CN118530357A - Rabbit monoclonal antibody against human lysosomal associated membrane protein 3, antibody conjugate and kit and application thereof - Google Patents
Rabbit monoclonal antibody against human lysosomal associated membrane protein 3, antibody conjugate and kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody resisting human lysosomal associated membrane protein 3, an antibody conjugate, a kit and application thereof. The amino acid sequences of the CDRs 1-3 of the light chain variable region of the rabbit monoclonal antibody are respectively shown as SEQ ID NO.5-7, and the amino acid sequences of the CDRs 1-3 of the heavy chain variable region are respectively shown as SEQ ID NO. 8-10. The anti-human lysosome related membrane protein 3 rabbit monoclonal antibody can specifically identify the recombinant expression and cell/tissue surface CD63 protein, has good specificity and high sensitivity, can effectively realize the purposes of tissue and cell parting and tumor tissue differential diagnosis, provides solid guarantee for high-quality immunodetection of CD63 protein, is particularly suitable for large-scale and large-sample multi-group immunohistochemical staining in cell smear research, and has great clinical application value.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody resisting human lysosomal associated membrane protein 3, an antibody conjugate, a kit and application thereof.
Background
CD63, also known as lysosomal associated membrane protein 3 (LAMP 3), consists of a short/long extracellular loop and a short N-/C-terminal cytoplasmic tail, forming a protein structure with four transmembrane protein enrichment domains (TEMs) on the cell surface. CD63 is widely expressed on the surface of many cell types and is associated with various life processes such as activation, adhesion, mutation, and invasion and metastasis of tumors. CD63 is expressed in endosomes and lysosomes, in late endosomes, CD63 is enriched on endoluminal vesicles, and by fusion of endosomes with plasma membranes, specialized cells secrete it as exosomes, an important exosome-specific marker protein suitable for non-invasive detection from biological fluids. In immune response, CD 63-expressing exosomes contribute to antigen presentation and activate cd4+ T cells for immune regulation, highlighting the importance of CD63 in immunotherapy and its potential as a target for small molecule inhibitors. In addition, it interacts with molecules such as integrins (α4β1, α3β1, α6β1, β2) and CD81, CD82, CD9, and CD15 to control cell adhesion and migration. Additional studies have shown that CD63 is expressed on activated platelets and may act as a platelet activation marker. In addition to its fundamental function in cell homeostasis, CD63 is expressed in a variety of tumors, with CD63 tetrameric protein highly expressed in early stages of melanoma and reduced expression in late stage lesions, suggesting that it may be an inhibitor of tumor progression, and may be one of the potential targets for cancer treatment. Moreover, research proves that CD63 exosomes are differentially expressed in specific cancers such as ovarian cancer, lung cancer and the like, and the potential application of the CD63 exosomes as biomarkers is shown, so that a new path is opened up for early diagnosis and continuous monitoring of related diseases.
In view of the importance of the multifaceted CD63 target in biomedical research and clinical treatment and diagnosis of related diseases, the development of a high-specificity human lysosome related membrane protein 3 detection method has wide application prospect. However, the current immunodetection technology for high sensitivity and reliability of human CD63 protein is not effectively researched and developed, and the main problem is the lack of antibodies with excellent performance for resisting human lysosome related membrane protein 3, so that the development of monoclonal antibodies with strong specificity and good anti-interference capability for human lysosome related membrane protein 3 has become a problem to be solved urgently.
Disclosure of Invention
Aiming at the problems that the anti-human-lysosomal related membrane protein 3 antibody in the prior art has poor specificity and is difficult to use for immunological detection and the like, the invention provides a rabbit monoclonal antibody and an antibody conjugate for the anti-human-lysosomal related membrane protein 3, and provides application of the rabbit monoclonal antibody and the antibody conjugate in preparing a human-lysosomal related membrane protein 3 immunological detection kit and a related kit. In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
The invention provides a rabbit monoclonal antibody for resisting human lysosomal associated membrane protein 3, which comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7; the amino acid sequences of complementarity determining regions 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.
Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4.
Further, the amino acid sequence of the light chain of the rabbit monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2.
Further, the rabbit monoclonal antibody is a full-length antibody or an antibody fragment having immunological activity; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
In a second aspect the invention provides an antibody conjugate comprising a rabbit monoclonal antibody as described above against human lysosomal associated membrane protein 3, and a detection label linked to said rabbit monoclonal antibody.
