CN115710315B - Recombinant antibody for resisting CA15-3 protein - Google Patents

Recombinant antibody for resisting CA15-3 protein Download PDF

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CN115710315B
CN115710315B CN202110966435.1A CN202110966435A CN115710315B CN 115710315 B CN115710315 B CN 115710315B CN 202110966435 A CN202110966435 A CN 202110966435A CN 115710315 B CN115710315 B CN 115710315B
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binding protein
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CN115710315A (en
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孟媛
钟冬梅
张嘉欣
何雯雯
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The application relates to an isolated binding protein comprising a CA15-3 protein antigen binding domain, and research on the preparation, application and the like of the binding protein. The binding protein has strong activity and high affinity with the CA15-3 protein, and can be widely applied to the detection field of the CA15-3 protein.

Description

Recombinant antibody for resisting CA15-3 protein
Technical Field
The application relates to the technical field of immunity, in particular to a recombinant antibody for resisting CA15-3.
Background
Breast Cancer (Breast Cancer) is one of the most common malignant tumors of women, constitutes a serious threat to human health and survival, and is one of the most important social problems facing countries around the world. Nowadays, breast cancer accounts for 7% -10% of the total body malignant tumors, the incidence rate of the breast cancer is in an increasing trend year by year, the incidence rate is increased by 3% every year since 1980, and the breast cancer is particularly obvious in developed countries such as North America and western Europe. Because the etiology and pathogenesis of the breast cancer are complex, the disease has the pathogenic characteristics of lower onset age, more asymptomatic early stage, relatively late disease period in diagnosis and shortened survival period caused by metastasis, and early diagnosis and treatment become the key for improving the survival rate of patients.
The method for detecting breast cancer comprises the methods of breast infrared ray, B ultrasonic, breast X-ray detection, MRI and the like, and the methods of tumor puncture biopsy, excision biopsy and the like are used for diagnosing the breast cancer. The pathology examination has a high accuracy but is often not acceptable to the patient because it is a traumatic examination. With the continuous application and popularization of clinical serum tumor markers, more and more tumor markers are gradually discovered and applied to clinical breast cancer diagnosis. Due to the noninvasive and rapid nature of serum tumor marker examination, patients are increasingly welcomed. Tumor markers are currently classified into carcinoembryonic antigen markers (CEA, AFP, etc.), carbohydrate antigens (CA 15-3, CA125, CA199, etc.), enzyme markers (PSA, NSE, etc.), hormone markers (HGH, HCG, etc.), and other protein markers (ferritin, etc.). The saccharide antigen marker is a substance secreted by tumor cells or tumor cells, and the substance is also a monoclonal antibody, so that the saccharide antigen marker is also called saccharide antigen, the content of the saccharide antigen in a normal human body is extremely low, and the abnormally-increased saccharide antigen can be detected in serum in a tumor state generally.
CA15-3 (MUC 1), a carbohydrate Antigen (CA15-3, carcinoem-bryonic anti-gen 153), is a monoclonal antibody against protein 1, belonging to the mucin family, the product encoded by the mvc-1 class of genes. The monoclonal antibody was designated CA15-3 in 1982 as a mouse monoclonal antibody (115-DB) prepared from glycoprotein MAM-6 on human milk fat globule (human milk fat globules, HMFG) membrane McAbll5D8 by Hikens et al, and as a monoclonal antibody (DF-3) prepared from liver metastasis breast cancer cell membrane fraction McAbDF3 by Kufu et al in 1984. CA15-3 is polymorphic epithelial mucin with molecular weight of 40kD, is breast cancer related antigen, is positioned on the surface of tumor cells, and when the cells are cancerous, the activities of protease and sialidase on the cell membranes are increased, and the cytoskeleton is destroyed, so that the cell antigens are withered, released into blood and the serum content is increased. CA15-3 antigen is present in malignant cells and normal epithelium of breast, lung, ovary, pancreas, so CA15-3 is elevated not only in malignant tumors such as breast cancer, ovarian cancer, lung cancer, but also to varying degrees in certain benign diseases. CA15-3 is one of the currently accepted serum markers that is better for breast cancer diagnosis, so the American clinical tumor society recommended CA15-3 as an important tumor marker for breast cancer prevention, diagnosis, treatment and follow-up since 1997.
