BCOADC binding protein, preparation method and application thereof

Abstract

The invention provides a BCOADC binding protein as well as a preparation method and application thereof, and relates to the technical field of biology. The BCOADC binding protein contains at least one CDR in the sequences as shown in SEQ ID NO.1-5 and the amino acid sequence VSS. The BCOADC binding protein has good specific binding capacity with BCOADC, and can be used for identification of an anti-BCOADC antibody, an AMA-M2 antibody or a BCOADC antigen and auxiliary diagnosis and detection of positive diseases of the AMA-M2 antibody.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a BCOADC binding protein, a preparation method and an application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Primary biliary cirrhosis (PBC) is a typical autoimmune disease (AID) characterized by non-suppurative progressive inflammatory damage to the small and medium-sized bile ducts in the liver, which eventually leads to cirrhosis and liver failure. A variety of autoantibodies can be detected in PBC patients, such as antinuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (SMA), anti-liver and kidney microsomal antibodies (LKM), etc., among which the most important antibody is AMA. AMA is divided into 9 subtypes from MI to M9, and only M2 is a PBC-specific antibody. The sensitivity of AMA-M2 antibody for PBC detection is more than 93%, and the specificity is almost 100%.

[0004] The target antigens recognized by AMA-M2 include the 2-oxo-acid dehydrogenase complex (2-0ADC) family members present on the inner membrane of mitochondria: ① the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), with a molecular weight of 70kDa; ② the E2 subunits of branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), with a molecular weight of 51kDa; ③) the E2 subunits of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2), with a molecular weight of 51kDa. Among them, the lactone acyl region of BCOADC is one of the antigenic epitopes recognized by M2 antibodies, and the positive rate of anti-BCOADC antibodies in the serum of PBC patients is relatively high.

[0005] At present, the coating antigen in the anti-M2 antibody detection kit developed based on the chemiluminescence method is usually derived from the naturally extracted or recombinantly expressed M2 protein (BCOADC, OGDC, PDC) protein. The calibrators and quality control products in the kit are mainly patient serum. Since it is difficult to obtain positive serum, the source is unstable, and the performance of each serum is difficult to be stable. BCOADC is the main target antigen of anti-M2 antibodies. Therefore, the development of anti-BCOADC recombinant humanized monoclonal antibodies that can be mass-produced, have small batch differences and high affinity is of great significance and value to the development of clinical detection kits.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a variety of BCOADC binding proteins, which have good specific binding ability with BCOADC antigens. Based on the BCOADC binding proteins provided by the present invention, the present invention also aims to provide applications thereof. Another object of the present invention is to develop BCOADC binding proteins with good binding activity in a simpler method, which can be used for the identification of BCOADC, and to assist in the diagnosis or auxiliary diagnosis of anti-BCOADC antibody-positive diseases or AMA-M2 antibodies.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In this article, BCOADC refers to branched chain 2-oxo-acid dehydrogenase complex, which is a multi-enzyme complex in mitochondria. Its main function is to participate in maintaining the redox state of cells. BCOADC is composed of multiple subunits, including E1, E2 and E3 subunits, among which E2 subunit is the core component of the complex.

[0010] The BCOADC binding protein provided by the present invention can specifically bind to BCOADC. The BCOADC bound by the BCOADC binding protein includes BCOADC produced from natural cells and variants thereof, wherein the variants include but are not limited to at least one subunit that has been mutated, truncated or fused with other domains. The variants of BCOADC retain the necessary antigenic epitopes that bind to the binding protein provided by the present invention.

[0011] Herein, "AMA-M2 antibody" refers to the anti-mitochondrial antibody (AMA) M2 subtype.

[0012] The technical term "binding protein" herein refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments that contain a complementary determining region (CDR region). The proteins and protein fragments may be antibodies, and the terms "antibody" and "full-length antibody" include polyclonal antibodies and monoclonal antibodies. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the term "antibody" can be used interchangeably with "immunoglobulin."

[0013] Proteins and protein fragments may be antigen-binding fragments that may also contain part or all of the antibody CDRs, which lack at least some of the amino acids present in the full-length chain of the antibody but are still able to specifically bind to the antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from, but not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody, VH and VL are connected by a connecting peptide), dsFv (disulfide stabilized Fv fragments, dsFv), bispecific antibodies, nanobodies, and any one of the minimum recognition units of antibodies. In addition to the above functional fragments, any fragments with increased half-life are also included.

[0014] The term "variable region" or "variable domain" refers to the domain of the amino terminus of the heavy chain or light chain of an antibody that recognizes and binds to an antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing an antigen. The heavy chain variable domain may be referred to as "VH". The light chain variable domain may be referred to as "VL". The variable domain contains an antigen binding site. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the framework region and CDR has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well known in the art, including a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. Generally, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0015] The term "constant region" or "constant domain" refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination. The heavy chain of an antibody has one variable domain (VH), followed by a number of constant domains or regions, such as one or more of hinge, CH1, CH2, CH3 and CH4, the CH1 domain being adjacent to the VH domain and at the amino terminus of the hinge region of the antibody heavy chain, and not forming part of the Fc region of the antibody; the hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is usually the CH3 domain, which usually forms the C-terminal portion of the antibody, and in some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, as well as subclasses and mutant forms of the constant regions thereof.

