CN112920275A

CN112920275A - Binding proteins, reagents and kits that specifically bind to sST2

Info

Publication number: CN112920275A
Application number: CN201911240782.5A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 陈小云; 覃婷
Original assignee: Fapon Biotech Inc
Current assignee: Fapon Biotech Inc
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2021-06-08
Anticipated expiration: 2039-12-06
Also published as: CN112920275B

Abstract

The invention discloses a binding protein, a reagent and a kit capable of specifically binding sST2, relating to the technical field of antibodies, wherein the binding protein comprises an antigen binding structural domain; the antigen binding domain includes at least one of the following complementarity determining regions: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL 3; the binding protein can be specifically bound with sST2, has better binding activity and affinity, can be used for detecting sST2 and diagnosing related diseases with sST2 as a marker, is favorable for improving the specificity and sensitivity of detection, and provides multiple protein selections for detection of sST 2.

Description

Binding proteins, reagents and kits that specifically bind to sST2

Technical Field

The invention relates to the technical field of antibodies, in particular to a binding protein, a reagent and a kit capable of specifically binding sST 2.

Background

Growth-stimulating expression of gene 2 protein (ST2) is one of the members of the interleukin 1(IL-1) receptor superfamily, and ST2 was first discovered in mouse fibroblasts by Tominaga et al in 1987; richard Lee, a female hospital affiliated with Harvard university Hospital, 2002, reported that myocardial stress and myocardial injury cause ST2 expression; in 2005, researchers such as Schmitz found that the ST2 gene not only participates in inflammatory reaction and pathological process of immune diseases, but also found that a specific functional ligand IL-33 of the ST2 gene; the us ACC/AHA heart failure guideline of 2013 and the heart failure diagnostic and therapeutic guideline of 2014 china accumulated the recommendation of the introduction of sST2 (soluble ST2) into myocardial fibrosis, a biomarker of heart failure, according to ST2 clinical study data.

The growth-stimulating expression of the gene 2 protein (ST2), also known as T1, IL1RL1 or Fit1, which is a gene located on human chromosome 2q12, has about 40KD, and the transcript of the ST2 gene has 4 subtypes, of which two are most important because of being regulated by different promoters, the first is transmembrane ST2(ST2L), which is composed of 3 extracellular immunoglobulin G domains, one transmembrane domain and one intracellular domain, and is a member of the membrane receptor of the interleukin-1 receptor family; the other is soluble ST2(sST2), sST2, which lacks transmembrane and intracellular domains and therefore flows freely in the blood, making it detectable in serum.

Research on growth stimulation expression gene 2 protein (ST2) shows that the protein is related to cardiac dysfunction, ST2L receptor has heart protection effect, and after being combined with specific ligand IL-33, the protein can reduce fibrosis and hypertrophic burden structure of cardiac myocytes, reduce myocardial damage degree after myocardial infarction, and relieve pathological symptoms such as myocardial apoptosis, inflammatory myocardial activation, myocardial remodeling and the like. Kakkar et al found that mechanical stretching of living cells could enhance IL-33 release from cytoplasmic vesicles, could induce cardiac remodeling of IL-33 due to volume or pressure overload, vasopressin II could cause damage to the heart by producing multiple Reactive Oxygen Species (ROS), while IL-33 exerts an anti-myocardial hypertrophy effect by blocking the effect of vasopressin II or phenylephrine on the myocardium, and ST2L in combination with IL33 activated nuclear transcription factor NF-kB and mitogen activated protein kinase MAPK pathway, resulting in the release of Th2 type cytokines IL-4, IL-5 from target cells, which could exert anti-myocardial hypertrophy myocardial fibrosis effect, thereby initiating repair protection. It has also been found that in vitro administration of IL-33 enhances the activation of ST2L and its own intracellular biochemical pathways, as well as reduces the ROS production induced by angiotensin II, thereby preventing or at least reducing the worsening of the clinical and structural state of the heart;

the sST2 is used as a decoy receptor of IL-33, is a marker of cardiovascular diseases, blocks a 1L-33/ST2L signal channel after being combined with the IL-33, thereby reducing the effectiveness of the IL-33 and a myomembrane receptor to interact, and in a cardiac muscle cell with heavy mechanical load, the sST2 is obviously induced, so that the heart failure or the worsening of the previous chronic heart failure state and myocardial infarction induced scar can not stretch the cardiac muscle well, and the conditions can be detected by measuring the blood sST2 level. The Weinberg et al study showed that myocardial infarction caused an increase in plasma levels of sST 2. Thus, an increase in sST2 may reduce the protective effect of IL-33 on cardiac myocytes and may have a negative impact on the overall cardiovascular risk profile.

