containing 1% BSA by volume to a volume ratio of 1:4500-5500, and obtaining the HRP-labeled anti-human IgG secondary antibody. The source of the anti-human IgG secondary antibody of the present invention is not particularly limited, and commercially available products in the art can be used. The enzyme substrate is tetramethylbenzidine. The washing solution is preferably a phosphate buffer containing 0.1% Tween by volume. The stop solution is preferably a 0.5M sulfuric acid solution. The standard is a PBS solution containing anti-AQP4 antibodies at concentrations of 81RU / mL, 27RU / mL, 9RU / mL, 3RU / mL and 1RU / mL, and the quality control includes a positive quality control and a negative quality control, the positive quality control is a sample diluent containing anti-AQP4 antibodies, with a concentration range of 5-10RU / mL; the negative quality control is a sample diluent not containing anti-AQP4 antibodies.

[0040] The present invention can rapidly detect AQP4-IgG by utilizing the kit, and the detection of AQP4-IgG has high sensitivity and accuracy and good repeatability.

[0041] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] In the following examples, the 100× protease inhibitor was purchased from Thermo Fisher Scientific with a catalog number of 87785; the NP-40 was purchased from Thermo Fisher Scientific with a catalog number of 85125; and the anti-human IgG secondary antibody was purchased from Merck with a catalog number of AP101P.

[0044] The preparation method of 250 mM imidazole eluent is as follows: 1.702 g of imidazole is weighed, dissolved in deionized water and the volume is made up to 100 mL.

[0045] The preparation method of the eluent containing 0.5 mM imidazole is to take 0.2 mL of 250 mM imidazole solution and dilute it to 100 mL with deionized water.

[0046] The sample diluent is PBS.

[0047] Example 1

[0048] The purpose of this example is to prepare a protein for accurately detecting AQP4-IgG. The specific steps of the method for preparing a protein for detecting AQP4-IgG are as follows:

[0049] 1. Preparation of recombinant vector pET-28a(+)-M1-M23: The coding sequences of the directly synthesized M1-type AQP4 and the M23-type AQP4 were double-digested with EcoRI and XhoI, respectively, and then connected to the pET-28a(+) vector to construct the recombinant vector pET-28a(+)-M1-M23. After PCR and double-enzyme digestion, the recombinant vector pET-28a(+)-M1-M23 was transformed into Escherichia coli DH5α, the plasmid was amplified, and the plasmid was extracted according to the instructions of the plasmid extraction kit.

[0050] Among them, the coding sequence of M1 type AQP4 is as follows:

[0051] aggcggtggggtaagtgtggacctttgtgtaccagagagaacatcatggtggctttcaaaggggtctggactcaag

[0052] ctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccaccatcaactggggtg

[0053] gaacagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaaccatggtgcagtgctttg

[0054] gccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatcagcatcgccaagtct

[0055] gtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacctcccagtgtggtgg

[0056] gaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataatcacatttcaattgg

[0057] tgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattggattttctgttgcaattg

[0058] gacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatcatgggaaattggg

[0059] aaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgtcttctgtccagatgt

[0060] tgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaggaagctacatggaggtggaggaca

[0061] acaggagtcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttgaccggggagagga

[0062] gaagaaggggaaagaccaatctggagaggtattgtcttcagtatga(SEQ ID NO.1).

[0063] The M1 protein sequence is as follows:

[0064] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIF

[0065] VLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTV

[0066] AMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLT

[0067] AGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGAS

[0068] MNPARSFAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQV

[0069] ETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV(SEQ ID NO.2)。

[0070] The coding sequence of M23 type AQP4 is as follows:

[0071] atggtggctttcaaaggggtctggactcaagctttctggaaagcagtcacagcggaatttctggccatgcttatttttg

[0072] ttctcctcagcctgggatccaccatcaactggggtggaacagaaaagcctttaccggtcgacatggttctcatctccctttgc

[0073] tttggactcagcattgcaaccatggtgcagtgctttggccatatcagcggtggccacatcaaccctgcagtgactgtggcca

