Anti-adiponectin antibodies, reagents and kits for detecting adiponectin

Abstract

The present invention discloses an anti-adiponectin antibody, a reagent and a kit for detecting adiponectin, and relates to the technical field of antibodies. The anti-adiponectin antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important source of raw materials for the detection of adiponectin and has improved affinity and activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-adiponectin antibody, and a reagent and a kit for detecting adiponectin. Background Art

[0002] Adiponectin (APN), also known as Acrp30, GBP28, AdipoQ or adiponectin, is a protein hormone mainly secreted by adipocytes. The human adiponectin monomer consists of 244 amino acids, including three regions: the amino-terminal signal sequence, the collagen domain and the globular domain of APN (gAd) at the carboxyl terminal. Adiponectin exists in the blood in three subtypes: trimer (65kDa), hexamer (150kDa) and high molecular weight (HMW, 18-36 monomers, >280kDa) polymer. Its concentration in serum ranges from 5 to 30 mg / L, accounting for about 0.01% of the total human serum protein.

[0003] The main biological function of adiponectin is to regulate the body's energy metabolism, promote the catabolism of glucose and fat, and improve insulin sensitivity. In recent years, more and more studies have shown that adiponectin levels are closely related to cardiovascular risk factors such as obesity, diabetes, hypertension, hyperlipidemia, atherosclerosis, and insulin resistance, and that serum adiponectin levels in patients are significantly lower than those in healthy people. Adiponectin has the effects of enhancing insulin sensitivity, resisting the formation of atherosclerosis (AS), anti-inflammation, and anti-intimal hyperplasia after vascular injury. Therefore, detecting the content of adiponectin in the blood is of great significance for understanding its physiological function, regulatory mechanism, and its relationship with the occurrence and development of diseases such as type II diabetes, coronary heart disease, and hypertension.

[0004] At present, the main detection method of adiponectin is fluorescent immunochromatography, which is an immunological detection method based on the specific reaction of antibodies and antigens, and uses fluorescence to amplify and display the detected signal. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc. The above immunological detection methods all require antibodies against adiponectin. Therefore, there is a strong demand in the art for antibodies that are effective and bind to adiponectin and detect it.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The present application provides an anti-adiponectin antibody with improved affinity and / or activity to improve the detection of adiponectin and provide an important source of raw materials for the detection of adiponectin.

[0007] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, an anti-adiponectin antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1 or 15, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, HCDR3 with an amino acid sequence as shown in SEQ ID NO:3, and LCDR1, LCDR2, LCDR3 with an amino acid sequence as shown in SEQ ID NO:4 to SEQ ID NO:6.

[0008] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, an anti-adiponectin antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are the amino acid sequences of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0009] In order to achieve the above object, according to the third aspect of the present invention, an anti-adiponectin antibody is provided, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises the above heavy chain variable region, and the light chain comprises the above light chain variable region.

[0010] In order to achieve the above object, according to the fourth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0011] In order to achieve the above object, according to the fifth aspect of the present invention, a reagent or kit for detecting adiponectin is provided, wherein the reagent or kit comprises the above antibody or its functional fragment or the above antibody conjugate.

[0012] To achieve the above object, according to a sixth aspect of the present invention, a method for detecting adiponectin is provided, comprising: contacting the above antibody or a functional fragment thereof with adiponectin in a sample to be detected to form an immune complex.

[0013] In order to achieve the above object, according to the seventh aspect of the present invention, there is provided the use of the above antibody or its functional fragment or conjugate in the preparation of a diagnostic reagent or a diagnostic kit for a disease related to adiponectin metabolism.

[0014] In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or its functional fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The results of reducing SDS-PAGE of Anti-APN 6A27mut1 to mut4 are shown. DETAILED DESCRIPTION

[0017] The present invention provides an anti-adiponectin antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1 or 15, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, and LCDR1, LCDR2, LCDR3 with an amino acid sequence as shown in SEQ ID NO: 4 to SEQ ID NO: 6. The above-mentioned antibody has improved affinity and / or activity.

