Anti-human CD146 rabbit monoclonal antibody and its application

Abstract

The present invention belongs to the technical field of antibody preparation, and particularly relates to anti-human CD146 rabbit monoclonal antibody and its application. The anti-human CD146 rabbit monoclonal antibody includes a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region both include complementary determining regions; the amino acid sequences of light chain complementary determining region 1, light chain complementary determining region 2 and light chain complementary determining region 3 are shown in SEQ ID NO.5-7 respectively; the amino acid sequences of heavy chain complementary determining region 1, heavy chain complementary determining region 2 and heavy chain complementary determining region 3 are shown in SEQ ID NO.8-10 respectively. The rabbit monoclonal antibody provided by the present invention can specifically recognize and bind to human CD146 protein and only recognize human target protein targets, has good specificity, high affinity with target, can be applied to immunological diagnosis and detection technology, is particularly suitable for flow cytometer detection, and has good adaptability to multiple fluorescence.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, in particular to anti-human CD146 rabbit monoclonal antibody and application thereof. Background Art

[0002] CD146, also known as melanoma cell adhesion molecule (MCAM) or cell surface glycoprotein MUC18, is a single-chain membrane-permeable glycoprotein that belongs to the immunoglobulin superfamily (Igsf) and has homology with many cell adhesion molecules. As a member of the adhesion molecule, CD146 mediates the interaction between cells and between cells and the matrix by interacting with its ligand. After activation, CD146 can form a complex with p59fyn through its cytoplasmic region. Activated p59fyn can further cause tyrosine kinase p125PAK phosphorylation and bind to paxillon, which also indicates that CD146 can transmit and communicate signals from the extracellular to the intracellular through its intracellular domain, which helps local adhesion aggregation, cytoskeleton reorganization, maintenance of cell structure and regulation of cell function. In addition to its adhesion molecule properties, recent studies have shown that CD146 is active in a variety of cellular physiological processes. Its expression is found in vascular endothelial cells, activated T lymphocytes, smooth muscle and bone marrow stromal cells, etc. It participates in development, signal transduction, cell migration, mesenchymal stem cell differentiation, angiogenesis and immune response, etc., and has a wide range of physiological and pathophysiological significance. As a surface receptor, CD146 can trigger the tyrosine phosphorylation of FYN and PTK2 / FAK1, causing a transient increase in intracellular calcium concentration, and participate in the signal transduction from extracellular signals to intracellular signals of endothelial cells; promote the polarization of actin by activating the p125FAK pathway, and participate in the rearrangement of the actin cytoskeleton; promote melanoma metastasis by activating the Id-1 pathway, and induce tumor angiogenesis by activating NFκB / P38. It has been confirmed that CD146 is abnormally expressed in melanoma, prostate cancer, breast cancer, lung cancer, liver cancer, breast cancer, urothelial carcinoma, choriocarcinoma, osteosarcoma, etc. and is closely related to the occurrence, development, metastasis, treatment and prognosis of these tumors. It has become a diagnostic marker molecule for many diseases and a candidate target for drug development.

[0003] By measuring the level of human plasma soluble CD146, a diagnosis and evaluation of cancer response and cancer treatment prognosis such as melanoma can be made. Related studies have confirmed that the plasma soluble CD146 level of normal people aged 62.64±10.44 years old ranges from 165.52±98.47ng / mL (male: 175.91±101.67ng / mL, female: 96.70±40.20ng / mL). Based on the diagnostic value of CD146, screening and preparing specific CD146 monoclonal antibodies to specifically identify human CD146 at the cellular molecular level is of great significance in providing a research basis for the early diagnosis and treatment of cancer. At present, patents have been published on the development of CD146 antibodies. The detection applications mainly meet the requirements of immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), etc. There are few antibodies that meet the requirements of flow cytometry. This is because during flow cytometry, the target antigen content is low and there are many interfering factors. The low specificity and / or affinity of the antibody can easily lead to non-specific binding. Therefore, there are few rabbit monoclonal antibodies on the market that meet the requirements of flow cytometry. Therefore, it is of great clinical significance to develop a highly specific and high-affinity humanized rabbit monoclonal antibody that can meet the requirements of flow cytometry application scenarios. Summary of the invention

[0004] In view of the problems of poor affinity and specificity of anti-human CD146 antibodies in the prior art, the present invention provides anti-human CD146 rabbit monoclonal antibodies and uses of the rabbit monoclonal antibodies in preparing human CD146 protein detection reagents and / or detection kits.

[0005] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides an anti-human CD146 rabbit monoclonal antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region both comprise complementary determining regions, and the amino acid sequences of the light chain complementary determining region 1, the light chain complementary determining region 2 and the light chain complementary determining region 3 are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; the amino acid sequences of the heavy chain complementary determining region 1, the heavy chain complementary determining region 2 and the heavy chain complementary determining region 3 are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4.

[0008] Furthermore, the anti-human CD146 rabbit monoclonal antibody also includes a light chain constant region and a heavy chain constant region, the light chain constant region and the light chain variable region constitute a complete light chain, and the amino acid sequence of the light chain is shown in SEQ ID NO.1; the heavy chain constant region and the heavy chain variable region constitute a complete heavy chain, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2.

[0009] Furthermore, the anti-human CD146 rabbit monoclonal antibody is a full-length antibody or an antibody fragment, and the antibody fragment 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 nucleotide molecule, which is used to encode the anti-human CD146 rabbit monoclonal antibody as described above.

