A neutralizing antibody for vitamin D binding protein and its preparation method and application

Abstract

The present invention discloses a neutralizing antibody of vitamin D binding protein and its preparation method and application, and relates to the field of antibody technology. The neutralizing antibody of the present invention comprises a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region, and the amino acid sequences of its complementary determining regions CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO.3 Nos. 25 to 32, 50 to 56, and 93 to 102, respectively; the light chain comprises a light chain variable region, and the amino acid sequences of its complementary determining regions CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO.5 Nos. 27 to 34, 52 to 54, and 91 to 101, respectively. The neutralizing antibody of the present invention can effectively neutralize vitamin D binding protein in cells, and can be used to treat nerve damage caused by vitamin D binding protein.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a neutralizing antibody of vitamin D binding protein and a preparation method and application thereof. Background Art

[0002] Vitamin D binding protein (VDBP) is a multifunctional protein. As the main transporter of vitamin D, it plays an important role in maintaining the total level of vitamin D in the body and regulating the level of free vitamin D. Related studies have shown that VDBP can bind fatty acids and actin monomers to prevent them from polymerizing in the circulatory system and playing a harmful role. At the same time, based on the immune regulation function of VDBP independent of vitamin D transport, further studies have found that VDBP has a certain relationship with the occurrence and development of inflammation and tumors.

[0003] Neutralizing antibodies are antibodies that can neutralize the activity of viruses, toxins or other pathogens, and are usually used to prevent and treat infectious diseases. In the related art, the techniques for preparing neutralizing antibodies mainly include methods such as immunizing animals, cell culture and protein expression. In the laboratory, neutralizing antibodies can be induced by injecting target antigens into immunized animals (such as mice, rabbits, etc.), and then the antibodies can be collected through serum or cell culture fluid. In addition, cell culture technology can also be used to introduce genes encoding specific antibodies into mammalian cells or eukaryotic expression systems, and neutralizing antibodies can be obtained by expression and purification. However, despite the diverse methods for preparing neutralizing antigens, neutralizing antibodies against vitamin D binding protein have not been reported.

[0004] Based on this, the present invention aims to provide a neutralizing antibody for vitamin D binding protein, which can effectively neutralize the expression level of VDBP in cells and help improve the protective effect on neurons. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an antibody or an antigen-binding fragment thereof of vitamin D binding protein, which has excellent affinity for vitamin D binding protein, can effectively neutralize the expression of VDBP in cells, and can improve the protective effect on neurons.

[0006] The present invention also provides biological materials related to the above antibody or antigen-binding fragment thereof.

[0007] The present invention also provides a conjugate.

[0008] The invention also provides a product.

[0009] The present invention also proposes the use of the above-mentioned antibody or its antigen-binding fragment and biological material in preparing a reagent for detecting vitamin D binding protein or preparing a drug for treating neuronal damage.

[0010] The present invention also provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof.

[0011] In a first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof to a vitamin D binding protein, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain;

[0012] The heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3;

[0013] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.3, positions 25 to 32;

[0014] The amino acid sequence of the CDR-H2 is shown in positions 50 to 56 of SEQ ID NO.3;

[0015] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.3, positions 93 to 102;

[0016] The light chain comprises a light chain variable region, wherein the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3;

[0017] The amino acid sequence of the CDR-L1 is shown in positions 27 to 34 of SEQ ID NO.5;

[0018] The amino acid sequence of the CDR-L2 is shown in positions 52 to 54 of SEQ ID NO.5;

[0019] The amino acid sequence of the CDR-L3 is shown in positions 91 to 101 of SEQ ID NO.5.

[0020] The antibody or antigen-binding fragment thereof of the vitamin D binding protein according to the embodiments of the present invention has at least the following beneficial effects:

[0021] The present invention screens and prepares, for the first time, an antibody or an antigen-binding fragment thereof of vitamin D binding protein, which can effectively neutralize the expression level of VDBP in cells and can be used to treat nerve damage caused by VDBP.

[0022] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region comprises any one of A1) to A3):

[0023] A1) the amino acid sequence shown in SEQ ID NO.3;

[0024] A2) an amino acid sequence having the same function as the sequence shown in SEQ ID NO.3 after one or more amino acids are substituted and / or deleted and / or added;

[0025] A3) an amino acid sequence having 80%, 85% or 90% or more homology with SEQ ID NO.3 and having the same function as the sequence shown in SEQ ID NO.3;

[0026] The amino acid sequence of the light chain variable region comprises any one of B1) to B3):

[0027] B1) the amino acid sequence shown in SEQ ID NO.5;

[0028] B2) an amino acid sequence in which one or more amino acids in SEQ ID NO.5 are substituted and / or deleted and / or added, and which has the same function as the sequence shown in SEQ ID NO.5;

[0029] B3) An amino acid sequence that has 80%, 85% or 90% or more homology with SEQ ID NO.5 and has the same function as the sequence shown in SEQ ID NO.5.

[0030] Herein, the term "homology" refers to the similarity between amino acid sequences or nucleotide sequences, which can be evaluated by computer software. It is understood that an amino acid sequence having more than 80% homology with SEQ ID NO.3 and having the same function as the sequence shown in SEQ ID NO.3 refers to an amino acid sequence that is identical to the neutralizing antibody heavy chain variable region in SEQ ID NO.3 and is obtained by adjusting the connecting peptide amino acids.

[0031] Herein, the 80% or more homology can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0032] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, and a multispecific antibody.

[0033] In some embodiments of the invention, the heavy chain further comprises a heavy chain constant region; and / or

[0034] The light chain also comprises a light chain constant region.