[0074] tggtgtgcaccaggaagatcagcatcgccaagtctgtcttctacatcgcagcccagtgcctgggggccatcattggagca

[0075] ggaatcctctatctggtcacacctcccagtgtggtgggaggcctgggagtcaccatggttcatggaaatcttaccgctggtc

[0076] atggtctcctggttgagttgataatcacatttcaattggtgtttactatctttgccagctgtgattccaaacggactgatgtcactg

[0077] gctcaatagctttagcaattggattttctgttgcaattggacatttatttgcaatcaattatactggtgccagcatgaatcccgcc

[0078] cgatcctttggacctgcagttatcatgggaaattgggaaaaccattggatatattgggttgggcccatcataggagctgtcct

[0079] cgctggtggcctttatgagtatgtcttctgtccagatgttgaattcaaacgtcgttttaaagaagccttcagcaaagctgccca

[0080] gcaaacaaaaggaagctacatggaggtggaggacaacaggagtcaggtagagacggatgacctgattctaaaacctgg

[0081] agtggtgcatgtgattgacgttgaccggggagaggagaagaaggggaaagaccaatctggagaggtattgtcttcagtat

[0082] ga(SEQ ID NO.3)。

[0083] The M23 protein sequence is as follows:

[0084] MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDM

[0085] VLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLG

[0086] AIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDS

[0087] KRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIY

[0088] WVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRS

[0089] QVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV(SEQ ID NO.4)。

[0090] The correct fusion protein sequence identified by PCR and double digestion is as follows:

[0091] aggcggtggggtaagtgtggacctttgtgtaccagagagaacatcatggtggctttcaaaggggtctggactcaag

[0092] ctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccaccatcaactggggtg

[0093] gaacagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaaccatggtgcagtgctttg

[0094] gccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatcagcatcgccaagtct

[0095] gtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacctcccagtgtggtgg

[0096] gaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataatcacatttcaattgg

[0097] tgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattggattttctgttgcaattg

[0098] gacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatcatgggaaattggg

[0099] aaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgtcttctgtccagatgt

[0100] tgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaagaagctacatggaggtggaggaca

[0101] acaggatcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttgaccggggagagga

[0102] gaaaggggaaagaccaatctggagaggtattgtcttcagtatgaggggsggggggggsatggtggctttcaaaggg

[0103] gtctggactcaagctttctggaaagcagtcacagcggaatttctggccatgcttatttttgttctcctcagcctgggatccacc

[0104] atcaactggggtggaacaagaaaagcctttaccggtcgacatggttctcatctccctttgctttggactcagcattgcaaccat

[0105] ggtgcagtgctttggccatatcagcggtggccacatcaaccctgcagtgactgtggccatggtgtgcaccaggaagatca

[0106] gcatcgccaagtctgtcttctacatcgcagcccagtgcctgggggccatcattggagcaggaatcctctatctggtcacacc

[0107] tcccagtgtggtgggaggcctgggagtcaccatggttcatggaaatcttaccgctggtcatggtctcctggttgagttgataa

[0108] tcacatttcaattggtgtttactatctttgccagctgtgattccaaacggactgatgtcactggctcaatagctttagcaattgga

[0109] ttttctgttgcaattggacatttatttgcaatcaattatactggtgccagcatgaatcccgcccgatcctttggacctgcagttatc

[0110] atgggaaattgggaaaaccattggatatattgggttgggcccatcataggagctgtcctcgctggtggcctttatgagtatgt

[0111] cttctgtccagatgttgaattcaaacgtcgttttaaagaagccttcagcaaagctgcccagcaaacaaaaggaagctacatg

[0112] gaggtggaggacaacaggagtcaggtagagacggatgacctgattctaaaacctggagtggtgcatgtgattgacgttga

[0113] ccggggagaggagaagaaggggaaagaccaatctggagaggtattgtcttcagtatga (SEQ ID NO. 5).

[0114] 2. Expression of fusion protein

[0115] (1) Cell seeding

[0116] HEK293 cells were seeded into culture dishes at a density of about 6 million cells per dish so that the density reached 70-80% confluence on the next day.