Further, the detection label is selected from one or more of biotin, fluorescein, chemiluminescent group, fluorescent protein, horseradish peroxidase, acid phosphatase, colloidal gold, colored magnetic beads, magnetic bead microspheres, latex particles, radionuclides, and detection antibodies.
In a third aspect the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody against human lysosomal associated membrane protein 3 as described above, a recombinant vector or host cell comprising said nucleic acid molecule.
In a fourth aspect, the invention provides the use of a rabbit monoclonal antibody or antibody conjugate directed against human lysosomal associated membrane protein 3 as described above in the preparation of a kit for immunodetection of human lysosomal associated membrane protein 3.
In a fifth aspect the invention provides a kit for the immunological detection of human lysosomal associated membrane protein 3, said kit comprising a rabbit monoclonal antibody or antibody conjugate as described above against human lysosomal associated membrane protein 3.
Further, the kit is an immunoblotting kit or an immunohistochemical kit.
The invention has the advantages and positive effects that:
The anti-human lysosome related membrane protein 3 rabbit monoclonal antibody can specifically identify the recombinant expression and the natural CD63 protein on the cell/tissue surface, has good specificity and high sensitivity, can effectively realize the purposes of tissue and cell typing and tumor tissue differential diagnosis, has the specificity and sensitivity up to 100 percent, provides firm guarantee for high-quality immunodetection of CD63 protein, is suitable for detection of various cell/tissue and pathological samples, is particularly suitable for large-scale and large-sample multi-group immunohistochemical staining in cell smear research, and has great clinical application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an SDS-PAGE gel of CD63 according to example 1 of the present invention;
FIG. 2 is an expression pattern of a mammalian pRB322 vector used for constructing an anti-human CD63 rabbit monoclonal antibody expression vector of example 2 of the present invention, from left to right, a vector carrying an antibody light chain constant region and a heavy chain constant region in advance, respectively;
FIG. 3 is a graph showing the WB results of the anti-human CD63 rabbit monoclonal antibody of example 3 of the present invention for detecting human CD63 in a sample of human malignant melanoma cells;
FIG. 4 is a graph showing IHC results of anti-human CD63 rabbit monoclonal antibody of example 4 of the present invention for detecting CD63 in human malignant melanoma tissue samples;
FIG. 5 is a graph showing IHC results of anti-human CD63 rabbit monoclonal antibody of example 4 of the present invention for detecting CD63 in human colon tissue samples;
FIG. 6 is a graph showing IHC results of anti-human CD63 rabbit monoclonal antibody of example 4 of the present invention for detecting CD63 in human breast cancer tissue samples.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" is considered to include the following: (i) A, (ii) B, and (iii) A and B.
The terms "monoclonal antibody", "mab", "antibody" and the like have the same meaning and are used interchangeably to refer to a rabbit monoclonal antibody that specifically binds to Human (Human) lysosomal associated membrane protein 3 (LAMP 3, also known as CD 63). The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity. Where "antibody fragment" refers to one or more portions or fragments of a full-length antibody, in typical examples, the antibody fragment comprises: fab, fab', F (ab) 2、F(ab')2、Fv、(Fv)2、scFv、sc(Fv)2.
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, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs may be identified according to Kabat definitions, chothia definitions, a combination of both Kabat and Chothia definitions, abM definitions, contact definitions, IMGT unique numbering definitions and/or conformational definitions, or any CDR determination method known in the art. As used herein, CDRs are defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. 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 antibody heavy and light chain constant regions are well known in the art.
Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.
The term "Antigen binding fragment (Fab)" is a region of an antibody molecule that binds Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain (variable and one constant region fragment), whereby fragments such as Fab, F (ab ') 2, fab' can be obtained by proteolytic cleavage of the full-length antibody. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab ') 2 fragment and a pFC' fragment by pepsin. The F (ab ') 2 fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has antibody-antigen affinity like a single chain antibody (scFv), excellent tissue penetrating power and the like, but also has a more stable structure, and is widely applied to clinical diagnosis and treatment.
The term "variable fragment (Fv)" is located at the N-terminus of an antibody Fab fragment, contains only the variable region, and consists of one variable region of one light chain and one heavy chain, is a dimer of one VH and one VL that are non-covalently bound (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than an intact antibody.
The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker, typically consisting of 10 to 25 amino acids), which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.
The full length sequences of the antibodies or antibody fragments of the invention may be from a single species, such as rabbit, or may be chimeric or humanized antibodies to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" antibody refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases, the variable regions of a non-human antibody bind to the constant regions of a human antibody, e.g., a human rabbit chimeric antibody; in other cases, the CDR regions of a non-human antibody bind to FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of a non-human antibody are grafted onto human antibody Framework (FR) sequences derived from single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.
