Monoclonal antibody of anti-Ki-67 protein and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-Ki-67 protein monoclonal antibody and application thereof. Amino acid sequences of complementary determining regions CDR1-3 on a light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.3-5; and the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10. The monoclonal antibody provided by the invention can effectively recognize Ki-67 protein expressed by cells / tissues, has good antigen binding specificity, relatively high affinity and excellent anti-interference capability, and is beneficial to greatly improving the accuracy, sensitivity and credibility of detecting whether the tissues / cells express Ki-67 or not; and the antibody has cross reactivity to homologous Ki-67 of human and mice (including rats and mice), and has wide detection applicability to different species of Ki-67 proteins.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to monoclonal antibodies against Ki-67 protein and applications thereof. Background Art

[0002] Ki-67 is a specific nuclear protein closely related to cell proliferation. Under normal physiological conditions, the expression level of Ki-67 gradually increases from the G1 phase of cell mitosis to the S phase and G2 phase, then rapidly decreases after the M phase, and finally stops expressing in the G0 phase (quiescent phase). Therefore, Ki-67 is an important cell proliferation marker, and its expression level reflects the proliferation rate and activity of cells.

[0003] Ki-67 has important clinical pathological diagnostic value and has become one of the most commonly used targets in clinical pathological diagnosis and is widely used in oncology research. In tumor tissues, high expression of Ki-67 often means that tumor cells have high proliferation activity, and the Ki-67 proliferation index is closely related to the degree of differentiation, infiltration and metastasis of the tumor, and prognosis. Therefore, Ki-67 can be used as an independent indicator in some cases to study tumor biological behavior, evaluate tumor deterioration and prognosis, and predict the risk of recurrence and metastasis. For example, in solid tumors such as breast cancer, prostate cancer, and gastrointestinal tumors, a high Ki-67 index is usually accompanied by a higher tumor grade, lymph node metastasis, and a poorer survival rate. Clinically, Ki-67 is also used to guide treatment decisions. In some cases, high Ki-67 levels may indicate the need for more aggressive treatment strategies; while for patients with low Ki-67 expression, relatively mild treatments such as endocrine therapy may be more suitable. This individualized treatment strategy based on Ki-67 levels helps optimize the treatment effect and reduce unnecessary treatment burden. The expression level of Ki-67 can also be used to predict treatment response. If the expression level of Ki-67 in tumor tissue is detected to be significantly decreased, it usually indicates a better treatment effect; if the expression level of Ki-67 is significantly increased, the treatment plan may need to be adjusted.

[0004] Based on the guiding role of Ki-67 in evaluating cell proliferation activity, assisting tumor diagnosis, judging tumor prognosis, predicting the risk of recurrence and metastasis, monitoring treatment effects, and guiding individualized treatment, the detection of Ki-67 in pathological cells or tissues can provide an important reference for the diagnosis and treatment of related tumors. With the popularization of immunohistochemistry in clinical pathological diagnosis, it is imperative to develop immunohistochemistry for Ki-67, and the key lies in the specificity and sensitivity of the antibody. When the content of the target antigen Ki-67 in cell or tissue biopsy is usually low or the antigen epitope is partially masked, if the antibody sensitivity is low, it may not be able to effectively identify the antigen, resulting in a false negative result. In addition, there are many interfering factors in cell components. If the antibody specificity is poor, it may cross-react with other antigens, produce nonspecific staining, enhance background staining, and produce false positive results, thereby interfering with the accurate judgment of Ki-67 positive cells. This is a challenge for detecting some tumors with low proliferation activity or tumor cells whose proliferation is inhibited after treatment. Therefore, it is of great value to develop anti-Ki-67 antibodies with good specificity and high sensitivity suitable for cell / tissue detection. Summary of the invention

[0005] In view of the problem that there is a lack of monoclonal antibodies suitable for cell / tissue Ki-67 detection in the prior art, the present invention provides a monoclonal antibody against Ki-67 protein, which can specifically recognize and bind to Ki-67 on the surface of cells / tissues, and has good antigen binding specificity, high affinity and excellent anti-interference ability. The present invention further provides the use of the monoclonal antibody in the preparation of a Ki-67 protein detection kit and related detection kits. To achieve the aforementioned purpose, the present invention is specifically implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides a monoclonal antibody against Ki-67 protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1-3 on the light chain variable region are respectively shown as SEQ ID NOs.3-5; and the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NOs.8-10.

[0007] Furthermore, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0010] The second aspect of the present invention provides a nucleic acid molecule, which encodes the monoclonal antibody against Ki-67 protein as described above.

[0011] The third aspect of the present invention provides a recombinant vector or a host cell, wherein the recombinant vector or the host cell comprises the nucleic acid molecule as described above.