Cardiovascular diseases (CVD) are the leading causes of death worldwide, Brain Natriuretic Peptide (BNP) and brain natriuretic peptide precursor NT-proBNP are the best known markers of Heart Failure (HF), and troponin markers improve the diagnosis of acute and chronic coronary artery diseases, however, a single biomarker not only reflects one side of the pathological mechanism of heart failure, but also is affected by pulmonary hypertension caused by kidney function, age, BMI, etc., and the accuracy of these biomarkers is reduced. With the increased awareness of the effects of sST2 and ST2L on the cardiovascular system, the assessment of plasma sST2 levels has begun to be considered as a new marker of cardiovascular events, in particular indices and clinical conditions associated with heart failure and ischemic heart disease, and sST2 is not affected by indices of renal function, age, body weight, etc. sST2 is also considered as a possible biomarker for acute heart failure and accompanying asthmatic patients. The research also proves the role of sST2 in predicting mortality, sST2 can be combined with other biomarkers to be used as a prognostic biomarker, and sST2 can be used for monitoring the pharmacological response of heart failure, has a relevant relation with the recommended treatment drugs of the heart failure, and can provide reference for clinical selection of the treatment drugs and schemes. At present, the main domestic methods for detecting ST2 include enzyme-linked immunosorbent assay (ELISA), magnetic particle chemiluminescence assay, gold labeling method and the like; different detection methods all require a specific monoclonal antibody directed against ST 2.

There are some defects in sensitivity, specificity and affinity of the monoclonal antibody currently used for detecting sST2, and there is a large room for improvement, and therefore, there is still a strong need in the art for monoclonal antibodies for detecting sST 2.

Disclosure of Invention

The invention aims to provide a binding protein, a reagent and a kit which can specifically bind to sST 2. The binding protein provided by the invention can be specifically bound with sST2, has better binding activity and affinity, can be used for detection of sST2 and diagnosis of related diseases with sST2 as a marker, is favorable for improving the specificity and sensitivity of detection, and provides multiple protein selections for detection of sST 2.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies, and "antibody functional fragments" include antigen-compound-binding fragments of these antibodies, including Fab, F (ab') 2, Fd, Fv, scFv, diabodies, and minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, 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 (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The "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 the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework region" or "FR" region means the region of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the terms "isolated" or "purified" mean that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention:

in a first aspect, embodiments of the invention provide a binding protein that specifically binds sST2, the binding protein comprising an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

a complementarity determining region CDR-VH1 having the amino acid sequence G-Y-X1-F-X2-S-W-M-X3, wherein: x1 is A or T, X2 is S or T, X3 is Q, H or N;

a complementarity determining region CDR-VH2 having the amino acid sequence R-I-X1-P-G-D-G-X2-T-N-Y-X3-G-N-F-K-G, wherein: x1 is H or Y, X2 is D or N, X3 is D or N;

a complementarity determining region CDR-VH3 having the amino acid sequence a-X1-E-R-F-X2-T-X3-V-D-a-M, wherein: x1 is S or T, X2 is I, V or L, X3 is I or L;

A complementarity determining region CDR-VL1 having the amino acid sequence R- A-S-X1-D-X2-S-N-Y-X3-N, wherein: x1 is Q or N, X2 is I, V or L, X3 is I, V or L;

a complementarity determining region CDR-VL2 having the amino acid sequence Y-X1-S-R-X2-H-S, wherein: x1 is T or S, X2 is I, V or L;

a complementarity determining region CDR-VL3 having the amino acid sequence Q-S-X1-E-X2-P-X3-T, wherein: x1 is I, V or L, X2 is D or H, and X3 is I, V or L.

The binding protein provided by the embodiment of the invention contains an antigen binding domain, the antigen binding domain comprises at least one of the complementarity determining regions, the amino acid sequence of the complementarity determining region is found and disclosed for the first time, the binding protein is a novel sequence, the binding protein can be endowed with the capability of specifically binding to the sST2 antigen, the binding protein has better binding activity and affinity, the detection of the sST2 by using the binding protein provided by the invention is favorable for improving the sensitivity and specificity of the detection of the sST2, the binding protein can be used for diagnosing related diseases taking the sST2 as a marker, such as cardiovascular diseases, and the invention provides more protein choices for the detection of the sST2 and the diagnosis of the related diseases taking the sST2 as the marker.