[0016] The present invention does not limit the method of obtaining the BCOADC binding protein. In some optional embodiments, the corresponding antibody can be obtained by connecting the polynucleotide encoding the BCOADC binding protein to the vector and expressing it in the cell. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct and obtain the BCOADC binding protein that can express it. In other optional embodiments, the binding protein can also be obtained by recombinant genetics techniques known to those skilled in the art or by peptide synthesis, such as an automatic peptide synthesizer (for example, an automatic peptide synthesizer sold by Applied BioSystems, etc.); the antigen-binding fragment can also be optionally produced by enzymatic cleavage of the antigen-binding molecule (including intact antibodies), such as pepsin or papain cleavage; or chemical cleavage, such as obtaining the above-mentioned antigen-binding fragment by chemical reduction splitting disulfide bonds.

[0017] The terms "specific recognition", "selective binding", "selectively binds" and "specifically binds" or similar expressions refer to the binding of a binding protein to an epitope on a predetermined antigen. Typically, the binding protein is expressed in an amount of less than about 10 -5 M, for example, less than about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D The K value of an antibody can be determined using methods well established in the art. D Other standard assays for evaluating the binding ability of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis.

[0018] The term "signal substance" herein refers to a substance that can provide a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art; the signal substance can directly provide a signal, such as color (such as colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation or luminescence; it can also provide a signal indirectly through a subsequent reaction in which the signal substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0019] The term "polynucleotide" herein refers to a polymer form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the above-mentioned BCOADC binding protein, and the encoding is optionally encoding a sense strand or an antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0020] The term "vector" as used herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the cell.

[0021] The vector is well known to those skilled in the art, including but not limited to: plasmid, episomal plasmid, minicircle DNA, phagemid, cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0022] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Progeny may not necessarily be identical to the original cell due to natural, accidental or deliberate mutation, and may differ from the original cell in morphology and / or in genomic DNA. "Transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom.

[0023] As used herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, such as if it is naturally present, from its natural environment. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally associated with it or are normally mixed with it or in solution. Isolated polypeptides include naturally produced polypeptides contained in cell lysates, polypeptides in purified or partially purified form, recombinant polypeptides, polypeptides expressed or secreted by cells, and polypeptides in heterologous cells or cultures. In connection with nucleic acids, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, connected to a promoter, or artificially introduced into a heterologous cell).

[0024] The term "antibody-positive disease" as used herein refers to a disease that can cause a patient's antibody level to be higher than that of a patient without the disease. Exemplary AMA-M2 antibody-positive diseases include, but are not limited to, primary biliary cirrhosis.

[0025] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbers (I), (II)...(VII).

[0026] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0027] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0028] In a first aspect, a BCOADC binding protein is provided.

[0029] In an optional embodiment, the BCOADC binding protein contains at least one complementary determining region selected from the following amino acid sequences:

[0030] The complementary determining region VH-CDR1 has an amino acid sequence of GF-X1-FT-X2-SC (SEQ ID NO.11), wherein

[0031] X1 is I or S, X2 is D or E;

[0032] The complementary determining region VH-CDR2 has an amino acid sequence of I-X3-PNTSLPTF (SEQ ID NO.12), wherein

[0033] X3 is D or N;

[0034] The complementary determining region VH-CDR3 has an amino acid sequence of Y-X4-TNECNY-X5-MDH (SEQ ID NO. 13), wherein

[0035] X4 is Q or W, X5 is G or F;

[0036] The complementary determining region VL-CDR1 has an amino acid sequence of QS-X6-LHSDG-X7-TNY (SEQ ID NO. 14), wherein

[0037] X6 is L or R, X7 is Q or Y;

[0038] The complementarity determining region VL-CDR2 has an amino acid sequence of VSS;

[0039] The complementary determining region VL-CDR3 has an amino acid sequence of S-X8-G-X9-FYPLT (SEQ ID NO. 15), wherein

[0040] X8 is G or K, X9 is L or D.

[0041] In an optional embodiment, the BCO ADC binding protein contains complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; and / or, the BCO ADC binding protein contains complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3.

[0042] In an optional embodiment, the mutation sites of the complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are combined according to the VH-CDR1, VH-CDR2 and VH-CDR3 combination in any one of the mutation combinations 1 to 32 in Table 1.

[0043] In an optional embodiment, the mutation sites of the complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3 are combined according to the VL-CDR1, VL-CDR2 and VL-CDR3 combination in any one of the mutation combinations 1 to 32 in Table 1.

[0044] Table 1

[0045]

[0046] In an optional embodiment, the BCO ADC binding protein comprises the complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region; and / or, the complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region.

[0047] The VH-CDR1 includes an amino acid sequence consistent with the VH-CDR1 of the heavy chain variable region shown in SEQ ID NO.16.