[0011] The third aspect of the present invention provides a recombinant vector, which carries the nucleotide molecule described above.

[0012] Furthermore, the starting vector of the recombinant vector is the mammalian expression vector pBR322.

[0013] A fourth aspect of the present invention provides a host cell, wherein the host cell carries the nucleotide molecule as described above or comprises the recombinant vector as described above.

[0014] Furthermore, the host cell is a 293F cell.

[0015] The fifth aspect of the present invention provides the use of the anti-human CD146 rabbit monoclonal antibody, the nucleotide molecule, the recombinant vector, and the host cell as described above in the preparation of a human CD146 protein detection reagent and / or a detection kit.

[0016] Furthermore, the detection method includes at least one of enzyme immunoassay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, immunohistochemistry, immunofluorescence, immunoblotting and flow cytometry.

[0017] The advantages and positive effects of the present invention are:

[0018] The rabbit monoclonal antibody provided by the present invention having the above complementary determining region sequence can specifically recognize and bind to human CD146 protein and only recognize human target protein target, has good specificity and high affinity with the target, can be applied to the field of immunological diagnosis and detection technology, especially suitable for the field of flow cytometer detection, and has good adaptability to various fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is a diagram showing the results of the ELISA test of the immune serum titer of New Zealand white rabbits in Example 1 of the present invention;

[0021] Figure 2 This is a flow cytometric analysis of the binding of New Zealand white rabbit immune serum to positive cells HUVEC in Example 1 of the present invention. From left to right, the serum dilutions are 1:500, 1:1000 and 1:2000, respectively. In the figure, the red curve is the negative control, the blue curve is the isotype control, and the yellow curve is the serum to be tested;

[0022] Figure 3 This is a flow cytometric analysis of the binding of New Zealand white rabbit immune serum to positive cells Hela in Example 1 of the present invention. From left to right, the serum dilutions are 1:500, 1:1000 and 1:2000, respectively. In the figure, the red curve is the negative control, the blue curve is the isotype control, and the yellow curve is the serum to be tested;

[0023] Figure 4 This is a flow cytometric analysis of the binding of New Zealand white rabbit immune serum to positive cells A431 in Example 1 of the present invention. From left to right, the serum dilutions are 1:500, 1:1000 and 1:2000, respectively. In the figure, the red curve is the negative control, the blue curve is the isotype control, and the yellow curve is the serum to be tested;

[0024] Figure 5 A map of the mammalian starting vector used to construct the rabbit monoclonal antibody expression vector in Example 1 of the present invention, from left to right, respectively, a map of the pRB322 vector carrying the heavy chain constant region and the antibody light chain constant region in advance;

[0025] Figure 6 This is a diagram showing the immunoblotting results of the rabbit monoclonal antibody 1E1 binding to different samples in Example 2 of the present invention;

[0026] Figure 7 This is a flow cytometric analysis of the binding of rabbit monoclonal antibody 1E1 to positive cells HUVEC in Example 3 of the present invention (fluorescent secondary antibody is 488), the red curve in the figure is the negative control, the blue curve is the isotype control, and the yellow curve is the antibody 1E1;

[0027] Figure 8This is a flow cytometric analysis of the binding of rabbit monoclonal antibody 1E1 to negative sample MCF-7 in Example 3 of the present invention (fluorescent secondary antibody is 488), the red curve in the figure is the negative control, the blue curve is the isotype control, and the yellow curve is the antibody 1E1;

[0028] Fig. 9 This is a flow cytometric analysis of the binding of rabbit monoclonal antibody 1E1 to positive cells HUVEC in Example 3 of the present invention (fluorescent secondary antibody is 647), the red curve in the figure is the negative control, the blue curve is the isotype control, and the yellow curve is the antibody 1E1;

[0029] Fig.10 This is a flow cytometric analysis of the binding of rabbit monoclonal antibody 1E1 to negative sample MCF-7 in Example 3 of the present invention (fluorescent secondary antibody is 647), the red curve is the negative control, the blue curve is the isotype control, and the yellow curve is the antibody 1E1. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] In order to better understand the present invention rather than limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention indicating dosage, percentage, etc., should be understood as modified by the word "approximately" 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 attempted to be obtained. Each numerical parameter should at least be regarded as being obtained according to the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "include", "comprise", "contain", "have" and the like is non-restrictive, and other steps and other ingredients that do not affect the results can be added.

[0033] 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.

[0034] The meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, i.e., 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" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0035] In the context of the present invention, the terms "rabbit monoclonal antibody", "monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably, all referring to antibodies that specifically bind to human (Human) CD146 protein. The modifier "rabbit" indicates that the complementary determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence.

[0036] 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 and antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen binding activity. Among them, "antibody fragment" refers to one or more parts or fragments of a full-length antibody. In a specific preferred embodiment, examples of antibody fragments include: Fab, Fab', (Fab)2, F(ab')2, Fv, (Fv)2, scFv, sc(Fv)2.

[0037] 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 kappa (κ) chain and lambda (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 CDRs and light chain CDRs are closely together and cooperate with each other through the framework region to form 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 framework 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.

[0038] CDRs 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 number definition and / or the conformational definition, or any CDR determination method well known in the art. As used herein, CDRs are defined by Kabat.

[0039] 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.

[0040] 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.