In alternative embodiments, in the complementarity determining region CDR-VH1, X1 is a; in the complementarity determining region CDR-VH2, X1 is Y; in the complementarity determining region CDR-VH3, X1 is S; in the complementarity determining region CDR-VL1, X1 is Q; in the complementarity determining region CDR-VL2, X1 is T; in the complementarity determining region CDR-VL3, X2 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is S.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X2 is T.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is Q.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is H.

In an alternative embodiment, in the complementarity determining region CDR-VH1, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X3 is D.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X3 is N.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X3 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X3 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X1 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X1 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X1 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is V.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X3 is L.

In alternative embodiments, the similar complementarity determining regions have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the complementarity determining regions described above.

In alternative embodiments, the antigen binding domain has a K with the sST2 protein_D≤9.88×10^-7Affinity of mol/L.

In alternative embodiments, the antigen binding domain has a K with the sST2 protein_D≤9×10^-7mol/L、8×10^-7mol/L、7×10^-7mol/L、6×10^-7mol/L、5×10^-7mol/L、4×10^-7mol/L、3×10^-7mol/L、2×10^-7mol/L、1×10^-7mol/L、9×10^-8mol/L、8×10^-8mol/L、7×10^-8mol/L、6×10^-8mol/L、5×10^- ⁸mol/L、4×10^-8mol/L、3×10^-8mol/L、2×10^-8mol/L、1×10^-8mol/L or 9X 10^-9Affinity of mol/L.

In an alternative embodiment, the antigen binding domain has 9.07 x 10 to sST2 protein^-9mol/L≤K_D≤9.88×10^-7Affinity of mol/L.

K_DThe detection of (2) is carried out with reference to the method in the examples of the present invention.

In an alternative embodiment, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 1-52:

in alternative embodiments, in the complementarity determining region CDR-VH1, X1 is T; in the CDR-VH2, X1 is H; in the complementarity determining region CDR-VH3, X1 is T; in the complementarity determining region CDR-VL1, X1 is N; in the complementarity determining region CDR-VL2, X1 is S; in the complementarity determining region CDR-VL3, X2 is H.

In alternative embodiments, the mutation site (i.e., Xn site, n ═ 1, 2, 3, or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 53-58:

in alternative embodiments, the binding protein includes at least 3 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain, or3 complementarity determining regions of a light chain); alternatively, the binding protein comprises at least 6 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain);

in alternative embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In alternative embodiments, the binding protein is a functional fragment of an antibody, such as any one of a nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. As will be readily understood by those skilled in the art based on the teachings of the present invention, functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In an alternative embodiment, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID Nos. 1-4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID Nos. 5-8.

In addition, based on the disclosure of the present invention, the species source of the heavy chain or light chain framework region of the binding protein may be human, so as to constitute a humanized antibody.

In alternative embodiments, the binding protein further comprises an antibody constant region.

In alternative embodiments, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the antibody constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the antibody constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 10.

The sequences of SEQ ID NOS: 1-10 are shown in the following table:

in a second aspect, the present embodiments provide the use of a binding protein according to any one of the preceding embodiments in a cardiovascular disease diagnostic reagent or kit.

In alternative embodiments, the cardiovascular disease is selected from one or more of heart failure and ischemic heart disease.

It should be noted that the binding protein provided by the present invention can detect sST2, and therefore, as long as the binding protein is any disease with sST2 as a marker, the binding protein provided by the present invention can be used for preparing diagnosis or prognosis evaluation of the disease, and the binding protein provided by the present invention is within the protection scope of the present invention.

In a third aspect, the embodiments of the present invention provide a cardiovascular disease diagnostic reagent or kit, which contains the binding protein as described above.

In a fourth aspect, an embodiment of the present invention provides a method for detecting sST2, including: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above detection method is aimed at the diagnosis of non-diseases.