[0018] 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, so long as they exhibit the desired biological activity.

[0019] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0020] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0021] 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 region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0022] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0023] In an optional embodiment, the antibody or its functional fragment further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences as shown in SEQ ID NO:7 to SEQ ID NO:10 and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences as shown in SEQ ID NO:11 to SEQ ID NO:14.

[0024] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0025] In an optional embodiment, the amino acid sequence of HFR3 of the antibody or its functional fragment is as shown in SEQ ID NO:16.

[0026] In an optional embodiment, the amino acid sequence of HFR4 of the antibody or its functional fragment is as shown in SEQ ID NO:17.

[0027] In an optional embodiment, the antibody or its functional fragment has a KD of less than 3.26×10 -08 Affinity binding of M to adiponectin.

[0028] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -08 M, KD≤10 -09 M, KD≤10 -10 M or KD≤10 -11 Affinity binding of M to adiponectin.

[0029] In an optional embodiment, the antibody or its functional fragment has a KD≤4.99×10 -09 Affinity binding of M to adiponectin.

[0030] In an optional embodiment, the antibody or its functional fragment has a KD≤3.46×10 -10 Affinity binding of M to adiponectin.

[0031] On the other hand, an embodiment of the present invention provides an anti-adiponectin antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, and the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the above-mentioned amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are the above-mentioned amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0032] In an optional embodiment, the heavy chain variable region amino acid sequence is shown in any one of SEQ ID NOs: 20 to 27.

[0033] In an optional embodiment, the light chain variable region amino acid sequence is as shown in SEQ ID NO:28.

[0034] In an alternative embodiment, the antibody further comprises a constant region.

[0035] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0036] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0037] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0038] In an alternative embodiment, the species origin of the constant region is mouse.

[0039] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:18, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:19.

[0040] 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-mentioned constant region (SEQ ID NO: 18 or 19).

[0041] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0042] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0043] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0044] On the other hand, the present invention provides an anti-adiponectin antibody, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; and the light chain comprises the above-mentioned light chain variable region and the above-mentioned light chain constant region.

[0045] In an optional embodiment, the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 29 to 36.

[0046] In an alternative embodiment, the amino acid sequence of the light chain is as shown in SEQ ID NO:37.

[0047] In another aspect, the present invention provides an antibody conjugate, comprising the above-mentioned antibody, wherein the antibody is directly or indirectly covalently coupled to the object to be coupled, or the antibody is coupled to the object to be coupled by non-covalent adsorption.

[0048] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to biotin or a biotin derivative.

[0049] In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to a label.

[0050] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0051] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0052] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0053] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0054] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0055] In an optional embodiment, the radioactive isotopes 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.

[0056] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0057] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0058] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0059] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0060] In an optional embodiment, the colloidal metal is colloidal gold.

[0061] In an alternative embodiment, the antibody in the above antibody conjugate is coupled to a solid phase.

[0062] In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

[0063] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0064] In an alternative embodiment, the solid phase is a nitrocellulose membrane.

[0065] On the other hand, the present invention provides a reagent or a kit for detecting adiponectin, wherein the reagent or the kit comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.

[0066] On the other hand, the present invention provides use of the above-mentioned antibody or its functional fragment, antibody conjugate or the above-mentioned reagent or kit in the detection of adiponectin.

[0067] On the other hand, the present invention provides the use of the above-mentioned antibody or its functional fragment or antibody conjugate in the preparation of a diagnostic reagent or a diagnostic kit for a disease related to adiponectin metabolism.

[0068] In an alternative embodiment, the disease includes, but is not limited to, diabetes, coronary heart disease, hypertension or atherosclerosis.

[0069] In another aspect, the present invention provides a method for detecting adiponectin, comprising: contacting the above-mentioned antibody or a functional fragment thereof with adiponectin in a sample to be detected to form an immune complex.

[0070] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0071] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to adiponectin.

[0072] In another aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0073] In another aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0074] In another aspect, the present invention provides a cell containing the above-mentioned vector.