[0012] The fourth aspect of the present invention provides the use of the monoclonal antibody against Ki-67 protein as described above in the preparation of a Ki-67 protein detection kit.

[0013] Furthermore, the Ki-67 protein is human, rat or mouse Ki-67 protein.

[0014] Furthermore, the detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot assay kit, an immunohistochemistry kit, an immunofluorescence assay kit, an immunoblotting kit or a flow cytometry kit.

[0015] In a fifth aspect, the present invention provides a Ki-67 protein detection kit, which comprises the anti-Ki-67 protein monoclonal antibody or its antibody conjugate as described above, wherein the antibody conjugate is formed by connecting the monoclonal antibody to a detection marker.

[0016] The advantages and positive effects of the present invention are as follows: the monoclonal antibody provided by the present invention can effectively identify Ki-67 protein expressed by cells / tissues, has good antigen binding specificity, high affinity and excellent anti-interference ability, is conducive to greatly improving the accuracy, sensitivity and credibility of detecting whether tissues / cells express Ki-67, effectively avoiding false positive and false negative results, and is particularly suitable for immunoblotting, immunofluorescence and immunohistochemical analysis, and has broad application prospects and good market prospects in clinical diagnosis and scientific research detection. In addition, the antibody has cross-reactivity to homologous Ki-67 of humans and mice (including rats and mice), and has wide applicability to the detection of Ki-67 proteins of different species. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 A map of the mammalian expression vector pRB322 used to construct an antibody expression vector against human Ki-67 protein in Example 1 of the present invention, from left to right, respectively, vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0019] Figure 2 This is an electrophoresis gel image of the expression of Ki-67 in positive and negative cells / tissues detected by immunoblotting using an anti-human Ki-67 protein antibody in Example 2 of the present invention;

[0020] Figure 3 This is a fluorescence staining image of the expression of Ki-67 in positive cells / tissue samples detected by immunofluorescence using an anti-human Ki-67 protein antibody according to Example 2 of the present invention;

[0021] Figure 4 This is a tissue staining diagram of Example 2 of the present invention, in which anti-human Ki-67 protein antibody is used to detect the expression of Ki-67 in positive and negative tissues through immunohistochemistry. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0024] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0025] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art.

[0026] The terms "include", "comprising", "containing", "having" and the like are not limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0027] The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" includes the following situations: (i) A, (ii) B, and (iii) A and B.

[0028] The terms "rabbit monoclonal antibody", "rabbit antibody" and "rabbit monoclonal antibody" and other similar terms have the same meaning and, unless otherwise specified, refer to rabbit antibodies that specifically bind to Ki-67 protein. The modifier "rabbit" indicates that the complementarity determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence.

[0029] An antibody is an immunoglobulin molecule that is able to specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity. An antibody fragment can be one or more parts or fragments of a full-length antibody that retain the ability of the antibody to specifically bind to a target antigen.

[0030] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, namely κ chain and λ chain; heavy chains can be classified into five types, namely μ, δ, γ, α and ε chains, and antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions near the N-terminus of the light and heavy chains where the amino acid sequences vary greatly are called variable regions (V), and the regions near the C-terminus where the amino acid sequences are relatively stable are called constant regions (C). The heavy chain variable region (VH) and the light chain variable region (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity determining regions (CDRs), which are ring-shaped structures. The heavy chain CDR and the light chain CDR are closely together and cooperate with each other through the FR region, forming a surface that can complement the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. The FR region is the more conservative part of VH and VL. They are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop. Each VH and VL is usually composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the accumulation of both the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define.

[0032] The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of antibodies. The length of CL of different types of Ig (κ or λ) is basically the same, but the length of CH of different classes of Ig is different, such as IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the heavy chain and light chain constant regions of antibodies are well known in the art and can be obtained by querying the IMGT database.

[0033] A full-length antibody is the most complete antibody molecular structure and has a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "complete antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0034] Antibody fragments are one or more parts or fragments of a full-length antibody, which substantially retain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR region as the full-length antibody, and more preferably have the same variable region, thereby retaining complete antigen recognition and binding sites, and can bind to the same antigen as the full-length antibody, especially to the same epitope. In typical examples, antibody fragments include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, sc(Fv)2, and these antibody fragments can be obtained by conventional techniques in the art.

[0035] (i) Fab: Antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable region and constant region) and a part of the heavy chain (variable region and the first constant region). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0036] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0037] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). The three CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the intact antibody.

[0038] (iv) (Fv)2: consists of two Fv fragments covalently linked together.