[0035] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is as shown at positions 133 to 455 in SEQ ID NO.8; and the light chain constant region is as shown at positions 133 to 236 in SEQ ID NO.9.

[0036] In some embodiments of the present invention, the heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown as positions 20 to 455 of SEQ ID NO.8; and the light chain amino acid sequence is shown as positions 21 to 236 of SEQ ID NO.9.

[0037] The second aspect of the present invention provides a biomaterial related to the antibody or antigen-binding fragment thereof according to any one of the first aspect, wherein the biomaterial comprises any one of C1) to C5);

[0038] C1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of the first aspects;

[0039] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0040] C3) a recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2);

[0041] C4) a recombinant microorganism containing the nucleic acid molecule described in C1) or the expression cassette described in C2) or the recombinant vector described in C3);

[0042] C5) A recombinant cell comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3), wherein the recombinant cell does not contain any propagation material.

[0043] Those skilled in the art can mutate the nucleotide sequence using known methods (such as directed evolution and point mutation). Those artificially modified nucleotide sequences that have a certain identity with the antibody or antigen-binding fragment thereof described in the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the nucleotide sequence of the present invention as long as they encode the antibody or antigen-binding fragment or variant thereof and have the same function as the antibody or antigen-binding fragment thereof.

[0044] In some embodiments of the present invention, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region;

[0045] Among them, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown as SEQ ID NO.2; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO.4.

[0046] In some embodiments of the present invention, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain;

[0047] Among them, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain is shown as SEQ ID NO.6; the nucleotide sequence of the nucleic acid molecule encoding the light chain is shown as SEQ ID NO.7.

[0048] In some embodiments of the present invention, the expression cassette refers to a DNA capable of expressing the antibody or antigen-binding fragment thereof in a host cell, and the DNA may include not only a promoter for initiating transcription of the DNA molecule encoding the antibody or antigen-binding fragment thereof, but also a terminator for terminating transcription of the DNA molecule encoding the antibody or antigen-binding fragment thereof. Further, the expression cassette may also include a replication initiation site, a transcription initiation sequence, an enhancer sequence, a selection element or a reporter gene.

[0049] In some embodiments of the present invention, the recombinant vector may be a plasmid, a phage, a lentivirus, an adenovirus, or an adeno-associated virus. The recombinant vector is used to achieve replication, integration, amplification, and / or expression of an exogenous target gene in a recipient cell; it may be a cloning vector or an expression vector. For example, the recombinant vector in C3) may specifically be a recombinant plasmid obtained by inserting the nucleic acid molecule into a CMV vector.

[0050] In some embodiments of the present invention, the recombinant microorganism may be a bacterium (such as Escherichia coli or Bacillus subtilis, etc.) or a fungus (such as yeast or Aspergillus, etc.). For example, the recombinant microorganism in C4) may specifically be Escherichia coli DN5α.

[0051] In some embodiments of the present invention, the recombinant cell may be an insect cell (such as S2 Drosophila cell or Sf9 cell, etc.), an animal cell (such as B lymphocyte, 293T cell, 293F cell, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell or HEK293 cell, etc.) or a plant cell (such as Arabidopsis or tobacco, etc.).

[0052] In some embodiments of the present invention, the recombinant cell may be a hybridoma cell obtained by fusion of spleen cells and SP2 / 0 cells.

[0053] The third aspect of the present invention provides a conjugate, comprising: the antibody or antigen-binding fragment thereof according to any one of the first aspect;

[0054] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a radionuclide, and an enzyme.

[0055] A fourth aspect of the present invention provides a product, the product comprising at least one of D1) to D3);

[0056] D1) The antibody or antigen-binding fragment thereof according to any one of the first aspects;

[0057] D2) The biomaterial according to any one of the second aspects;

[0058] D3) the conjugate as described in the third aspect;

[0059] The product is selected from at least one of a drug, a test plate, a chip, a test paper and a test kit.

[0060] The fifth aspect of the present invention provides the use of any one of E1) to E3) in the preparation of a reagent for detecting vitamin D binding protein or a drug for treating neuronal damage:

[0061] E1) The antibody or antigen-binding fragment thereof according to any one of the first aspects;

[0062] E2) The biomaterial according to any one of the second aspects;

[0063] E3) The conjugate as described in the third aspect.

[0064] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient, and the dosage used is non-toxic to the recipient.

[0065] In some embodiments of the present invention, the auxiliary material includes at least one of a solvent, a dispersant, a diluent, a filler, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier, an excipient, a stabilizer, a buffer, an isotonic agent, a flavoring agent, and a carrier. The auxiliary material is used to promote the absorption of the antibody or its antigen-binding fragment to exert a preventive and / or therapeutic effect.

[0066] The sixth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising: obtaining the antibody or the antigen-binding fragment thereof by culturing the recombinant microorganism or the recombinant cell in the biological material as described in the second aspect.

[0067] In some embodiments of the present invention, the antibodies or antigen-binding fragments thereof of the present invention can be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), or can also be obtained using recombinant DNA technology (see, for example, Journal of Virological Methods, 2009, 158(1-2):171-179).

[0068] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0070] Figure 1 The SDS-PAGE result diagram of each sample during the purification process of the target protein of the present invention;

[0071] Figure 2 This is the SDS-PAGE result after 80ml XtenCHO was transfected with the target protein of the present invention;

[0072] Figure 3 This is the SDS-PAGE result after 330ml XtenCHO was transfected with the target protein of the present invention;

[0073] Figure 4 This is the SDS-PAGE result after transfection of 480ml XtenCHO with the target protein of the present invention;

[0074] Figure 5 This is a diagram showing the SDS-PAGE results of the 6A2 antibody of the present invention;

[0075] Figure 6 The Western Blot analysis results of different cell treatment groups of the present invention;

[0076] Figure 7 These are the results of the effect of the VDBP neutralizing antibody 6A2 of the present invention on the complexity of neuronal dendrites, wherein A is the result of immunofluorescence staining, and B is the result of statistical analysis. DETAILED DESCRIPTION

[0077] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0078] In the description of the present invention, reference terms such as "preferably", "more preferably", etc. refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0079] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0080] In the description of the present invention, the description with reference to terms such as "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.