[0075] In another aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0076] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0077] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0079] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0080] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0081] Example 1 Preparation of Anti-APN 6A27 Monoclonal Antibody

[0082] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen.

[0083] 1 Construction of recombinant plasmid

[0084] (1) Antibody gene preparation

[0085] mRNA was extracted from the hybridoma cell line secreting anti-adiponectin monoclonal antibodies, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0086] (2) Sequence analysis of the variable region gene of Anti-APN 6A27 antibody

[0087] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, 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. In the gene fragment amplified by the Light Chain, the VL gene sequence was 321 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 354 bp, belonging to the VH1 gene family, preceded by a 57 bp leader peptide sequence.

[0088] (3) Construction of recombinant antibody expression plasmid

[0089] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.73kb Light Chain gene fragment and the 1.42kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0090] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0091] 2. Screening of stable cell lines

[0092] (1) Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

[0093] The plasmid was diluted to 40ug / 100ul with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100 μL of plasmid with 700 μL of cells, transfer to an electroporation cup, electroporate, take samples and count on the 3rd, 5th and 7th days, and collect samples for detection on the 7th day.

[0094] Coating solution (main component NaHCO3) was used to dilute adiponectin antigen (purchased from HYTEST, 8AN7) to 0.25ug / ml, 100μL per well, overnight at 4℃; the next day, wash twice with washing solution and pat dry; add blocking solution (20% BSA + 80% PBS), 120μL per well, 37℃, 1h, pat dry; add diluted cell supernatant, 100μL / well, 37℃, 30min (part of the supernatant for 1h); wash 5 times with washing solution and pat dry; add goat anti-mouse Ig G-HRP, 100 μL per well, 37°C, 30 min; wash 5 times with washing solution, pat dry; add colorimetric solution A (50 μL / well, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), add colorimetric solution B (50 μL / well, containing citric acid + EDTA·2Na + TMB + concentrated HCL), 10 min; add stop solution (50 μL / well, containing EDTA·2Na + concentrated H2SO4); read OD value at 450nm (reference 630nm) on the microplate reader. The results showed that the reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the well without cell supernatant was less than 0.1, indicating that the antibody produced after transient plasmid transfection was active against adiponectin antigen.

[0095] (2) Linearization of recombinant antibody expression plasmid

[0096] Prepare the following reagents: 50 μL of Buffer, 100 μg of DNA / tube, 10 μL of PuvⅠ enzyme, and make up to 500 μL with sterile water. Perform enzymatic digestion in a 37°C water bath overnight. First, use an equal volume of phenol / chloroform / isoamyl alcohol (lower layer) 25:24:1, and then use chloroform (aqueous phase) for extraction in sequence. Precipitate on ice with 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volumes of ethanol, rinse the precipitate with 70% ethanol, remove the organic solvent, and reconstitute with an appropriate amount of sterile water after the ethanol has completely evaporated. Finally, measure the concentration.

[0097] (3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0098] The plasmid was diluted to 40ug / 100ul with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100μL plasmid with 700μL cells, transfer to an electroporation cup, electrotransform, and count the next day; culture under pressure in 25umol / L MSX 96-well for about 25 days.

[0099] Observe the clone wells with cells under a microscope and record the confluence; take the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration and transfer them to 24 wells, and transfer them to 6 wells after about 3 days; after 3 days, keep the seed batch culture and adjust the cell density to 0.5×10 6 cells / ml, 2.2 ml for batch culture, cell density 0.3×10 6 cells / ml, 2ml for seed preservation; 7-day 6-well batch culture supernatant samples were sent for testing, and cell lines with smaller antibody concentrations and cell diameters were selected for TPP seed preservation and subculture.

