An antibody against dengue virus or its NS1 protein and its application

Abstract

The present invention discloses an antibody against dengue virus or its NS1 protein and its application, relating to the field of antibodies. The antibody against dengue virus or its NS1 protein disclosed by the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. This antibody provides an important source of raw materials for the detection of dengue virus or its NS1 protein and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an antibody against dengue virus or its NS1 protein and its applications. Background Art

[0002] Dengue fever (DF) is an acute mosquito-borne infectious disease caused by four serotype viruses (DENV-1, DENV-2, DENV-3, DENV-4), mainly transmitted by Aedes aegypti and Aedes albopictus. DF is an arboviral disease with the widest distribution, the highest incidence, and relatively great harm, widely prevalent in more than 100 countries and regions in the tropical and subtropical regions of Africa, the Americas, Southeast Asia, and the Western Pacific.

[0003] Clinically, DF is a severe influenza-like disease. The main manifestations are sudden onset, high fever, severe headache, retro-orbital pain, muscle and joint pain, which may be accompanied by rash, lymphadenopathy, and leukopenia. It can affect all populations, but the symptoms may vary depending on the age of the patient. Generally, this disease type is called classical dengue fever, which spreads rapidly and can cause large-scale epidemics. During the dengue fever epidemic period, the morbidity rate of susceptible populations is usually 40%-50%, and can be as high as 80%-90%, but the fatality rate is very low. Dengue hemorrhagic fever is characterized by high fever, hemorrhage, hepatomegaly, and circulatory failure in severe cases, with a high fatality rate, and is a relatively severe clinical type. The one accompanied by shock syndrome is called dengue shock syndrome.

[0004] There is no specific treatment method for dengue fever. Without appropriate treatment, the fatality rate of dengue hemorrhagic fever can exceed 20%, and after effective supportive therapy, the fatality rate can be less than 1%. Key points for the diagnosis of dengue fever: 1) Epidemiological data, the activity situation 15 days before the onset, whether having been to the epidemic area, and the mosquito bite history; 2) Clinical characteristics, sudden onset, fever, "three pains and three rednesses", and rash; 3) Laboratory tests, decrease in white blood cells and platelets; positive detection of serum-specific IgM; a four-fold increase in IgG in the convalescent period compared with the acute phase; isolation of the virus or specific antigen.

[0005] Currently, the detection methods for dengue virus include virus culture, serological detection, immunoassay, etc. The time required for virus isolation is relatively long and cannot achieve the purpose of rapid diagnosis, while conventional serological diagnosis is interfered due to extensive cross-reactions. The immunoassay method has the characteristics of being rapid, simple, not relying on important equipment, and being able to achieve on-site detection, and has become a research hotspot in the rapid diagnosis of infectious diseases. The principle of the immunoassay method is a detection method based on antigen-antibody reaction. The NS1 protein is a glycoprotein in the non-structural proteins of dengue virus, with extremely strong antigenicity and not causing ADE, so it is often used as the target of the immunoassay method.

[0006] Immunoassays require antibodies against dengue virus or its NS1 protein. Therefore, there is a strong need in the art for antibodies against dengue virus or its NS1 protein with good performance. Summary of the Invention

[0007] The present application provides an antibody against dengue virus or its NS1 protein or an antigen-binding fragment thereof, which provides an important source of raw materials for the detection of dengue virus or its NS1 protein and has good activity or affinity.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided an antibody against dengue virus or its NS1 protein or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises three complementarity-determining regions of a heavy-chain variable region having the amino acid sequence SEQ ID NO: 20 and three complementarity-determining regions of any one of the light-chain variable regions having the amino acid sequences SEQ ID NO: 22, 23, 24, 25.

[0009] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody against dengue virus or its NS1 protein or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises the following complementarity-determining regions:

[0010] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0011] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0012] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0013] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4 or 17, or consists of the same;

[0014] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; and

[0015] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0016] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody against dengue virus or its NS1 protein or an antigen-binding fragment thereof, comprising a heavy-chain variable region and / or a light-chain variable region, wherein the amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO: 20; the amino acid sequence of the light-chain variable region is as shown in any one of SEQ ID NO: 22, 23, 24, 25.