The tumor markers have the advantages of simplicity, no wound, low cost and the like, and become the key point in the field of tumor research in recent years. The research layers of tumor marker detection methods are endless, and include a Radioimmunoassay (RIA), an enzyme-linked immunoassay (ELA), a time-resolved immunoassay (TFIA) and a chemiluminescent immunoassay (CLIA), a protein chip detection method, a gene chip detection method and the like. Among them, the detection method of CA15-3 is usually chemiluminescence immunoassay (CLIA), enzyme-linked immunoassay (ELA), etc. The chemiluminescence and enzyme-linked immunosorbent assay can realize automatic, large-batch and quantitative detection. Both of the above methods for detecting CA15-3 require a specific monoclonal antibody directed against CA15-3.
The existing CA15-3 antibodies cannot be well applied to detection of CA15-3 proteins due to low activity and poor affinity, so that there is a strong need in the art for antibodies that effectively and specifically bind to and detect CA15-3.
In view of this, the present application has been made.
Disclosure of Invention
The present application relates to a novel isolated binding protein comprising a CA15-3 antigen binding domain, and studies have been made on the preparation, use, etc. of the binding protein.
Wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of:
a complementarity determining region CDR-VH1 comprising SEQ ID NO:1 or consists of the amino acid sequence shown in 1;
a complementarity determining region CDR-VH2 comprising SEQ ID NO:2 or consists of the amino acid sequence shown in the figure;
a complementarity determining region CDR-VH3 comprising SEQ ID NO:3 or consists of the amino acid sequence shown in 3;
a complementarity determining region CDR-VL1 comprising SEQ ID NO:4 or consists of the amino acid sequence shown in figure 4;
a complementarity determining region CDR-VL2 comprising SEQ ID NO:5 or consists of the amino acid sequence shown in figure 5;
a complementarity determining region CDR-VL3 comprising SEQ ID NO:6 or consists of the amino acid sequence shown in figure 6.
The beneficial technical effects of the application include that the binding protein has strong activity and very high affinity with CA15-3.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electrophoresis chart of a monoclonal antibody against CA15-3 in example 1 of the present application.
Detailed Description
The application may be understood more readily by reference to the following detailed description of some embodiments of the application and the examples included therein.
Before the present application is further described, it is to be understood that this application is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.
Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present application are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.
In order that the application may be more readily understood, selected terms are defined below.
The term "amino acid" refers to a naturally occurring or non-naturally occurring spindle alpha-amino acid. The term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: A1a, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1N, Q), glutamic acid (G1 u, E), glycine (G1Y, G), histidine (His, H), isoleucine (I1E, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Va 1, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.
The term "isolated binding protein" is a protein that, due to its origin or source of derivation, does not bind to a naturally bound component that accompanies it in its natural state; substantially free of other proteins from the same species; expressed by cells from different species; or not present in nature. Thus, a protein that is chemically synthesized or synthesized in a cellular system other than the cell from which it naturally originates will be "isolated" from the component with which it naturally binds. Protein purification techniques well known in the art can also be used by isolation so that the protein is substantially free of substantially bound components.
The term "isolated binding protein comprising an antigen binding domain" broadly refers to all proteins/protein fragments comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies and these antibodiesAntigen compound binding fragments, including Fab, F (ab') 2 Fd, fv, scFv, bispecific antibodies and antibody minimal recognition units, and single chain derivatives of these antibodies and fragments. The type of antibody may be selected from the group consisting of IgG1, igG2, igG3, igG4, and IgA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions and CDR ranges have been precisely defined, for example, in Kabat (see sequence of immunologically important proteins (Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of health and human services (U.S. device of Health and Human Services), (1983)) and Chothia. The framework regions of antibodies, i.e., the framework regions that make up the combination of the essential light and heavy chains, function to locate and align the CDRs, which are primarily responsible for binding to the antigen.