[0100] 3. Recombinant Antibody Production

[0101] (1) Cell expansion

[0102] After cell recovery, the cells are first cultured in a 125ml shake flask with an inoculation volume of 30ml and a culture medium of 100% Dynamis medium. The cells are placed in a shaker at a speed of 120r / min, a temperature of 37°C, and a carbon dioxide content of 8%. After 72 hours of culture, the cells are inoculated and expanded at an inoculation density of 500,000 cells / ml. The expansion volume is calculated according to production requirements, and the culture medium is 100% Dynamis medium. The cells are expanded every 72 hours. When the cell volume meets the production requirements, the inoculation density is strictly controlled to be around 500,000 cells / ml for production.

[0103] (2) Shake flask production and purification

[0104] Shake flask parameters: speed 120r / min, temperature 37℃, carbon dioxide 8%. Feeding: Start feeding every day when the culture reaches 72h in the shake flask. HyCloneTM Cell BoostTM Feed 7a is fed daily at 3% of the initial culture volume, and Feed 7b is fed daily at one thousandth of the initial culture volume until the 12th day (feeding on the 12th day). Glucose is added at 3g / L on the sixth day. Samples are collected on the 13th day. Affinity purification is performed using a proteinA affinity chromatography column. Take 3μg of the purified antibody for reducing SDS-PAGE. The electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD and the other Mr is 28KD.

[0105] Example 2 Affinity and activity optimization

[0106] Although the Anti-APN 6A27 monoclonal antibody obtained in Example 1 has the ability to bind to the adiponectin antigen, its affinity and antibody activity are not ideal. Therefore, the applicant carried out directed mutation of the light chain CDR and heavy chain CDR of the antibody. That is, the antibody variable region structure simulation, the antigen and antibody variable region complex structure simulation, the antibody key amino acid analysis and mutation design were performed by computer, and bidirectional primers covering the mutation site were designed and synthesized according to the mutation scheme, and primers at both ends of the target DNA were synthesized. A high-fidelity PCR reaction was performed, and the PCR product was cloned into a vector, and then the mutant antibody was prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained, and were named: Anti-APN 6A27mut1 to Anti-APN 6A27mut4. The heavy chain and light chain amino acid sequences of each monoclonal antibody are shown in the following table.

[0107] Table 1 Antibody sequences

[0108] Sample name Heavy chain number Light chain number Anti-APN 6A27mut1 SEQ ID NO:29 SEQ ID NO:37 Anti-APN 6A27mut2 SEQ ID NO:32 SEQ ID NO:37 Anti-APN 6A27mut3 SEQ ID NO:36 SEQ ID NO:37 Anti-APN 6A27mut4 SEQ ID NO:30 SEQ ID NO:37

[0109] Example 3 Performance Testing of Antibodies

[0110] 1 Affinity analysis

[0111] Using the AMC sensor, the purified antibody was diluted to 10 ug / ml with PBST, and the adiponectin antigen (purchased from HYTEST, 8AN7) was graded diluted with PBST.

[0112] Operation process: 60s of equilibrium in buffer 1 (PBST), 300s of antibody fixation in antibody solution, 180s of incubation in buffer 2 (PBST), 420s of binding in antigen solution, 1200s of dissociation in buffer 2, sensor regeneration with 10mM pH 1.69 GLY solution and buffer 3 (PBST), and data output. (KD represents equilibrium dissociation constant, i.e. affinity; kon represents binding rate; kdis represents dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0113] Table 2 Affinity data

[0114] Sample name KD(M) kon(1 / Ms) kdis(1 / s) Comparison 3.26E-08 1.22E+05 3.98E-03 Anti-APN 6A27mut1 3.46E-10 4.59E+06 1.59E-03 Anti-APN 6A27mut2 4.99E-09 4.23E+05 2.11E-03 Anti-APN 6A27mut3 3.55E-09 3.52E+05 1.25E-03 Anti-APN 6A27mut4 9.70E-10 3.62E+06 3.51E-03