[0017] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody against dengue virus or its NS1 protein or an antigen-binding fragment thereof, including a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 21; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 26, 27, 28, 29.

[0018] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above-mentioned antibody or its antigen-binding fragment.

[0019] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0020] To achieve the above object, according to the seventh aspect of the present invention, there is provided a method for detecting dengue virus or its NS1 protein, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, or reagent or kit with dengue virus or its NS1 protein in a test sample to form an immune complex under conditions sufficient for an antibody / antigen binding reaction; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0021] To achieve the above object, according to the eighth aspect of the present invention, there is provided the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of a product for detecting dengue virus or its NS1 protein.

[0022] To achieve the above object, the present invention also provides a nucleic acid, a vector, a cell, and a method for preparing the above-mentioned antibody or its antigen-binding fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 Results of reducing SDS-PAGE for Anti-DN 8G12 Rmb1 to Anti-DN 8G12 Rmb4. DETAILED DESCRIPTION OF THE INVENTION

[0025] In a first aspect, embodiments of the present invention provide an antibody against dengue virus or its NS1 protein, or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises three complementary determining regions of a heavy chain variable region having the amino acid sequence SEQ ID NO: 20 and three complementary determining regions of a light chain variable region having any one of the amino acid sequences SEQ ID NO: 22, 23, 24, 25.

[0026] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody or the antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody or the antigen-binding fragment thereof described in the first aspect.

[0027] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 20; LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 22.

[0028] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0029] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the main amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0030] In the present invention, the heavy chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3.

[0031] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents may vary slightly. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0032] Table 1: CDR Definitions 1

[0033]

[0034]

[0035] 1 The numbers for all CDR definitions in Table 1 are based on the Kabat numbering system (see below), where the amino acid numbers on the heavy chain are represented by "H + number" and those on the light chain are represented by "L + number". A person of ordinary skill in the art can clearly map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0036] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0037] 3If neither H35A nor H35B exists, then CDR-H1 ends at position 35; if only H35A exists, then CDR-H1 ends at position 35A; if both H35A and H35B exist, then CDR-H1 ends at position 35B.

[0038] 4 If neither H35A nor H35B exists, then CDR-H1 ends at position 32; if only H35A exists, then CDR-H1 ends at position 33; if both H35A and H35B exist, then CDR-H1 ends at position 34.

[0039] 5 If neither H35A nor H35B exists, then CDR-H1 ends at position 33; if only H35A exists, then CDR-H1 ends at position 34; if both H35A and H35B exist, then CDR-H1 ends at position 35.

[0040] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0041] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0042] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0045] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0047] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0048]

[0049]

[0050] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof against dengue virus or its NS1 protein, and the antibody or the antigen-binding fragment thereof comprises the following complementarity-determining regions:

[0051] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same;

[0052] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same;

[0053] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same;

[0054] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4 or 17, or consists of the same;

[0055] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same; and

[0056] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0057] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0058] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, and refers to the regions other than the CDRs in the variable regions of the heavy chain and the light chain of the antibody; wherein, the heavy-chain framework region can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region can be further divided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0059] In the present invention, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0060] In an alternative embodiment, the antibody or its antigen-binding fragment according to the first or second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0061] The HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0062] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0063] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0064] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;

[0065] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0066] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.

[0069] It should be noted that in other embodiments, the amino acid sequences of the framework regions of the antibody or its antigen-binding fragment against dengue virus or its NS1 protein provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0070] In an alternative embodiment, the LFR1 comprises the amino acid sequence as shown in SEQ ID NO: 18 or 19.

[0071] In a third aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment against dengue virus or its NS1 protein, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is any one of SEQ ID NO: 22, 23, 24, 25.

[0072] In an alternative embodiment, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect above are selected from any one of the following combinations:

[0073] Combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:20 SEQ ID NO:22 2 SEQ ID NO:20 SEQ ID NO:24 3 SEQ ID NO:20 SEQ ID NO:25 4 SEQ ID NO:20 SEQ ID NO:23 。

[0074] In an alternative embodiment, the antibody or its antigen-binding fragment described in the first aspect, the second aspect, and the third aspect above further comprises a constant region.

