Antibodies against human CD127 protein, antibody conjugates and their applications

Abstract

The present invention belongs to the technical field of antibody preparation, and more particularly to antibodies, antibody conjugates and applications thereof against human CD127 proteins. The amino acid sequences of CDR1‑3 on the light chain variable region of the antibody are shown in SEQ ID NO.3‑5, respectively, and the amino acid sequences of CDR1‑3 on the heavy chain variable region are shown in SEQ ID NO.8‑10, respectively. The antibody provided by the present invention can specifically recognize the recombinant CD127 protein obtained by genetic engineering expression and the CD127 protein naturally expressed in cells or tissues, has good specificity and high affinity, can effectively resist interference from cell components, effectively reduce false positive or false negative results caused by nonspecific binding during immunodetection of CD127 protein, etc., improve the accuracy, sensitivity and reliability of the test results, and has broad application prospects in immunodetection fields such as flow cytometry.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to an antibody against human CD127 protein, an antibody conjugate and applications thereof. Background Art

[0002] IL-7 receptor alpha chain (IL-7Rα), also known as CD127, plays an important role in maintaining the homeostasis of the internal environment of T lymphocytes (T cells for short). Interleukin IL-7 has been identified as the main homeostatic cytokine for the survival of various mature T cells. Its receptor is composed of α chain and γ chain. The γ chain receptor is widely expressed in lymphocytes and is a universal receptor for IL-2, IL-4, IL-9, IL-15 and IL-21. Therefore, the specific response of lymphocytes to IL-7 is mainly regulated by the expression of CD127. CD127 is mainly expressed by lymphoid lineage cells that play an important role in lymphocyte differentiation, proliferation and survival, including developing and mature T cells, especially in naive T cells ( It is expressed in primary T cells and memory T cells, and in bone marrow stromal cells and B cell progenitors, but not in mature B cells.

[0003] The body's regulation of the expression of CD127 on the surface of T cells runs through the entire life cycle of T cells. The IL-7-CD127 regulatory axis synergizes with other growth factors to amplify T cell precursors. In addition, the IL-7-CD127 regulatory axis promotes T cell co-stimulation through proliferation and cytokine secretion, and can regulate the homeostasis of initial T cells and memory CD4 and CD8 T cells and their lymphocyte-induced proliferation. In addition, the IL-7-CD127 regulatory axis also promotes the survival and reconstitution of T cells through anti-apoptosis effects, maintaining the homeostasis of the internal environment of T lymphocytes. In addition to being found to be highly expressed in lymphocytes, CD127 has also been reported in recent years to be highly expressed in some monocytes. A considerable number of CD127-highly expressed monocytes were found in the lungs of COVID-19 patients and in the peripheral blood and joint tissues of patients with rheumatoid arthritis, revealing that the difference in the expression level of CD127 between monocyte subsets indicates different degrees of mononuclear inflammatory phenotypes. Moreover, it was discovered for the first time that the IL-7 receptor-mediated CD127-STAT5 signaling pathway on monocytes has an anti-inflammatory regulatory effect, suggesting the possibility that the CD127-mediated monocyte immune phenotype may serve as a relevant target for future disease treatment.

[0004] In terms of pathology, CD127 is associated with autoimmune diseases such as rheumatoid arthritis (RA). In clinical models, blocking IL-7 or CD127 effectively impairs joint inflammation, osteoclast formation, and neovascularization mainly by hindering the infiltration of monocytes and endothelial cells and inhibiting the differentiation of pro-inflammatory macrophages and Th1 / Th17 cells. In addition, CD127 plays an important role in the immune pathogenesis of HIV infection. CD8+T cells highly expressed in the blood of HIV-infected patients have been shown to be CD127 - Subtype, CD127 - CD8 + The expansion of T cells is directly related to the severity of viral infection and the increase in the level of T cell activation, and CD8 + The reduced expression of CD127 on the surface of T cells can cause the differentiation of effector T cells into memory T cells, leading to the inhibition of HIV-specific CD8 + T cells cannot survive for long periods of time. Therefore, CD127 is increasingly considered an important marker for the progression of HIV infection. In addition, CD127 is amplified in different solid cancers, including breast and lung cancer, and its high levels are associated with poor prognosis.