[0039] (v) scFv: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (generally composed of 10-25 amino acids), which retains the binding specificity of the original antibody to the antigen. The linker in the present invention is not particularly limited as long as it does not hinder the expression of the antibody variable regions connected to its two ends. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side effects.

[0040] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected via a linker or the like.

[0041] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a complementary determining region (CDR) and a framework region (FR) from a rabbit immunoglobulin sequence. In other embodiments, the antibody may include amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to a portion of an antibody derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" is a chimeric antibody of a non-human antibody such as a rabbit antibody's CDR region and a human FR region. In some cases, the variable region of a non-human antibody is combined with a constant region of a human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is combined with a FR region and a constant region derived from a human antibody sequence, that is, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, and this framework sequence is derived from a single or multiple FR sequences of other human antibody variable regions. In the present invention, the CDR region in the chimeric antibody or humanized antibody is derived from a rabbit-derived CDR region.

[0042] The terms "monoclonal antibody" or "single antibody" and the like are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a small amount of mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitopes on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0043] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0044] In order to make the above-mentioned objects and advantages of the present invention more obvious and understandable, the specific implementation methods of the present invention are described in detail below.

[0045] An embodiment of the present invention provides a monoclonal antibody against Ki-67 protein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each comprise three complementary determining regions (CDRs), respectively named CDR1, CDR2 and CDR3, wherein the amino acid (AA) sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the amino acid (AA) sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0046] The antibody provided by the present invention is suitable for a variety of immunological detection methods. Specifically, by immunoblotting, positive cells or tissues expressing Ki-67 protein and negative cells or tissues not expressing Ki-67 protein were detected, and only bands were detected in the protein lysate of the positive sample and were consistent with expectations, indicating that the antibody has good specificity and good reactivity to Ki-67 proteins of different species. By immunofluorescence, multiple positive samples such as Hela cell line, A431 cell line, mouse spleen, C2C12 cell line, rat spleen and PC-12 cell line were detected, and obvious specific positive staining was also presented in the positive samples, and the localization of the antigen Ki-67 was consistent with the theory. Positive and negative tissue samples were detected by immunohistochemistry, and there was no coloring in the negative tissue samples, while the staining positioning was accurate in the positive tissue samples, there was no non-specific staining, and the positive staining was clear and bright, the background was clean, and the detection sensitivity and specificity could reach 100%. Combined with the above results, it can be seen that the antibody of the present invention can effectively identify Ki-67 protein expressed by cells / tissues, has good antigen binding specificity, high affinity and excellent anti-interference ability, and the test results are highly consistent with the actual situation, which is conducive to greatly improving the accuracy, sensitivity and credibility of immunoassay to directly detect whether tissues / cells express Ki-67, effectively avoiding false positive and false negative results, and is particularly suitable for immunoblotting, immunofluorescence and immunohistochemical analysis, and has broad application prospects and good market prospects in clinical diagnosis and scientific research detection. In addition, the antibody has cross-reactivity to homologous Ki-67 of humans and mice (including rats and mice), and has wide applicability to the detection of Ki-67 proteins of different species.

[0047] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), and the four FRs and three CDRs are arranged alternately in sequence to form a variable region. The amino acid sequence of the light chain variable region (VL) of the monoclonal antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.7.

[0048] Optionally, the monoclonal antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, CL and VL constitute the light chain, and CH and VH constitute the heavy chain. The constant region of the antibody is usually obtained by querying the IMGT online database.

[0049] Specifically, the amino acid sequence of the monoclonal antibody light chain (FL) is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.6, and the heavy chain is of IgG type.

[0050] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen binding region of the full-length antibody; the antigen binding region refers to a polypeptide that substantially maintains the same biological function or activity as the full-length form. Specifically, the antigen binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen binding regions can be obtained by conventional techniques in the art.

[0051] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the aforementioned nucleic acid molecule, or a host cell comprising the aforementioned nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal antibody against Ki-67 protein as described above.

[0052] The nucleic acid molecule can be in the form of DNA (such as cDNA or genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be derived based on the antibody amino acid sequence by conventional means such as codon coding rules.

[0053] The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0054] The original vector for constructing the recombinant vector is any conventional vector in the art, as long as it can hold the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and mini-chromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules to a host and multiply in a host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecules inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, and then introduced into a host cell to culture and obtain the antibody. This is a well-known technology in the art and will not be described in detail here.

[0055] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two vectors respectively and introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into a vector, and each nucleic acid sequence is connected to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to different promoters, or the nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to a single promoter, so that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0056] The recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth period and treated with CaCl2 or MgCl2; they can also be transfected by microinjection, electroporation or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging or particle bombardment to achieve gene introduction.