[0048] The VH-CDR2 includes an amino acid sequence consistent with the VH-CDR2 of the heavy chain variable region shown in SEQ ID NO.16.

[0049] The VH-CDR3 comprises an amino acid sequence consistent with the VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.16.

[0050] SEQ ID NO.16 is as follows:

[0051] QVQIEESGAGLVQPGASMKFSCAASGFX1FTX2SCNGLGRAPEKGLEWIAEIX3PNTSLPTFYD TDSVKGRFTVSKSDSKTNVHLHMMTSEDDDVDFCYX4TNECNYX5MDHWGPGTSITVSS.

[0052] Among them, X1 is I or S, X2 is D or E, X3 is D or N, X4 is Q or W, and X5 is G or F.

[0053] The VL-CDR1 includes an amino acid sequence consistent with the VL-CDR1 of the light chain variable region shown in SEQ ID NO.17.

[0054] The VL-CDR2 comprises an amino acid sequence consistent with the VL-CDR2 of the light chain variable region shown in SEQ ID NO.17.

[0055] The VL-CDR3 comprises an amino acid sequence consistent with the VL-CDR3 of the light chain variable region shown in SEQ ID NO.17.

[0056] SEQ ID NO.17 is as follows:

[0057] DIAVTQTPSSLTVTIGQKVTVSCKSSQSX6LHSDGX7TNYMSWYQKPPHPPHNLFILVSSLDESG VPDLFAGGSSGNYFSLIISIVAADYLSLLLLSX8GX9FYPLTFGAGTLLDLK

[0058] Among them, X6 is L or R, X7 is Q or Y, X8 is G or K, and X9 is L or D.

[0059] It is understood that the amino acid sequences of other regions in the variable region shown in SEQ ID NO. 16 or 17 except the CDR region are not used to limit the BCOADC binding protein provided by the present invention. For example, if the BCOADC binding protein contains a framework region, it may be different from the framework region in the variable region shown in SEQ ID NO. 16 or 17. The CDR region in the variable region shown in SEQ ID NO. 16 or 17 can be divided according to an optional method known in the art.

[0060] Optionally, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the variable region are defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems. Taking the definition of Kabat, Chothia, IMGT or Contact as an example, the distribution of VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.16 in the sequence shown in SEQ ID NO.16 is shown in Table 2; the distribution of VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO.17 in the sequence shown in SEQ ID NO.17 is shown in Table 3.

[0061] Table 2 Distribution of CDRs in VH shown in SEQ ID NO.16 according to various definitions

[0062] definition VH-CDR1 VH-CDR2 VH-CDR3 Chothia No. 26-32 No. 52-59 No. 99-108 Kabat No. 31-35 No. 50-68 No. 99-108 Contact 30th to 35th No. 47-61 No. 97-107 IMGT No. 26-33 No. 51-60 No. 97-108

[0063] Table 3 Distribution of CDRs in VL of SEQ ID NO. 17 according to various definitions

[0064] definition VL-CDR1 VL-CDR2 VL-CDR3 Chothia 24th to 40th No. 56-62 No. 95-103 Kabat 24th to 40th No. 56-62 No. 95-103 Contact No. 36-42 No. 52-61 No. 95-102 IMGT No. 27-38 No. 56-58 No. 95-103

[0065] In an optional embodiment, each mutation site of VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region shown in SEQ ID NO.16 is combined according to the VH-CDR1, VH-CDR2 and VH-CDR3 combination mode in any one of the mutation combinations 1 to 32 in Table 1. Optionally, the mutation combination mode of X1 to X5 in VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.16 defined by IMGT conforms to the VH-CDR1, VH-CDR2 and VH-CDR3 combination mode in any one of the mutation combinations 1 to 32 in Table 1.

[0066] In an optional embodiment, each mutation site of VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region shown in SEQ ID NO.17 is combined according to the VL-CDR1, VL-CDR2 and VL-CDR3 combination mode in any one of the mutation combinations 1 to 32 in Table 1. Optionally, the mutation combination mode of X6 to X9 in VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO.17 defined by IMGT conforms to the VL-CDR1, VL-CDR2 and VL-CDR3 combination mode in any one of the mutation combinations 1 to 32 in Table 1.

[0067] In an optional embodiment, the BCOADC binding protein has a KD≤8.2×10 -9 mol / L affinity.

[0068] In an optional embodiment, the heavy chain variable region further contains at least one framework region, such as one framework region, two framework regions, three framework regions or four framework regions.

[0069] In an alternative embodiment, the light chain variable region further comprises at least one framework region, such as one framework region, two framework regions, three framework regions or four framework regions.

[0070] In an optional embodiment, the species origin of each of the framework regions independently includes, but is not limited to, one or more of rabbits, cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks, humans, and their mutants.