[0005] With the development of CD127 research, it also plays an important role as a marker of certain cell specificity. The most common is that CD127 is a negative surface marker of regulatory T cells (Treg T Cell). The homeostasis of regulatory T cells mainly depends on IL-2 rather than IL-7, and their surface expresses low levels of CD127. Therefore, CD127 can help identify Treg cells (CD4 + CD25 + CD127 low ) and activated T cells (CD4 + CD25 + CD127 high ), through CD4 + CD25 + CD127 low The method of purifying subpopulations plays an important role in the study of Treg cell function. In addition, CD127 is also used as a surface marker of memory T cells (Memory TCell). According to the expression of CD127 and CD62L, CD8+T cells stimulated by antigens are divided into three subtypes: central memory T cells (CD62L + CD127 + ), effector memory T cells (CD62L - CD127 + ) and effector T cells (CD62L - CD127- ). This shows that CD127 is of great significance for the research on the typing of immune cells such as T cells.

[0006] Based on the expression characteristics of CD127 and its marking value, the detection of CD127 is very important. At the level of live cell detection, cell subpopulations are mainly identified by flow cytometry (FC), which has an increasing demand for CD127 flow cytometry antibodies and an increasing challenge for antibody research and development. Currently, most CD127 flow cytometry antibodies on the market use mouse anti-human monoclonal antibodies, which have low affinity and specificity. There are challenges such as low sensitivity and large background interference in flow cytometry detection. Therefore, the development of monoclonal antibodies against human CD127 protein with strong specificity and good anti-interference ability has become an urgent problem to be solved. Summary of the invention

[0007] In view of the problems that the anti-human CD127 protein antibodies in the prior art have poor specificity and anti-interference ability and are difficult to use for immunological detection, especially flow cytometry detection, the present invention provides antibodies and antibody conjugates of anti-human CD127 protein, and further provides the use of the antibody or antibody conjugate in the preparation of a human CD127 protein immunoassay kit and related immunoassay kits. To achieve the aforementioned purpose, the present invention is specifically implemented by the following technical solutions:

[0008] In a first aspect, the present invention provides an antibody against human CD127 protein, comprising 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; and 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.

[0009] 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.

[0010] Furthermore, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0011] Furthermore, the 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.

[0012] The second aspect of the present invention provides an antibody conjugate comprising the above-mentioned anti-human CD127 protein antibody and a detection label connected to the antibody.

[0013] The third aspect of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody against human CD127 protein as described above.

[0014] Furthermore, the nucleic acid sequence of the antibody light chain variable region is as shown in SEQ ID NO.12 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a sequence complementary thereto.

[0015] Furthermore, the nucleic acid sequence of the antibody light chain is as shown in SEQ ID NO.11 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or a sequence complementary thereto.

[0016] A fourth aspect of the present invention provides the use of the above-mentioned anti-human CD127 protein antibody or antibody conjugate in the preparation of a human CD127 protein immunoassay kit.

[0017] A fifth aspect of the present invention provides a human CD127 protein immunoassay kit, the kit comprising the anti-human CD127 protein antibody or antibody conjugate as described above.

[0018] Furthermore, the immunoassay kit is a flow cytometry assay kit.

[0019] Furthermore, the CD127 protein is a full-length protein or a 21-236aa fragment of the CD127 protein.

[0020] The advantages and positive effects of the present invention are:

[0021] The anti-human CD127 protein antibody with the above-mentioned CDR sequence provided by the present invention can specifically recognize the recombinant CD127 protein expressed by genetic engineering and the CD127 protein naturally expressed in cells or tissues, has good specificity and high affinity, can effectively resist the interference of cellular components, effectively reduce the false positive or false negative results caused by nonspecific binding during immunodetection of CD127 protein, improve the accuracy, sensitivity and reliability of the detection results, and has broad application prospects in the field of immunodetection such as flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is a graph showing the result of detecting the immune serum titer using enzyme-linked immunosorbent assay (ELISA) in Example 1 of the present invention;