[0057] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining a host cell transfected or transformed with a recombinant vector as described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain a purified antibody.

[0058] In the present invention, the recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl2 or MgCl2. If necessary, microinjection, electroporation or liposome packaging can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, as well as microinjection, electroporation, liposome packaging, etc.

[0059] Preferably, the recombinant vector is the expression vector pBR322, and the host cell is a human kidney epithelial (293F) cell.

[0060] Another embodiment of the present invention provides the use of the monoclonal antibody against Ki-67 protein as described above in the preparation of a Ki-67 protein detection kit.

[0061] The advantages of using the monoclonal antibody against Ki-67 protein in preparing a Ki-67 protein detection kit are the same as the advantages of the monoclonal antibody against Ki-67 protein over the prior art as described above, and will not be repeated here.

[0062] The monoclonal antibody of the present invention has cross-recognition effect on Ki-67 proteins of multiple species. Specifically, the Ki-67 protein can be human, rat or mouse Ki-67 protein. The amino acid and nucleotide sequences of Ki-67 protein can be obtained by conventional techniques. Among them, the information of human Ki-67 protein can be found in Uniprot protein number: P46013, GeneID number: 4288; or, NCBI protein accession number: NP_002408.3, gene accession number: NM_002417.5. The information of mouse Ki-67 protein can be found in Uniprot protein number: E9PVX6, GeneID number: 17345; or, NCBI protein accession number: NP_001074586.2, NCBI gene accession number: NM_001081117.2. For information on rat Ki-67 protein, please refer to Uniprot protein number: Q5RJMO, GeneID number: 246042; or, NCBI protein accession number: NP_631925.2, NCBI gene accession number: NM_139186.2.

[0063] Based on the same inventive concept, an embodiment of the present invention further provides a Ki-67 protein detection kit, which comprises the anti-Ki-67 protein monoclonal antibody or antibody conjugate as described above, wherein the antibody conjugate is formed by connecting the monoclonal antibody to a detection marker.

[0064] The advantages of the Ki-67 protein detection kit over the prior art are the same as the advantages of the monoclonal antibody against Ki-67 protein over the prior art as described above, and will not be repeated here.

[0065] It should be noted that the antibodies of the present invention can be used alone or combined or coupled with a detection marker to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding antibodies, which specifically recognize and bind to Ki-67 in the sample to be tested, and then qualitative or quantitative detection of Ki-67 is achieved by analyzing the detection marker signal connected thereto; in other embodiments, the anti-Ki-67 antibody (as a primary antibody or capture antibody) is not labeled, and the detection marker is coupled to a secondary antibody (as a detection antibody) or other molecules that can bind to the primary antibody. For example, if the anti-Ki-67 antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby qualitatively or quantitatively detecting Ki-67 by analyzing the changes in the detection marker signal generated after the secondary antibody specifically binds to the antibody of the present invention.

[0066] The detection label is used to generate a recognizable signal change to identify the sample to be tested according to the signal change. The detection label includes, but is not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0067] Immunoassay methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), immunoprecipitation (IP), and flow cytometry (FC). The detection kit may be an ELISA kit, an ELISPOT kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.

[0068] Preferably, the detection kit is an immunoblotting kit, an immunohistochemistry kit or an immunofluorescence kit.

[0069] The test samples include serum, plasma, urine, cell or tissue samples, wherein cells include but are not limited to: human Hela cell line, human A431 cell line, mouse C2C12 cell line or rat PC-12 cell line; tissues include but are not limited to: human spleen, human colon, human colon cancer, human tonsil, human breast cancer, human invasive ductal carcinoma of the breast, human brain, human appendix, human kidney, human lung cancer, mouse colon, mouse spleen, mouse testicle, rat colon, rat spleen or rat testicle.

[0070] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0071] Example 1 Preparation of rabbit antibodies against Ki-67 protein

[0072] The immunogen used to prepare the rabbit monoclonal antibody in this example is a polypeptide fragment of 42 amino acids from position 2295 to position 2336 of the human Ki-67 protein. The polypeptide was synthesized by Suzhou Modif Biotechnology Co., Ltd. and the initial AA residue C was added. The sequence of the immunogen is: C-NLPGSKRWPQTPKEKAQALEDLAGFKELFQTPGTDKPTTDEK (see SEQ ID NO.11).

[0073] This example is based on B cell labeling and sorting technology, directly enriching and separating B lymphocytes that can recognize human Ki-67 protein from the spleen of New Zealand white rabbits immunized with the immunogen, and then culturing the separated B lymphocytes in the form of single cells to obtain monoclonal antibodies. For large-scale production, naturally paired antibody heavy chain variable region (VH) and light chain variable region (VL) genes are obtained by PCR amplification from B lymphocytes that secrete monoclonal antibodies, recombinantly expressed, and finally the target antibody 4E5 is isolated and purified in large quantities from the host cell culture supernatant.