[0075] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0076] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0077] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0078] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0079] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0080] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, murine, rat, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

[0081] In an alternative embodiment, the species origin of the constant region is murine.

[0082] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0083] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO:15 or 16).

[0084] In an alternative embodiment, the antigen-binding fragment is selected from any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0085] The antigen-binding fragments of the above antibodies generally have the same binding specificity as their source antibodies. Those skilled in the art can easily understand from the content recorded in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0086] The antigen-binding fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0087] In a fourth aspect, the present invention provides an antibody or its antigen-binding fragment against dengue virus or its NS1 protein, including a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO:21, and the amino acid sequence of the light chain is any one of SEQ ID NO:26, 27, 28, 29.

[0088] In an alternative embodiment, the antibody or its antigen-binding fragment described in the first, second, third or fourth aspect above includes heavy and light chains in any of the following combinations:

[0089] Combination Heavy chain Light chain 1 SEQ ID NO:21 SEQ ID NO:26 2 SEQ ID NO:21 SEQ ID NO:28 3 SEQ ID NO:21 SEQ ID NO:29 4 SEQ ID NO:21 SEQ ID NO:27 。

[0090] In a fifth aspect, the present invention provides an antibody conjugate, which comprises the above-mentioned antibody or its antigen-binding fragment.

[0091] In an alternative embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0092] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody or its antigen-binding fragment.

[0093] In an alternative embodiment, the above-mentioned label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through this characteristic.

[0094] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0095] In the actual use process, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0096] In an alternative embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as but not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0097] In an alternative embodiment, the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0098] In alternative embodiments, the radioisotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0099] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0100] In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0101] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0102] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0103] In alternative embodiments, the colloidal metal is colloidal gold.

[0104] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0105] In alternative embodiments, the solid-phase carriers include, but are not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0106] In a sixth aspect, the present invention provides a reagent or kit, which includes the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0107] As described above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to dengue virus or its NS1 protein. Therefore, reagents or kits containing the anti-dengue virus or its NS1 protein antibodies or antigen-binding fragments thereof can effectively qualitatively or quantitatively detect dengue virus or its NS1 protein. Using the reagents or kits provided by the present invention, for example, they can be used in immunoblotting, immunoprecipitation, etc., which involve detections utilizing the specific binding property between dengue virus or its NS1 protein and its antibody. As described above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity with dengue virus or its NS1 protein. Therefore, the reagents or kits containing the antibodies or antigen-binding fragments thereof have higher detection sensitivity or specificity.

[0108] In a seventh aspect, the present invention provides a method for detecting dengue virus or its NS1 protein, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with dengue virus or its NS1 protein in a test sample under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample;

[0109] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or its antigen-binding fragment.

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to dengue virus or its NS1 protein.

[0111] In an eighth aspect, the present invention provides the use of the above-mentioned anti-dengue virus or its NS1 protein antibody or its antigen-binding fragment, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting dengue virus or its NS1 protein.

[0112] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0113] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0114] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0115] In a twelfth aspect, the present invention provides a method for preparing an anti-dengue virus or its NS1 protein antibody or its antigen-binding fragment, which comprises: culturing the cells as described above.

[0116] Based on the disclosure of the amino acid sequences of antibodies or antigen-binding fragments thereof against dengue virus or its NS1 protein in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibodies or antigen-binding fragments thereof against dengue virus or its NS1 protein. For example, the antibodies or antigen-binding fragments thereof can be isolated and purified from the culture products of recombinant cells capable of recombinantly expressing the antibodies or antigen-binding fragments thereof described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the antibodies or antigen-binding fragments thereof against dengue virus or its NS1 protein in the present invention, they all fall within the protection scope of the present invention.

[0117] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by purchasing commercially.

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0119] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0120] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0121] Example 1 Preparation of Anti-DN 8G12 Monoclonal Antibody

[0122] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Corporation. The MagExtractor-RNA extraction kit was purchased from TOYOBO Corporation. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-DN 8G12 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0123] (1) Preparation of antibody genes

[0124] mRNA was extracted from the hybridoma cell line secreting the Anti-DN 8G12 monoclonal antibody, and DNA products were obtained by RT-PCR. After the A-addition reaction of the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew out, 4 clones of the Heavy Chain and Light Chain genes were respectively taken for cloning and sent to a gene sequencing company for sequencing.