[0081] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0082] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0083] 1. Acquisition of target antigens

[0084] 1. Construction of pATX2-VDBP recombinant vector

[0085] The gene sequence of vitamin D binding protein was obtained from the National Center for Biotechnology Information (NCBI) database, and the gene was optimized according to the codon preference of Escherichia coli. The optimized VDBP nucleotide sequence information is as follows:

[0086]

[0087] Among them, positions 1 to 15 in SEQ ID NO.1 are connection sequences, positions 16 to 1445 are VDBP gene coding sequences, and positions 1446 to 1481 are connection sequences containing His tag sequences.

[0088] The optimized VDBP gene sequence was cloned into pATX2 vector EcoR I and Not I restriction site was recombined into the pATX2 vector to obtain the recombinant plasmid pATX2-VDBP. The obtained recombinant plasmid pATX2-VDBP was sequenced and verified, and the results were consistent with expectations.

[0089] 2. Small sample purification

[0090] (1) Pre-purification analysis

[0091] Using FectoPRO ® The transfection kit was used to transfect 80 mL (1×10 6 Cells / mL) were cultured in FreeStyle™ CHO Expression Medium. On day 6 after transfection, 1.0 mL of sample was taken and purified using the following steps:

[0092] S1. Place the sample in a high-speed refrigerated centrifuge (model: Neofuge 15R) and centrifuge at 10,000 g to separate the culture supernatant and bacterial precipitate, and obtain the culture supernatant sample (labeled as Medium) and bacterial precipitate respectively;

[0093] S2. Add protease inhibitors to the bacterial pellet, resuspend and mix, place on crushed ice, and use an ultrasonic crusher to ultrasonically disrupt the bacteria (specific parameters are: 250W, ultrasonic 4s, interval 7s, 99 cycles), centrifuge at 13000g, 4℃ for 5min, separate the supernatant and the pellet, resuspend the pellet with an equal volume of PBS to obtain the supernatant sample (marked as NPE) and the pellet sample (marked as DPE) after bacterial disruption, respectively.

[0094] S3. Add 10 μL of 6× Protein Loading Buffer (provided by Beijing Quanshijin Biotechnology Co., Ltd.) to the culture supernatant sample, supernatant sample after bacterial cell disruption, and precipitate sample after bacterial cell disruption, vortex mix, centrifuge instantaneously, and place in a metal bath at 100°C for 10 min. Perform SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining on the samples after the metal bath.

[0095] The results of SDS-PAGE were as follows: Figure 1As shown, the theoretical molecular weight of the target antigen is 54.77 kDa (including His tag), which is mainly expressed in the culture supernatant. The target antigen is subsequently obtained by purifying the culture supernatant.

[0096] (2) Small sample purification process

[0097] Based on the above transfection system, the culture was continued until the 9th day, and then the culture supernatant was collected and purified by Ni-NTA affinity chromatography using the His tag. The specific steps are as follows:

[0098] The collected culture supernatant was loaded onto a chromatography column (Ni-NTA resin) equilibrated with an equilibrium solution (PBS, pH=7.5, 30mM Imidazole). After loading, the column was first rinsed with a washing solution, and then a gradient elution of 0%-100% was performed with an eluent. The eluted components were tested by SDS-PAGE protein electrophoresis. The eluent was collected according to the test results, and the eluent was dialyzed into an equilibrium solution, and the dialyzate was collected for SDS-PAGE electrophoresis. Among them, the equilibrium solution contained PBS (pH=7.5) and 30mM Imidazole; the washing solution contained PBS (pH=7.5) and 50mM Imidazole; and the eluent contained PBS (pH=7.5) and 300mM Imidazole.

[0099] The results of SDS-PAGE electrophoresis are as follows Figure 2 As shown, the band position is consistent with the expectation. Further, quantitative analysis showed that 80 mL of XtenCHO cells were transfected with the recombinant plasmid pATX2-VDBP, and about 1.04 mg of the target antigen was obtained after 9 days of culture. The target antigen obtained by the above purification was stored in a PBS buffer with a pH of 7.5 for future use.

[0100] 3. Scale-up and purification

[0101] The FectoPRO® transfection kit was used to transfect 330 mL (1×10 6 cells / mL) XtenCHO cells were cultured in FreeStyle™ CHO expression medium. The culture supernatant was collected on the 9th day after transfection for purification and SDS-PAGE protein electrophoresis detection. The specific purification method refers to the above sample purification process. The SDS-PAGE protein electrophoresis detection results are shown in Figure 3As shown, the band position is consistent with the expectation, and quantitative analysis shows that 330mL XtenCHO cells can be transfected with the recombinant plasmid pATX2-VDBP and about 1.76mg of the target antigen can be obtained after 9 days of culture. The purified target antigen is stored in a PBS buffer with a pH of 7.5 for later use.