[0041] The term "antigen-binding fragment (Fab)" refers to the region of an antibody molecule that can bind to an antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment). By enzymatically digesting the full-length antibody with proteases, 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 an Fc fragment; under the action of pepsin, IgG can be degraded into a F(ab')2 fragment and a pFc' fragment. The F(ab')2 fragment can be further reduced to form two Fab' fragments. Because Fab has an antigen-binding region and a partial constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as a single-chain antibody (scF v), but also has a more stable structure, thus playing a huge role in clinical diagnosis and treatment.

[0042] The term "variable fragment (Fv)" refers to the smallest antibody fragment containing only the variable region, which is located in the N-terminal part of the antibody Fab fragment 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.

[0043] The term "single-chain variable fragment (scFv)" refers to the smallest antibody fragment formed by connecting the heavy chain variable region (VH) and the light chain variable region (VL) through a flexible linker consisting 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 antibody molecules, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side effects.

[0044] The full-length sequence of the antibody or antibody fragment of the present invention can be from a single species, such as a rabbit, or can be a chimeric or humanized antibody to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion of the antibody is derived from a specific source or species (rabbit), 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 human antibody's constant region, 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 sequence 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 other human antibody variable region framework sequences. In the present invention, the CDR region in a chimeric antibody or humanized antibody is derived from the rabbit CDR region of the present invention.

[0045] The term "monoclonal antibody" refers to a homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigen or epitope. "Monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population and should not be construed as limiting the structure, source, or preparation of the antibody. In certain embodiments, monoclonal antibodies are prepared by hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0046] 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.

[0047] 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.

[0048] The embodiment of the present invention provides an anti-human CD146 rabbit monoclonal antibody, which includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region both include a complementary determining region (CDR), and the amino acid sequences of the light chain complementary determining region 1 (LCDR1), the light chain complementary determining region 2 (LCDR2), and the light chain complementary determining region 3 (LCDR3) are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the amino acid sequences of the heavy chain complementary determining region 1 (HCDR1), the heavy chain complementary determining region 2 (HCDR2), and the heavy chain complementary determining region 3 (HCDR3) are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0049] The specificity of the antibody lies in the structural complementarity between the site where the antibody recognizes and binds to the antigen and the antigenic determinant, which is mainly composed of residues from the amino acid sequence of the complementary determining region (CDR), that is, the affinity and specificity of the antibody depend largely on the amino acid sequence of the complementary determining region (CDR) of the antibody. The rabbit monoclonal antibody of the present invention having the above CDR sequence can specifically recognize and bind to the human CD146 protein, and detect positive cell samples expressing CD146 protein (human umbilical vein endothelial cells HUVEC, human cervical cancer cells Hela, rat adrenal pheochromocytoma cells PC-12) and positive tissue samples of different species (Mouse Lung, Mouse Testis, Rat) by Western blot. Lung), and found that only a single band was detected in human positive samples and the single band was clear, while the relevant cells or tissue samples of mice and rats were not recognized, confirming that the antibody only recognized relevant targets of human species with good specificity. Further, flow cytometry was used to detect positive cells human umbilical vein endothelial cells HUVEC and negative cells human breast cancer cells MCF-7. The fluorescence transition was good when the positive cells were detected, but there was no fluorescence transition in the negative cells. It can be seen that the antibody has high affinity and good specificity for target recognition, that is, the anti-human CD146 rabbit monoclonal antibody provided by the present invention has a strong affinity and high specificity to the human CD146 protein, and can be applied to antibody raw materials in the field of immunological diagnosis and treatment technology, especially suitable for the field of flow cytometer detection.

[0050] Optionally, the light chain variable region and the heavy chain variable region both include a framework region (FR), and four FRs and three CDRs are arranged alternately in sequence to constitute a variable region. Correspondingly, the amino acid sequence of the light chain variable region (VL) of the anti-human CD146 rabbit monoclonal antibody of the present invention is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.4.

[0051] Optionally, the anti-human CD146 rabbit monoclonal antibody further comprises a light chain constant region and a heavy chain constant region, wherein the light chain constant region and the light chain variable region constitute a complete light chain, and the heavy chain constant region and the heavy chain variable region constitute a complete heavy chain. The constant region of an antibody can usually be obtained by public query, such as: searching rabbit IgG gammaCreign to obtain the heavy chain constant region through the IMGT online database (www.imgt.org), and searching rabbit IgG Kappa C reign to obtain the light chain constant region.

[0052] Illustratively, the amino acid sequence of the light chain comprising the light chain constant region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain comprising the heavy chain constant region is shown as SEQ ID NO.2.

[0053] Optionally, the anti-human CD146 rabbit monoclonal antibody is a full-length antibody or an antibody fragment, the antibody fragment including but not limited to: (i) Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy chain and light chain; (ii) F(ab)2 fragment, a bivalent fragment comprising two Fab fragments connected by a hinge region disulfide bridge; (iii) Fv fragment, consisting of a heavy chain variable region and a light chain variable region of an antibody; (iv) (Fv)2 fragment, consisting of two covalently linked Fv fragments; (v) scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region connected by a linker; (vi) sc(Fv)2 fragment, is formed by connecting two heavy chain variable regions and two light chain variable regions by a linker, etc. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0054] The antibody fragment of the present invention has the CDR region (SEQ ID NO.5-10) as described above, and more preferably has the variable region (SEQ ID NO.3-4) as described above, retains complete antigen recognition and binding sites, and can bind to the same antigen as the full-length antibody, especially to the same epitope.