[0074] The amino acid (AA) sequence of antibody 4E5 is shown in Table 1. For ease of description, LCDR1-3 in the table represent light chain complementary determining regions CDR1-3, and HCDR1-3 represent heavy chain complementary determining regions CDR1-3, respectively.

[0075] Table 1 Sequence information of monoclonal antibody 4E5 in this example

[0076]

[0077] 1. Animal immunization: 4 Japanese white rabbits were immunized with the above-mentioned immunogen; 300 μg was immunized for each white rabbit. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to make an emulsifier, and multiple injections were made subcutaneously in the abdomen and back of the rabbit. After the first immunization, 150 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to make an emulsifier every 2 weeks, and multiple injections were made subcutaneously in the abdomen and back of the rabbit for two booster immunizations. After three immunizations, rabbit serum samples were collected, and the titer against human Ki-67 protein was determined by ELISA. The serum after the last immunization was purified into antibodies, and the specificity and sensitivity of the antibodies against endogenous detection samples were detected by WB, IF and IHC. Take the rabbit with high serum titer and the best endogenous detection, and use 300 μg of immunogen for multiple injections subcutaneously to boost immunization once. The animal was sacrificed three days later and the spleen was taken.

[0078] 2. Isolate B lymphocytes from the spleen and sort B lymphocytes: Use conventional methods to isolate B lymphocytes from the spleen, and sort out antigen-specific B lymphocytes. For related methods, please refer to the patents "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication number: CN110016462A, publication date: 2019-07-16)" and "A B lymphocyte in vitro culture system and application (publication number: CN111518765A, publication date: 2020-08-11)".

[0079] 3. Cloning of the gene encoding rabbit monoclonal antibody: The supernatant of cultured B lymphocytes was used to identify positive clones by ELISA coated with biotinylated screening peptide (sequence: NLPGSKRWPQTPKEKAQALEDLAGFKELFQTPGTDKPTTDEK-Biotin, synthesized by Suzhou Modifu Biotechnology Co., Ltd.). The screening criteria were OD 450 Value greater than 0.7.

[0080] The positive clones of single B lymphocytes were collected and lysed and then analyzed by Quick-RNA TMRNA was extracted using MicroPrep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using cDNA as template, the naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified from the cDNA of the corresponding positive clone by PCR method; PCR reaction system: 4μL cDNA, 1μL forward primer (10mM), 1μL reverse primer (10mM), 12.5μL 2×Gloria HiFi (from ABclonal, catalog number RK20717) and 6.5μL H2O; PCR amplification program: 98℃ pre-denaturation for 30s, followed by 40 cycles of 98℃ for 10s, 64℃ for 30s, and 72℃ for 30s, and finally kept at 72℃ for 5min, and the resulting reaction solution was stored at 4℃. The primer sequences (5'-3') for amplifying VL and VH genes are as follows, F and R represent forward and reverse primers, respectively:

[0081] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO. 12);

[0082] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO. 13);

[0083] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO. 14);

[0084] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO. 15).

[0085] The amplified DNA product was sequenced by Jin Kairui Biotechnology Co., Ltd. to obtain the VL amino acid sequence shown in SEQ ID NO.2 and the VH amino acid sequence shown in SEQ ID NO.7; then the IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, and the complete light chain (FL) amino acid sequence shown in SEQ ID NO.1 and the complete heavy chain (FH) amino acid sequence shown in SEQ ID NO.6 were obtained, thereby obtaining a rabbit monoclonal antibody against Ki-67 protein, named 4E5.

[0086] 4. Production and purification of rabbit monoclonal antibodies: In order to obtain multiple antibodies that recognize human Ki-67, the heavy chain and light chain genes of the selected antibody 4E5 were loaded on expression vectors, and the plasmids were transfected into 293F cells. The mammalian expression vector pBR322 used pre-carried the light chain constant region (CL) and heavy chain constant region genes (CH), and its expression profile is shown in Figure 1 In the figure, pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of CL (left figure), and Heavy chain constant is the nucleic acid sequence of CH (right figure). Then, the VL and VH genes are respectively inserted into the upstream of the CL and CH genes of the pBR322 vector. The construction process of the light chain and heavy chain gene expression vectors is as follows: the mammalian cell expression vector pBR322 containing rabbit monoclonal antibodies CH and CL is conventionally linearized with NheI and XbaI restriction endonucleases, respectively, and the PCR products amplified above are purified. Then, the VL and VH genes with signal peptide encoding genes are constructed into the expression vector pBR322 by homologous recombination, and the vector construction is successfully verified by sequencing.