[0117] (2) Cell transfection

[0118] Before transfection, each dish needs to be replaced with 12mL of complete culture medium. Take two clean sterile centrifuge tubes, add 500μL DMEM high-glucose culture medium to each, then add 10μg of plasmid to one tube, gently blow and mix with a gun, add 20μL PEI transfection agent to the other tube (the mass volume ratio of plasmid to transfection agent is 1:2), mix thoroughly, then use a pipette to gently add the culture solution containing plasmid to the culture solution containing transfection agent, gently blow and mix, let stand at room temperature for 15min to obtain a plasmid-PEI mixed solution; add the prepared plasmid-PEI mixed solution dropwise to the culture dish, gently mix, and place in a cell culture incubator for 48 hours.

[0119] 3. Fusion protein extraction

[0120] A. Observe the transfection efficiency under an inverted microscope and it should be above 90%. Remove the culture medium as cleanly as possible and be careful not to perform any digestion treatment on the cells. (If not used immediately, store in a -80℃ refrigerator).

[0121] B. Add 5 mL of cell lysis solution to the cell culture plate and use a pipette to transfer all the cells to a centrifuge tube. The preparation method of 5 mL of cell lysis solution is to mix 4.95 mL of 1×PBS and 50 μL of 100× protease inhibitor (PI) evenly.

[0122] C. Place the entire tube of cells in liquid nitrogen for 1 min, and freeze and thaw repeatedly three times to destroy the cells.

[0123] D. Removal of cell nuclei and unbroken cells: Centrifuge the cells at 4°C, 1000 g for 10 min and keep the supernatant.

[0124] E. Precipitate cell membrane fragments: Centrifuge the supernatant obtained in step D at 4°C, 14,000 g for 30 min and keep the precipitate.

[0125] F. The precipitated cells obtained in step E are resuspended and dissolved with 500 μL of membrane protein extract to obtain a cell membrane protein solution; wherein, the preparation method of 1 mL of cell membrane protein extract is to mix 50 μL of 1M Tris-HCl with pH 7.4, 150 μL of 1M NaCl, 10 μL of NP-40, 5 μL of Triton-X-100, 10 μL of 100×PI and 775 μL of H2O evenly.

[0126] G. Take 250 μL of His tag purification resin, rinse it with PBS for 3 times, add 500 μL of the obtained cell membrane protein solution, and shake it on an oscillator at room temperature for 60 minutes to obtain a mixed solution.

[0127] H. Centrifuge the mixture obtained in step G at 4°C and 1000g for 5 minutes to obtain the first precipitation resin. Wash the retained first precipitation resin three times with 1×PBS precooled at 4°C to obtain the second precipitation resin. Each washing method is centrifugation at 4°C and 1000g for 5 minutes to obtain the precipitation resin.

[0128] I. The second precipitation resin was washed with 1 mL of 0.5 mM imidazole-containing eluent and 500 μL of 250 mM imidazole-containing eluent, respectively, mixed at 4° C. for 60 min, and centrifuged at 1000 g for 5 min to obtain the supernatant to obtain the fusion protein.

[0129] The expression of fusion protein was detected by SDS-PAGE. Figure 1 At the same time, the fusion protein sequence obtained by sequencing is shown in SEQ ID NO.5.

[0130] Figure 1 The results showed that the present invention successfully obtained the fusion protein shown in SEQ ID NO.5.

[0131] Example 2

[0132] An ELISA kit for improving the accuracy of detecting AQP4-IgG comprises: a microplate coated with a fusion protein, a secondary antibody against human IgG labeled with HRP, a standard substance, a quality control substance, a phosphate buffer with a volume fraction of 0.1% Tween, tetramethylbenzidine (TMB) and a 0.5M sulfuric acid solution.