[0125] (2) Sequence analysis of the variable region genes of Anti-DN 8G12 antibody

[0126] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 321 bp, and there was a 57-bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 342 bp, belonging to the VH1 gene family, and there was a 57-bp leader peptide sequence in front of it.

[0127] (3) Construction of recombinant antibody expression plasmid

[0128] pcDNA TM 3.4 The pcDNA 3.4 vector was the recombinant antibody eukaryotic expression vector constructed. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and 0.70-kb Light Chain gene fragments and 1.37-kb Heavy Chain gene fragments were amplified by PCR amplification.

[0129] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0130] 2. Recombinant antibody production

[0131] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select antibodies with the appropriate concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the culture medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the culture medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture at a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0132] The obtained antibody was named Anti-DN 8G12Rmb1, and the Anti-DN 8G12Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0133] Table 2 Antibody sequences

[0134] Antibody name Heavy chain Light chain Anti-DN 8G12Rmb1 SEQ ID NO:21 SEQ ID NO:26 Anti-DN 8G12Rmb2 SEQ ID NO:21 SEQ ID NO:28 Anti-DN 8G12Rmb3 SEQ ID NO:21 SEQ ID NO:29 Anti-DN 8G12Rmb4 SEQ ID NO:21 SEQ ID NO:27

[0135] Example 2 Performance detection of the antibody

[0136] 1. Activity identification

[0137] Dilute goat anti-mouse IgG at 1 μg / ml with coating buffer (main component: NaHCO₃) and coat the microplate, 100 μL per well, overnight at 4°C; the next day, wash twice with washing buffer (main components: Na₂HPO₄ + NaCl), pat dry; add blocking buffer (20% BSA + 80% PBS), 120 μL per well, incubate at 37°C for 1 h, pat dry; add the diluted purified antibody, 100 μL per well, incubate at 37°C for 60 min; discard the liquid in the plate, pat dry, add 20% mouse negative blood for blocking, 120 μl per well, incubate at 37°C for 1 h; discard the liquid in the plate, pat dry, add the diluted dengue NS1 antigen (from PhyGenesys), 100 μL per well, incubate at 37°C for 40 min; wash 5 times with washing buffer, pat dry; add the anti-dengue NS1 antibody labeled with HRP (from PhyGenesys, which can pair with the purified antibody), 100 μL per well, incubate at 37°C for 30 min; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), incubate for 10 min; add stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0138] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid

[0139] + EDTA·2Na + TMB

[0140] + concentrated HCl); Stop solution (EDTA·2Na + concentrated H₂SO₄)

[0141] Table 3 Activity data

[0142] Concentration (ng / ml) 125.00 62.50 31.25 15.63 7.81 0.00 Control 1.239 0.765 0.401 0.227 0.117 0.023 Anti-DN 8G12Rmb1 1.810 1.567 0.871 0.429 0.252 0.099 Anti-DN 8G12Rmb2 1.805 1.467 0.778 0.459 0.227 0.098 Anti-DN 8G12Rmb3 1.833 1.496 0.811 0.415 0.212 0.098 Anti-DN 8G12Rmb4 1.957 1.608 0.917 0.550 0.368 0.097

[0143] 2. Application of antibodies in colloidal gold detection

[0144] 2.1 Preparation of colloidal gold detection strips

[0145] 2.1.1 Preparation of nitrocellulose membrane

[0146] Preparation of nitrocellulose membrane: Dilute the anti-dengue NS1 protein antibody Anti-DN (purchased from PhyGenesys) to 1 - 5 mg / mL with coating buffer, adjust the machine, and draw a line as the T line, which is the test line, and the T line is close to the gold conjugate pad end; dilute the goat anti-mouse IgG antibody to 1 - 5 mg / mL with coating buffer, adjust the machine, and draw a line as the C line, which is the control line, and the C line is close to the absorbent pad. The distance between the two lines is 5 - 8 mm and uniform. Dry at 37°C and store for later use.