[0102] The FectoPRO® transfection kit was used to transfect 480 mL (1×10 6 cells / mL) XtenCHO cells (6 groups, 80 mL) were cultured in FreeStyle™ CHO expression medium. The culture supernatant was collected on day 9 after transfection for purification and SDS-PAGE protein electrophoresis detection. The specific purification method refers to the small sample purification process mentioned above. The results of SDS-PAGE protein electrophoresis are shown in Figure 4 As shown, the band position is consistent with the expectation, and quantitative analysis shows that 330mL XtenCHO cells can be transfected with the recombinant plasmid pATX2-VDBP and about 2.70mg of the target antigen (VDBP) can be obtained after 9 days of culture. The purified target antigen is stored in a PBS buffer with a pH of 7.5 for later use.

[0103] 2. Antigen Immunization and Serum Titer Determination

[0104] (1) VDBP immunization of New Zealand white rabbits

[0105] Three female New Zealand white rabbits aged 4 to 5 months were randomly divided into two groups: one group was the control group, which included one New Zealand white rabbit (not immunized); the other group was the immunization group, which included two New Zealand white rabbits.

[0106] Pierce™ BCA Protein Assay Kits (Cat. No.: 23227, Thermo) were used to determine the VDBP concentration. VDBP was then diluted with PBS to the target concentration and mixed with Freund's adjuvant at a volume ratio of 1:1. After sufficient emulsification, New Zealand white rabbits were immunized. The specific immunization scheme is shown in Table 1.

[0107] Table 1: Immunization schedule

[0108]

[0109] According to the above immunization scheme, the emulsified reagent was injected intradermally (2 points) and subcutaneously (3 points) in the abdomen of New Zealand white rabbits, with 200 μL injected at each site. Blood was collected after the third and fourth immunizations to obtain New Zealand white rabbit serum.

[0110] (2) ELISA test for serum titer

[0111] The above-collected New Zealand white rabbit serum was tested by ELISA method, and the specific steps are as follows:

[0112] S1. Coating: Dilute VDBP to a concentration of 5 μg / mL with PBS, add 100 μL / well to the ELISA plate, and incubate at 37°C for 2 h.

[0113] S2, blocking: spin dry the coating solution in the ELISA plate, add 300 μL PBS (containing 3% BSA) to each well, cover the plate and incubate in a 37°C incubator for 1 hour (or 4°C overnight);

[0114] S3, washing: take out the coated and blocked ELISA plate, throw the blocking solution into the water pool, add 300 μL PBST to each well, and wash 3 times;

[0115] S4. Primary antibody incubation: dilute the serum with blocking solution by 2000, 4000, 8000, 16000, 32000, 64000, and 128000 times, add 100 μL / well to the ELISA plate, incubate at 37°C for 1 hour, spin dry, wash 3 times with PBST (300 μL per well), and then pat dry on absorbent paper towels;

[0116] S5. Secondary antibody incubation: dilute the enzyme-labeled antibody Anti-Rabbit IgG (H+L) (min X Hu SrProt) (brand AntibodySystem, catalog number PTB96431) at 1:10000 with blocking solution, add 100 μL / well to the ELISA plate, incubate at 37°C for 1 hour, shake off the secondary antibody, wash three times with PBST (300 μL per well), and then pat dry on absorbent paper towels;

[0117] S6, TMB color development: Pour the prepared TMB color development solution into a clean sample loading tank covered with PE gloves, and use a dispenser to add 100 μL per well to the ELISA plate in sequence, cover the lid and react at 37°C in the dark for 15 min, and observe the color development;

[0118] S7, termination: turn on the microplate reader, preheat for 1 minute, take out the microplate from the incubator, and add 2 M HCl to each well of the microplate in turn at a volume of 50 μL;

[0119] S8. Reading: Open the Thermo ELISA reader software, select 450nm wavelength, wipe the bottom of the ELISA plate with an absorbent towel and put it into the card slot of the ELISA reader, click start reading to determine the OD value. Use the blocking solution without serum as the blank control.

[0120] The rabbit serum titer was the maximum dilution when (immunization group rabbit serum OD value - blank control OD value) / (control group rabbit serum OD value - blank control OD value) ≥ 2.1. The test results are shown in Table 2.

[0121] Table 2: Serum titers after the third and fourth immunizations

[0122]

[0123] The test results showed that as the number of immunizations increased, the rabbit serum titer also continued to increase, and after the third immunization, its highest titer reached 1:128000.

[0124] 3. Monoclonal cell screening

[0125] 1. Single B lymphocyte sorting

[0126] The New Zealand white rabbit with the highest titer after four immunizations was intraperitoneally injected with 500 μL of PBS containing 100 μg VDBP three days in advance for booster immunization; spleen tissue was aseptically separated and peripheral blood was obtained, and PBMC cells were separated from the two materials using lymphocyte separation fluid. Then, about 960 single B lymphocyte clones (10*96-well plates, enriched by antigen) were isolated and cultured using B lymphocyte staining and flow sorting. After the 7th day of culture, the supernatant was taken to detect the titer of each monoclonal cell line according to the above-mentioned ELISA method for detecting rabbit serum titer, and positive monoclonal cells with relatively high titers were screened out.

[0127] 2. Monoclonal cell culture

[0128] The positive monoclonal cells with higher titer were blown off, washed once with complete medium, resuspended, and the cell density was adjusted to 10 4 Cells were diluted 1000 times by limiting dilution method and added to 96-well plates at 100 μL / well for monoclonal cloning. After 7-10 days of culture, the cells were observed under a microscope to see if there were monoclonal cell lines and stored for later use.