[0133] Wherein, the microplate coated with the target protein is prepared as follows: the fusion protein prepared in Example 1 is diluted to 10 μg / mL with phosphate buffer (pH 7.4), and the obtained 10 μg / mL fusion protein solution is used to coat a 96-well microplate, 100 μL of the 10 μg / mL fusion protein solution is added to each well, incubated at 4°C overnight, and blocked with 200 μL of phosphate buffer (PBS) containing 1% bovine serum albumin (BSA) by volume per well, incubated at 37°C for 1 hour, and washed 3 times with PBS buffer to obtain a microplate coated with the fusion protein;

[0134] Preparation of the HRP-labeled anti-human IgG secondary antibody: dilute the horseradish peroxidase (HRP)-labeled anti-human IgG secondary antibody with a PBS buffer containing 1% BSA by volume to a volume ratio of 1:5000 to obtain the HRP-labeled anti-human IgG secondary antibody. The HRP-labeled anti-human IgG secondary antibody is used to form a complex with AQP4-IgG in the sample and generate a signal.

[0135] TMB is used as an enzyme substrate to react with horseradish peroxidase to produce a measurable color change, thereby obtaining an ELISA kit for improving the accuracy of detecting AQP4-IgG.

[0136] The standard is a sample diluent containing different concentrations of anti-AQP4 antibodies, and the five concentrations are 81RU / mL, 27RU / mL, 9RU / mL, 3RU / mL and 1RU / mL.

[0137] The quality control products include positive quality control and negative quality control. The positive quality control is a sample diluent containing anti-AQP4 antibodies with a concentration range of 5 to 10 RU / mL; the negative quality control is a sample diluent not containing anti-AQP4 antibodies.

[0138] Comparative Example 1

[0139] The difference between this comparative example and Example 2 is that the target protein of this comparative example is the M1 type target protein with a nucleotide sequence of SEQ ID NO.1 to replace the fusion protein prepared in Example 1, and a microplate coated with the M1 type target protein alone is obtained, and finally an ELISA kit with the M1 type alone as the coated antigen is obtained, and the other components of the kit are the same as those in Example 2, and an ELISA kit with the M1 type alone as the coated antigen is obtained.

[0140] Example 3

[0141] The purpose of this example is to simultaneously detect AQP4-IgG in 40 clinical sera using the ELISA kit provided in Example 2 of the present invention and the ELISA kit with M1 type alone as the coated antigen in Comparative Example 1, and to compare with the indirect fluorescence detection method (CBA) based on cell transfection, which is considered to be the "gold standard". The specific steps are as follows:

[0142] 1. Detect AQP4-IgG in serum using the ELISA kit of Example 2 or Comparative Example 1

[0143] (1) Place the kit at room temperature, take out the microplate coated with the fusion protein or the microplate coated with the M1 target protein, add 100 μL (1:100 dilution) of the serum sample to be tested diluted with the sample diluent, the standard, and the negative and positive quality controls to each well, and incubate at 37°C for 30 min to allow the AQP4-IgG in the serum sample to be tested to bind to the coated fusion protein or the M1 target protein (antigen);

[0144] (2) removing the sample from the microplate wells, adding 0.1% Tween phosphate buffer, 300 μL per well, letting stand for 10 s, removing the liquid from the wells, repeating three times to wash away the unbound sample and obtain the AQP4-IgG complex;

[0145] (3) Add anti-human IgG secondary antibody labeled with horseradish peroxidase (HRP) to the microplate, add 100 μL to each well, and incubate at 37°C for 30 min to allow the secondary antibody to bind to the AQP4-IgG complex;

[0146] (4) Add 0.1% Tween phosphate buffer, 300 μL per well, let stand for 10 s, remove the liquid in the well, repeat three times to wash away the unbound complex;

[0147] (5) Add TMB enzyme substrate to the wells, add 100 μL to each well, incubate at room temperature for 15 min, allow the enzyme to react with the substrate, and produce a color change; add stop solution (0.5 M sulfuric acid solution) to the microplate, add 50 μL to each well to terminate the reaction, and the color of the solution changes from blue to yellow; read the absorbance (OD) of the microplate with an ELISA reader at a wavelength of 450 nm, and calculate the concentration of AQP4-IgG. The depth of the solution color is proportional to the concentration of the antibody;

[0148] (6) Draw a standard curve: Use the concentration of the calibrator as the horizontal axis (X-axis) and the absorbance OD value of the corresponding calibrator as the vertical axis (Y-axis), and use double logarithmic (LOG10) linear regression or other appropriate mathematical models to fit the dose-response curve to make a standard curve;

[0149] (7) Substitute the absorbance OD value of the sample to be tested into the linear regression equation of the standard curve to calculate the AQP4-IgG concentration value of the sample to be tested.