[0147] 2.1.2 Preparation of colloidal gold and gold-labeled monoclonal antibody

[0148] Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, place it on an electric furnace and boil. Add 2 mL of 1% trisodium citrate to every 100 mL of 0.01% chloroauric acid, continue boiling until the liquid turns bright red, then stop heating. After cooling to room temperature, make up for the lost water. The prepared colloidal gold should be pure, transparent, without precipitation and floating substances.

[0149] Adjust the pH value of the colloidal gold to 8.2 with 0.1 M potassium carbonate. Add the above-mentioned antibodies Anti-DN 8G12Rmb1 - Anti-DN 8G12Rmb4 and mainstream raw materials in the market respectively, mix well with a magnetic stirrer for 30 min, add BSA to a final concentration of 1% under stirring and let it stand for 1 hour. Centrifuge at 13000 rpm and 4 °C for 30 min, discard the supernatant, wash the precipitate twice with the labeled washing solution, resuspend the precipitate with the labeled washing solution with one-tenth of the initial volume of the colloidal gold, and store the prepared colloidal gold-labeled antibodies Anti-DN 8G12Rmb1 - Anti-DN 8G12Rmb4 at 4 °C for later use.

[0150] 2.1.3 Preparation of the gold-labeled pad

[0151] Soak the gold-labeled pad in the blocking solution for 30 min, then dry it at 37 °C. Then evenly spread the prepared gold-labeled antibody on the gold-labeled pad, spreading 20 square centimeters per milliliter of the solution, freeze-dry, encapsulate, and store at 4 °C for later use.

[0152] 2.1.4 Assembly of the test strip

[0153] Place the absorbent pad (purchased from Millipore), nitrocellulose membrane, gold-labeled pad, and sample pad on a non-absorbent support sheet, cut into small strips 3 mm wide. Pack every ten small strips together, add a desiccant, and vacuum encapsulate to obtain the test strip.

[0154] 2.2 Application of antibodies in colloidal gold detection

[0155] Use the above-assembled test strip to detect whether the tested material contains dengue NS1 protein, so as to determine the effectiveness of the antibodies Anti-DN 8G12Rmb1 - Anti-DN 8G12Rmb4 in detecting dengue NS1 protein.

[0156] The double antibody sandwich method is used to detect whether dengue NS1 protein is contained in the test material. During the detection, the dengue NS1 protein first binds to the colloidal gold-labeled antibody to form a dengue NS1 protein-colloidal gold-labeled antibody complex. Due to capillary action, the dengue NS1 protein-colloidal gold-labeled antibody complex migrates forward along the nitrocellulose membrane. When it reaches the test line, the dengue NS1 protein-colloidal gold-labeled antibody complex will bind to the anti-dengue NS1 protein antibody Anti-DN prepared in step 3.1.1 of the above example to form an Anti-DN-dengue NS1 protein-colloidal gold-labeled antibody complex, thus being enriched on the test line to form a red precipitation line. The dengue NS1 protein-colloidal gold-labeled antibody complex that does not bind to the dengue NS1 protein antibody on the test line passes through the test line and is captured by the goat anti-mouse IgG antibody, being enriched on the quality control line to form a red precipitation line. When there are red precipitation lines on both the test line and the quality control line, it is judged as a positive result. If the sample does not contain dengue NS1 protein, when the colloidal gold-labeled antibody that does not bind to the dengue NS1 protein reaches the test line, an Anti-DN-dengue NS1 protein-colloidal gold-labeled antibody complex will not be formed. The colloidal gold-labeled antibody complex that does not bind to the dengue NS1 protein passes through the test line and is only enriched on the quality control line to form a red precipitation line, and it is judged as a negative result at this time.

[0157] Note: The gold label color development is composed of C plus a number. The smaller the number after C, the stronger the color development and the higher the activity; the higher the number after C, the weaker the color development and the lower the activity; a number with a "+" after it means slightly stronger than 0.5C, a number with a "-" after it means slightly weaker than 0.5C, and "B" represents negative.

[0158] Table 4 Performance evaluation data

[0159]

[0160]

[0161] 3. Stability assessment

[0162] The above antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples at 7 days, 14 days, and 21 days were taken for state observation, and the 21-day samples were subjected to activity detection. The results showed that there were no obvious protein state changes after the antibody was placed for 21 days under the three assessment conditions, and the activity did not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-DN 8G12Rmb4 after 21 days of assessment.