[0024] Figure 2 This is a histogram of the binding of immune serum to 293T cells detected by flow cytometry in Example 1 of the present invention, wherein, from left to right, the serum dilutions are 1:500 and 1:2000 respectively;

[0025] Figure 3 This is a statistical result diagram of the mean fluorescence intensity of the binding of immune serum to 293T cells detected by flow cytometry in Example 1 of the present invention;

[0026] Figure 4 A map of the mammalian expression vector pRB322 used to construct an antibody against human CD127 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;

[0027] Figure 5 This is a histogram of the binding between the anti-human CD127 protein antibody and 293T cells detected by flow cytometry in Example 2 of the present invention;

[0028] Figure 6 This is a histogram of the binding between the anti-human CD127 protein antibody and PBMC cells detected by flow cytometry in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the result may be added. 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" is regarded as including the following situations: (i) A, (ii) B and (iii) A and B.

[0034] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit antibody" and "rabbit monoclonal antibody" and other similar terms have the same meaning and, unless otherwise specified, refer to antibodies that specifically bind to the human interleukin IL-7 receptor α chain. The modifier "rabbit" indicates that the complementary determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. The terms "interleukin IL-7 receptor α chain", "CD127", "IL-7Rα" and "CD127 / IL-7Rα" have the same meaning and can be used interchangeably.

[0035] An antibody is an immunoglobulin molecule that can 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] (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.

[0042] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0043] (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.

[0044] (iv) (Fv)2: consists of two Fv fragments covalently linked together.

[0045] (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.

[0046] (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.

[0047] 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 mouse 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 mouse-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 mouse antibody framework (FR) sequence, and this framework sequence is derived from a single or multiple other mouse antibody variable region framework sequences. In the present invention, the CDR region in the chimeric antibody or humanized antibody is derived from a rabbit-derived CDR region.

[0048] 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.

[0049] 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.

[0050] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0051] An embodiment of the present invention provides an antibody against human CD127 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 include three complementary determining regions (CDRs), respectively named CDR1, CDR2 and CDR3, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0052] The rabbit monoclonal antibody against human CD127 protein with the above CDR sequence provided by the present invention can specifically recognize the recombinant CD127 protein expressed by genetic engineering and the CD127 protein naturally expressed in cells or tissues. By flow cytometry, immunoassay is performed, and there is an obvious fluorescence transition and a single signal intensity on the CD127 protein-positive cells expressing the CD127 protein, while there is no fluorescence signal transition in the negative cells that do not express the CD127 protein, which proves that the antibody has good specificity and high affinity for the CD127 protein, can effectively resist the interference of cellular components, effectively reduce the false positive or false negative results caused by nonspecific binding during immunoassay of CD127 protein, improve the accuracy, sensitivity and reliability of the detection results, and has broad application prospects in the field of immunoassay such as flow cytometry.

[0053] 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 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.

[0054] Optionally, the antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, CL and VL constitute a complete light chain (FL), and CH and VH constitute a complete heavy chain (FH). The constant region of an antibody can usually be obtained by public query, such as: searching for rabbit IgG gamma C reign to obtain CH through the IMGT online database (www.imgt.org), and searching for rabbit IgG Kappa C reign to obtain CL. Specifically, the amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0055] Optionally, the first antibody and / or the second antibody is 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 of the antibody, 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.

[0056] Another embodiment of the present invention provides an antibody conjugate, comprising the above-mentioned anti-human CD127 protein antibody, and a detection label connected to the antibody.

[0057] 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.

[0058] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule is used to encode the antibody against human CD127 protein as described above.

[0059] Nucleic acid molecules 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.

[0060] The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. The obtained nucleic acid molecule is inserted into an expression vector, then introduced into a host cell, and cultured under specific conditions to express the antibody.

[0061] Illustratively, the nucleic acid sequence of the antibody light chain variable region is as shown in SEQ ID NO.12 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a sequence complementary thereto.

[0062] Illustratively, the nucleic acid sequence of the antibody light chain is as shown in SEQ ID NO.11 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or a sequence complementary thereto.