The term "monoclonal antibody" refers to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the structure, source, or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a rabbit monoclonal antibody for resisting human lysosomal associated membrane protein 3, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs) respectively named as CDR1, CDR2 and CDR3, and the amino acid sequences of the CDR1, the CDR2 and the CDR3 on the light chain variable region are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7; the amino acid sequences of CDR1, CDR2 and CDR3 on the variable region of the heavy chain are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.
The invention relates to a human lysosome related membrane protein 3 (CD 63) protein complete extracellular domain is an antigen immune rabbit, a rabbit monoclonal antibody of the human lysosome related membrane protein 3 is prepared, the obtained antibody can specifically identify and recombine and express the natural CD63 protein on the surface of cells/tissues, the antibody has good specificity and high sensitivity, a target strip can be accurately detected by the antibody through a western blotting method for detecting a human malignant melanoma cell A-375 sample expressing the CD63 protein, a plurality of tissues expressing the CD63 protein can be accurately distinguished by an immunohistochemical method, 7 positive samples and 6 negative samples can be accurately distinguished by the antibody, the positive samples are obviously dyed, the background is clean, no nonspecific dyeing can be effectively realized, the aim of tissue and cell parting and tumor tissue differential diagnosis can be effectively achieved, the specificity and the sensitivity are up to 100%, the antibody has higher accuracy, good reliability and excellent anti-interference capability when the CD63 protein is detected through immunodetection, solid guarantee is provided for high-quality immunodetection of the CD63 protein, the antibody is suitable for detecting a plurality of cells/tissues and has great clinical application values in a plurality of cell/tissue samples, especially in clinical research, and a plurality of clinical tissue samples are applied to a large-scale tissue samples.
Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of VL of the rabbit monoclonal antibody is shown as SEQ ID NO.3, and the amino acid sequence of VH is shown as SEQ ID NO. 4.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region and a heavy chain constant region, CL and VL comprise the complete light chain, and CH and VH comprise the complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA CREIGN was searched for CL via IMGT online database (www.imgt.org). Specifically, the amino acid sequence of the light chain comprising the light chain constant region is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain comprising the heavy chain constant region is shown in SEQ ID NO. 2.
It should be noted that the rabbit monoclonal antibody of the present invention may be a full-length antibody (having a typical Y-type molecular structure) or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full-length form of the rabbit monoclonal antibody, specifically, an antibody fragment includes CDR regions as described above, more preferably has variable regions as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, particularly to the same epitope, as the full-length antibody. Such antibody fragments include, but are not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) 2 fragment consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) The sc (Fv) 2 fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via a linker or the like. These antibody fragments can be obtained by conventional techniques in the art.
In yet another embodiment, the invention provides an antibody conjugate comprising a rabbit monoclonal antibody directed against human lysosomal associated membrane protein 3 as described above, and a detection label attached to said rabbit monoclonal antibody.
The detection mark is used for generating identifiable signal changes so as to identify a sample to be detected according to the signal changes. Detection labels include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, magnetic bead microspheres, latex particles, radionuclides, detection antibodies, or combinations thereof.
In another embodiment the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody against human lysosomal associated membrane protein 3 as described above, a recombinant vector or host cell comprising said nucleic acid molecule.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.
The starting vector for constructing the recombinant vector may be any vector as long as it can contain the nucleic acid molecule, and various vectors conventional in the art can be used, and typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). Thus, a cloning vector may comprise a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector comprises regulatory elements (e.g., promoters, enhancers) for expression in a specified host cell. The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector. This is well known in the art and will not be described in detail herein.
Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the rabbit monoclonal antibodies of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.
Transfection or transformation of the recombinant vector into a host cell according to the present invention is carried out using conventional techniques. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after the exponential growth phase and treated with CaCl 2 or MgCl 2. Microinjection, electroporation, or liposome encapsulation may also be used if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomycete, salmonella typhimurium, yeast, drosophila S2 or Sf9 cells, CHO, COS7, 293 series cells, etc. After obtaining host cells transfected or transformed with the vectors described above, monoclonal antibodies can be expressed by culturing under appropriate conditions and then isolating to obtain purified antibodies.
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).
In a further embodiment, the invention provides the use of a rabbit monoclonal antibody or antibody conjugate against human lysosomal associated membrane protein 3 as described above for the preparation of a kit for immunodetection of human lysosomal associated membrane protein 3.