Although the 2 domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made into a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv), "single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, in some embodiments the Fv polypeptide additionally comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The light or heavy chain variable region is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions of antibodies, i.e., the framework regions that make up the combination of the essential light and heavy chains, function to locate and align the CDRs, which are primarily responsible for binding to the antigen.
As used herein, "framework," "framework," or "FR" regions mean regions of an antibody variable domain that are excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR 1, FR2, FR3, and FR 4) separated by CDRs.
Typically, the variable regions VL/VH of the heavy and light chains are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT, chothia and Lesk numbering schemes, and the 1997 Lefranc et al, were introduced as a new standardized numbering system for all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The application adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the application.
As used herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes polypeptides or nucleic acids that are removed from their original environment, e.g., if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some protein or other cellular component that is normally associated therewith or that is normally admixed therewith or in solution. Isolated polypeptides include naturally produced said polypeptides contained in cell lysates, purified or partially purified forms of said polypeptides, recombinant polypeptides, said polypeptides expressed or secreted by cells, and said polypeptides in heterologous host cells or cultures. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
The term "affinity" as used herein refers to the equilibrium constant of reversible binding of 2 agents and is expressed as KD. The affinity of the binding protein for the ligand, such as the affinity of the antibody for the epitope, may be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomole (fM). The term "affinity" as used herein means the resistance of a complex of 2 or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art.
The term "homology" or "identity" or "similarity" as used herein refers to sequence similarity between two peptides or between two nucleic acid molecules. Each of homology and identity may be determined by comparing the positions in each sequence that are aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when an equivalent position is occupied by the same or a similar amino acid residue (e.g., similar in terms of steric and/or electronic properties), then the molecules may be said to be homologous (similar) at that position. The percent homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of additional residues also reduces identity and homology/similarity.
The present application relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of:
a complementarity determining region CDR-VH1 comprising SEQ ID NO:1 or consists of the amino acid sequence shown in 1;
a complementarity determining region CDR-VH2 comprising SEQ ID NO:2 or consists of the amino acid sequence shown in the figure;
a complementarity determining region CDR-VH3 comprising SEQ ID NO:3 or consists of the amino acid sequence shown in 3;
a complementarity determining region CDR-VL1 comprising SEQ ID NO:4 or consists of the amino acid sequence shown in figure 4;
a complementarity determining region CDR-VL2 comprising SEQ ID NO:5 or consists of the amino acid sequence shown in figure 5;
a complementarity determining region CDR-VL3 comprising SEQ ID NO:6 or consists of the amino acid sequence shown in figure 6.
In some embodiments, the binding protein comprises at least 3 CDRs (e.g., 3 light chain CDRs or 3 heavy chain CDRs).
In some embodiments, the binding protein comprises at least 6 CDRs.
In some embodiments, the binding protein is an intact antibody comprising a variable region and a constant region.
In some embodiments, the binding protein is an antibody, F (ab') 2 One of Fab', fab, fv, scFv, bispecific antibodies and antibody minimal recognition units.
In some embodiments, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 that are shown in sequence SEQ ID NO. 11-14 or have at least 90% homology thereto, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 that are shown in sequence SEQ ID NO. 7-10 or have at least 90% homology thereto.
In alternative embodiments, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 that are shown in SEQ ID NO. 11-14 in sequence or have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 that are shown in SEQ ID NO. 7-10 in sequence or have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
In some embodiments, the binding protein further comprises an antibody constant region sequence.
In some embodiments, the constant region sequence is selected from the group consisting of the sequence of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, igD.
In some embodiments, the constant region is of a species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
In some embodiments, the constant region is derived from a mouse;
the light chain constant region sequence is shown in SEQ ID NO: shown at 16;
the heavy chain constant region sequence is shown in SEQ ID NO: 15.
In another aspect, the application also provides an isolated nucleic acid molecule, which is DNA or RNA, encoding a binding protein as described above.