[0071] In an optional embodiment, the framework region VH-FR1 of the heavy chain variable region includes an amino acid sequence consistent with VH-FR1 of the heavy chain variable region shown in SEQ ID NO.16; and / or the framework region VH-FR2 includes an amino acid sequence consistent with VH-FR2 of the heavy chain variable region shown in SEQ ID NO.16; and / or the framework region VH-FR3 includes an amino acid sequence consistent with VH-FR3 of the heavy chain variable region shown in SEQ ID NO.16; and / or the framework region VH-FR4 includes an amino acid sequence consistent with VH-FR4 of the heavy chain variable region shown in SEQ ID NO.16.

[0072] In an optional embodiment, the framework region VL-FR1 of the light chain variable region includes an amino acid sequence consistent with VL-FR1 of the light chain variable region shown in SEQ ID NO.17; and / or the framework region VL-FR2 includes an amino acid sequence consistent with VL-FR2 of the light chain variable region shown in SEQ ID NO.17; and / or the framework region VL-FR3 includes an amino acid sequence consistent with VL-FR3 of the light chain variable region shown in SEQ ID NO.17; and / or the framework region VL-FR4 includes an amino acid sequence consistent with VL-FR4 of the light chain variable region shown in SEQ ID NO.17.

[0073] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown in SEQ ID NO.1, 3 or 5.

[0074] In an optional embodiment, the amino acid sequence of the light chain variable region of the BCOADC binding protein is shown in SEQ ID NO. 2, 4 or 6.

[0075] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2; and / or, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.4; and / or, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.6.

[0076] In an alternative embodiment, the BCOADC binding protein is an antibody or antigen binding fragment comprising a constant region.

[0077] In an alternative embodiment, at least a portion of the constant region sequence of the BCOADC binding protein is a human constant region sequence.

[0078] In an optional embodiment, the constant region sequence of the BCOADC binding protein is selected from the sequence of a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, wherein the IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD includes their subclasses and mutant forms.

[0079] In an alternative embodiment, the BCOADC binding protein contains a heavy chain constant region.

[0080] In an optional embodiment, the heavy chain constant region sequence of the BCOADC binding protein is selected from part or all of the constant region sequence of human IgG1, preferably including CH1, CH2 and CH3 of the constant region of human IgG1.

[0081] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.7.

[0082] In an alternative embodiment, the BCOADC binding protein contains a light chain constant region.

[0083] In an optional embodiment, the light chain constant region sequence is selected from the light chain constant region of a mouse.

[0084] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.8.

[0085] In an optional embodiment, the BCOADC binding protein is a human-mouse chimeric antibody.

[0086] In a second aspect, a biological material is also provided, comprising a polynucleotide, a vector or a cell; wherein the polynucleotide encodes the above-mentioned BCOADC binding protein; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector or is capable of expressing the BCOADC binding protein.

[0087] After the polynucleotide encoding the BCOADC binding protein is connected to the vector, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line capable of expressing the BCOADC binding protein, and the corresponding protein can be obtained by cell expression.

[0088] In an optional embodiment, the cells used to express the BCOADC binding protein are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0089] In an alternative embodiment, the cells used to express the BCOADC binding protein are CHO cells (Chinese Hamster Ovary cells).

[0090] In a third aspect, a method for preparing the BCOADC binding protein of the first aspect is also provided, comprising culturing cells capable of expressing the BCOADC binding protein.

[0091] In an optional embodiment, the preparation method further comprises transforming the polynucleotide encoding the BCOADC binding protein into cells and expressing the polynucleotide, and obtaining the BCOADC binding protein by purification.

[0092] In an optional embodiment, the preparation method further comprises synthesizing a polynucleotide containing the BCOADC binding protein encoding gene as required, and / or preparing a suitable expression vector as required, transforming the expression vector into a desired cell and expressing it, and obtaining the BCOADC binding protein by purification.

[0093] In an optional embodiment, the cell is obtained by transforming a polynucleotide encoding the BCO ADC binding protein as described in the first aspect into a cell, the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.

[0094] In an optional embodiment, the constant region fragment is fused to the C-terminus of the heavy chain variable region to construct a complete heavy chain expression plasmid.

[0095] In an optional embodiment, the constant region fragment includes one or more of CH1, CH2 and CH3, preferably includes CH1, CH2 and CH3.

[0096] In an optional embodiment, the cell is a eukaryotic cell, preferably a mammalian cell.

[0097] In an optional embodiment, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

[0098] In a fourth aspect, there is also provided a use of the BCOADC binding protein of the first aspect or the biomaterial of the second aspect in any one of the following (I) to (VII):

[0099] (I) Detection of anti-BCOADC antibodies or AMA-M2 antibodies for non-diagnostic and therapeutic purposes;

[0100] (II) preparing products for detecting anti-BCOADC antibodies or AMA-M2 antibodies;

[0101] (III) Preparation of products for diagnosis and / or auxiliary diagnosis of AMA-M2 antibody positive diseases;

[0102] (IV) detection of BCOADC for non-diagnostic and therapeutic purposes;

[0103] (V) preparing products for detecting BCOADC;

[0104] (VI) for separating, enriching and / or purifying BCOADC;

[0105] (VII) preparing products for separation, enrichment and / or purification of BCOADC;

[0106] In the applications of aspects (I) to (III) above, the BCOADC binding protein can be used as a standard and / or quality control product during detection to provide a reliable reference for the detection results, and can also be used to construct a standard curve.