The application advantages of the rabbit monoclonal antibody or antibody conjugate of the anti-human-lysosomal-associated membrane protein 3 in preparing the immune detection kit of the human-lysosomal-associated membrane protein 3 are the same as those of the rabbit monoclonal antibody of the anti-human-lysosomal-associated membrane protein 3 in the prior art, and are not repeated herein.
Based on the same inventive concept, the embodiment of the invention also provides a human lysosomal associated membrane protein 3 immunoassay kit comprising the rabbit monoclonal antibody or antibody conjugate against human lysosomal associated membrane protein 3 as described above.
The antibody of the present invention may be used alone or may be conjugated or coupled to a detection label to form an antibody conjugate. In some embodiments, the antibodies of the invention are used as antigen binding antibodies that specifically recognize and bind to CD63 protein in a sample to be tested, and qualitative or quantitative detection of CD63 protein is then achieved by producing a recognizable signal change by a detection label attached thereto, in other embodiments, the anti-human CD63 protein antibody (as primary or capture antibody) is not labeled, and the detection label is conjugated to a primary antibody (as detection antibody) or other molecule, e.g., if the anti-human CD63 protein antibody is a rabbit IgG antibody, the secondary antibody may be an anti-rabbit IgG antibody, whereby a recognizable signal change is produced after specific binding of the antibody by the conjugated detection label, thereby achieving qualitative or quantitative detection.
The above-described immunoassay methods include, but are not limited to: enzyme immunoassay (Enzyme immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunosorbent assay (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (Immunohistochemistry, IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), immunoprecipitation (Immunoprecipitation, IP) and Flow Cytometry (FC).
In a preferred embodiment, the immunodetection method is immunoblotting or immunohistochemistry, and the kit is an immunoblotting kit or an immunohistochemical kit.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
Example 1 preparation of human CD63 protein antigen
The invention selects a complete domain of Human (Human) lysosome related membrane protein 3 (LAMP 3, also called CD 63) extracellular region as an antigen, NCBI number of CD63 is referred to NP-001771.1,Uniprot ID and is referred to P08962, the complete domain of extracellular region is amino acid fragments from 103 rd position to 203 th position, a gene sequence (NCBI number is referred to NM-001780.6) corresponding to Human CD63 protein 103-203AA is constructed into a pGEX-4T-AB1 vector, and the gene sequence is transfected into escherichia coli Rosetta for expression, so that Human CD63 recombinant protein with higher purity is obtained and is used as an antigen for immunizing animals.
1. Construction of protein expression plasmid
Using a plasmid (from martial arts, inc.) containing the human CD63 CDs sequence as a template, the human CD63 protein 103-203AA fragment was amplified with a pair of specific primers; the PCR reaction system is as follows: 1. Mu.L of template, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 25. Mu.L of 2X Gloria Hi-FI PCR MASTER Mix with GC Buffer (from Wuhan Aibotake Biotechnology Co., ltd., cat# RK 20717) and 22. Mu.L of N.F H 2 O; PCR amplification procedure: the reaction solution is subjected to pre-denaturation at 98 ℃ for 30s, then 30 times of circulation are carried out according to the conditions of 10s at 98 ℃, 30s at 64 ℃ and 30s at 72 ℃, and finally the reaction solution is kept at 72 ℃ for 5min, and the obtained reaction solution is kept at 4 ℃.
The sequence of the fragment of human CD63 protein from amino acid 103 to 203 (see SEQ ID NO. 11) is as follows:
AGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIP SMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNV.
The forward and reverse primer nucleotide sequences (see SEQ ID NOS.12-13) are:
CD63-F：GGTTCCGCGTGGATCCCCGGAAGCTGGCTATGTGTTTAGAGATAAGG；
CD63-R：TGGTGATGATGATGCGGCCGCTCCACATTTTTCCTCAGCCAGCC。
after the amplified PCR product is purified, the PCR product is loaded on an expression vector in a homologous recombination mode, the prokaryotic expression vector is pGEX-4T-1 (commercial vector), and plasmid is preserved after sequencing verification, and sequencing work is completed by Jin Kairui biotechnology Co.