In this context, a nucleic acid comprises variants of its conservative substitutions (e.g., substitutions of degenerate codons) and the complementary sequence. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof, e.g., oligonucleotides.
According to an aspect of the present application, there is also provided a vector comprising the nucleic acid molecule described above.
Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" refers to a coding sequence being linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in a suitable host cell, and include promoters, enhancers and other expression control elements.
Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the application or a fragment thereof, which is capable of carrying genetic information and which may deliver the genetic information into a cell. Typical vectors include plasmids, viruses, phages, cosmids, and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, the cell may be propagated and the cell may be selected for the presence or absence of the genetic information) or an expression vector (i.e., a vector comprising the necessary genetic elements to allow expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements necessary to effect expression in the specified target cell.
The nucleic acid of the application or a fragment thereof may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid fragment of the application. Such novel vectors are also part of the present application. The vector may include a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning vectors and expression vectors of the present application are capable of spontaneous replication and thus can provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may include a promoter for driving expression of the nucleic acid fragment of the application, optionally a nucleic acid sequence encoding a signal peptide that causes secretion or integration of the peptide expression product onto a membrane, the nucleic acid fragment of the application, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a producer strain or cell line, the vector may or may not be integrated into the host cell genome when introduced into the host cell. The vector typically carries a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.
In another aspect, the application also provides a host cell transformed with a vector as described above.
The expression vectors of the application are useful for transforming host cells. Such transformed cells are also part of the application and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the application, or for recombinant production of the polypeptides of the application. Transformed cells of the application include microorganisms such as bacteria (e.g., E.coli, bacillus, etc.). Host cells also include cells derived from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably mammalian derived cells, e.g., CHO cells. The transformed cell is capable of replicating the nucleic acid fragment of the application. When the peptide combinations of the application are recombinantly produced, the expression product may be exported into culture medium or carried on the surface of the transformed cells.
According to an aspect of the present application, there is also provided a method for producing the above binding protein, comprising the steps of:
the above-described host cells are cultured in a suitable culture condition, and the produced binding protein is recovered from the culture medium or from the cultured host cells.
The method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions to express the binding protein. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The binding proteins may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.
Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the application, and these mutations may comprise deletions or insertions or substitutions, etc.
According to one aspect of the application, the application also provides the use of a binding protein as described above for the preparation of a diagnostic agent or kit for diagnosing heart failure and evaluating heart function.
According to one aspect of the present application there is also provided a method of detecting CA15-3 in a test sample comprising:
a) Contacting the CA15-3 antigen in the test sample with a binding protein as described above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and
b) Detecting the presence of the immune complex.
In the above detection method, the presence of said complex in step b) is indicative of the presence of said CA15-3 in said test sample.
In some embodiments, in step a), a second antibody is also included in the immunocomplex, the second antibody binding to the binding protein.
In some embodiments, in step a), a second antibody is also included in the immunocomplex, the second antibody binding to the CA 15-3;
in this embodiment, the binding protein forms a partner antibody in the form of a first antibody with the second antibody for binding to a different epitope of CA15-3.
In some embodiments, the binding protein may be labeled with an indicator that shows signal strength to allow the complex to be readily detected.
In some embodiments, in step a), a second antibody is also included in the immune complex, which binds to the CA15-3 antigen;
in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator that shows signal strength, so that the complex is easily detected.
In some embodiments, the indicator that displays signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.
In some embodiments, the fluorescent material comprises Alexa 350, alexa 405, alexa 430, alexa488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosin, phthalocyanine, cyanine, alternet-2-oxa-3-diazole azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyl diamine europium, europium cryptate or chelate, diamine, bis anthocyanin, la Jolla Blue dye, allophycocyanin b, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine and texas red.
In some embodiments, the radioisotope comprises 110 In、 111 In、 177 Lu、 18 F、 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc、 94 Tc、 99 mTc、 120 I、 123 I、 124 I、 125 I、 131 I、 154-158 Gd、 32 P、 11 C、 13 N、 15 O、 186 Re、 188 Re、 51 Mn、 52 mMn、 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and is provided with 83 Sr.