It should be noted that, those skilled in the art can perform qualitative or quantitative detection of sST2 protein in a test sample based on the characteristics of immune complex formed by antibody/antigen binding. The method for detecting an antigen or an antibody based on the formation of an immune complex upon binding of the antibody to the antigen comprises:

(1) the detection purpose is realized by a precipitation reaction, which comprises the following steps: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoblotting, and the like;

(2) the detection purpose is realized by marking an indicator for displaying the signal intensity, and the method comprises the following steps: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay (e.g., double antibody sandwich, indirect or competitive assay), and chemiluminescence immunoassay.

The indicator may be selected appropriately according to different detection methods, including but not limited to the indicators described below:

(1) in the immunofluorescence method, the indicator may be a fluorescent dye, for example, a fluorescein dye (fluorescein isothiocyanate (FITC), hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or an analog thereof), a rhodamine dye (including red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine b (tritc), etc. or an analog thereof), a Cy series dye (including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or an analog thereof), an Alexa series dye (including Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or an analog thereof), a protein dye (including Phycoerythrin (PE), Phycocyanin (PC), allophycocyanin (phycocyanin), polymetaxanthin-chlorophyll protein (preCP), etc.);

(2) in radioimmunoassays, the indicator may be a radioisotope, for example: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, 18F, and the like.

(3) In enzyme-linked immunoassays, the indicator may be an enzyme that catalyzes the development of a substrate (e.g., horseradish peroxidase, alkaline phosphatase, or glucose oxidase, etc.).

(4) In chemiluminescent immunoassays, the indicator may be a chemiluminescent reagent such as acridinium ester, horseradish peroxidase and luminol, alkaline phosphatase and AMPPD, electrochemiluminescent agents ruthenium and tripropylamine, and the like.

Based on the disclosure of the above binding protein, those skilled in the art can easily think of using any one of the above methods or a combination of several methods or other methods to achieve quantitative or qualitative detection of sST2 in a sample to be detected, and it is within the scope of the present invention to use the binding protein disclosed in the present invention to detect sST2 by any method.

In alternative embodiments, the binding protein is labeled with an indicator that indicates the intensity of the signal, such that a complex in which the binding protein binds to sST2 protein is detected.

In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding a binding protein according to any one of the preceding embodiments; in alternative embodiments, the nucleic acid is DNA or RNA.

Based on the disclosure of the amino acid sequence of the binding protein, one skilled in the art can easily obtain the nucleic acid sequence encoding the binding protein according to the codon corresponding to the amino acid, and obtain various nucleic acid sequences encoding the binding protein according to the degeneracy of the codon, which are within the protection scope of the present invention as long as they encode the binding protein.

Embodiments of the invention provide a hybridoma cell expressing a binding protein according to any one of the preceding embodiments;

in a sixth aspect, embodiments of the invention provide a vector comprising a nucleic acid according to the previous embodiments.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the nucleic acid sequence is linked to the regulatory sequence in a manner that allows expression. Regulatory sequences, including promoters, enhancers and other expression control elements, are selected to direct the expression of the protein of interest in a suitable host cell.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, which may be propagated and in which the genetic information may be present or absent) or an expression vector (i.e., a vector which comprises the necessary genetic elements to permit expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the invention are capable of autonomous replication and thus provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a signal peptide sequence encoding for secretion or integration of the protein expression product into a membrane, and optionally a nucleic acid sequence encoding for a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In a seventh aspect, embodiments of the present invention provide a host cell comprising a vector according to the previous embodiments.

The expression vectors of the invention are useful for transforming host cells. Such transformed host cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the binding proteins of the invention. Host cells of the present invention include microorganisms such as bacteria (e.g., Escherichia coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells. In an eighth aspect, embodiments of the invention provide a method of producing a binding protein of any one of the preceding embodiments, comprising:

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production 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 such that the binding protein is expressed. 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 bound protein 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 suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows a reduced SDS-PAGE of the monoclonal antibody to sST2 from example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a laboratory Manual (Molecular Cloning: available Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of activities or events, as some activities may occur in different orders and/or concurrently with other activities or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

This example provides a method for the preparation of recombinant antibodies against sST2

1 construction of recombinant plasmid

(1) Primer and method for producing the same

Amplification of Heavy Chain and Light Chain 5' RACE primers:

(2) antibody variable region gene cloning and sequencing

RNA extracted from a hybridoma cell line secreting a monoclonal antibody against sST2 is subjected to first strand cDNA synthesis by using a SMARTERTM RACE cDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and an obtained first strand cDNA product is used as a PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIgG CHR primers. The primer pair of Light Chain can amplify a target band about 0.8KB, and the primer pair of Heavy Chain can amplify a target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH 5. alpha. competent cells, and after colonies were grown, the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen for sequencing.