[0115] 2. Activity identification

[0116] The coating solution (main component NaHCO3) was used to dilute the adiponectin antigen (purchased from HYTEST, 8AN7) to 0.25ug / ml, 100μL per well, and incubated at 4℃ overnight; the next day, the cells were washed twice with the washing solution and patted dry; the blocking solution (20% BSA + 80% PBS) was added, 120μL per well, incubated at 37℃ for 1h, and patted dry; the diluted monoclonal antibody was added, 100μL / well, incubated at 37℃ for 30min-60min; the cells were washed 5 times with the washing solution and patted dry. Dry; add goat anti-mouse IgG-HRP, 100μl per well, 37℃, 30min; wash 5 times with washing solution (PBS), pat dry; add color solution A solution (50μl / well, containing 2.1g / L citric acid, 12.25g / L citric acid, 0.07g / L acetanilide and 0.5g / L urea peroxide), add color solution B solution (50μl / well, containing 1.05g / L citric acid, 0.186g / EDTA·2Na, 0.45g / L TMB and 0.2ml / L concentrated HCl), 10min; add stop solution (50μl / well, containing 0.75g / EDTA·2Na and 10.2ml / L concentrated H2SO4); read OD value at 450nm (reference 630nm) on the microplate reader. The results are shown in the table below.

[0117] Table 3 Activity data

[0118] Sample concentration (ng / ml) 62.50 31.25 15.63 7.81 3.91 0 Comparison 1.723 1.289 0.897 0.514 0.295 0.014 Anti-APN 6A27mut1 2.113 1.438 1.075 0.757 0.429 0.096 Anti-APN 6A27mut2 1.948 1.823 1.302 0.785 0.461 0.013 Anti-APN 6A27mut3 2.215 1.822 1.376 0.713 0.436 0.011 Anti-APN 6A27mut4 2.181 2.113 1.924 1.285 0.661 0.071

[0119] 3. Stability Assessment

[0120] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. The following table shows the OD results of the enzyme immunoassay activity test for 21 days of antibody test.

[0121] Table 4 Stability data

[0122] Sample concentration (ng / ml) 62.5 15.625 0 4℃, 21-day samples 1.652 0.692 0.31 -80℃, 21 days sample 1.678 0.702 0.22 37℃, 21 days sample 1.631 0.673 0.26

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0124] The partial amino acid sequences involved in this application are as follows:

[0125] Sequence number Sequence fragment SEQ ID NO:1 LYVMS SEQ ID NO:2 TVNGGGSYTYYPDSVKG SEQ ID NO:3 QS SEQ ID NO:4 KASQDVIAAVA SEQ ID NO:5 SASNRYI SEQ ID NO:6 QQQHYTNP SEQ ID NO:15 YV SEQUENCE LISTING <110> Dongguan Pengzhi Biotechnology Co., Ltd. <120> Anti-adiponectin antibodies, reagents and kits for detecting adiponectin <130> P2022068CN01 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 1 The Tyr Val Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 2 Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 6 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 3 Gln Ser Thr Phe Tyr Ala 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 4 Lys Ala Ser Gln Asp Val Ile Ala Ala Val Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 5 Ser Ala Ser Asn Arg Tyr Ile 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 6 Gln Gln His Tyr Thr Asn Pro 1 5 <210> 7 <211> 30 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 8 <211> 14 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 8 Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala 1 5 10 <210> 9 <211> 32 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 9 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr Leu Gln 1 5 10 15 Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 <210> 10 <211> 14 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 10 Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 11 <211> twenty three <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 11 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys 20 <210> 12 <211> 15 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 12 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 13 <211> 32 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 13 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Phe Thr Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 20 25 30 <210> 14 <211> 12 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 14 Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 15 <211> 5 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 15 Ile Tyr Val Met Ser 1 5 <210> 16 <211> 32 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 16 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr Leu Gln 1 5 10 15 Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 <210> 17 <211> 14 <212> PRT <213> Artificial <220> <223> Artificial Sequence <400> 17 Ile Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 18 <211> 329 <212> PRT <213> Artificial <220> <223> Artificial Sequence <400> 18 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 100 105 110 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 145 150 155 160 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 165 170 175 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 180 185 190 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 195 200 205 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 210 215 220 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 225 230 235 240 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280 285 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 290 295 300 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr 305 310 315 320 Thr Lys Ser Phe Ser Arg Thr Pro Gly 325 <210> 19 <211> 107 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 19 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 20 <211> 118 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 21 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 22 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 23 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 24 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 25 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 26 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 27 <211> 118 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 28 <211> 107 <212> PRT <213> Artificial <220> <223> artificial sequence <400> 28 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Ala Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Asn Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 30 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 31 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 32 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 33 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 34 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 35 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 36 <211> 447 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Val Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gln Ser Thr Phe Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 37 <211> 214 <212> PRT <213> Artificial <220> <223> Artificial sequence <400> 37 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Ala Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Asn Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210