[0107] In an optional embodiment, the AMA-M2 antibody-positive disease includes type II autoimmune hepatitis.

[0108] The applications of aspects (IV) to (VII) above can utilize the ability of BCOADC binding protein to specifically target and bind to BCOADC to achieve detection, separation, enrichment and / or purification of BCOADC. In an optional embodiment, when the BCOADC binding protein is an immunoconjugate, for example, connected with a signal substance, the positioning or real-time detection of BCOADC can be achieved by detecting the signal substance. In an optional embodiment, the detection, separation, enrichment and / or purification of BCOADC is achieved by separating the anti-BCOADC binding protein-BCOADC immune complex.

[0109] In an optional embodiment, in the above aspects (II), (III), (V) or (VII), a person skilled in the art can prepare a corresponding product (such as the above-mentioned immunoconjugate) according to actual use, and select other reagents in the product, including but not limited to one or more of a signal substance, a solid phase carrier, a buffer reagent, a salt, a secondary antibody, a chromogenic substrate, a blocking solution, a washing solution, a solvent, an eluent, a coupling agent, a negative control, a positive control, a standard, a quality control product and a marker.

[0110] In a fifth aspect, a reagent or a kit is also provided, wherein the reagent or the kit comprises the BCOADC binding protein of the first aspect, or the biological material of the second aspect.

[0111] In an optional embodiment, the kit is used to detect anti-BCOADC antibodies, AMA-M2 antibodies or AMA-M2 antibody-positive diseases; the kit includes standards and / or quality control products, and the standards and / or quality control products contain the BCOADC binding protein.

[0112] In an optional embodiment, the kit is used to detect primary biliary cirrhosis.

[0113] In an optional embodiment, the kit further comprises a detection reagent, and the detection reagent comprises an anti-BCOADC antibody or an AMA-M2 antibody detection reagent.

[0114] In an optional embodiment, the kit further comprises a detection reagent for detecting antibodies binding to PDC (pyruvate dehydrogenase complex) and / or OGDC (2-oxoglutarate dehydrogenase complex).

[0115] In an optional embodiment, the kit further comprises a reagent for detecting at least one of anti-nuclear antibodies (ANA), anti-smooth muscle antibodies (SMA) and anti-liver and kidney microsomal antibodies (LKM).

[0116] In an optional embodiment, the kit further comprises a detection reagent of an antibody of at least one of the following substances: dsDNA, Histone, Nucleosome, Ribosomal P, SSA / Ro52, SSA / Ro60, SSB / La, PM-scl, scl-70, Jo-1, U1-snRNP, Sm, CENP-B, Mi-2 and PCNA.

[0117] In an optional embodiment, the kit further comprises a solid phase carrier.

[0118] In an optional embodiment, the BCOADC binding protein in the reagent or kit is coupled to a solid phase carrier; or the BCOADC binding protein in the reagent or kit and the solid phase carrier are packaged separately. By coupling the BCOADC binding protein to a solid phase carrier, it can be used to capture BCOADC in a sample to be tested. Alternatively, by coupling the BCOADC binding protein to a solid phase carrier, it can be used to purify BCOADC.

[0119] In an optional embodiment, the BCOADC antibody or AMA-M2 antibody detection reagent includes a solid phase carrier coupled with BCOADC.

[0120] In an optional embodiment, the kit further comprises a signal substance.

[0121] In an optional embodiment, the BCOADC binding protein in the reagent or kit is coupled to a signal substance; or the BCOADC binding protein and the signal substance in the reagent or kit are packaged separately. By coupling the BCOADC binding protein to the signal substance, it can be used to locate and detect BCOADC or detect BCOADC in a sample by Western blotting.

[0122] The above reagents or kits may also optionally include reagents and / or consumables used in the art for detection reactions or protein purification, which are well known to those skilled in the art, including but not limited to one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality control products and markers.

[0123] The above reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow sorting, immunoblotting, immunohistochemistry, ELISA, immunochromatography or immunomagnetic beads. Those skilled in the art can prepare reagents or other reagents in the kit according to the corresponding detection means, and the present invention is not limited to this.

[0124] The signal substance in any of the above embodiments includes, but is not limited to, one or more of enzymes, luminescent labels, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals and photosensitizers.

[0125] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Examples of luminescent markers include, but are not limited to, fluorescent proteins, synthetic small molecules or polymer dyes, and specific examples include, but are not limited to, Alexa350, Alexa 405, Alexa430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyl diamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin One or more of B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine and Texas Red. Fluorescent microspheres, colored microspheres and latex microspheres are independently selected from products acceptable in the art, such as commercially available products. Radionuclides include but are not limited to 110In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I. 154-158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co. 72 As, 75 Br, 76 Br, 82 mRb and 83 Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0126] The solid phase carrier in any of the above embodiments includes but is not limited to microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices or lateral flow devices; more specifically, for example, but not limited to, enzyme-labeled wells, immunochromatographic test strips or magnetic beads. The chromatography matrix can be any known chromatography matrix acceptable in the art, including but not limited to polystyrene, polysaccharide polymers or silica gel. In an optional embodiment, the chromatography matrix includes gel particles.