(3) Sequence analysis of variable region Gene of monoclonal antibody against sST2

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by 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 324bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 366bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(4) Construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the anti-sST 2 antibody are designed, wherein two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and the primers are as follows:

a0.75 KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Diluting plasmid with ultrapure water to 400ng/ml, adjustingNode CHO cell 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

Diluting goat anti-mouse IgG1 mu g/ml with the coating solution to coat the microplate, wherein each well is 100 mu l, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60 min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting to dry, adding diluted ST2 protein, 100 mul per hole, 37 ℃, 40 min; washing with washing solution for 5 times, and drying; adding HRP-labeled ST2 monoclonal antibody 100 μ l per well, 37 deg.C, 30 min; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results showed that the OD of the reaction after the dilution of the cell supernatant by 1000 times was still greater than 1.0, and that the OD of the reaction in the wells without the addition of the cell supernatant was less than 0.1, indicating that the antibodies produced after the transient transformation of the plasmid were both active against the ST2 protein.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme, and sterile water to 500 mul, water bath enzyme digestion overnight at 37 ℃; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Microscopic observation of the cell-marked clonal wells and recording of confluencyDegree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody (i.e., sST2 monoclonal antibody) was subjected to reducing SDS-PAGE, and the electrophoretogram is shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (i.e., heavy chain, SEQ ID NO:14) and 28kD of Mr (i.e., light chain, SEQ ID NO: 12).

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Further analysis revealed that the heavy chain variable region of the sST2 monoclonal antibody (WT) of example 1 is shown in SEQ ID NO:13, wherein the amino acid sequences of the complementarity determining regions of the heavy chain are as follows:

CDR-VH1：G-Y-T(X1)-F-T(X2)-S-S-W-M-H(X3)；

CDR-VH2：R-I-H(X1)-P-G-D-G-N(X2)-T-N-Y-N(X3)-G-N-F-K-G；

CDR-VH3：A-T(X1)-E-R-F-V(X2)-T-T-I(X3)-V-D-A-M；

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：R-A-S-N(X1)-D-V(X2)-S-N-Y-L(X3)-N；

CDR-VL2：Y-S(X1)-S-R-I(X2)-H-S；

CDR-VL3：Q-Q-S-I(X1)-E-H(X2)-P-I(X3)-T。

on the basis of the sST2 monoclonal antibody of example 1, mutations were made in the complementarity determining regions at sites relevant for antibody activity, wherein X1, X2, X3, and X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

And (3) detecting the binding activity: coating solution (PBS) dilutes goat anti-mouse IgG1 mug/ml for coating a microplate, each well is 100 mug, and the temperature is kept overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1 hr, and adding blocking solution (120 μ l per well); adding diluted ST2 monoclonal antibody, 100 μ l/well, 37 deg.C, 60 min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off liquid in the plate, patting dry, adding diluted sST2 protein, 100 mul per hole, 37 ℃, 40 min; washing with washing solution (PBS) for 5 times, and drying; adding another sST2 monoclonal antibody marked with HRP, wherein each hole is 100 mu l, 37 ℃ and 30 min; adding color development liquid A (50 μ L/well containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide) and adding color development liquid B (50 μ L/well containing 1.05g/L citric acid, 0.186g/L LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10 min; add stop solution (50. mu.l/well, 0.75 g/EDTA-2 Na and10.2ml/L concentrated H₂SO₄) (ii) a OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2.

TABLE 2 Activity data of antibodies and mutants thereof

As can be seen from the data in Table 2, the binding of WT and mutations 1-4 to antigen was detected at different concentrations, indicating that the binding activity was better, indicating that the amino acid mutation pattern at the mutation sites listed in Table 1 was unpredictable.

(2) Example 1 affinity assays for antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

And (3) affinity detection:

using AMC sensors, purified antibodies (each of the mutant antibodies in table 3) were diluted with PBST at a 2-fold gradient starting at 50 μ g/ml and sST2 protein was diluted with PBST at a gradient;

the operation flow is as follows: equilibrating in buffer 1(PBST) for 60s, immobilizing antibody in antibody solution for 300s, incubating in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociating in buffer 2 for 1200s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data. (KD represents the equilibrium dissociation constant, i.e.affinity; kon represents the association rate; kdis represents the dissociation rate.)