[0129] IV. Amplification and sequencing analysis of variable regions of positive monoclonal antibodies

[0130] The antibody variable region gene VH and VL fragments of the above positive monoclonal cells were amplified by PCR, and then subcloned into an expression vector containing the rabbit IgG constant region for sequencing analysis. Sequence alignment analysis was performed in the IMGT database to obtain the sequence information of the VH and VL fragments of the positive cloned antibody. The results showed that the nucleotide sequence information of the heavy chain variable region VH was as follows:

[0131] CAGGCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAACTTCTACATGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGCTCGGAGTTATTGCTACTGGTGGTAACACATAC TACGCGAGCTGGGCGAAAGGCCGATTCACCATTTCCAAAACCTCGACCACGGTAGATCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGTCAGAGGTGATCCTGCTTATCCTTACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO.2);

[0132] The amino acid sequence information of the heavy chain variable region VH is as follows:

[0133] QAVEESGGRLVTPGTPLTLTCTVSGFSLSNFYMQWVRQAPGKGLEWLGVIATGGNTYYASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCVRGDPAYPYLWGQGTLVTVSS (SEQ ID NO.3). Among them, the amino acid sequence of the heavy chain variable region CDR-H1 is GFSLSNFY (as shown in SEQ ID NO.3, positions 25 to 32); the amino acid sequence of the heavy chain variable region CDR-H2 is IATGGNT (as shown in SEQ ID NO.3, positions 50 to 56); and the amino acid sequence of the heavy chain variable region CDR-H3 is VRGDPAYPYL (as shown in SEQ ID NO.3, positions 93 to 102).

[0134] The nucleotide sequence information of the light chain variable region VL is as follows:

[0135] GCGCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTCGTAGTAGTAACAATTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACTGGGCATCCAAACTGGT ATCTGGGGTCCCATCCCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCACTTATTATAATAGTGGTTGGTCTAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA (SEQ ID NO.4).

[0136] The amino acid sequence information of the light chain variable region VL is as follows:

[0137] AQVLTQTPSSVSAAVGGTVTISCQASQSVRSSNNLAWFQQKPGQPPKLLIYWASKLVSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGTYYNSGWSNTFGGGTEVVVK (SEQ ID NO.5). Among them, the amino acid sequence of the light chain variable region CDR-L1 is QSVRSSNN (as shown in SEQ ID NO.5, positions 27 to 34); the amino acid sequence of the light chain variable region CDR-L2 is WAS (as shown in SEQ ID NO.5, positions 52 to 54); and the amino acid sequence of the light chain variable region CDR-L3 is QGTYYNSGWSN (as shown in SEQ ID NO.5, positions 91 to 101).

[0138] 5. Expression, purification and sequencing analysis of neutralizing antibodies

[0139] 1. Antibody expression

[0140] Based on the VH and VL fragment information obtained above, a recombinant vector containing heavy chain and light chain sequences was constructed and then co-transfected into XtenCHO cells, wherein the heavy chain encoding nucleotide sequence information is as follows:

[0141]

[0142] Among them, positions 1 to 57 in SEQ ID NO.6 are the nucleotide sequence encoding the heavy chain signal peptide, positions 58 to 396 are the nucleotide sequence encoding the heavy chain variable region, and positions 397 to 1368 are the nucleotide sequence encoding the heavy chain constant region.

[0143] The light chain encoding nucleotide sequence information is as follows:

[0144] (SEQ ID NO.7).

[0145] Among them, positions 1 to 60 in SEQ ID NO.7 are the nucleotide sequence encoding the light chain signal peptide, positions 61 to 396 are the nucleotide sequence encoding the light chain variable region, and positions 397 to 711 are the nucleotide sequence encoding the light chain constant region.

[0146] After transfection of XtenCHO cells, recombinant cells were obtained, and after culture, the culture supernatant and bacterial precipitate were separated and set aside.

[0147] 2. Antibody purification and sequencing analysis

[0148] (1) Purification sample pretreatment: collect the above cell culture supernatant, further clarify it by high-speed centrifugation at 8500 rpm for 10 min, then filter and clarify the culture medium that has been centrifuged by high-speed centrifugation again using a pre-treated hollow fiber filter device, and finally concentrate the filtered culture medium using a pre-treated tangential flow concentrator for later use. During this period, take 80 μl of small sample (IN) for SDS-PAGE detection;

[0149] (2) Column: Add the purified filler (rProtein A filler, purchased from GE healthcare, catalog number 17547402) to the gravity column, and after the 20% ethanol in the preservation solution has dripped off, add 10 times the column volume of ultrapure water to rinse the ethanol, and finally add 10 times the column volume of PBS pH 7.5 to balance the gravity column; plug the lower end of the gravity column with a plug, and suspend the filler with the sample to be purified, mix it with the sample, put it into a binding shaker, and place the binding shaker in a 4°C refrigerator. The binding time must not be less than 60 min; remove the bound sample from the binding shaker, let it stand in a 4°C refrigerator for 5-10 min, then use a pipette to add the mixture of the sample and filler to the empty column, collect the flow-through sample (FT), and take a 40 μl sample (FT) for SDS-PAGE detection;

[0150] (3) Elution: Wash the column with PBS pH 7.5 to elute the non-specifically bound host proteins, collect the sample (W1), and take a 40 μl sample (W1) for SDS-PAGE detection; then wash one tube with Citric acid for each column volume, add the amount of neutralizing solution tested before, and gently invert to mix. Wash a total of 11 tubes, take a 40 μl sample from each tube, and record it as E1~E11 for SDS-PAGE detection;

[0151] (4) The protein of the purified antibody (labeled as 6A2 antibody) was quantified by the BCA method, and the purified antibody was detected by electrophoresis using SDS-PAGE.

[0152] The results of SDS-PAGE were as follows: Figure 5 As shown, it was consistent with expectations, indicating that the 6A2 antibody was purified successfully.