2. CD63 protein expression
PGEX-4T-AB1 plasmid containing the 103-203AA fragment of human CD63 protein was transformed into E.coli Rosetta strain and incubated overnight at 37℃on LB plate containing 100. Mu.g/mL ampicillin to obtain several single colony transformants. Single colony transformants were inoculated in 2mL of LB medium (containing 100. Mu.g/mL ampicillin) and cultured at 37℃and 220rpm for 3-4 hours until OD 600nm was about 0.4-0.6, and then 2mL of the culture of each strain was transferred to 400mL of LB expression medium and further cultured at 37℃and 220rpm for 3-4 hours. When OD 600nm reaches about 0.45-0.55, 0.8mM IPTG is added, induction is carried out for 3-4h at 37 ℃, the induced bacterial liquid is centrifuged at 4000rpm for 10min, the supernatant is discarded, and the bacterial cells are stored in a freezer at-20 ℃.
30ML of the bacterial suspension (50 mM Tris-300mM NaCl) was used to suspend the cells in a centrifuge bottle. The suspended bacterial liquid was transferred to a 50mL round bottom centrifuge tube, placed in an ice box and fixed with ice. Selecting an amplitude transformer, placing the amplitude transformer into a bacteria breaking cabin, wherein the power is 350W, the bacteria breaking time is 3s, the interval time is 3s, the time is counted down for 5min, then placing the amplitude transformer into an ice-water mixture for cooling for 5min, and repeating the steps for breaking bacteria for 5min. After the completion of the sterilization, the mixture was centrifuged at 9000rpm for 10min to obtain a supernatant and a precipitate.
Crushing for the second time: 30mL of bacteria breaking liquid (2M urea-PBS buffer solution) is measured and poured into the sediment, the sediment is uniformly blown, the sediment is transferred into a 50mL round bottom centrifuge tube, the power is 350W, the bacteria breaking time is 3s, the interval time is 3s, the bacteria breaking time is 0-5 min (the breaking time is determined according to the sediment amount and the texture, the bacteria liquid is placed in ice cubes), the supernatant and the inclusion body are obtained by centrifugation at 9000rpm for 10min, and the inclusion body is subjected to 5-time dilution (short for 5 "). SDS-PAGE patterns of inclusion bodies are shown to the left of Marker (MK) in FIG. 1, and it can be seen that: CD63 (103-203 AA) -GST protein was expressed in inclusion bodies.
3. CD63 protein purification
CD63 protein was purified by affinity chromatography from inclusion bodies using GST affinity chromatography resin (available from Huiyan under the trade designation HQ 030307001L) and the SDS-PAGE electrophoresis of the purified protein was shown on the right side of Marker (MK) in FIG. 1. In FIG. 1, lanes "bag" represent inclusion bodies, lanes "0.4" and "0.2" represent 0.4mg/mL and 0.2mg/mL BSA, lane "MK" represents marker, lane "FT" represents flow-through during purification, lane ". Times.5" represents inclusion body purification product, 300-1 represents protein concentration of 0.8mg/mL,300-2 represents protein concentration of 0.1mg/mL, and 500 represents protein concentration of 0.5mg/mL. The purity of the recombinant CD63 protein is more than 85% from the SDS-PAGE gel diagram of FIG. 1, and the recombinant CD63 protein comprises a CD63 protein fragment and a glutathione-transferase (GST) tag.
Example 2 preparation of Rabbit monoclonal antibodies against human lysosomal associated Membrane protein 3
1. Immunization of animals
2 New Zealand white rabbits were immunized with recombinant human CD63 protein (purified in example 1) as an immunogen; immunization of each white rabbit with 300 μg of immunogen, mixing the immunogen with an equivalent amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifier before the first immunization, and subcutaneously injecting the emulsifier into the abdomen and back of the rabbits at multiple points; 150 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) at 3 weeks intervals after the first immunization to prepare an emulsifier, which was injected subcutaneously in the abdomen and back of rabbits in multiple spots to boost the immunization twice. After three immunizations, serum samples of rabbits were collected, and serum was taken at a ratio of 1: after 243000 dilution, the titer against human CD63 protein was determined by ELISA, rabbits with OD 450nm exceeding 0.2 were boosted by subcutaneous multipoint injection with 200. Mu.g of immunogen, and after three days the animals were sacrificed and spleens were obtained.
2. Separation of B lymphocytes from spleen and B lymphocyte sorting
The B lymphocytes in the spleen are separated by adopting a conventional method, and then antigen-specific B lymphocytes are obtained by sorting, and related methods are referred to a patent (publication number: CN110016462A, publication date: 2019-07-16) for efficient separation of single antigen-specific B lymphocytes from spleen cells and a patent (publication number: CN111518765A, publication date: 2020-08-11) for an in vitro culture system and application of B lymphocytes.
3. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected and lysed, and RNA was extracted using Quick-RNA TM MicroPrep kit (available from ZYMO, cat. No. R1100-250) and reverse transcribed into cDNA. The cDNA is used as a template, and the natural paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) genes are amplified from the cDNA of the corresponding positive clone by adopting a PCR method. The PCR reaction system comprises: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2X GloriaHiFi (available from Wuhan Aibotac Biotechnology Co., ltd., product No. RK 20717) and 6.5. Mu. L H 2 O. The PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃. Primer pair sequences (5 '-3') for amplifying the VL and VH genes are shown below (see SEQ ID NOS.14-17), F and R representing forward and reverse primers, respectively:
VL-Primer-F：tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC；
VL-Primer-R：cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG；
VH-Primer-F：tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG；
VH-Primer-R：gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG。
Sequencing the amplified cDNA, wherein the sequencing work is completed by Jin Kairui Biotechnology Inc., so as to obtain variable region (VL and VH) amino acid sequences; the IMGT online database (www.imgt.org) was then queried to obtain the constant region amino acid sequence, the antibody sequence is shown in table 1.
TABLE 1 sequence information of Rabbit monoclonal antibodies of this example
4. Production and purification of Rabbit monoclonal antibodies
The heavy chain genes and the light chain genes of the selected rabbit monoclonal antibodies are respectively loaded on an expression vector, the used mammal expression vector pBR322 carries the light chain constant region (CL) and the heavy chain constant region (CH) genes of the rabbit monoclonal antibodies, the expression patterns are shown in figure 2, pRB322 origin and f1 origin are replication promoters in escherichia coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a promoter in eukaryote, SV40PA terminator is a tailing signal, LIGHT CHAIN constant is a nucleic acid sequence of the light chain constant region (left graph), HEAVY CHAIN constant is a nucleic acid sequence of the heavy chain constant region (right graph). The variable region genes containing the signal peptide obtained by amplification in the steps are respectively constructed to the upstream of CL and CH genes corresponding to the expression vector pBR322, so that the light chain gene and heavy chain gene expression vectors are obtained, and the successful construction of the expression vectors is verified by sequencing.
The signal peptide of this example uses antibodies commonly used in the art to express signal peptide sequences, such as the light chain variable region upstream signal peptide "MDTRAPTQLLGLLLLWLPGARC" of the patent "anti-Human interferon alpha 2 rabbit monoclonal antibody and its use (publication number: CN116063487A, publication date: 2023-05-05)", and the patent "high affinity Human IL-5 rabbit monoclonal antibody and its use (publication number: CN115819578A, publication date: 2023-03-21)", with the heavy chain variable region upstream signal peptide "METGLRWLLLVAVLKGVQC".
The expression vector containing the light chain gene and the heavy chain gene is transfected into 293F cells together, and the recombinant human CD 63-recognizing antibody is obtained from the culture supernatant after the transfection for 72 to 96 hours. Recombinant rabbit monoclonal antibodies recognizing human CD63 protein were purified from the transfected culture medium supernatant using a proteona affinity gel resin (purchased from world and cat No. SA 023100). The purity of the antibody is more than or equal to 95 percent by using 12 percent SDS-PAGE gel electrophoresis, the concentration is 0.4mg/mL, and the purified antibody is split-packed and stored at a low temperature of minus 20 ℃ for standby.
Example 3 immunoblot analysis (WB) against CD63 rabbit monoclonal antibody
Taking human malignant melanoma cells A-375 with high expression of CD63, subculturing the cells in a 48-well cell culture plate, sucking out culture solution supernatant when the confluency is 70-80%, carefully cleaning the cells twice by using PBS solution, performing 7% polyacrylamide gel electrophoresis after cell lysis, transferring protein bands on the gel onto a PVDF membrane in an electrotransfer system according to the conventional operation, placing the membrane in TBST sealing solution containing 3% skimmed milk powder for incubation for 1h at room temperature, adding purified CD63 rabbit monoclonal antibody (1:1000 dilution), and incubating overnight at 4 ℃; after washing the membrane with TBST, add 1: goat anti-rabbit secondary antibody (from martial arts, botec biotechnology, inc., cat No. AS 014) diluted with 100000 was incubated at room temperature for 1h; the membrane was again washed with TBST, ECL-hypersensitive developer was added, and developed, and the results are shown in FIG. 3.