[0063] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, nucleic acid molecules other than those exemplified above can also encode the antibodies of the present invention. Therefore, the nucleic acid molecules exemplified above should not be used as a limitation on the scope of protection of the present invention.

[0064] The original vector for constructing the recombinant vector is any conventional vector in the art, as long as it can carry 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 molecules of the present invention can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is a well-known technique in the art and will not be described in detail herein.

[0065] The nucleic acid molecules encoding the antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be 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.

[0066] 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.

[0067] 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.

[0068] In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0069] Another embodiment of the present invention provides the use of the above-mentioned anti-human CD127 protein antibody or antibody conjugate in the preparation of a human CD127 protein immunoassay kit.

[0070] The application advantages of the anti-human CD127 protein antibody or antibody conjugate in the preparation of a human CD127 protein immunoassay kit are the same as the advantages of the anti-human CD127 protein antibody over the prior art as described above, and will not be repeated here.

[0071] Based on the same inventive concept, an embodiment of the present invention further provides a human CD127 protein immunoassay kit, the kit comprising the anti-human CD127 protein antibody or antibody conjugate as described above.

[0072] It should be noted that the antibody of the present invention can be used alone, or it can be combined or coupled with a detection label to form an antibody conjugate. In some embodiments, the antibody of the present invention is used as an antigen-binding antibody, which specifically recognizes and binds to the CD127 protein in the sample to be tested, and then produces a recognizable signal change by the detection label connected thereto to achieve qualitative or quantitative detection of CD127 protein, In other embodiments, the antibody against human CD127 protein is not labeled (as a primary antibody or capture antibody), and the detection label is coupled to a secondary antibody (as a detection antibody) or other molecules that can be combined with a primary antibody, For example, if the anti-human CD127 protein antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby generating a change in a recognizable signal after the secondary antibody of the coupling detection label is specifically combined with the antibody of the present invention, thereby achieving qualitative or quantitative detection.

[0073] The above-mentioned immunoassay method includes but is not limited to: enzyme immunoassay (Enzyme immunoassay, EIA), enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunospot (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (Immunohistochemistry, IHC), immunofluorescence (Immunofluorescence, IF), immunoblotting (Western blot, WB), immunoprecipitation (Immunoprecipitation, IP) and flow cytometry (Flow Cytometry, FC). The detection object includes recombinantly expressed CD127 protein and naturally secreted or expressed CD127 protein, and the CD127 protein can be a full-length form or a 21-236aa fragment thereof. The detection sample includes but is not limited to serum, plasma, urine, cell culture fluid and tissue homogenate.

[0074] In a preferred embodiment, the immunoassay method is flow cytometry, and the kit is a flow cytometry kit.

[0075] 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.

[0076] Example 1 Preparation of rabbit monoclonal antibody against human CD127 protein

[0077] In order to obtain rabbit monoclonal antibodies that recognize human CD127 / IL-7Rα protein, the immunogen used was commercial human recombinant CD127 / IL-7Rα protein (from ABclonal, catalog number RP02407), and New Zealand white rabbits were immunized to obtain immune-enhanced rabbit B lymphocytes. Then, based on the monoclonal antibody development technology of single B lymphocyte screening and culture, single antigen-specific B lymphocytes were sorted out, and then the antibody encoding genes of antigen-specific B lymphocytes were cloned by PCR amplification to obtain nucleic acid sequences encoding heavy chain and light chain amino acids. After the nucleic acids encoding heavy chain and light chain amino acids were connected to the expression vector, recombinant expression was performed, and the target antibody strain 2D11 was screened. The antibody sequencing work was completed by Jinkairui Biotechnology Co., Ltd. The amino acid (AA) and nucleic acid (DNA) sequences of the antibodies are shown in Table 1, in which LCDR1-3 respectively represent the light chain complementary determining regions CDR1-3, and HCDR1-3 respectively represent the heavy chain complementary determining regions CDR1-3.