Claims

1. An anti-adiponectin antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1 or 15, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, HCDR3 with an amino acid sequence as shown in SEQ ID NO:3, and LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:4 to SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or its antigen-binding fragment also includes HFR1, HFR2, HFR3, HFR4 whose amino acid sequences are shown in SEQ ID NO:7 to SEQ ID NO:10 and LFR1, LFR2, LFR3 and LFR4 whose amino acid sequences are shown in SEQ ID NO:11 to SEQ ID NO:

14.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof includes HFR1, HFR2, HFR3, HFR4 whose amino acid sequences are at least 80% identical to SEQ ID NO:7 to SEQ ID NO:10, and LFR1, LFR2, LFR3 and LFR4 whose amino acid sequences are at least 80% identical to SEQ ID NO:11 to SEQ ID NO:14, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The amino acid sequence of HFR3 of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

16.

5. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The amino acid sequence of HFR4 of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

17.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The antibody or antigen-binding fragment thereof has a KD of less than 3.26×10 -08 Affinity binding of M to adiponectin.

7. An anti-adiponectin antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described in claim 1, and the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 described in any one of claims 2 to 5.

8. An anti-adiponectin antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 20 to 27; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

28.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, 7 to 8, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

10. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The constant region includes a heavy chain constant region and / or a light chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

12. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

13. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The species origin of the constant region is mouse.

14. The antibody or antigen-binding fragment thereof according to claim 10, characterized in that: The heavy chain constant region sequence is as shown in SEQ ID NO: 18 or has at least 80% identity thereto; the light chain constant region sequence is as shown in SEQ ID NO: 19 or has at least 80% identity thereto.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, 7-8, 10-14, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

16. An anti-adiponectin antibody comprising a heavy chain and a light chain, characterized in that: The heavy chain comprises the heavy chain variable region of claim 7 or 8 and the heavy chain constant region of claim 14; the light chain comprises the light chain variable region of claim 7 or 8 and the light chain constant region of claim 14.

17. An anti-adiponectin antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 29 to 36; the amino acid sequence of the light chain is shown in SEQ ID NO:

37.

18. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16 or 17.

19. The antibody conjugate according to claim 18, characterized in that The antibody is conjugated to biotin or a biotin derivative.

20. The antibody conjugate according to claim 18, characterized in that The antibody is coupled to a solid phase.

21. The antibody conjugate according to claim 18, characterized in that The antibody is conjugated to a label.

22. The antibody conjugate according to claim 21, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

23. The antibody conjugate according to claim 21, characterized in that The label is colloidal gold.

24. A reagent or kit for detecting adiponectin, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the antibody according to claim 16 or 17, or the antibody conjugate according to any one of claims 18 to 23.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the antibody according to claim 16 or 17, or the antibody conjugate according to any one of claims 18 to 23, or the reagent or kit according to claim 24 in the preparation of a product for detecting adiponectin, characterized in that: include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the antibody according to claim 16 or 17, is contacted with adiponectin in a sample to be detected to form an immune complex.

26. The use according to claim 25, characterized in that The immune complex also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

27. The use according to claim 25, characterized in that The immune complex also includes a second antibody that binds to adiponectin.

28. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16 or 17.

29. A carrier, characterized in that It contains the nucleic acid according to claim 28.

30. A cell, characterized in that It contains the nucleic acid according to claim 28 or the vector according to claim 29.

31. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody according to claim 16 or 17, characterized in that: It includes: Cultivate the cell of claim 30.

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