[0153] Furthermore, the purified 6A2 antibody was sequenced and analyzed, and the results showed that the heavy chain amino acid sequence information of the 6A2 antibody was as follows:

[0154] MKHLWFFLLLVAAPRWVLSQAVEESGGRLVTPGTPLTLTCTVSGFSLSNFYMQWVRQAPGKGLEWLGVIATGGNTYYASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCVRG DPAYPYLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAP STCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTI SKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO.8).

[0155] The sequence shown at positions 1 to 19 in SEQ ID NO.8 is the heavy chain signal peptide sequence, the sequence shown at positions 20 to 132 is the heavy chain variable region sequence, and the sequence shown at positions 133 to 455 is the heavy chain constant region sequence.

[0156] The amino acid sequence information of the light chain of 6A2 antibody is as follows:

[0157] MVLQTQVFISLLLWISGAYGAQVLTQTPSSVSAAVGGTVTISCQASQSVRSSNNLAWFQQKPGQPPKLLIYWASKLVSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGTYYNSGWS NTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO.9).

[0158] The sequence shown at positions 1 to 20 in SEQ ID NO.9 is the light chain signal peptide sequence, the sequence shown at positions 21 to 132 is the light chain variable region sequence, and the sequence shown at positions 133 to 236 is the light chain constant region sequence.

[0159] VI. Neutralizing Antibody Titer Detection

[0160] ELISA was used to detect the binding ability of cell supernatant containing 6A2 antibody to antigen protein. The specific detection method is as follows:

[0161] S1. Coating: Dilute VDBP to a concentration of 5 μg / mL with PBS to obtain coating solution, add 100 μL / well to the ELISA plate, and incubate at 37°C for 2 h;

[0162] S2, blocking: spin dry the coating solution in the ELISA plate, add 300 μL PBS (containing 3% BSA) to each well, cover the plate and incubate in a 37°C incubator for 1 hour (or 4°C overnight);

[0163] S3, washing: take out the coated and blocked ELISA plate, throw the blocking solution into the water pool, add 300 μL PBST to each well, and wash 3 times;

[0164] S4. Primary antibody incubation: The cell supernatant containing 6A2 antibody obtained above was diluted with blocking solution into 7 gradients (the concentrations of 6A2 antibody were 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 μg / mL, respectively). The negative control group did not contain 6A2 antibody (0 μg / mL). 100 μL / well was added to the ELISA plate. After incubation at 37°C for 1 hour, the plate was dried and washed 3 times with PBST (300 μL per well), and then patted dry on absorbent paper towels.

[0165] S5. Secondary antibody incubation: Dilute the enzyme-labeled antibody Anti-Rabbit IgG (H+L) (min X Hu SrProt) at 1:10000 with blocking solution, add 100 μL / well to the ELISA plate, incubate at 37°C for 30 min, shake off the secondary antibody, wash three times with PBST (300 μL per well), and then pat dry on absorbent paper towels;

[0166] S6, TMB color development: Pour the prepared TMB color development solution into a clean sample loading tank covered with PE gloves, and add 100 μL per well to the ELISA plate in sequence using a dispenser, cover the plate and incubate at 37°C in the dark for 10-15 min, and observe the color development;

[0167] S7, termination: turn on the microplate reader, preheat for 1 minute, take out the microplate from the incubator, and add 2 M HCl to each well of the microplate in turn at a volume of 50 μL;

[0168] S8. Reading: Open the Thermo microplate reader software, select 450nm wavelength, wipe the bottom of the microplate with an absorbent towel and put it into the microplate reader slot, click start reading to determine the OD value. Use the cell supernatant without VDBP neutralizing antibody as the negative control. The sample OD450 value is 2.1 times greater than the negative control OD450 value as a positive judgment.

[0169] The test results are shown in Table 3:

[0170] Table 3: Antibody titer test results

[0171]

[0172] The results showed that as the concentration of 6A2 antibody increased, the OD450 value also increased, proving that the titer became higher.

[0173] VII. Western Blot Analysis of Neutralizing Antibodies

[0174] The cells were treated with the VDBP neutralizing antibody (6A2 antibody) obtained above and subjected to Western Blot analysis, wherein the cells were treated and grouped as follows:

[0175] Control group: primary neuronal cells were cultured in normal culture medium without any treatment.

[0176] VDBP-treated group: primary neuronal cells were cultured in normal medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) was added.

[0177] VDBP+6A2 antibody treatment group: primary neuronal cells were cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) and 6A2 neutralizing antibody were added.

[0178] The steps of protein extraction and Western Blot detection are as follows:

[0179] (1) Total protein extraction and quantification:

[0180] ① Sample preparation and protein extraction: Collect cell samples from each group, discard the culture medium and rinse twice with PBS, add RIPA lysis buffer supplemented with protease inhibitors, place the culture flask / dish on ice, use a cell scraper to scrape the cells, and pipette the mixture into a centrifuge tube.

[0181] ② Protein quantification: Refer to the instructions of the BCA protein quantification kit to perform the corresponding operations and determine the protein concentration. Specifically, dilute the protein standard with a concentration of 2µg / µL to 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125µg / µL with ddH2O, and use 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0µg / µL as the detection points of the protein standard curve. Dilute the protein sample to be tested 10 times with ddH2O for use. Mix the A solution and B solution in the kit at a ratio of 50:1, and add 200µL to each well of the 96-well plate. Add the diluted protein standard and the sample to be tested to the AB mixture in the 96-well plate in turn and incubate at 37℃ for 30min. Use an enzyme reader to read the absorbance value at 562nm. A standard curve was drawn based on the concentration of the protein standard and the corresponding absorbance value, and the concentration of each sample to be tested was calculated using the curve.