[0087] In order to achieve secretory expression of antibodies, signal peptide sequences need to be added to the front end of their VL and VH genes. Signal peptides can use antibody expression signal peptides commonly used in the art, such as the patent "Rabbit monoclonal antibody against human interferon α2 and its application (publication number: CN116063487A, publication date: 2023-05-05)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)" VL upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGARC", and VH upstream has a signal peptide "METGLRWLLLVAVLKGVQC". Of course, those skilled in the art can also replace other signal peptides for antibody expression after obtaining the antibody sequence of the present invention. Therefore, the signal peptide sequence is not reflected in the antibody sequence of Table 1 of this embodiment.

[0088] The constructed FL and FH expression vectors were transfected into 293F cells together, and cultured for 72-96 hours after transfection to obtain the culture supernatant containing recombinant rabbit monoclonal antibodies that recognize human Ki-67 protein. Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100) was used to purify the target antibody from the culture supernatant. Using 12% SDS-PAGE gel electrophoresis, the antibody concentration was 0.55 mg / mL and the purity was greater than 90%.

[0089] Example 2 Reaction Specificity and Application Analysis of Monoclonal Antibodies Against Ki-67 Protein

[0090] 1. Western blot analysis of monoclonal antibody 4E5 (Western blot, WB)

[0091] Positive cell / tissue samples with high Ki-67 expression and negative cell / tissue samples without Ki-67 expression were lysed separately to obtain protein extracts, and subjected to 10% polyacrylamide gel electrophoresis. The gel protein bands were transferred to a PVDF membrane in an electrotransfer system according to conventional methods; the membrane was placed in a TBST blocking solution containing 5% skimmed milk powder and incubated at room temperature for 1 hour, and antibody 4E5 (primary antibody dilution ratio 1:1000, working concentration 0.55 μg / mL) was added and incubated at room temperature for 1 hour; then the membrane was washed with TBST, and goat anti-rabbit secondary antibody (purchased from Jackson, product number AS014, secondary antibody dilution ratio 1:10000) was added and incubated at room temperature for 1 hour; the membrane was washed again with TBST; finally, ECL supersensitive colorimetric solution was added for development.

[0092] The positive cell / tissue samples in this embodiment include: HeLa cell line, and the negative cell / tissue samples include: MKI67 (Ki67 encoding gene) homozygous knockout HeLa cell line (HeLa-MKI67-1E6), mouse embryonic fibroblast cell line NIH / 3T3, mouse liver tissue and rat liver tissue. The aforementioned Hela cell line is derived from the cell bank of the Chinese Academy of Sciences in Shanghai. The Hela cell line was starved and stimulated with nocodazole before detection, wherein the starvation group used serum starvation for 18 hours, and the nocadazole stimulation group used 1μg / mL nocadazole to stimulate the cells for 16 hours. Nocadazole is a fast and reversible microtubule inhibitor that binds to β-tubulin and disrupts microtubule assembly / disassembly dynamics, thereby blocking the cells in the G2 / M phase. Ki-67 is most highly expressed in the G2 phase, so the expression of Ki-67 in cells treated with nocadazole is enhanced. The test results of the corresponding samples are shown in Figure 2 .

[0093] As can be seen from the figure, there are obvious Ki-67 protein bands in the positive samples. Because Ki-67 will be rapidly degraded while being expressed, the detected bands are tailing and diffuse, which is a normal phenomenon, and the band positions are all below 359kDa, which is consistent with the diffuse distribution characteristics of Ki-67 protein. In addition, it can be seen from the figure that the expression of the Nocadazole stimulation group is significantly enhanced compared with the starvation treatment group, which is consistent with the expected results. At the same time, the homozygous knockout of the Ki-67 encoding gene has no bands regardless of stimulation or not, so it can be determined that the tailing bands in the positive samples are all different degradation bands of the target protein. No bands were detected in the remaining negative samples, indicating that the antibody 4E5 can uniquely identify the Ki-67 protein in the cell lysate, the antibody has high specificity, and the affinity for binding to the Ki-67 protein is good, the detection accuracy is high, and false positive results can be effectively avoided. And from the results, it can be seen that the antibody can be used for the specific detection of Ki-67 proteins expressed in multiple species such as humans, mice and rats.