[0055] Optionally, the immunogen of the anti-human CD146 rabbit monoclonal antibody is a human recombinant CD146 protein, which is expressed using a mammalian cell expression system. The protein sequence of the human CD146 protein is shown in NP_006491.2 (Uniprot ID: P43121), and the nucleic acid sequence is shown in NM_006500.3.

[0056] Another embodiment of the present invention provides a nucleotide molecule, which is used to encode the anti-human CD146 rabbit monoclonal antibody as described above.

[0057] The nucleotide molecule is usually DNA (e.g., cDNA, genomic DNA, or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA), the nucleic acid molecule can be single-stranded or double-stranded, and the DNA can be a coding strand or a non-coding strand. The sequence of the nucleotide molecule can be derived from the amino acid sequence of the antibody by conventional means such as codon coding rules.

[0058] The full-length sequence of a nucleotide molecule or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0059] Another embodiment of the present invention provides a recombinant vector, which carries the nucleotide molecule described above.

[0060] Typical vectors of the present invention include plasmids, viruses, bacteriophages, cosmids and minichromosomes. Plasmids are the most common form of vectors, and therefore, in the context of the present invention, vectors and plasmids are used interchangeably. Vectors can be cloning vectors (i.e., used to transfer nucleotide molecules to a host and multiply in a host cell) or expression vectors (i.e., containing necessary genetic elements to allow the nucleotide molecules inserted into the vector to be expressed in the cell). Therefore, the cloning vector can include a selection marker, and a replication origin that matches the cell type specified by the cloning vector, while the expression vector includes regulatory elements (such as promoters, enhancers) expressed in the specified host cell. The nucleotide molecules of the present invention or fragments thereof can be inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleotide molecules of the present invention. This is a well-known technique in the art and will not be described in detail herein.

[0061] It should be noted that the nucleotide molecules encoding the heavy chain and light chain of the antibody of the present invention can be cloned into a vector, and each nucleotide sequence is connected to a suitable promoter downstream. For example, each nucleotide sequence encoding the heavy chain and light chain can be operably connected to different promoters, or the nucleotide sequence encoding the heavy chain and light chain can be operably connected to a single promoter so that the heavy chain and light chain can be expressed by the same promoter. In other specific embodiments, the nucleotide molecules encoding the heavy chain and light chain of the antibody of the present invention can also be constructed into two vectors, which can be introduced into the same or different host cells. When the heavy chain and 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. The selection of the aforementioned expression vector / promoter depends on the type of host cell used to produce the antibody.

[0062] Optionally, the recombinant vector includes a prokaryotic vector (such as an Escherichia coli vector, a Bacillus subtilis vector), a eukaryotic vector (such as a yeast vector) or a viral vector (such as a lentivirus, adenovirus).

[0063] In a preferred embodiment, the starting vector of the recombinant vector is the mammalian expression vector pBR322.

[0064] Still another embodiment of the present invention provides a host cell, wherein the host cell carries the nucleotide molecule as described above or comprises the recombinant vector as described above.

[0065] Host cells include prokaryotic cells and eukaryotic cells, which are selected according to the type of recombinant vector. For example, when it is a prokaryotic vector, the host cell is selected from prokaryotic cells, and commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc. When it is a eukaryotic vector, the host cell is selected from eukaryotic cells, and commonly used eukaryotic host cells include yeast, mammalian cells, etc. After obtaining the host cell transformed with the nucleotide molecule as described above or containing the expression vector as described above, the cell is cultured under suitable conditions to express the monoclonal antibody, and then the expressed monoclonal antibody is isolated.

[0066] In a preferred embodiment, the host cell is a 293F cell (human embryonic kidney cell).

[0067] The embodiments of the present invention also provide the use of the anti-human CD146 rabbit monoclonal antibody, the nucleotide molecule, the recombinant vector, and the host cell described above in the preparation of a human CD146 protein detection reagent and / or a detection kit.

[0068] The anti-human CD146 rabbit monoclonal antibody of the present invention has high affinity and good specificity for human CD146 protein. It is used as an antigen-binding antibody (capture antibody) to specifically identify and bind to the target protein in the sample to be tested. At the same time, the antigen-binding antibody can be connected to a luminescent marker or specifically bind to a detection antibody coupled with a luminescent marker. The luminescent marker can be one of colloidal gold, chemical dyes, fluorescent dyes, etc. The luminescent marker is used to generate a recognizable signal change, thereby realizing qualitative or quantitative detection of human CD146 protein. The anti-human CD146 rabbit monoclonal antibody of the present invention can be used to establish an immunoassay analysis method for Human CD146 protein with good specificity and high sensitivity, especially multi-fluorescence flow analysis.

[0069] Optionally, the immunoassay analysis method includes but is not limited to: enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB) and flow cytometry (FC).

[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 monoclonal antibody against human CD146 protein

[0072] The immunogen used is human recombinant CD146 protein, and the sequence of Human CD146 protein 24aa-559aa is constructed into the pYURK-Chis vector. The nucleic acid sequence of Human CD146 protein is shown in NM_006500.3, and the protein sequence is shown in NP_006491.2. The Uniprot number is P43121. The pYURK-Chis vector is then transferred into 293F cells, and high-quality recombinant Human CD146 mature protein 24aa-559aa with biological activity is expressed in 293F cells. New Zealand white rabbits are immunized with it as an immunogen, and monoclonal antibody development technology based on single B lymphocyte screening and culture is used to obtain rabbit monoclonal antibodies against human CD146 protein. The aforementioned CD146 protein expression and purification method adopts conventional operations in the art.