[0182] ③ Protein denaturation: According to the measured concentration of each sample to be tested, adjust the concentration of each sample to the same with RIPA lysis buffer, add 1 / 4 volume of 5×SDS loading buffer, shake and mix, and heat in a metal bath at 100℃ for 5 minutes to denature the protein. After denaturation, the sample can be used for subsequent experiments or stored at -20℃.

[0183] (2) Protein immunoblotting experiment:

[0184] S1. Gel preparation: Prepare the separation gel of corresponding concentration according to the molecular weight of the detected protein in the above ratio, add appropriate amount of isopropanol to press the gel. After the lower gel solidifies, prepare the upper concentrated gel and insert the comb. Place the prepared gel in the electrophoresis tank, add electrophoresis solution, and remove the comb.

[0185] S2, SDS-PAGE electrophoresis: Take out the sample at -20℃, rewarm and shake to mix. Add protein marker indicator and protein sample in sequence according to the experimental purpose. Perform electrophoresis at 80V constant voltage. After the bromophenol blue indicator enters the separation gel, adjust to 120V constant voltage and continue electrophoresis until the bromophenol blue reaches the bottom edge of the separation gel and the protein marker indicator is completely separated. The electrophoresis is completed.

[0186] S3, transfer: Pre-cool the transfer solution, and place the sponge and filter paper in it for later use. Use methanol to activate the PVDF membrane for 30 seconds. Take out the gel and cut off the concentrated gel. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clip in order, making sure there are no bubbles between the layers, and place them in the transfer tank after clamping. Adjust the current intensity and transfer time of the transfer according to the molecular weight of the protein to be tested.

[0187] S4. Blocking: Place the transferred PVDF membrane in 5% skim milk and shake it slowly on a shaker for 1-2 hours to completely remove the influence of non-specific antigens.

[0188] S5. Primary antibody incubation: Wash the membrane three times with TBST, prepare the primary antibody with 5% BSA-TBST, place the PVDF membrane in the primary antibody, and incubate overnight at 4°C. The antibody dilution ratio is: VDBP (1:1000), β-Actin (1:1000).

[0189] S6, secondary antibody incubation: Remove the PVDF membrane from the primary antibody and wash the membrane 3 times with TBST on a shaker for 10 min each time. Place the PVDF membrane in the corresponding secondary antibody prepared in 5% skim milk-TBST and incubate at room temperature for 1-1.5 hours. Wash the membrane again on a shaker with TBST for 3 times for 10 min each time.

[0190] S7. Exposure: Referring to the ECL reagent instructions, mix solution A and solution B in a ratio of 1:1 to prepare a developer. Add the developer evenly onto the PVDF membrane and use a chemiluminescence imaging system for exposure.

[0191] Test results such as Figure 6 As shown, A is the WB bands of each group, and B is the statistical results of each group, showing that compared with the control group, the expression of synaptic proteins PSD95 and Synapsin I in the VDBP-treated group was significantly decreased, and compared with the VDBP-treated group, the synaptic proteins PSD95 and Synapsin I in the neutralizing antibody 6A2-treated group were restored, indicating that VDBP can damage the synaptic plasticity of neurons, and the neutralizing antibody 6A2 can block VDBP and thus avoid the damage to the synaptic plasticity of neurons.

[0192] 8. Evaluation of Neutralizing Activity of Neutralizing Antibodies

[0193] The neutralizing effect of the VDBP neutralizing antibody (6A2 antibody) obtained above was evaluated by MAP2 immunofluorescence staining, wherein a control group, a VDBP treatment group and a VDBP+6A2 antibody treatment group were set up respectively, wherein:

[0194] Control group: primary neuronal cells were cultured in normal culture medium without any treatment.

[0195] VDBP-treated group: primary neuronal cells were cultured in normal medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) was added.

[0196] VDBP+6A2 antibody treatment group: primary neuronal cells were cultured in normal culture medium, but VDBP (recombinant mouse vitamin D binding protein, purchased from Wuhan Huamei Company) and 6A2 neutralizing antibody were added.

[0197] The specific method of MAP2 immunofluorescence staining is as follows:

[0198] (1) Preparation of reagents and consumables

[0199] Rabbit anti-MAP2 antibody (primary antibody), CoraLite488 labeled goat anti-rabbit IgG (secondary antibody, purchased from Proteintech), 4% paraformaldehyde, Triton (membrane permeabilization agent), BSA or serum from the animal where the secondary antibody comes from, generally goat serum (for blocking), PBS, DAPI and anti-fluorescence quenching mounting medium (anti-fluorescence quenching mounting medium containing DAPI can be used), coverslip or special cell slide, slide, etc.

[0200] (2) Preparation of cell slides

[0201] Pre-treatment of slides: Soak the cut slides or purchased finished slides in concentrated sulfuric acid overnight, rinse with tap water the next day and place in a sterilized lunch box, put gauze on the bottom of the lunch box, and place the slide against the side wall of the lunch box, with the front of the slide not touching the gauze. Then sterilize with high-pressure steam and dry in an oven.

[0202] Treat the slides with poly-lysine, laminin or collagen solution to increase cell adhesion before primary cells or cells that are not firmly attached to the surface can prevent the cells from being washed away when washing with PBS later. After treating the slides with the above cell adhesion-promoting solutions, rinse them with culture medium 1 to 3 times before placing them in culture medium.