In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
In some embodiments, the fluorescent microsphere is: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.
According to one aspect of the application, the application also relates to a detection reagent or kit comprising a binding protein as described above.
In some embodiments, the kit or kit further comprises one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, diluent, or carrier.
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Example 1
This example provides an exemplary method of preparing a recombinant antibody against CA15-3.
1. Construction of expression plasmids
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation. The hybridoma cell strain secreting the Anti-CA 15-3F 10 monoclonal antibody is an existing hybridoma cell strain, and is recovered for later use.
1.1 preparation of Anti-CA15-3 antibody Gene
mRNA is extracted from hybridoma cell strains secreting Anti-CA15-3 monoclonal antibodies, DNA products are obtained through an RT-PCR method, rTaq DNA polymerase is used for carrying out an A adding reaction on the products, the products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain gene and the Light Chain gene are respectively taken for cloning 4 clones, and the 4 clones are sent to a gene sequencing company for sequencing.
1.2 sequence analysis of the variable region Gene of the Anti-CA 15-3F 10 antibody
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the amplified genes of the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 336bp, belongs to the VkII gene family, and the front part of the VL gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 348bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene sequence.
1.3 construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the Anti-CA 15-3F 10 antibody are designed, hindIII, ecoRI enzyme cutting sites and protective bases are respectively arranged at two ends, and a Light Chain gene fragment of 0.74KB and a Heavy Chain gene fragment of 1.4KB are amplified by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
2. Stable cell line selection
2.1 transient transfection of recombinant antibody expression plasmids into CHO cells, determination of expression plasmid Activity
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 100ul of plasmid is mixed with 700ul of cells in a centrifuge tube, transferred into an electrorotating cup, electrorotated, sampled and counted on days 3, 5 and 7, and collected and detected on day 7.
Coating liquid (mainly composed ofSplit NaHCO 3 ) Diluting sheep anti-mouse IgG 1ug/ml, coating a microplate, and performing 100 uL/well overnight at 4 ℃; the next day, the washing liquid (main component Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted cell supernatant at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing at 37deg.C for 1 hr; removing liquid in the plate, beating, adding diluted CA15-3 antigen, 100uL per hole, 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; adding HRP-labeled CA15-3 monoclonal antibody, 100uL per well at 37 ℃ for 30min; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient transformation of the plasmid were active against the CA15-3 antigen.
Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCl); stop solution (EDTA.2Na+ concentrated H) 2 SO 4 )
2.2 linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50ul, DNA 100 ug/tube, pvuI enzyme 10ul, sterile water to 500ul, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
2.3 stable transfection of recombinant antibody expression plasmid, pressure screening of stable cell lines
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100ul of plasmid with 700ul of cells, transferring into an electric rotating cup, carrying out electric rotation, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Microscope lower viewExamining the clone holes marked with cells and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 6 Performing seed preservation by using cells/ml and 2 ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3. Recombinant antibody production
3.1 cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
3.2 shaking flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 4. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of external control antibody was used as a control, and the electrophoresis pattern was shown in FIG. 1. Two bands are shown after reducing SDS-PAGE, 1 Mr is 50KD (heavy chain), and the sequence is shown as SEQ ID NO. 18; the other Mr is 28KD (light chain), and the sequence is shown as SEQ ID NO. 20.
Example 2
Antibody affinity assay and Activity characterization
The resulting antibody of example 1, anti-CA 15-3F 10 (having the heavy and light chain variable regions shown in SEQ ID NO:17 and SEQ ID NO: 19)
The complementarity determining regions of the heavy chain were analyzed:
CDR-VH1, SEQ ID NO:1, and a polypeptide sequence shown in the specification;
CDR-VH2, SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);
CDR-VH3, SEQ ID NO:3, an amino acid sequence shown in 3;
complementarity determining regions of the light chain:
CDR-VL1, SEQ ID NO:4, and a polypeptide sequence shown in the figure;
CDR-VL2, SEQ ID NO:5, and a polypeptide sequence shown in the figure;
CDR-VL3, SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.