[0127] The present invention discloses a BCOADC binding protein, and obtains multiple BCOADC binding proteins with BCOADC binding ability by mutating the CDR region, and also prepares human-mouse chimeric recombinant monoclonal antibodies. The present invention has the following beneficial effects:

[0128] (1) This method prepares BCOADC binding proteins with known sequences, can be recombinantly expressed in vitro, is simple to operate, takes a short time, has controllable production processes, small product batch-to-batch differences, and good stability, and has good application prospects.

[0129] (2) The BCOADC binding protein prepared by the present invention can effectively bind to the BCOADC protein, K D The maximum affinity can reach 6.4E-10M, with good affinity. It can be used for BCOADC detection, including but not limited to the use of liquid chips, immunofluorescence staining, flow sorting, fluorescent microspheres and other technologies to identify BCOADC; the BCOADC binding protein can also be coupled to a chromatography matrix for immunoaffinity chromatography purification of BCOADC to improve the purity of the antigen.

[0130] (3) The BCOADC binding protein can be used for anti-BCOADC antibody or AMA-M2 antibody detection, and can be used in diagnostic products for anti-BCOADC antibody or AMA-M2 antibody positive diseases, for example, as a quality control product and / or standard product for anti-BCOADC antibody or AMA-M2 antibody detection products; when the BCOADC binding protein is used as a quality control product in the test kit, it can alleviate the problems of cumbersome immune polyclonal antibody operation and low subsequent coupling efficiency; it can reduce production costs, stabilize product quality, and significantly improve reaction values; on the other hand, compared with the direct use of human serum, it can also avoid the problems of difficult sample sources and high costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0132] Figure 1 This is the SDS-PAGE protein electrophoresis diagram of the recombinant monoclonal antibody (mutation combination 1) BCOADC in Example 1, M represents Marker, reduced represents the reduced SDS-PAGE electrophoresis result of the recombinant monoclonal antibody BCOADC-Ab, and the heavy chain and light chain are 50 kDa and 25 kDa, respectively, and non-reduced represents the non-reduced SDS-PAGE electrophoresis result of the recombinant monoclonal antibody BCOADC-Ab, and the size is about 150 kDa. DETAILED DESCRIPTION

[0133] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0134] Example 1 Construction of BCOADC chimeric antibody expression plasmid and expression and purification of recombinant monoclonal antibody

[0135] Phage display technology was used to screen and obtain BCOADC-specific high-affinity monoclonal strains. After sequencing, the Fab region sequence was obtained and gene synthesis was performed. The synthesized light chain gene and heavy chain gene were constructed on the vector PTT5 respectively. The heavy chain gene expression plasmid and the light chain gene expression plasmid were mixed with PEI at a molar ratio of 1:2 and transfected into 293F suspension cells in the logarithmic growth phase. The cells were cultured in a shaking incubator at 37°C at a speed of 120 rpm. After 5 days, the cell supernatant was collected, the antibody was purified with Protein A, eluted with 0.1M Glycine (pH3.0), and neutralized by adding 1M Tris (pH8.0). After the elution was completed, the ultrafiltration centrifuge tube was replaced with PBS buffer and concentrated, the protein concentration was determined, and its purity was verified by SDS-PAGE. Figure 1 .

[0136] The VH of the monoclonal antibody is shown in SEQ ID NO: 1, the VL is shown in SEQ ID NO: 2, the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO: 1 are shown in Table N, and the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable region shown in SEQ ID NO: 2 are shown in Table N.

[0137] A human-mouse chimeric recombinant antibody containing a human constant region sequence was further prepared, the heavy chain sequence was as shown in SEQ ID NO:9; the light chain sequence was as shown in SEQ ID NO:10; wherein the heavy chain constant region sequence was as shown in SEQ ID NO:7; and the light chain constant region sequence was as shown in SEQ ID NO:8.

[0138] Table 4

[0139]

[0140] Example 2 Antibody affinity analysis and activity identification

[0141] The antibody obtained in Example 1 was analyzed and the heavy chain complementary determining region and light chain complementary determining region were obtained according to the IMGT definition as follows:

[0142] Complementarity determining region VH-CDR1, amino acid sequence GF-X1-FT-X2-SC (SEQ ID NO.11)

[0143] The complementary determining region VH-CDR2 has an amino acid sequence of I-X3-PNTSLPTF (SEQ ID NO. 12),

[0144] The complementary determining region VH-CDR3, the amino acid sequence is Y-X4-TNECNY-X5-MDH (SEQ ID NO.13),

[0145] The complementary determining region VL-CDR1, the amino acid sequence is QS-X6-LHSDG-X7-TNY (SEQ ID NO.14),

[0146] The complementarity determining region VL-CDR2 has an amino acid sequence of VSS;

[0147] The complementary determining region VL-CDR3, the amino acid sequence is S-X8-G-X9-FYPLT (SEQ ID NO.15),

[0148] It contains a total of 9 mutable sites, X1 to X9. The complementary determining region of the antibody prepared in Example 1 is used as the skeleton, and X1 to X9 are used as mutation sites for mutation to prepare antibodies having the mutation sites in Table 5 (except for the different mutation sites, the rest are the same as the monoclonal antibody provided in Example 1).