[0078] Table 1 Sequence information of rabbit monoclonal antibody 2D11 in this example

[0079]

[0080]

[0081] 1.1 Animal Immunization

[0082] Commercial human recombinant CD127 / IL-7Rα protein (from ABclonal, catalog number RP02407) was used to immunize New Zealand white rabbits at 200 μg per rabbit. For the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple points in the abdomen and back. At intervals of 3 weeks, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple points in the abdomen and back. The booster immunization was performed twice. After three immunizations, the immune serum was diluted 1:24300 and the serum titer was determined by enzyme-linked immunosorbent assay (ELISA). The OD 450For rabbits with titers greater than 0.2, the final immunization serum was tested by flow cytometry. Rabbits with high serum titers that met the flow cytometry requirements were selected and boosted with 200 μg of the immunogen by subcutaneous injection at multiple points. The animals were sacrificed three days later and the spleen was removed.

[0083] 1.2. Serum titer ELISA test

[0084] The enzyme-linked immunosorbent assay (ELISA) for serum titer detection includes the following steps: 1) Coating: dilute human CD127 / IL-7Rα protein (final concentration of 1 μg / mL) with 1× PBS buffer, add coating solution at 25 μL / well in a 384-well plate, level the 384-well plate, briefly centrifuge (stop after rpm reaches 1000), and incubate at 4°C overnight; 2) Blocking: Take out the plate overnight at 4°C, add 75 μL of washing buffer to each well and wash 5 times with a plate washer; use a spray gun to add 50 μL / well of blocking buffer to the plate, and incubate at room temperature for 1 hour to block irrelevant binding sites; 3) Serial dilution and sample loading of the serum to be tested: repeat 2) to wash the plate, serially dilute the serum to be tested in a 96-well plate, and dilute the serum three times starting from 1:1000 with dilution buffer, for a total of 8 gradients, the dilution ratio can be adjusted according to actual conditions; 4) Secondary antibody incubation: Repeat 2) plate washing process to clean the plate, dilute the horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG ((purchased from Jackson, product number 111-035-045)) with dilution buffer, the dilution ratio is 1:5000, add the diluted goat anti-rabbit IgG-HRP to the plate at 25 μL / well to bind the target protein in the plate, and incubate at room temperature in the dark for 1 hour; 5) Termination reaction and color development: Repeat 2) plate washing process to clean the plate, add TMB color development at 100 μL / well, incubate at 37°C in the dark for 10 minutes, add 100 μL / well diluted hydrochloric acid solution to terminate the reaction, measure the absorption value at 450nm, use the rabbit serum before immunization as the negative control, and judge the titer of the immune serum by the ratio of the measured value to the control value ≥2.1 as positive.

[0085] The formulas of the above solutions are as follows: 1) Washing buffer: Mix 1.8L 1×PBS (purchased from biosharp, product number BL601A) and 2mL Tween-20 (purchased from Sinopharm, product number 30189328) thoroughly, and add ddH2O to a total volume of 2L; 2) Blocking buffer: Mix 90mL 1×PBS, 5g Skim milk, 1g BSA (purchased from Biofrox, product number 4240GR500) and 0.05mL Tween-20 thoroughly, and add ddH2O to a total volume of 100mL, and prepare and use it immediately; 3) Dilution buffer: Mix 90mL 1×PBS, 1g BSA and 0.05mL Tween-20 thoroughly, and add ddH2O to a total volume of 100mL; 4) TMB colorimetric solution: Add 71.628g disodium hydrogen phosphate dodecahydrate and 29.41g trisodium citrate dihydrate to 800mL Mix thoroughly with ddH2O, adjust pH to 5.0 with monohydrated citric acid, and add ddH2O to a total volume of 1 L; 5) Stop solution: Mix 79 mL of pure water and 21 mL of hydrochloric acid thoroughly and store at room temperature before use.

[0086] Serum titer test results are shown in Figure 1 It can be seen that a stronger immune response was produced in the rabbit during the fourth booster immunization, with a high antibody titer, and the serum titer of the fourth immunization was optimal and could be used to isolate antibodies.