The sequences of the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 are shown as SEQ ID NO. 11-14 in sequence;
the sequences of the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 are shown as SEQ ID NO. 7-10.
1. Affinity analysis
The purified antibody is diluted to 10ug/ml by PBST by using an AMC sensor, and the CA15-3 antigen is subjected to gradient dilution by using the PBST;
the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate.) Table 1 shows the obtained affinity detection data.
Table 1 affinity assay data
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control 8.45E-08 4.44E+04 3.75E-03
Anti-CA15-3 3F10 5.17E-09 3.11E+04 1.61E-04
2. Activity assay
Coating liquid (main component NaHCO) 3 ) Diluting sheep anti-mouse IgG 1ug/ml, coating a microplate, and performing 100 uL/well overnight at 4 ℃; the next day, the washing liquid (main component Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted purified antibody at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing at 37deg.C for 1 hr; removing liquid in the plate, beating, adding diluted CA15-3 antigen, 100uL per hole, 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; the HRP-labeled CA15-3 monoclonal antibody (available from the Figpeng organism paired with purified antibody) was added at 100uL per well at 37℃for 30min; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; OD values read at 450nm (reference 630 nm) on a microplate reader, and the obtained antibody activity analysis data are shown in Table 2.
Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCl); stop solution (EDTA.2Na+ concentrated H) 2 SO 4 )
TABLE 2 data from antibody Activity analysis
3. Stability analysis
Bare stability assessment:
the self-produced antibody is placed at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, 7 days, 14 days and 21 days are taken for state observation, and activity detection is carried out on the 21 days, so that the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, the activity is not in a descending trend along with the increase of the examination temperature, and the stability of the self-produced antibody is indicated. Table 3 shows the results of the detection of OD after 21 days of enzyme-free activity.
TABLE 3 antibody stability test data
Sample concentration (ng/ml) 125 31.25 0
4 ℃,21 days sample 1.887 0.968 0.021
Sample at-80℃for 21 days 1.901 0.999 0.041
37 ℃ and 21 days of sample 1.925 0.955 0.038
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Sequence listing
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> a recombinant antibody against CA15-3 protein
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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
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Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
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Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
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Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
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Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val
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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
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Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
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Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
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Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
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Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Asn Tyr
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Pro Met Ser Trp Val Arg Gln Thr Pro Glu Met Arg Leu Glu Trp Val
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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ala Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Phe Trp Phe Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
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Thr Val Ser Ser
115
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Asn Tyr
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35 40 45
Ala Tyr Ile Ser Ser Gly Gly Asp Ser Thr Tyr Tyr Pro Gly Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ala Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Phe Trp Phe Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala
115 120 125
Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu
130 135 140
Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
145 150 155 160
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp
165 170 175
Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro
180 185 190
Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys
195 200 205
Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile
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Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro
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Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val
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Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val
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Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln
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Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln
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Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala
305 310 315 320
Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro
325 330 335
Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala
340 345 350
Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu
355 360 365
Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr
370 375 380
Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr
385 390 395 400
Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe
405 410 415
Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys
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Ser Leu Ser His Ser Pro Gly
435
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
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145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
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Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (23)

1. An isolated binding protein comprising a CA15-3 antigen binding domain, wherein said antigen binding domain comprises a complementarity determining region of the amino acid sequence:
the amino acid sequence of the CDR-VH1 of the complementarity determining region is shown in SEQ ID NO:1 is shown in the specification;
the amino acid sequence of the complementarity determining region CDR-VH2 is shown in SEQ ID NO:2 is shown in the figure;
the amino acid sequence of the CDR-VH3 of the complementarity determining region is shown in SEQ ID NO:3 is shown in the figure;
the amino acid sequence of CDR-VL1 of the complementarity determining region is shown in SEQ ID NO:4 is shown in the figure;
the amino acid sequence of CDR-VL2 of the complementarity determining region is shown in SEQ ID NO:5 is shown in the figure;
the amino acid sequence of CDR-VL3 of the complementarity determining region is shown in SEQ ID NO: shown at 6.