[0149] Table 5

[0150]

[0151]

[0152] Example 3 ELISA determination of the binding activity of recombinant monoclonal antibodies to BCOADC antigen

[0153] (1) BCOADC protein (reference sequence NP_001909.4, 2 μg / mL) was coated on an ELISA plate and BSA was coated as a nonspecific binding control and incubated at 4°C overnight.

[0154] (2) Discard the coating solution, wash the plate with PBST, pat dry, add 3% milk, and block at 37°C for 2 h.

[0155] (3) The blocking solution was discarded, the plate was washed with PBST, patted dry, and each antibody in Example 2 was added in a 3-fold serial dilution starting from 1 μM, and incubated at 37° C. for 1.5 h.

[0156] (4) Discard the primary antibody, wash the plate with PBST, pat dry, add horseradish peroxidase (HRP)-labeled mouse anti-human IgG secondary antibody, and incubate at 37°C for 1 h.

[0157] (5) Discard the secondary antibody, wash the plate with PBST, pat dry, add the colorimetric substrate for color development, add the stop solution after 15 min to stop the reaction, and measure the OD value with an enzyme-labeled instrument.

[0158] (6) A nonlinear fitting plot was performed with the OD value as the ordinate and the logarithm of the antibody molar concentration as the abscissa.

[0159] The affinity of the recombinant mutant antibodies prepared in Example 2 was measured according to the above method, and the results are shown in the following table.

[0160] Table 6 Affinity analysis data of various mutation combinations

[0161] Location <![CDATA[K D (M)]]> Location <![CDATA[K D (M)]]> Mutation combination 1 2.5E-9 Mutation combination 17 2.9E-7 Mutation combination 2 4.3E-8 Mutation combination 18 9.5E-6 Mutation combination 3 5.3E-8 Mutation combination 19 5.6E-8 Mutation combination 4 4.3E-9 Mutation combination 20 6.5E-7 Mutation combination 5 7.2E-7 Mutation combination 21 3.8E-8 Mutation combination 6 7.3E-8 Mutation combination 22 4.2E-7 Mutation combination 7 5.3E-9 Mutation combination 23 4.4E-8 Mutation combination 8 6.4E-10 Mutation combination 24 6.2E-6 Mutation combination 9 3.5E-8 Mutation combination 25 6.8E-7 Mutation combination 10 8.2E-9 Mutation combination 26 2.3E-9 Mutation combination 11 3.4E-7 Mutation combination 27 3.5E-8 Mutation combination 12 1.7E-9 Mutation combination 28 7.3E-6 Mutation combination 13 2.3E-7 Mutation combination 29 5.6E-8 Mutation combination 14 3.7E-8 Mutation combination 30 3.2E-7 Mutation combination 15 5.2E-8 Mutation combination 31 3.6E-6 Mutation combination 16 5.7E-9 Mutation combination 32 8.4E-8

[0162] From the above table, we can see that the K values ​​of mutation combinations 1, 4, 7, 8, 10, 12, 16, and 26 are D Up to E-9M; among them, the recombinant mutant antibody K with mutation combination 8 D The smallest one is 6.4E-10M, with the highest affinity.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A BCOADC binding protein, characterized in that Contains at least one complementarity determining region selected from the following amino acid sequences: The complementary determining region VH-CDR1 has an amino acid sequence of GF-X1-FT-X2-SC (SEQ ID NO.11), wherein X1 is I or S, X2 is D or E; The complementary determining region VH-CDR2 has an amino acid sequence of I-X3-PNTSLPTF (SEQ ID NO.12), wherein X3 is D or N; The complementary determining region VH-CDR3 has an amino acid sequence of Y-X4-TNECNY-X5-MDH (SEQ ID NO. 13), wherein X4 is Q or W, X5 is G or F; The complementary determining region VL-CDR1 has an amino acid sequence of QS-X6-LHSDG-X7-TNY (SEQ ID NO. 14), wherein X6 is L or R, X7 is Q or Y; The complementarity determining region VL-CDR2 has an amino acid sequence of VSS; The complementary determining region VL-CDR3 has an amino acid sequence of S-X8-G-X9-FYPLT (SEQ ID NO. 15), wherein X8 is G or K, X9 is L or D.