[0087] 1.3 Serum flow cytometry detection

[0088] Flow cytometry (FC) analysis of serum and cell samples included the following steps: 1) UV-irradiated and sterilized the clean bench for 15-20 min, turned on the fan for 5 min, and prepared for sterile work; 2) collected and washed the cells, and then the total number of cells and viability were determined. The specific operation was as follows: the cell culture medium was discarded, the cells were washed with 10 mL 1× PBS (purchased from biosharp, product number BL601A) (pH 7.4), and then 3 mL Digestion was performed at room temperature for 3 min with 0.05% Trypsin-EDTA (purchased from Gibco, catalog number 25300062). A large number of cells were observed to fall off under a microscope. The reaction was terminated with 15 mL of culture medium. Finally, the adherent cells were repeatedly washed with a pipette and collected in a 50 mL centrifuge tube. The cells were centrifuged at 400 g for 5 min. After the cells were collected, the total number of cells was determined and the cell viability was checked to be not less than 90%. 3) The cells were resuspended in PBS solution (0.5% BSA / PBS) containing 0.5% BSA (purchased from Biofrox, catalog number 4240GR500) to about 3 × 10 6 -5×10 6cells / mL, and distribute the cells to each well of a 96-well V-shaped plate at 50 μL; 4) distribute 50 μL of immune serum (as primary antibody) diluted with 0.5% BSA / PBS to a 96-well V-shaped plate, resuspend the cells in each well, incubate at room temperature for 20 minutes, and gently mix on a microplate shaker to ensure uniform and sufficient staining. The serum to be tested and the negative serum were diluted at 1:500 and 1:2000, respectively; 5) centrifuge at 400g for 5 minutes, discard the supernatant, and use 200 μL Wash twice with 0.5% BSA / PBS, centrifuge at 400g for 5 minutes, and discard the supernatant; 6) Dispense the fluorescent secondary antibody (purchased from Jackson, 111-136-144) diluted 1:200 with 0.5% BSA / PBS into a 96-well V-shaped plate at a volume of 100 μL per well, resuspend the cells in each well, incubate at room temperature and away from light for 20 minutes, and gently mix on a microplate shaker to ensure uniform and sufficient staining; 7) Wash twice with 200 μL 0.5% BSA / PBS, and finally resuspend the cells in each well with 200 μL 0.5% BSA / PBS and store in the dark; 8) Flow analysis: perform analysis according to Beckmancytoflex flow analyzer use and maintenance SOP-105-AND-CA-008. The isotype control used Rabbit Isotype Control antibody (from ABclonal, catalog number A22070), the positive control antibody was purchased from Biolegend, catalog number 351315, and the blank control was added with 100 μL 0.5% BSA / PBS.

[0089] The cell samples include human renal epithelial (293T) cells that do not express CD127 (negative samples) and 293T cells that overexpress CD127 (positive samples). The preparation method of positive cell samples includes: using the gene sequence corresponding to the Glu21-Gly236aa fragment of the Human CD127 protein sequence to construct a pCDNA TM 3.1 vector (purchased from Thermo, V38520) to form a recombinant plasmid; the 293T (purchased from Thermo) in culture was subcultured at a cell density of 5×10 5 / mL plate, inoculate into a TC-treated cell culture dish with a diameter of 10cm (purchased from JET, catalog number TCD010100) for culture, the system is 10mL. After 24h of culture, the 293T culture medium was replaced with an equal system of opti-MEM (purchased from gibco, catalog number 31985088), 10μg of the constructed target plasmid was taken, and the transfection reagent PeiMAX (purchased from polysciences, catalog number 24765-1) was mixed at a ratio of 1μg: 3μL for 10min. The mixture was dripped into the cell culture dish and mixed. After 48h of culture, the cells in the dish were digested and blown by 0.05% trypsin (purchased from gibco, 25300062) to drop and collect in a centrifuge tube to obtain the desired CD127 overexpression cell sample. The CD127 protein sequence can be found in NCBI accession number: NP_000871.1 or Uniprot ID: P16871, and the gene sequence can be found in NM_000880.4, wherein CD127 protein 21-236aa corresponds to 542-1075bp bases of NM_000880.4.