2. The isolated binding protein comprising an antigen binding domain of claim 1, wherein the binding protein is an antibody.
3. The isolated binding protein comprising an antigen binding domain of claim 1, wherein the binding protein is F (ab') 2 Fab', fab, fv, scFv, bispecific antibody.
4. The isolated binding protein comprising an antigen binding domain according to claim 2, wherein the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having the sequence shown in SEQ ID NOs 11-14 in sequence or at least 90% homology thereto, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having the sequence shown in SEQ ID NOs 7-10 in sequence or at least 90% homology thereto.
5. An isolated binding protein comprising a CA15-3 antigen binding domain, wherein said binding protein has a heavy chain variable region of sequence set forth in SEQ ID No. 17 and a light chain variable region set forth in SEQ ID No. 19.
6. The isolated binding protein comprising an antigen binding domain of any one of claims 1-5, wherein the binding protein further comprises an antibody constant region sequence.
7. The isolated binding protein comprising an antigen binding domain of claim 6, wherein the constant region sequence is selected from the group consisting of the sequence of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, igD.
8. The isolated binding protein comprising an antigen binding domain of claim 6, wherein the constant region is of a species source of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
9. The isolated binding protein comprising an antigen binding domain of claim 8, wherein the cow is a dairy cow.
10. The isolated binding protein comprising an antigen binding domain of claim 8, wherein the constant region is of mouse origin;
the light chain constant region sequence is shown in SEQ ID NO: shown at 16;
the heavy chain constant region sequence is shown in SEQ ID NO: 15.
11. An isolated nucleic acid molecule, wherein the nucleic acid molecule is DNA or RNA encoding the binding protein of any one of claims 1 to 10.
12. A vector comprising the nucleic acid molecule of claim 11, which is DNA.
13. A host cell transformed with the vector of claim 12.
14. The host cell of claim 13, wherein the host cell is a mammalian cell.
15. A method of producing a binding protein according to any one of claims 1 to 10, comprising the steps of: culturing the host cell of claim 13 or 14 under suitable culture conditions and recovering the produced binding protein from the culture medium or from the cultured host cell.
16. Use of a binding protein according to any one of claims 1 to 10 in the manufacture of a diagnostic agent or kit for the diagnosis of breast, lung, ovarian, lung adenocarcinoma or colorectal cancer.
17. A method of detecting CA15-3 in a test sample, comprising:
a) Contacting the CA15-3 antigen in the test sample with the binding protein of any one of claims 1-10 under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and
b) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the CA15-3 in the test sample;
the method is not directed to the diagnosis or treatment of a disease.
18. The method of claim 17, wherein in step a) the immunocomplex further comprises a second antibody, the second antibody binding to the binding protein.
19. The method of claim 17, wherein in step a) the immune complex further comprises a second antibody, which binds to the CA15-3.
20. A reagent or kit comprising the binding protein of any one of claims 1 to 10.
21. The reagent or kit of claim 20, further comprising a pharmaceutically acceptable excipient.
22. The reagent or kit of claim 20, wherein the reagent or kit further comprises: one or more of a buffer, a stabilizer, and a diluent.
23. The reagent or kit of claim 20, further comprising a pharmaceutically acceptable carrier.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008014458A2 (en) * 2006-07-27 2008-01-31 Ludwig Institute For Cancer Research Protein markers for the diagnosis and prognosis of ovarian and breast cancer
CN110818796A (en) * 2018-08-09 2020-02-21 东莞市朋志生物科技有限公司 Recombinant antibody of anti-human CA153 protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008014458A2 (en) * 2006-07-27 2008-01-31 Ludwig Institute For Cancer Research Protein markers for the diagnosis and prognosis of ovarian and breast cancer
CN110818796A (en) * 2018-08-09 2020-02-21 东莞市朋志生物科技有限公司 Recombinant antibody of anti-human CA153 protein

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