[0163] Table 5 Stability data

[0164] Sample concentration (ng / ml) 62.50 31.25 0.00 Sample at 4°C for 21 days 1.667 0.991 0.055 Sample at -80°C for 21 days 1.658 0.972 0.056 Sample at 37°C for 21 days 1.635 0.955 0.053

[0165] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0166] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0167]

[0168]

Claims

1. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, wherein the antibody or antigen-binding fragment thereof comprises three complementarity-determining regions of a heavy chain variable region having the amino acid sequence SEQ ID NO: 20 and three complementarity-determining regions of a light chain variable region having any one of the amino acid sequences SEQ ID NO: 22, 23, 24, 25; the complementarity-determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM systems.

2. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, characterized in that The antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3: HCDR1, with the amino acid sequence shown in SEQ ID NO:1; HCDR2, with the amino acid sequence shown in SEQ ID NO:2; HCDR3, with the amino acid sequence shown in SEQ ID NO:3; LCDR1, with the amino acid sequence shown in SEQ ID NO:4 or 17; LCDR2, with the amino acid sequence shown in SEQ ID NO:5; and LCDR3, with the amino acid sequence shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; The HFR1 comprises the amino acid sequence SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; The HFR2 comprises the amino acid sequence SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; The HFR3 comprises the amino acid sequence SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; The HFR4 comprises the amino acid sequence SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; The LFR1 comprises the amino acid sequence SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; The LFR2 comprises the amino acid sequence SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; The LFR3 comprises the amino acid sequence SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; The LFR4 comprises the amino acid sequence SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith.

4. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain variable region and a light chain variable region, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:20; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:22, 23, 24, 25.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-2 and 4, characterized in that The antibody or its antigen-binding fragment further comprises a constant region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The constant region includes a heavy chain constant region and / or a light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that The heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

8. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

9. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

10. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that The species origin of the constant region is mouse.

11. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that The sequence of the heavy chain constant region is shown in SEQ ID NO:15 or has at least 80% identity therewith.

12. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that The sequence of the light chain constant region is shown in SEQ ID NO:16 or has at least 80% identity therewith.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, 4, and 6-12, characterized in that The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

14. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain and a light chain, characterized in that The amino acid sequence of the heavy chain is shown in SEQ ID NO:21; the amino acid sequence of the light chain is shown in any one of SEQ ID NO:26, 27, 28, 29.

15. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or its antigen-binding fragment according to any one of claims 1-14, and the antibody conjugate further comprises a marker conjugated to the antibody or its antigen-binding fragment.

16. The antibody conjugate according to claim 15, characterized in that The marker is selected from biotin or a biotin derivative.

17. The antibody conjugate according to claim 15, characterized in that The marker is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based markers.

18. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further comprises a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

19. A reagent or kit, characterized in that The reagent or kit comprises the antibody or its antigen-binding fragment according to any one of claims 1-14 or the antibody conjugate according to any one of claims 15-18.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14, the antibody conjugate according to any one of claims 15-18, or the reagent or kit according to claim 19 in the preparation of a product for detecting dengue virus or its NS1 protein, characterized in that Comprising: a) contacting the antibody or its antigen-binding fragment according to any one of claims 1-14, the antibody conjugate according to any one of claims 15-18 or the reagent or kit according to claim 19 with dengue virus or its NS1 protein in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

21. The application according to claim 20, wherein The immune complex further comprises a second antibody that binds to the antibody or an antigen-binding fragment thereof.

22. The application according to claim 20, wherein The immune complex further comprises a second antibody that binds to dengue virus or its NS1 protein.

23. Use of the antibody or its antigen-binding fragment according to any one of claims 1-14, the antibody conjugate according to any one of claims 15-18, or the reagent or kit according to claim 19 in the preparation of a product for detecting dengue virus or its NS1 protein.

24. A nucleic acid, wherein The nucleic acid encodes the antibody or an antigen-binding fragment thereof according to any one of claims 1-14.

25. A vector, wherein The vector contains the nucleic acid according to claim 24.

26. A cell, wherein The cell contains the nucleic acid according to claim 24 or the vector according to claim 25.

27. A method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-14, wherein The method comprises culturing the cell according to claim 26.

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