[0073] 1. Animal immunization

[0074] Four New Zealand white rabbits were immunized with recombinant human CD146 mature protein as immunogen; each rabbit was immunized with 200 μg of immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to make an emulsifier, which was injected subcutaneously at multiple points in the rabbit's abdomen and back; after the first immunization, 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to make an emulsifier every 3 weeks, and injected subcutaneously at multiple points in the rabbit's abdomen and back for two booster immunizations. After three immunizations, rabbit serum samples were collected, and the titer against human CD146 was determined by ELISA. The serum was diluted 1:243K and the titer was measured by ELISA. Rabbits with OD450nm exceeding 0.2 were selected and boosted with 200 μg of immunogen subcutaneously at multiple points. The animals were sacrificed three days later and the spleen was taken. The specificity of serum and positive samples was determined by FC method, and the affinity of serum and the sample to be tested was detected by FC after the serum was diluted.

[0075] The immune serum titer was tested by indirect enzyme-linked immunosorbent assay (ELISA), and the specific operation included:

[0076] 1) Coating: dilute the recombinant human CD146 protein (final concentration 1 μg / mL) with 1× PBS buffer, add the coating solution to the 384-well plate, 25 μL / well, level the 384-well plate, centrifuge briefly (stop after rpm reaches 1000), and incubate at 4°C overnight;

[0077] 2) Blocking: Take out the plate that has been kept overnight at 4°C, add 75 μL of washing buffer (PBS containing 0.05% (v / v) Tween-20) to each well using a plate washer and wash 5 times; use a spray gun to add 50 μL / well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) to the plate and incubate at room temperature for 1 hour to block irrelevant binding sites;

[0078] 3) Serial dilution and sample loading of the test serum: Repeat the plate washing process to clean the well plate, and serially dilute the test serum in a 96-well plate. Use dilution buffer (PBS containing 1% BSA) to dilute the serum three times starting from 1:1000, for a total of 8 serial dilutions. The dilution ratio can be adjusted according to the actual situation.

[0079] 4) Secondary antibody incubation: Repeat the plate washing process to clean the well plate, dilute the horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG with dilution buffer at a dilution ratio of 1:5000, use a dispenser to 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;

[0080] 5) Termination of reaction and color development: Repeat the plate washing process to clean the well plate, add TMB for color development at 100 μL / well, incubate at 37°C in dark for 10 min, add 100 μL / well 0.5 M oxalic acid solution to terminate the reaction, measure the absorbance at 450 nm, use pre-immune rabbit serum as a negative control, and determine the titer of the immune serum when the ratio of the measured value to the control value ≥ 2.1 is positive.

[0081] Serum titer test chart Figure 1 It can be seen that after the third immunization and the fourth booster immunization, a stronger immune response was produced in the rabbits, and the titer of neutralizing antibodies was higher.

[0082] The specificity of immune serum and the sample to be tested was analyzed by flow cytometry (FC), which includes the following steps:

[0083] 1) Experimental preparation: sterilize the clean bench with ultraviolet irradiation for 15-20 minutes, turn on the fan for 5 minutes, and prepare for sterile work;

[0084] 2) Staining method: Collect and wash the cells, then determine the total number of cells, check whether the cell viability is around 95% and not less than 90%, and resuspend the cells in 1× PBS solution to about 3×10 6 -5×10 6cells / mL, the cells were distributed into a 96-well V-shaped plate, 100 μL per well, washed once with 1× PBS, and then stained with Live / Dead (Zombie NIR Fixable Viability Kit, purchased from Biolegend, Cat. No. 423106), L / D staining solution (L / D staining solution) was diluted with 1× PBS at a ratio of 1:1500, and the L / D staining solution was distributed into a 96-well V-shaped plate as designed, 100 μL per well, the cells in each well were resuspended, and mixed; wrapped with aluminum foil, gently mixed on a microplate shaker for 15 minutes; centrifuged at 400g for 5 minutes, discarded the supernatant, and washed twice with FACS buffer; 1× Intracell uLar fixative (Fixation 1×Permeablization buffer) was dispensed into a 96-well V-shaped plate, 100 μL per well, the cells in each well were resuspended, and the cells were mixed; wrapped with aluminum foil, and gently mixed on a microplate shaker for 30 min; centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were washed twice with 1×Permeablization buffer; as designed, the immune serum diluted with 1×Perm buffer was dispensed into a 96-well V-shaped plate, 100 μL per well, the cells in each well were resuspended, and the cells were mixed; wrapped with aluminum foil, and gently mixed on a microplate shaker for 30 min; centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were washed twice with 1×Permeablization buffer; as designed, the Fluorescein(FITC)AffiniPure F(ab')2Fragment Goat Anti-Rabbit diluted 1:200 with 1×Permeablization buffer was IgG (purchased from Jackson, Cat#111-096-046) fluorescent secondary antibody was distributed into a 96-well V-shaped plate, 100 μL per well, and the cells in each well were resuspended and mixed; wrapped with aluminum foil, and gently mixed on a microplate shaker for 30 min; washed twice with 1×Permeablizationbuffer; resuspended the cells in each well with 200 μL FACS buffer and stored in the dark.

[0085] 3) Flow analysis: The analysis was performed according to Beckman cytoflex flow analyzer use and maintenance SOP-105-AND-CA-008.