Table 4 affinity assay data

As can be seen from the data in Table 4, K for mutation 1 and from mutation 1-1 to mutation 1-51_DThe lower value indicates that the antibodies all have better affinity, and also indicates that the mutation sites and the mutation modes listed in the table 3 have no negative influence on the affinity of the antibodies, and the better affinity can be obtained after mutation according to the mutation modes in the table 3. Correspondingly, the kit has higher specificity and sensitivity when being applied to a cardiovascular disease diagnostic kit.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 results of affinity assay of mutations with WT as backbone

	K_D(M)	kon(1/Ms)	kdis(1/s)
				WT	7.06E-07	1.84E+03	1.30E-03
WT 1-1	9.88E-07	2.53E+03	2.50E-03
				WT 1-2	2.74E-07	3.65E+03	1.00E-03
WT 1-3	1.81E-07	4.52E+03	8.20E-04
				WT 1-4	1.72E-07	5.54E+03	9.52E-04
WT 1-5	2.13E-07	3.43E+03	7.31E-04

As seen from Table 6, WT1-1 to WT1-6 all had better affinity, indicating that the mutation pattern of the mutation sites listed in Table 5 has little effect on the affinity of the antibody, and better affinity can be obtained by mutation.

(3) Evaluation of stability against naked antibody

Placing the antibodies in the table at 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples at 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples at 21 days, wherein the result shows that no obvious protein state change is seen in 21 days of placing the antibodies under three examination conditions, and the activity is more prone to decrease with the increase of the examination temperature, which indicates that the self-produced antibodies are stable. The following table 7 shows the OD results of the enzyme immunity activity assay for 21 days of the mutation 1 antibody test.

TABLE 7

Antibody concentration (ng/ml)	31.25	3.906	0
				Samples at 4 ℃ for 21 days	2.215	0.846	0.062
21 days samples at-80 deg.C	2.294	0.892	0.094
				21 day samples at 37 deg.C	2.206	0.855	0.087

As can be seen from Table 7, after the antibody provided by the embodiment of the invention is stored for 21 days at different temperatures, the antigen can still be detected by the antibody provided by the embodiment of the invention, the change of the binding activity is small, and the antibody provided by the embodiment of the invention has better stability.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Fenpeng biological products Ltd

<120> binding proteins, reagents and kits that specifically bind to sST2

<160> 14

<170> PatentIn version 3.5

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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

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Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile Gly

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Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

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Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

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Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

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Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

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Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

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Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

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His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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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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Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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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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Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

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Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

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Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val

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Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

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Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

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Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ile Glu His Pro Ile

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

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Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

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Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

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Phe Asn Arg Asn Glu Cys

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

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Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

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Gly Arg Ile His Pro Gly Asp Gly Asn Thr Asn Tyr Asn Gly Asn Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Thr Glu Arg Phe Val Thr Thr Ile Val Asp Ala Met Asp Tyr Trp

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Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

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Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

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Gly Arg Ile His Pro Gly Asp Gly Asn Thr Asn Tyr Asn Gly Asn Phe

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Thr Glu Arg Phe Val Thr Thr Ile Val Asp Ala Met Asp Tyr Trp

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Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

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Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met

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Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

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Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

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Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

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Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His

195 200 205

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

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Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe

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Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

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Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

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Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

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Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

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Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

305 310 315 320

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

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Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

340 345 350

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

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Thr Asn Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

370 375 380

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

385 390 395 400

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

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Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

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Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

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Claims

1. A binding protein that specifically binds sST2, wherein the binding protein comprises an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

2. The binding protein of claim 1,

in the CDR-VH1, X1 is A;

in the complementarity determining region CDR-VH2, X1 is Y;

in the complementarity determining region CDR-VH3, X1 is S;

in the complementarity determining region CDR-VL1, X1 is Q;

in the complementarity determining region CDR-VL2, X1 is T;

in the complementarity determining region CDR-VL3, X2 is D;

preferably, in the complementarity determining region CDR-VH1, X2 is S;

preferably, in the complementarity determining region CDR-VH1, X2 is T;

preferably, in the complementarity determining region CDR-VH1, X3 is Q;

preferably, in the complementarity determining region CDR-VH1, X3 is H;

preferably, in the complementarity determining region CDR-VH1, X3 is N;

preferably, in the complementarity determining region CDR-VH2, X2 is D;