[0150] When the ELISA kit provided in Example 2 of the present invention is used for detection, when the AQP4-IgG concentration of the serum sample to be tested is greater than 3RU / mL, it can be judged as positive, that is, the serum sample to be tested contains the antibody AQP4-IgG.

[0151] 2. Use CBA test kit for testing

[0152] (1) Place the kit at room temperature, take out the slide, add 30 μL of the serum sample to be tested diluted with sample diluent (1:10 dilution) to the reaction area of ​​the slide, and incubate at 37°C for 1 h to allow the AQP4-IgG in the sample to bind to the cell antigen on the slide;

[0153] (2) adding washing solution and repeating three times to wash away the unbound sample to obtain the AQP4-IgG complex;

[0154] (3) Drain the slide thoroughly, add 30 μL of fluorescent secondary antibody working solution to the slide reaction area, and incubate at 37°C for 30 min to allow the secondary antibody to bind to the AQP4-IgG complex;

[0155] (4) Add washing solution and repeat three times;

[0156] (5) Add 30 μL of washing solution (phosphate buffer containing 0.1% Tween by volume) to each reaction zone;

[0157] (6) Observe with a fluorescence microscope and interpret the results. Green fluorescence indicates that the serum sample to be tested contains the antibody AQP4-IgG.

[0158] 3. Results Analysis

[0159] Table 1 Comparison of results between the ELSIA kit of the present invention and the CBA kit

[0160]

[0161] Note: “+” indicates that the serum sample to be tested contains antibody AQP4-IgG, and “-” indicates that the serum sample to be tested does not contain antibody AQP4-IgG.

[0162] Table 2 Comparison of results between M1 ELSIA kit and CBA kit

[0163]

[0164] The results of Table 1 and Table 2 show that the detection consistency of the ELSIA kit of the present invention and the CBA kit is 100%, while the detection consistency of the M1 type ELSIA reagent alone and the CBA reagent is not completely consistent, with a consistency rate of 95%. Therefore, the detection accuracy of the ELSIA kit of the present invention is higher.

[0165] Example 4

[0166] The evaluation method and results of the analytical performance of the kit of Example 2 of the present invention are as follows, including the evaluation method and results of the minimum detection limit, linear range and repeatability.

[0167] 1. Minimum detection limit

[0168] 1.1. Detection method: The sample diluent (PBS) was used as the sample to be tested. The detection method was carried out by referring to the method for detecting AQP4-IgG in serum in part "1" of Example 3. The measurement was repeated 20 times to obtain the absorbance value (OD value) of the 20 measurement results. The mean value (M) and standard deviation (SD) were calculated to obtain the OD value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the calibration curve equation of the calibrator used in the kit or the concentration-OD value results between the zero-concentration calibrator and the adjacent calibrator. The OD value corresponding to M+2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit.

[0169] 1.2 Acceptance criteria: no more than 0.5RU / mL.

[0170] Table 3 Calibrator results

[0171] Concentration (RU / mL) OD value 1 OD value 2 Average OD 81 1.3253 1.3776 1.351 27 0.576 0.5974 0.587 9 0.2315 0.2532 0.242 3 0.1013 0.1073 0.104 1 0.0418 0.0466 0.044

[0172] Based on the results in Table 3, a standard curve for calculating sample concentrations was drawn, and the detection limit concentration was obtained according to the minimum detection limit results in Table 4.

[0173] Table 4 Minimum detection limit results

[0174]

[0175]

[0176] The results in Table 4 show that the minimum detection limit of the kit of the present invention is 0.24 RU / mL.

[0177] 2. Linear range

[0178] 2.1 Inspection methods

[0179] The calibrators of 5 concentrations were tested for linearity, and each concentration sample was tested twice, and the average concentration was calculated. The average result and the dilution ratio were fitted with a linear line using the least squares method. The linear correlation coefficient r was calculated. The test method was carried out according to the method for detecting AQP4-IgG in serum in part "1" of Example 3.