[0086] The cells used in this example include positive cells human umbilical vein endothelial cells HUVEC, human cervical cancer cells Hela, and human epidermal cancer cells A431. The FC detection chart of rabbit immune serum diluted with Human CD146 recombinant protein is shown in Figure 2-4 ,in, Figure 2-4The flow cytometric analysis of positive cells HUVEC, Hela and A431, from left to right, the serum dilutions are 1:500, 1:1000 and 1:2000, respectively. The horizontal axis represents the fluorescence brightness of APC (647), the vertical axis represents the number of cells, the red curve is the negative control, the blue curve is the isotype control, and the yellow curve is the serum to be tested. It can be seen from the figure that the fluorescence transition of each serum dilution is good, which confirms that the animal is well immunized and has produced antibodies that can be used for flow cytometry detection in the body, and the animal can enter the stage of isolating spleen cells.

[0087] 2. Isolation and culture of B lymphocytes in spleen

[0088] Separation of spleen cells: Take out a culture dish in a safety cabinet aseptically, add 30-40mL of basal culture medium, put a cell sieve, take out the spleen and put it in the cell sieve, cut off the excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into pieces and put it into the cell sieve for grinding, take a clean grinding rod, and use the end of the pressing part to crush and grind the tissue. The cells in the membrane will slowly come out, pass through the cell sieve, and suspend in the culture dish solution; wash the cell sieve with 10mL of basal culture medium, and collect the basal culture medium outside the cell sieve. Centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 13 mL of RBC lysis buffer (BD Pharm lyseTM Lysing Buffer, Catalog No. BD555899) at room temperature, gently blow away the cell clusters with a pipette, then time for 1 min to lyse the red blood cells, add 37 mL of basal culture medium, mix well, terminate the red blood cell lysis, centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 40 mL of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, resuspend the cells, complete the first wash, centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 20 mL of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, and resuspend the cells; filter the resuspended cells again through a cell sieve to remove clumping cells, and then count the cells.

[0089] For methods of sorting and culturing B lymphocytes in the spleen, please refer to the patent "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A)" and the patent "A B lymphocyte in vitro culture system and application (Publication No.: CN111518765A)".

[0090] 3. Cloning of the gene encoding rabbit monoclonal antibody

[0091] After culture, the supernatant of 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 TMMicro Prep kit (purchased from ZYMO, catalog number R1051) was used to extract RNA and reverse transcribe it into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of the corresponding positive clones by PCR, and several clones were selected for sequencing. Among them, the PCR reaction system was as follows: 4μL cDNA, 1μL forward primer (10mM), 1μL reverse primer (10mM), 12.5μL 2×GloriaHiFi (purchased from Wuhan Aibotek Biotechnology Co., Ltd.) and 6.5μL H2O. The PCR amplification program included: pre-denaturation at 98℃ for 30s, followed by 40 cycles of 98℃ for 10s, 64℃ for 30s, and 72℃ for 30s, and finally kept at 72℃ for 5min. The reaction solution was stored at 4℃.

[0092] The nucleotide sequences of the forward primer and reverse primer described above are as follows:

[0093] VL-F: 5'-tgaattcgagctcggtacccatggacacgagggcccccac-3' (see SEQ ID NO: 11);

[0094] VL-R: 5'-cacacacacgatggtgactgttccagttgccacctgatcag-3' (see SEQ ID NO: 12);

[0095] VH-F: 5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3' (see SEQ ID NO: 13);

[0096] VH-R: 5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3' (see SEQ ID NO: 14).

[0097] The amplified cDNA was sequenced by Jinkairui Biotechnology Co., Ltd. to obtain the light chain variable region (VL) amino acid sequence shown in SEQ ID NO: 3 and the heavy chain variable region (VH) amino acid sequence shown in SEQ ID NO: 4; then the IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, and the light chain amino acid sequence shown in SEQ ID NO: 1 and the heavy chain amino acid sequence shown in SEQ ID NO: 2 were obtained. The obtained rabbit monoclonal antibody against HumanCD146 protein was named 1E1. The specific sequence involved in 1E1 is shown in Table 1.

[0098] Table 1 Summary of amino acid sequence information of rabbit monoclonal antibody 1E1 of the present invention

[0099]

[0100] 4. Production and purification of rabbit monoclonal antibody 1E1

[0101] In order to obtain multiple rabbit monoclonal antibodies that recognize Human CD146 protein, the heavy chain genes and light chain genes of several rabbit monoclonal antibodies selected above were loaded on expression vectors respectively. The mammalian expression vector pBR322 used was pre-carried with the light chain and heavy chain constant regions. The expression profile is shown in Figure 6 In the figure, pRB322origin and f1origin are replication promoters in Escherichia coli (E.Coli), Ampcillin is a plasmid resistance gene, CMV immearlypromotor is a promoter in eukaryotes, SV40 PA ter minator is a tailing signal, Heavy chain constant is the nucleotide sequence of the rabbit heavy chain constant region (left figure), and Light chain constant is the nucleotide sequence of the rabbit monoclonal antibody light chain constant region (right figure). The two expression vectors were linearized with NheI and XbaI restriction endonucleases respectively, and the PCR products after the above amplification were purified. Then, the heavy chain variable region gene and the light chain variable region gene with the upstream signal peptide encoding gene were constructed into the corresponding mammalian expression vector by homologous recombination; after sequencing verification, the expression vectors containing the corresponding rabbit monoclonal antibody light chain gene and heavy chain gene were transfected into 293F cells together; after transfection, the cells were cultured for 72-96 hours to obtain the supernatant containing the Human CD146 rabbit monoclonal antibody. Rabbit monoclonal antibody 1E1 that recognizes human CD146 protein was purified from the culture supernatant using protein A affinity gel resin. After the antibody was verified to be qualified, it was packaged and stored at -20°C for future use. The obtained antibody concentration was 2 mg / mL and the purity was greater than 95%.