[0090] Serum flow cytometry histogram data are shown in Figure 2 , where the horizontal axis represents the relative fluorescence intensity, the vertical axis represents the relative cell number, the red curve is the blank control, the blue curve is the isotype control, the yellow curve is the immune serum to be tested, and the green curve is the positive control; the statistical results of the MFI mean fluorescence intensity are shown in Figure 3 . The results showed that the competitor's positive antibody (green) had a signal transition difference in the positive sample, indicating that the CD127 overexpressing cell sample was successfully prepared. The serum to be tested had a higher signal than the negative sample in the positive sample of CD127 overexpressing cells, and the positive signal of the serum to be tested was significantly stronger than that of the negative serum, indicating that there were specific antibodies against CD127 / IL-7Rα in the serum, so the N28519 New Zealand white rabbit corresponding to the N28519-4 serum was selected for the final immunization.

[0091] 1.4. Isolation and sorting of B lymphocytes from spleen

[0092] Conventional methods are used to isolate B lymphocytes from the spleen, and antigen-specific B lymphocytes are sorted. For related methods, please refer to the published patents "Method for efficiently isolating 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)".

[0093] 1.5. Cloning of the gene encoding rabbit monoclonal antibody

[0094] The supernatant of cultured B cells was used to identify positive clones by antigen-coated ELISA. The cells of positive clones were collected and lysed and then analyzed by Quick-RNA TM RNA was extracted using MicroPrep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA.

[0095] Using the aforementioned cDNA as a 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 clones by PCR and sequenced. The PCR reaction system included: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from ABclonal, catalog number RK20717) and 6.5 μL H2O; the PCR amplification program included: 98°C pre-denaturation for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally kept at 72°C for 5 min, and the resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying VL and VH genes are shown below, where F and R represent forward primers and reverse primers, respectively.

[0096] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 15);

[0097] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 16);

[0098] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 17);

[0099] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 18).

[0100] 1.6 Expression and large-scale production of rabbit monoclonal antibodies

[0101] In order to obtain multiple antibodies that recognize human CD127 protein, the mammalian expression vector pBR322 carrying the light chain constant region (CL) and heavy chain constant region (CH) genes was linearized with XbaI and NheI restriction enzymes respectively, and the above-amplified VL and VH genes containing signal peptides were loaded on the expression vector pBR322 carrying CL and CH genes respectively to obtain light chain gene and heavy chain gene expression vectors. The expression vector was successfully constructed and verified by sequencing. The expression map of the vector used is shown in Figure 4 In the figure, pBR322origin and f1origin are replication promoters, Ampcillin is a resistance gene, CMVpromoter is a transcription promoter, SV40PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of the light chain constant region (left figure), and Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right figure). CL and CH genes were obtained by searching the IMGT online database (www.imgt.org) to search for rabbit IgG gamma C reign to obtain CH, and rabbit IgG Kappa C reign to obtain CL.

[0102] The signal peptide of this embodiment can adopt the antibody expression signal peptide sequence 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)" The light chain variable region upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGARC", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

[0103] The constructed expression vector containing the light chain (FL) gene and the heavy chain (FH) gene was transfected into 293F cells together. After transfection, the cells were cultured for 72-96 hours to obtain the culture supernatant containing the recombinant rabbit monoclonal antibody that recognizes the human CD127 protein. The target antibody 2D11 was purified from the culture supernatant using proteinA affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100). The antibody purity was greater than 95% using 12% SDS-PAGE gel electrophoresis. The purified antibody was aliquoted and stored at -20°C for later use.

[0104] Example 2 Identification of Antigen Recognition Epitopes by Rabbit Monoclonal Antibody 2D11

[0105] 2.1. Verification of 293T positive cells overexpressing CD127 protein

[0106] After obtaining human CD127 / IL-7Rα rabbit monoclonal antibody, the antibody affinity and antigen recognition epitope were identified by flow cytometry, as shown in step “1.3, serum flow cytometry detection” in Example 1. The only difference is that the immune serum is replaced with antibody 2D11, the working concentration of antibody 2D11 is 2 μg / mL, the fluorescent secondary antibody is purchased from Jackson (Cat. No. 111-136-144), 100 μL of diluted Rabbit Isotype Control (from ABclonal, Cat. No. A22070) is added to the isotype control, 100 μL of 0.5% BSA / PBS is added to the blank control, and 100 μL of diluted positive antibody (purchased from Biolegend, Cat. No. 351315) is added to the positive control.