[0094] 2. Immunofluorescence analysis (IF) of monoclonal antibody 4E5

[0095] Take the positive samples expressing Ki-67 protein, completely cover the aforementioned cell / tissue samples with 5% blank goat serum, incubate in a 37°C constant temperature and humidity incubator for 30 minutes, remove the blocking solution, then add the antibody 4E5 working solution prepared in TBS buffer (primary antibody dilution ratio 1:200, working concentration 2.75 μg / mL), and incubate at 4°C overnight; take out the sample and rewarm it at room temperature for 15 minutes, remove the primary antibody working solution, wash with TBST buffer, then add goat anti-rabbit secondary antibody working solution (secondary antibody dilution ratio 1:10000, from ABclonal, product number AS007), incubate at 37°C for 1 hour in dark, remove the secondary antibody working solution, wash with TBST buffer, add DAPI working solution on the sample, and incubate for 10 minutes; remove the DAPI working solution, add anti-fluorescence attenuation mounting medium, and observe and collect images under a fluorescence microscope. Using α-Tubulin skeleton protein as a reference, the primary antibody and secondary antibody were both from ABclonal, with catalog numbers AC012 (dilution ratio 1:200) and AS076 (dilution ratio 1:500) respectively.

[0096] The positive cell / tissue samples in this example include: human cervical cancer cell line (Hela) and human epidermal cancer cell line (A431), mouse spleen tissue and mouse myoblast cell line (C2C12), rat spleen tissue sample and rat adrenal pheochromocytoma cell line (PC-12). The corresponding sample results are shown in Figure 3Among them, the upper figure from left to right is the fluorescence imaging of the human Hela cell line, human A431 cell line, mouse spleen tissue, mouse C2C12 cell line, rat spleen tissue and rat PC-12 cell line samples using antibody 4E5. After antibody 4E5 binds to Ki-67 protein, it is labeled in red with fluorescent secondary antibody. The lower figure from left to right is the comparison of the corresponding samples counterstained with DAPI nuclei and the red, blue and green merge. DAPI counterstained cell nuclei are blue, and green is the reference of α-Tubulin skeleton protein.

[0097] As can be seen from the figure, the cell nuclei of human Hela cell line and A431 cell line, mouse C2C12 cell line and rat PC-12 cell line are stained with clear and bright fluorescence signals, strong positive signals and accurate antibody positioning; the proliferating cell nuclei of mouse and rat spleen tissue samples are positive and other cell components are negative, and the specificity is qualified. It can be seen that the antibody 4E5 of the present invention has high specificity for Ki-67 protein, the actual IF detection results are consistent with the theoretical positioning, the detection results are highly credible, and have good applicability to Ki-67 proteins of different species.

[0098] 3. Immunohistochemistry (IHC) of monoclonal antibody 4E5

[0099] Ki-67 protein is expressed in proliferating cells in all active phases of the cell cycle (late G1, S, G2 and M phases), so most normal and tumor tissues express it to varying degrees, and subcellular localization is in the nucleus. The tissue section samples used in this embodiment include: 1) human samples: spleen, liver, hepatocellular carcinoma, tonsil, lung, lung cancer, colon, colon cancer, kidney, thyroid cancer, breast, breast cancer, esophagus, pancreas; 2) rat samples: heart, liver, spleen, lung, kidney, brain, colon, testicle; 3) mouse samples: heart, liver, spleen, lung, kidney, brain, colon, testicle, a total of 30 samples. Tissue samples with high expression of Ki-67 include: human tonsil, human colon, human lung cancer, rat colon, rat spleen, rat testicle, mouse colon, mouse spleen, mouse testicle; rat liver, mouse liver and human liver are almost not expressed, and other tissues express to varying degrees.