[0102] It should be noted that the signal peptide of this embodiment can adopt the antibody expression signal peptide sequence commonly used in the art. For details, please refer to the patent "Rabbit monoclonal antibody against human interferon α2 and its application (publication number: CN116063487A, publication date: 2023-05-05)", the light chain variable region upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGATF", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

[0103] Example 2 Western blot analysis (WB) of rabbit monoclonal antibody 1E1

[0104] Cell culture: Collection of adherent cells: remove the culture medium of adherent cells, wash once with PBS, NS or serum-free medium, centrifuge at low speed, discard the supernatant, and keep the precipitate. Collection of suspended cells: centrifuge suspended cells at low speed, discard the supernatant, collect the precipitate, and flick the cells with your fingers to loosen them. Collection of tissue samples: cut the tissue into small pieces, the smaller the better. Take the tissue frozen in liquid nitrogen or ultra-low temperature refrigerator for more than 30 minutes, and quickly grind it with liquid nitrogen. Try to control the grinding process within 1-2 minutes to reduce protein degradation.

[0105] The cell samples used in this example include positive cells HUVEC, Hela and PC-12; the tissue samples include positive tissues Mouse Lung, Mouse Testis and Rar Lung, and positive means that the corresponding cells or tissues express CD146 protein.

[0106] Total protein extraction: 1) Cell / tissue lysis: Place the container containing cell pellets or tissue fragments completely in ice. 7 1) Add the corresponding volume of lysis buffer (3 mL is added when the cell volume is sufficient, and calculated according to the cell volume if insufficient), lyse for 20 minutes, and place the centrifuge tube on a vortex shaker for 10 seconds every 5 minutes. Add protein lysis buffer to the homogenizer at the rate of 0.5 mL lysis buffer / 100 mg tissue, grind once every 3 minutes, and repeat 5 times to crush the tissue as much as possible (add or not add protease inhibitors to the lysis buffer as needed); 2) Centrifugation: After the lysed sample is balanced, place it in a pre-cooled high-speed refrigerated centrifuge and centrifuge at 12000 rpm for 15 minutes; 3) Protein denaturation: After centrifugation, the supernatant is the protein extract. Take a small amount of protein extract and use the BCA method to determine the protein concentration. Add 1 / 5 of the supernatant volume of 5× Loading Buffer to the remaining protein extract centrifuge tube (the final working solution is 1×). After the temperature of the dry thermostat rises to 95°C, insert the 1.5mL centrifuge tube into the heating hole and heat at 95°C for 10 minutes for denaturation. After the liquid is completely cooled, store it at -20°C.

[0107] Immunoblotting experiment: 1) Preparation of separation gel: Select the appropriate separation gel according to the size of the protein; Inject the prepared gel solution into the pre-installed gel caster and add isopropanol to seal the gel. Place it horizontally at room temperature for about 30 minutes. Be careful to prevent the gel solution from generating bubbles and injecting it into the gel caster; Add TEMED before injecting the gel to prevent solidification before injection; When injecting isopropanol, slowly drag it along the side of the glass plate to add it; 2) Preparation of concentrated gel: After the separation gel solidifies, pour out the isopropanol along the side of the glass plate and dry it with filter paper. Prepare concentrated gel according to the required volume. Inject the prepared gel solution into the gel caster, and slowly insert the prepared sample comb into the gel solution along one end of the glass plate, and place it horizontally at room temperature for 20-30 minutes. Pay attention to avoid bubbles between the sample comb and the gel solution; 3) Loading: After the concentrated gel solidifies, pull out the sample comb vertically with both hands, and inject Tris-Glycine SDSRunning Buffer into the inner and outer grooves to form a closed loop. Use a pipette to draw the sample vertically comb hole for sample loading, protein content 25ug / well. Note that the inner tank Tris-Glycine SDS Running Buffer must be filled, and the outer tank Tris-Glycine SDS Running Buffer should be 3-5cm above the bottom; 4) Electrophoresis: After loading the sample, connect the electrophoresis instrument power supply and set appropriate electrophoresis parameters. The concentrated gel electrophoresis parameter is a constant voltage of 80V. After the sample enters the separation gel, adjust the voltage to 120V. When bromophenol blue electrophoresis reaches the bottom of the gel, stop electrophoresis and turn off the power of the electrophoresis instrument; 5) Transfer: Soak the 0.45 μm NC membrane in Western Transfer Buffer for standby use, and transfer the protein band on the gel to the mNC membrane in the electrotransfer system according to the conventional method; 6) Blocking: After the electrotransfer is completed, put the mNC membrane into a suitable antibody incubation tank, add 1 mL of Western Blocking Buffer and incubate at room temperature; 7) HRP-labeled primary antibody incubation: Dilute the HRP-labeled primary antibody (i.e., the rabbit monoclonal antibody 1E1 of the present invention) with Western Transfer Buffer at 1:217000, pour the diluted primary antibody working solution (the final antibody concentration is 0.01 μg / mL) into the incubation tank, and incubate at room temperature for 2 hours or at 4°C overnight; 8) Washing: After the primary antibody incubation is completed, add TBST Buffer and wash 4 times, 5 minutes / time; 9) Exposure: According to the size of the membrane, every 10 cm 2Use 1-2mL of working solution for the membrane, draw equal volumes of Solution I and Solution II in proportion and mix them to prepare the luminescence detection working solution. Use flat-tipped tweezers to remove the membrane, and gently touch the lower edge of the membrane to absorbent paper to remove excess liquid on the membrane. Use a pipette to add the working solution to the transfer membrane so that it is evenly covered, and incubate at room temperature for 1-2 minutes. This step can be completed on a clean plastic wrap or in a plastic box. Pressing test: Fix the membrane in the film clip, press the film in a dark room for 1 minute, and immediately develop and fix it. Adjust the pressing time according to the results. Or directly press the film for 30s, 1, 3, and 5min respectively, and then develop and fix it together to observe the results; 10) Fluorescence imager detection: Place the membrane in the fluorescence imager and refer to the instrument manual for detection.