CD63 protein belongs to four transmembrane proteins, the total length is 238AA, 3 glycosylation sites are arranged on the surface, and the size of the protein can be influenced after glycosylation, so that the theoretical molecular weight of CD63 is between 30 KD and 60 KD. As can be seen from FIG. 3, the CD63 rabbit monoclonal antibody obtained by the screening has better specificity, and can detect a crude target band which is consistent with the size of the target protein.
Example 4 immunohistochemical analysis (IHC) of a rabbit monoclonal antibody directed against CD63
The human CD63 protein is highly expressed in cytoplasm and cell membrane of monocyte and cytoplasm and cell membrane of macrophage, and is also used for auxiliary diagnosis of malignant melanoma. The positive samples are 1 case of lung cancer, 1 case of colon cancer, 2 cases of colon cancer, 1 case of tonsil, 1 case of malignant melanoma, the negative samples are 1 case of esophagus, 1 case of esophagus cancer, 1 case of placenta, 1 case of liver cancer and 1 case of breast cancer, wherein only macrophages and monoclonal antibody cells of the breast cancer are positive, the rest cells are negative, and the breast cancer is judged to be negative.
Immunohistochemical (IHC) staining is performed with reference to a pathological chip-09-03, which is manufactured by Wohai Vir Biotechnology Co., ltd., and specifically includes the steps of: 1) Sample preparation and baking: placing paraffin slices on a slice rack in the same direction, and baking the slices in a constant temperature box at 56 ℃ for 30min; simultaneously placing the dewaxing liquid (purchased from the original industry and trade Co., ltd.) 1 jar together into a constant temperature box at 56 ℃; 2) Dewaxing to water: placing paraffin slices and a slice frame into a dewaxing liquid 1 jar together, taking out the paraffin slices from an incubator and placing the paraffin slices at normal temperature for 5min, taking out the paraffin slices and immersing the paraffin slices into a normal temperature dewaxing liquid 2 jar, and sequentially placing the paraffin slices into the jar according to the sequence of dewaxing liquid 2, dewaxing liquid 3, absolute ethyl alcohol 1, absolute ethyl alcohol 2 and absolute ethyl alcohol 3, wherein each jar is placed for 5min when placed into a dewaxing liquid reagent jar, and each jar is placed for 3min when placed into an absolute ethyl alcohol reagent jar; Washing the slices with running water for 3min; 3) Antigen retrieval: performing high-pressure thermal restoration (high-pressure thermal restoration is to put a slice cover and a pot cover after the restoration liquid is heated to boiling, and to adjust to medium fire after the pressure pot starts to jet air, to stop fire for 2min, to cover after the pressure drops, and to cool naturally to room temperature) by using 0.01M sodium citrate restoration liquid (pH 6.0); 4) Inactivation of endogenous peroxidases: immersing and washing for 3 times by using PBS buffer solution for 1min each time, and removing the buffer solution on the slice; immersing the slices into 3% hydrogen peroxide solution completely, and incubating for 10min at room temperature; 5) Closing: immersing and washing for 3 times by using PBS buffer solution for 3min each time, and removing the buffer solution on the slice; An immunohistochemical water pen is used for delineating a tissue region to be detected on a slide, and PBS blocking solution is dripped into the delineating region; horizontally placing the slices in an incubation wet box with water at the bottom, incubating for 30min at normal temperature, and starting timing from dripping the sealing liquid; 6) Incubation resistance: removing the blocking solution, and dripping antibody diluent-PBS working solution 1 on the tissue slice: the 25 diluted anti-CD 63 rabbit monoclonal antibody is horizontally placed in an incubation wet box and incubated for 60min at normal temperature; removing antibody working solution, quickly rinsing with PBS buffer solution for 1 time, soaking and washing with PBS buffer solution for 3 times, and 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing; 7) Secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (Dako REAL EnVision Detection System, peroxidase/DAB, rabbit/Mouse, HRP; purchased from Dako company, cat# K5007) on the tissue slice, horizontally placing in an incubation wet box, and incubating for 25min at normal temperature; removing the secondary antibody working solution on the slice, quickly rinsing the slice for 1 time by using PBS buffer solution, soaking and washing the slice for 3 times by using the PBS buffer solution for 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing; 8) Color development: dropwise adding a color development liquid working solution on the slice, closely observing the color change condition under a microscope to obtain proper dyeing intensity, immersing the slice in a large amount of distilled water to terminate color development, and washing the slice in running water for 10min after the color development is terminated; 9) Counterstaining: immersing the slightly drained tissue slice into Mayer's hematoxylin for counterstaining for 1min, and then washing with running water for 3min;10 Blue-returning: immersing the slightly drained slice into a saturated aqueous solution of lithium carbonate to blu for 3s, and cleaning the slice with running water for 3min;11 Dewatering: soaking the cleaned slice in absolute ethyl alcohol for 1 time, lifting up and down for several times during the soaking period, and taking out after timing for 10 seconds; completely drying at high temperature (54-58 deg.C) in constant temperature blast drying oven; 12 Sealing plate): dripping a proper amount of neutral gum at the center of the slice, covering a cover glass, wherein the glue adding amount is proper, and the cover glass is required to cover tissues completely and cannot overflow the glue; 13 Finally, slice scanning is performed.