preferably, in the complementarity determining region CDR-VH2, X2 is N;

preferably, in the complementarity determining region CDR-VH2, X3 is D;

preferably, in the complementarity determining region CDR-VH2, X3 is N;

preferably, in the complementarity determining region CDR-VH3, X2 is I;

preferably, in the complementarity determining region CDR-VH3, X2 is V;

preferably, in the complementarity determining region CDR-VH3, X2 is L;

preferably, in the complementarity determining region CDR-VH3, X3 is I;

preferably, in the complementarity determining region CDR-VH3, X3 is L;

preferably, in the complementarity determining region CDR-VL1, X2 is I;

preferably, in the complementarity determining region CDR-VL1, X2 is V;

preferably, in the complementarity determining region CDR-VL1, X2 is L;

preferably, in the complementarity determining region CDR-VL1, X3 is I;

preferably, in the complementarity determining region CDR-VL1, X3 is V;

preferably, in the complementarity determining region CDR-VL1, X3 is L;

preferably, in the complementarity determining region CDR-VL2, X2 is I;

preferably, in the complementarity determining region CDR-VL2, X2 is V;

preferably, in the complementarity determining region CDR-VL2, X2 is L;

preferably, in the complementarity determining region CDR-VL3, X1 is I;

preferably, in the complementarity determining region CDR-VL3, X1 is V;

preferably, in the complementarity determining region CDR-VL3, X1 is L;

preferably, in the complementarity determining region CDR-VL3, X3 is I;

preferably, in the complementarity determining region CDR-VL3, X3 is V;

preferably, in the complementarity determining region CDR-VL3, X3 is L;

preferably, the antigen binding domain has a K with sST2 protein_D≤9.88×10^-7Affinity of mol/L;

preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following mutation combinations 1-52:

3. the binding protein of claim 1,

in the complementarity determining region CDR-VH1, X1 is T;

in the CDR-VH2, X1 is H;

in the complementarity determining region CDR-VH3, X1 is T;

in the complementarity determining region CDR-VL1, X1 is N;

in the complementarity determining region CDR-VL2, X1 is S;

in the complementarity determining region CDR-VL3, X2 is H;

preferably, the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following combinations of mutations 53-58:

4. the binding protein according to any one of claims 1 to 3, wherein at least 3 complementarity determining regions are included in the binding protein; alternatively, the binding protein comprises at least 6 complementarity determining regions;

preferably, the binding protein is an antibody or a functional fragment thereof;

preferably, the binding protein is selected from any one of nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit;

preferably, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 which have the sequences shown in SEQ ID NO. 1-4 in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 which have the sequences shown in SEQ ID NO. 5-8 in sequence;

preferably, the binding protein further comprises an antibody constant region;

preferably, the antibody constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the antibody constant region is from cattle, horses, dairy cattle, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, bangles, or humans;

preferably, the antibody constant region is derived from a mouse;

preferably, the light chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 10.

5. Use of a binding protein according to any one of claims 1 to 4 in the manufacture of a cardiovascular disease diagnostic reagent or kit;

preferably, the cardiovascular disease is selected from one or more of heart failure and ischemic heart disease.

6. A cardiovascular disease diagnostic reagent or kit comprising the binding protein of any one of claims 1 to 4;

7. A method of detecting sST2, comprising: mixing the binding protein of any one of claims 1-4 with a sample to be tested;

preferably, the method is a method for realizing the detection of sST2 by a precipitation reaction or a method for realizing the detection of sST2 by marking an indicator showing signal intensity;

preferably, the method for detecting sST2 by precipitation reaction is selected from any one or more of the following methods: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoelectrophoresis, and an immunoblotting method;

preferably, the method for detecting the sST2 by marking the indicator for displaying the signal intensity is selected from any one or more of the following methods: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay, and chemiluminescent immunoassay;

preferably, the indicator is selected from any one of a fluorescent dye, a radioisotope, an enzyme catalyzing color development of a substrate, and a chemiluminescent reagent.

8. A hybridoma cell expressing a binding protein according to any one of claims 1 to 4.

9. A vector comprising a nucleic acid encoding the binding protein of any one of claims 1 to 4;

preferably, the nucleic acid is DNA.

10. A host cell comprising the vector of claim 9.

11. A method of producing the binding protein of any one of claims 1 to 4, comprising:

culturing the host cell of claim 10, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.