[0108] The WB test results of rabbit monoclonal antibody 1E1 against human CD146 protein are shown in Figure 6 , 72 and 120KD in the figure refer to 72kD and 120kD, both of which can be used as references, 1:217000 refers to the antibody dilution ratio of 1:217000, the working concentration is the concentration of the antibody after final dilution, T=30s, which is 30s exposure. The detection samples HUVEC and Hela in this embodiment are human-related samples, the detection samples Mouse Lung and Mouse Testis are mouse-related samples, and the detection samples PC-12 and Rar Lung are rat-related samples. It can be seen from the figure that only human-related positive samples have a single clear band, proving that antibody 1E1 recognizes human-related targets, mice and rats do not recognize them, and there are no mixed bands in the swimming lane, proving that rabbit monoclonal antibody 1E1 has high specificity and good affinity.

[0109] Example 3 Flow Cytometry (FC) Analysis of Rabbit Monoclonal Antibody 1E1

[0110] The cell samples of this example include positive sample HUVEC and negative sample MCF-7, wherein positive means that the corresponding cells or tissues express CD146 protein, and negative means that the corresponding cells or tissues do not express CD146 protein. The determination method is shown in Example 1, the primary antibody is the rabbit monoclonal antibody 1E1 of the present invention, the antibody concentration is 0.2 μg / mL, and the fluorescent secondary antibody is 488 (Fluorescein (FITC) AffiniPure F(ab')2Fragment Goat Anti-Mouse IgG, Fcγfragmentspecific, purchased from Jackson, catalog number 115-096-071) and 647 (Alexa 647AffiniPureGoat Anti-Mouse IgG, Fcγfragment specific, purchased from Jackson, catalog number 115-605-071), was used to verify the adaptability of the antibody to different fluorescence. Antibody 1E1 of the present invention recognizes CD146 protein, and the fluorescent secondary antibody recognizes antibody 1E1. By identifying the fluorescence intensity (fluorescence transition) and the percentage of fluorescent cells, the specificity and affinity of the primary antibody can be determined. The results are shown in Figure 7-10 ,in, Figure 7-8 HUVEC and MCF-7 488 fluorescence FC detection diagram, Figure 9-10 HUVEC and MCF-7 FC detection diagram of 647 fluorescence.

[0111] As can be seen from the figure, the fluorescence transition is good, indicating that the antibody has a high affinity for recognizing the target human CD146 protein, and there is a unique peak for positivity, indicating that the antibody has high specificity, and the results of both 488 and 647 fluorescence detection are good, indicating that the antibody of the present invention can meet the detection characteristics of multiple fluorescence.

[0112] 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 anti-human CD146 rabbit monoclonal antibody, characterized in that: comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region both comprise complementarity determining regions; The amino acid sequences of the light chain complementary determining region 1, the light chain complementary determining region 2 and the light chain complementary determining region 3 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; The amino acid sequences of the heavy chain complementary determining region 1, the heavy chain complementary determining region 2 and the heavy chain complementary determining region 3 are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The anti-human CD146 rabbit monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. The anti-human CD146 rabbit monoclonal antibody according to claim 2, characterized in that: It also includes a light chain constant region and a heavy chain constant region, wherein the light chain constant region and the light chain variable region constitute a complete light chain, and the amino acid sequence of the light chain is shown in SEQ ID NO.1; the heavy chain constant region and the heavy chain variable region constitute a complete heavy chain, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

2.

4. The anti-human CD146 rabbit monoclonal antibody according to claim 1 or 2, characterized in that: The anti-human CD146 rabbit monoclonal antibody is a full-length antibody or an antibody fragment, and the antibody fragment 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 nucleotide molecule, characterized in that Used to encode the anti-human CD146 rabbit monoclonal antibody as described in any one of claims 1-4.

6. A recombinant vector, characterized in that: Carrying the nucleotide molecule as claimed in claim 5.

7. The recombinant vector according to claim 6, characterized in that The starting vector of the recombinant vector is the mammalian expression vector pBR322.

8. A host cell, characterized in that Carrying the nucleotide molecule as described in claim 5 or comprising the recombinant vector as described in claim 6.

9. The host cell according to claim 8, characterized in that The host cell is 293F cell.

10. Use of the anti-human CD146 rabbit monoclonal antibody according to any one of claims 1 to 4 in the preparation of a human CD146 protein detection reagent and / or a detection kit, characterized in that: The detection method includes at least one of enzyme immunoassay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, immunohistochemistry, immunofluorescence, immunoblotting and flow cytometry.

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