Immunohistochemical staining results were divided into: positive (tan staining in specific tissues and cells) and negative (no tan staining in specific tissues and cells), where positive expression requires tan staining in specific antigen sites of tissues and cells and no background staining of antigen sites (cells and tissues that should not be stained in theory) is low, i.e. no non-specific staining. For example, positive samples were highly expressed in human melanoma cells with CD63 protein in the cytoplasm and cell membrane, the corresponding sites should be tan, and the rest were not tan colored (fig. 4); in human colon tissue samples, the cytoplasm and cell membrane of monocytes and macrophages highly expressed CD63 protein, showing a tan positive staining, colon epithelial cells and other cells negative, no tan staining (fig. 5). The nuclei were blue after hematoxylin counterstain and showed a background profile of cells stained with specific immunity for determining subcellular localization of the target protein CD 63.
In this embodiment, 7 positive samples are all positively colored, 6 samples in 6 negative samples are negatively detected, the sensitivity is 13/13=100%, and the specific positive control samples are: 1/1 human lung, 1/1 human lung cancer, 1/1 human colon, 2/2 human colon cancer, 1/1 human tonsil, 1/1 human malignant melanoma scattered on lymphocyte positive staining with sensitivity of 7/7=100%; negative samples: 1/1 human esophagus, 1/1 human esophagus cancer, 1/1 human placenta, 1/1 human liver cancer, 1/1 human breast cancer, only macrophages and monocytes are positive, the rest cells are negative, the cells are consistent with theoretical localization, the cells are recorded as negative, and the specificity is 6/6=100%. Part of the positive results are shown in fig. 4-5, wherein fig. 4 shows the immunohistochemical staining results for human malignant melanoma, fig. 5 shows the immunohistochemical staining results for human colon, and part of the negative results are shown in fig. 6, which shows the immunohistochemical staining results for human breast cancer. The result shows that the anti-human CD63 rabbit monoclonal antibody prepared by the invention has accurate dyeing positioning, clear dyeing, no nonspecific dyeing and clean background.
The anti-human CD63 rabbit monoclonal antibody prepared by the invention can specifically identify human CD63 protein expressed in cells and tissues, has no non-specific binding and staining during immunodetection, can effectively avoid false positive results, and can meet the requirements of high-accuracy and high-precision detection of pathological samples.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
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| WO2011029823A1 (en) * | 2009-09-09 | 2011-03-17 | Novartis Ag | Monoclonal antibody reactive with cd63 when expressed at the surface of degranulated mast cells |
| CN116041520A (en) * | 2022-12-28 | 2023-05-02 | 武汉爱博泰克生物科技有限公司 | Rabbit monoclonal antibody aiming at Human CD63, and preparation method and application thereof |
| CN117736326A (en) * | 2023-09-27 | 2024-03-22 | 苏州志恒生物科技有限公司 | anti-CD63 single domain antibody and application thereof |
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| WO2011029823A1 (en) * | 2009-09-09 | 2011-03-17 | Novartis Ag | Monoclonal antibody reactive with cd63 when expressed at the surface of degranulated mast cells |
| CN116041520A (en) * | 2022-12-28 | 2023-05-02 | 武汉爱博泰克生物科技有限公司 | Rabbit monoclonal antibody aiming at Human CD63, and preparation method and application thereof |
| CN117736326A (en) * | 2023-09-27 | 2024-03-22 | 苏州志恒生物科技有限公司 | anti-CD63 single domain antibody and application thereof |
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| CN119613542A (en) * | 2025-02-13 | 2025-03-14 | 北京索莱宝科技有限公司 | Antibody 6G12 of human Golgi membrane protein GP73, antibody composition and application |
| CN119613542B (en) * | 2025-02-13 | 2025-05-13 | 北京索莱宝科技有限公司 | Antibody 6G12 of human Golgi membrane protein GP73, antibody composition and application |
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