2. The BCOADC binding protein according to claim 1, characterized in that The BCOADC binding protein contains complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; and / or, the BCOADC binding protein contains complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3; Optionally, the mutation sites of the complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are combined according to any one of the following mutation combinations 1 to 32, and / or the mutation sites of the complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 are combined according to any one of the following mutation combinations 1 to 32:

3. The BCOADC binding protein according to claim 1, characterized in that The BCOADC binding protein has a KD of ≤8.2×10 -9 mol / L affinity.

4. A BCOADC binding protein, characterized in that Complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region; and / or, complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region; The complementary determining regions VH-CDR1, VH-CDR2 and VH-CDR3 include amino acid sequences consistent with VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.16; The complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3 include amino acid sequences consistent with VL-CDR1, VL-CDR2 and VL-CDR3 of the heavy chain variable region shown in SEQ ID NO.

17.

5. The BCOADC binding protein according to any one of claims 1 to 4, characterized in that: The heavy chain variable region further comprises a framework region, and / or the light chain variable region further comprises a framework region; Optionally, the species origin of each of the framework regions independently includes, but is not limited to, one or more of rabbit, cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human and their mutants; Optionally, at least one framework region of the heavy chain variable region comprises an amino acid sequence consistent with the framework region of the heavy chain variable region shown in SEQ ID NO.16; and / or, at least one framework region of the light chain variable region comprises an amino acid sequence consistent with the framework region of the light chain variable region shown in SEQ ID NO.17; Optionally, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is as shown in SEQ ID NO.1, 3 or 5, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2, 4 or 6; Optionally, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; and / or, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; and / or, the amino acid sequence of the heavy chain variable region of the BCOADC binding protein is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6; Optionally, the BCOADC binding protein is a complete antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody or antibody minimum recognition unit; Optionally, the species of origin of the remaining sequence of the antibody minus the CDR region include one or more of cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks and humans; Optionally, the BCOADC binding protein further comprises a constant region; Optionally, at least a portion of the constant region sequence is a human constant region sequence; Optionally, the constant region sequence is selected from a sequence of a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the BCOADC binding protein contains a heavy chain constant region, and the heavy chain constant region sequence is selected from a human heavy chain constant region; Optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.7; Optionally, the BCOADC binding protein contains a light chain constant region; Optionally, the light chain constant region sequence is selected from the light chain constant region of a mouse, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

8.

6. Biomaterial, characterized in that including a polynucleotide, a vector or a cell; The polynucleotide encodes the BCOADC binding protein according to any one of claims 1 to 5; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the BCOADC binding protein according to any one of claims 1 to 5.

7. The method for preparing the BCOADC binding protein according to any one of claims 1 to 5, characterized in that: Comprising culturing the cell as claimed in claim 6; Optionally, the cell is obtained by transforming a polynucleotide encoding a BCOADC binding protein according to any one of claims 1 to 5 into a cell, wherein the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell; Optionally, the cell is a eukaryotic cell, preferably a mammalian cell; Optionally, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

8. Use of the BCOADC binding protein according to any one of claims 1 to 5, or the biomaterial according to claim 6 in any one of the following (I) to (VII): (I) Detection of anti-BCOADC antibodies or AMA-M2 antibodies for non-diagnostic and therapeutic purposes; (II) preparing products for detecting anti-BCOADC antibodies or AMA-M2 antibodies; (III) Preparation of products for diagnosis and / or auxiliary diagnosis of anti-BCOADC antibody-positive diseases or AMA-M2 antibody-positive diseases; (IV) detection of BCOADC for non-diagnostic and therapeutic purposes; (V) preparing products for detecting BCOADC; (VI) for separating, enriching and / or purifying BCOADC; (VII) preparing products for separation, enrichment and / or purification of BCOADC; Optionally, the anti-BCOADC antibody-positive disease or AMA-M2 antibody-positive disease includes primary biliary cirrhosis; Optionally, in any one of (I) to (III), the BCOADC binding protein is used as a standard or quality control product.

9. A reagent or a kit, characterized in that The reagent or kit comprises the BCOADC binding protein according to any one of claims 1 to 5, or the biomaterial according to claim 6.

10. The reagent or kit according to claim 9, characterized in that The kit is used to detect anti-BCOADC antibodies, AMA-M2 antibodies, anti-BCOADC antibody-positive diseases or AMA-M2 antibody-positive diseases; The kit includes a standard substance and / or a quality control substance, and the standard substance and / or the quality control substance contain the BCOADC binding protein; Optionally, the kit further comprises an anti-BCOADC antibody or AMA-M2 antibody detection reagent; Optionally, the kit further comprises a detection reagent for detecting antibodies binding to PDC and / or OGDC; and / or, a reagent for detecting at least one of anti-nuclear antibodies, anti-smooth muscle antibodies and anti-liver and kidney microsomal antibodies; Optionally, the kit further comprises a detection reagent of an antibody of at least one of the following substances: dsDNA, Histone, Nucleosome, Ribosomal P, SSA / Ro52, SSA / Ro60, SSB / La, PM-scl, scl-70, Jo-1, U1-snRNP, Sm, CENP-B, Mi-2 and PCNA.

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