[0180] 2.2 Acceptance criteria: r not less than 0.9900.

[0181] Table 5 Linear range results

[0182]

[0183] According to the results in Table 5, the calculated linear correlation coefficient r is 0.9996. Therefore, the linearity of AQP4-IgG detection using the kit of the present invention is good.

[0184] 3. Repeatability

[0185] 3.1 Inspection methods

[0186] The same batch of the kit of Example 2 of the present invention was used to test AQP4 antibody samples at two concentration levels of 15 RU / mL and 27 RU / mL (respectively recorded as sample 1 and sample 2), and each test was repeated 10 times. The mean M and standard deviation SD of the 10 test concentrations were calculated, and the coefficient of variation CV was obtained according to the formula CV=SD / M×100%.

[0187] 3.2 Acceptance criteria: CV no greater than 12%.

[0188] 3.3 Results:

[0189] Table 6 Repeatability results

[0190]

[0191]

[0192] The results in Table 6 show that the CV of AQP4-IgG detected by the kit of the present invention is no more than 12%, that is, the kit of the present invention has good repeatability in detecting AQP4-IgG.

[0193] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fusion protein for detecting aquaporin 4 antibody, characterized in that: The nucleotide sequence of the fusion protein is shown in SEQ ID NO.

5.

2. A method for preparing the fusion protein according to claim 1, characterized in that: The following steps are involved: The coding gene sequences of M1-type AQP4 and M23-type AQP4 were double-digested and then connected to the backbone vector to obtain a recombinant vector; transforming the recombinant vector into Escherichia coli, amplifying the recombinant vector, and obtaining the amplified recombinant vector; The amplified recombinant vector is transfected into human embryonic kidney cells HEK293, and cultured to obtain transfected cells; Finally, the fusion protein in the transfected cells was extracted.

3. The preparation method according to claim 2, characterized in that The coding gene sequence of the M1 type AQP4 is shown in SEQ ID NO.1, and the coding gene sequence of the M23 type AQP4 is shown in SEQ ID NO.3; The double enzymes are EcoRI enzyme and XhoI enzyme; the backbone vector is pET-28a(+) vector.

4. The preparation method according to claim 2, characterized in that The Escherichia coli includes Escherichia coli DH5α.

5. The preparation method according to claim 2, characterized in that The transfection method comprises: adding the amplified recombinant vector and PEI transfection reagent into human embryonic kidney cells HEK293, mixing, standing, culturing, and obtaining transfected cells.

6. The preparation method according to claim 2, characterized in that The extraction method comprises: mixing the transfected cells with a cell lysate, repeatedly freezing and thawing, centrifuging at 2-6°C and 800-1200g for 5-15min to obtain a supernatant, centrifuging the supernatant at 2-6°C and 12000-16000g for 25-35min to obtain a precipitate, dissolving the precipitate with a membrane protein extract to obtain a cell membrane protein solution; mixing a His purification resin with the cell membrane protein solution to obtain a mixed solution, centrifuging the mixed solution at 2-6°C and 800-1200g for 5-15min, obtaining a first precipitation resin, washing the first precipitation resin to obtain a second precipitation resin, eluting with an elution solution containing imidazole, centrifuging to obtain a supernatant to obtain a fusion protein.

7. Use of the fusion protein according to claim 1 or the fusion protein obtained by the preparation method according to any one of claims 2 to 6 in the preparation of an antibody product for detecting aquaporin 4.

8. A kit for detecting aquaporin 4 antibodies, characterized in that: The kit comprises the fusion protein according to claim 1 or the fusion protein obtained by the preparation method according to any one of claims 2 to 6.

9. The kit according to claim 7, characterized in that The kit includes an ELISA kit.

10. The kit according to claim 9, characterized in that The kit also includes a secondary antibody labeled with horseradish peroxidase, a standard substance, a quality control substance, an enzyme substrate, a washing solution and a stop solution.

Images