[0203] (3) Climbing

[0204] After digesting the cells with trypsin, count the primary neuronal cells and resuspend them in complete culture medium. Before adding cells, according to the size of the slide, first drop 100 μL of culture medium in each well where the slide is to be placed, so that the slide and the culture dish are bonded together by the tension of the culture medium, and then place the slide to prevent the slide from floating up when the cell suspension is added, resulting in a double-layer cell patch (pay attention to aseptic operation during the whole process). After the cells adhere to the wall, they are grouped and treated according to the above-mentioned control group, VDBP treatment group and VDBP + 6A2 antibody treatment group. The amount of VDBP added to the culture medium in the VDBP treatment group and VDBP + 6A2 antibody treatment group is 20 μL (concentration is 50 ng / mL); the amount of 6A2 neutralizing antibody added to the culture medium in the VDBP + 6A2 antibody treatment group is 10 μL (concentration is 0.56 mg / mL), and the slides are taken after 24 hours of treatment.

[0205] (4) Fixation and permeabilization

[0206] Remove the slides, wash with PBS and fix. For the confocal dish, remove the culture medium and add PBS to wash 1-3 times. Be careful not to shake violently during washing. After washing, add a certain volume of 4% paraformaldehyde for fixation. Usually fix at room temperature (RT) for 15-20 minutes. After fixation, wash three times with pre-cooled PBS, and then treat with 0.1% Triton X-100 (permeabilizing agent) for 10 minutes.

[0207] (5) Closed

[0208] The concentration of BSA only needs to be 1 or 5% (dissolved in PBS), and the concentration of serum only needs to be 10%. Block for 30 minutes at RT. If your primary antibody binds strongly or the secondary antibody has non-specific binding, you can add 0.1% Tween20.

[0209] (6) Incubation

[0210] Primary antibody incubation: After blocking, wash with PBS three times and then incubate with primary antibody. The primary antibody can be diluted with blocking solution, or with PBS or antibody diluent, and incubated overnight at 4°C. The dilution ratio of the primary rabbit anti-MAP2 antibody is (1:250);

[0211] Secondary antibody incubation: After aspirating the primary antibody (recoverable) the next day, wash with PBS three times, 5-10 minutes each time, and then incubate with secondary antibody for 1 hour in the dark.

[0212] (7) DAPI staining and sealing

[0213] Remove the secondary antibody, wash three times with PBS, add DAPI, let stand at room temperature for 5 minutes, remove DAPI, and wash three times with PBS. Add a drop of sealing agent to the slide, slowly buckle the cover glass on the slide, with the cell side close to the slide, remove the excess sealing agent with absorbent paper, and then perform fluorescence microscopy observation.

[0214] Neutralization effect of VDBP neutralizing antibody (6A2 antibody) Figure 7 As shown, A is the MAP2 immunofluorescence staining of the control group, the VDBP treatment group and the VDBP+6A2 neutralizing antibody treatment group, and B is the corresponding statistical analysis. The results show that the complexity of the neuronal dendrites in the VDBP treatment group decreased, indicating that VDBP can damage the dendritic structure of neurons, and the use of the 6A2 neutralizing antibody of the present invention can offset the effect of VDBP on neuronal damage and play a protective role in neuronal damage.

[0215] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An antibody or antigen-binding fragment thereof to vitamin D binding protein, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; The heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.3, positions 25 to 32; The amino acid sequence of the CDR-H2 is shown in positions 50 to 56 of SEQ ID NO.3; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.3, positions 93 to 102; The light chain comprises a light chain variable region, wherein the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-L1 is shown in positions 27 to 34 of SEQ ID NO.5; The amino acid sequence of the CDR-L2 is shown in positions 52 to 54 of SEQ ID NO.5; The amino acid sequence of the CDR-L3 is shown in positions 91 to 101 of SEQ ID NO.

5.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region comprises any one of A1) to A3): A1) the amino acid sequence shown in SEQ ID NO.3; A2) an amino acid sequence having the same function as the sequence shown in SEQ ID NO.3 after one or more amino acids are substituted and / or deleted and / or added; A3) an amino acid sequence having 80%, 85% or 90% or more homology with SEQ ID NO.3 and having the same function as the sequence shown in SEQ ID NO.3; The amino acid sequence of the light chain variable region comprises any one of B1) to B3): B1) the amino acid sequence shown in SEQ ID NO.5; B2) an amino acid sequence in which one or more amino acids in SEQ ID NO.5 are substituted and / or deleted and / or added, and which has the same function as the sequence shown in SEQ ID NO.5; B3) An amino acid sequence that has 80%, 85% or 90% or more homology with SEQ ID NO.5 and has the same function as the sequence shown in SEQ ID NO.

5.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, and a multispecific antibody.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The heavy chain further comprises a heavy chain constant region; and / or The light chain also comprises a light chain constant region.

5. A biomaterial related to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The biological material comprises any one of C1) to C5); C1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1) or the expression cassette described in C2) or the recombinant vector described in C3); C5) A recombinant cell comprising the nucleic acid molecule of C1) or the expression cassette of C2) or the recombinant vector of C3), wherein the recombinant cell does not contain any propagation material.

6. The biomaterial according to claim 5, characterized in that The nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region; Among them, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown as SEQ ID NO.2; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO.

4.

7. A conjugate, characterized in that: Comprising: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; and a coupling portion, wherein the coupling portion comprises at least one of a drug, a toxin, a radionuclide, and an enzyme.

8. A product, characterized in that The product comprises at least one of D1) to D3): D1) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; D2) The biomaterial according to claim 5 or 6; D3) The conjugate according to claim 7; The product is selected from at least one of a drug, a test plate, a chip, a test paper and a test kit.

9. Use of any one of E1) to E3) in the preparation of a reagent for detecting vitamin D binding protein or a drug for treating neuronal damage: E1) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; E2) The biomaterial according to claim 5 or 6; E3) The conjugate according to claim 7.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: Obtained by culturing the recombinant microorganism or recombinant cell as claimed in claim 5 or 6.

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