[0100] IHC staining was performed as follows: 1) Sample preparation and baking: paraffin sections were placed on the slice rack in the same direction and placed in a 56°C thermostat for 30 min; at the same time, dewaxing solution 1 was placed in a 56°C thermostat; paraffin sections were placed in dewaxing solution 1 together with the slice rack, and then taken out of the thermostat and placed at room temperature. After 5 min, the sections were taken out and immersed in room temperature dewaxing solution 2, and the paraffin sections were placed in the cylinders in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. When placed in the dewaxing solution reagent cylinder, each cylinder was placed for 5 min, and when placed in the anhydrous ethanol reagent cylinder, each cylinder was placed for 3 min; the sections were washed with running water for 3 min; dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology Trade Co., Ltd.; 2) Antigen repair: 0.01 M High-pressure heat repair with Tris-EDTA repair solution (pH 9.0); 3) Inactivation of endogenous peroxidase: Wash with PBS buffer 3 times, 1 min each time, remove the buffer on the slice; immerse the slice completely in 3% hydrogen peroxide solution and incubate at room temperature for 10 min; 4) Blocking: Wash with PBS buffer 3 times, 3 min each time, remove the buffer on the slice; circle the tissue area to be tested on the slide with an immunohistochemical pen, and add PBS blocking solution to the circled area; place the slice horizontally in an incubation humid box with water at the bottom, start timing from the addition of the blocking solution, and incubate at room temperature for 30 min; 5) Primary antibody incubation: remove the blocking solution, and add anti-Ki-67 protein antibody 4E5 dilution solution (primary antibody dilution ratio 1:1000, working concentration 0.55μg / mL), placed horizontally in an incubation wet box, and incubated at room temperature for 60min; remove the antibody working solution, rinse quickly once with PBS buffer, and soak and wash three times with PBS buffer, each time for 3min; 6) Secondary antibody incubation: add ready-to-use secondary antibody working solution (from ABclonal ready-to-use immunohistochemistry pika and rabbit universal secondary antibody kit, item number RK50015) to the tissue section and place it horizontally in an incubation wet box, incubate at room temperature for 25min; remove the secondary antibody working solution on the section, rinse quickly once with PBS buffer, and soak and wash three times with PBS buffer, each time for 3min; 7) Color development: add the color development solution of ABclonal ready-to-use immunohistochemistry pika and rabbit universal secondary antibody kit to the section and incubate at room temperature for 5min After that, immerse the slices in a large amount of distilled water to stop the color development. After the color development is stopped, wash them in running water for 10 minutes; 8) Re-staining: immerse the slightly drained tissue slices in Mayer's hematoxylin for 1 minute, and wash them with running water for 3 minutes; 9) Re-blueing: immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, and wash them with running water for 3 minutes; 10) Dehydration: Soak the washed slices in anhydrous ethanol once, and pull them up and down several times during the soaking period. Take them out after 10 seconds; place them in a constant temperature blast drying oven at high temperature (54-58℃) to completely dry; 11) Sealing: Add an appropriate amount of neutral gum to the center of the slice, and cover it with a cover glass. The amount of glue added should be appropriate. After sealing the cover glass, the tissue should be completely covered, and no glue should overflow. Finally, scan the slices. .

[0101] Immunohistochemical staining results are divided into: positive and negative, where positive expression must be brown-colored in the specific antigenic sites of the corresponding cells and tissues with low or no background to be considered positive, and negative refers to the absence of brown-colored staining in specific tissues and cells. For example, human tonsil samples can be used as both positive and negative controls. Almost all B cells in the dark zone of the germinal center of human tonsils must show moderate to strong nuclear staining reactions, while at least weak to moderate staining reactions must be seen in most B cells in the bright zone, and the vast majority of mantle zone B cells have no staining reactions. In addition, the liver or pancreas can be used mainly as a supplementary negative tissue control, in which <1% of hepatocytes and exocrine pancreatic epithelial cells should be positive (inflammation conditions may induce Ki-67 elevation). Figure 4From left to right and from top to bottom, the IHC staining results of human colon, human tonsil, human breast cancer, mouse colon, mouse spleen, mouse testis, rat spleen, and rat testis are representatively shown. It shows that the antibody 4E5 is accurately positioned in the nucleus of proliferating cells, with clear staining, no nonspecific staining, and a clean background, indicating that the antibody has high specificity; and the IHC detection results of the present invention for 30 tissue samples are consistent with the actual expression and positioning of Ki-67 in the corresponding tissue samples and theoretical expectations, further illustrating that the anti-Ki-67 antibody has high specificity, strong anti-interference ability, good detection accuracy and reliability, good detection sensitivity and specificity, and no false positive and false negative results occur.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against Ki-67 protein, characterized in that: It comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The monoclonal antibody against Ki-67 protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against Ki-67 protein according to claim 2, characterized in that: The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against Ki-67 protein according to claim 1, characterized in that: The monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against Ki-67 protein according to any one of claims 1 to 4.

6. A recombinant vector or host cell, characterized in that: The recombinant vector or the host cell comprises the nucleic acid molecule according to claim 5.

7. Use of the anti-Ki-67 protein monoclonal antibody according to any one of claims 1 to 4 in the preparation of a Ki-67 protein detection kit.

8. Use of the anti-Ki-67 protein monoclonal antibody according to claim 7 in the preparation of a Ki-67 protein detection kit, characterized in that: The Ki-67 protein is human, rat or mouse Ki-67 protein.

9. Use of the anti-Ki-67 protein monoclonal antibody according to claim 7 in the preparation of a Ki-67 protein detection kit, characterized in that: The detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot assay kit, an immunohistochemistry kit, an immunofluorescence assay kit, an immunoblotting kit or a flow cytometry kit.

10. A Ki-67 protein detection kit, characterized in that: The detection kit comprises the anti-Ki-67 protein monoclonal antibody or its antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate is formed by connecting the monoclonal antibody to a detection marker.

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