[0107] The histogram data of rabbit monoclonal antibody 2D11 detecting 293T positive cells by flow cytometry is shown in Figure 5 As shown, the horizontal axis represents the relative fluorescence intensity, the vertical axis represents the relative number of cells, the red curve is the blank control, the blue curve is the isotype control, and the yellow curve is the antibody to be tested 2D11. The results showed that the antibody to be tested 2D11 (yellow curve) had an obvious high expression group in the 293T positive cell sample overexpressing CD127 protein (the double peak of the positive peak here is mainly due to the transfection efficiency of the overexpression sample), there was no background signal in the negative sample, and there was no obvious signal transition in the blank control (red curve) and the isotype control (blue curve), indicating that the 2D11 antibody strain can meet the detection requirements of flow cytometry.

[0108] 2.2. Verification of primary PBMC cells with high expression of CD127 protein

[0109] The positive sample used in the above test is 293T cell. This embodiment uses human peripheral blood mononuclear cells (PBMC) (purchased from Aoneng Bio, item number PB003F-C-10M) to perform primary verification of the antibody. PBMC includes lymphocytes and monocytes. CD127 is a molecular marker for related immune phenotypes such as regulatory T cells (Treg TCell). In clinical and other practical application scenarios, it is more suitable for samples such as blood, tissue, and PBMC. Therefore, PBMC primary samples are added for verification, and compared with competing antibodies at the same time to demonstrate the performance of the antibody in practical applications. The antibody flow cytometry detection method is the same as above.

[0110] The histogram data of PBMC cells detected by rabbit monoclonal antibody 2D11 by flow cytometry are shown in Figure 6As shown, the horizontal axis represents the relative fluorescence intensity, the vertical axis represents the relative cell number, the red curve is the blank control, the blue curve is the isotype control, and the yellow curve is the antibody to be tested 2D11. The results show that the antibody of the present invention has an obvious high expression group (yellow curve) on PBMC, which can clearly distinguish CD127 - and CD127 + In the cell population, the positive signal is stronger than that of the competitor antibody (green curve) and the signal is single. There is no signal transition in the blank (red curve) and the isotype control (blue curve), which proves that the antibody 2D11 can recognize the CD127 protein in its natural state and has the advantages of high specificity and strong anti-interference ability.

[0111] 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. An antibody against human CD127 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 anti-human CD127 protein antibody 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 anti-human CD127 protein antibody according to claim 2, characterized in that: The amino acid sequence of the antibody light chain 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 anti-human CD127 protein antibody according to claim 1, characterized in that The 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. An antibody conjugate, characterized in that: An antibody comprising an anti-human CD127 protein as described in any one of claims 1 to 4, and a detection label connected to the antibody.

6. A nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule, characterized in that: The nucleic acid molecule encodes an antibody against human CD127 protein as described in any one of claims 1-4.

7. The nucleic acid molecule, the recombinant vector comprising the nucleic acid molecule or the host cell comprising the nucleic acid molecule according to claim 6, characterized in that: The nucleic acid sequence of the antibody light chain variable region is shown as SEQ ID NO.12 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO.14 or a sequence complementary thereto.

8. The nucleic acid molecule, the recombinant vector comprising the nucleic acid molecule or the host cell comprising the nucleic acid molecule according to claim 7, characterized in that: The nucleic acid sequence of the antibody light chain is shown as SEQ ID NO.11 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain is shown as SEQ ID NO.13 or a sequence complementary thereto.

9. A human CD127 protein immunoassay kit, characterized in that: An antibody comprising an anti-human CD127 protein as described in any one of claims 1 to 4, or an antibody conjugate as described in claim 5.

10. The human CD127 protein immunoassay kit according to claim 9, characterized in that: The kit is a flow cytometry detection kit.

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