[go: up one dir, main page]

CN113912729B - Single-domain antibody aiming at sST2, and derivative protein and application thereof - Google Patents

Single-domain antibody aiming at sST2, and derivative protein and application thereof Download PDF

Info

Publication number
CN113912729B
CN113912729B CN202111491678.0A CN202111491678A CN113912729B CN 113912729 B CN113912729 B CN 113912729B CN 202111491678 A CN202111491678 A CN 202111491678A CN 113912729 B CN113912729 B CN 113912729B
Authority
CN
China
Prior art keywords
gly
ser
ala
thr
tyr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111491678.0A
Other languages
Chinese (zh)
Other versions
CN113912729A (en
Inventor
苏志鹏
孟巾果
王乐飞
谢维
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Rongjiekang Biotechnology Co ltd
Original Assignee
Nanjing Rongjiekang Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Rongjiekang Biotechnology Co ltd filed Critical Nanjing Rongjiekang Biotechnology Co ltd
Priority to CN202111491678.0A priority Critical patent/CN113912729B/en
Publication of CN113912729A publication Critical patent/CN113912729A/en
Application granted granted Critical
Publication of CN113912729B publication Critical patent/CN113912729B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention belongs to the field of immunology, and relates to a single domain antibody aiming at sST2, and a derivative protein and application thereof. The single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1 shown in SEQ ID NO. 41-SEQ ID NO. 59, a heavy chain CDR2 shown in SEQ ID NO. 60-SEQ ID NO. 79 and a heavy chain CDR3 shown in SEQ ID NO. 80-SEQ ID NO. 99; compared with the polyclonal antibody in the existing detection kit, the single domain antibody has more single component and better batch stability as the sST2 protein detection antibody, so that the actual detection result of the sST2 protein content is more reliable; and the sST2 fusion protein can be detected, and the target protein in cells or culture medium can be further purified, so that the sST2 fusion protein with high purity can be obtained.

Description

Single-domain antibody aiming at sST2, and derivative protein and application thereof
Technical Field
The invention relates to the technical field of biotechnology or immunology, and relates to a single domain antibody aiming at soluble growth stimulation expression gene 2 protein (sST 2) and a derivative protein thereof.
Background
Antibodies (abs), i.e., immunoglobulins (immunoglobulins, igs), are glycoproteins in blood and tissue fluid, are produced by plasma cells generated by proliferation and differentiation of B cells after stimulation by antigens, are mainly present in body fluids such as serum, can be specifically bound to the corresponding antigens, and are important effector molecules for mediating humoral immunity. In addition to the fact that antibodies mediate specific humoral immune responses as important effector molecules, antibodies play an important role in the prevention and treatment of diseases, particularly infectious diseases, and Behring created serotherapy has thus gained a medical and physiological nobel prize. Thereafter, antibodies artificially prepared by polyclonal, monoclonal and genetically engineered antibody techniques are gradually applied to clinical use.
It is also obvious that an antibody, which is a molecule capable of specifically recognizing a certain protein (antigen), has a strong effect in the prevention and treatment of diseases, and is also useful as a detection tool in the field of examination and scientific research. Enzyme-linked immunosorbent assay (ELISA) is an important method for detecting by using antibody/antigen combination. At present, the commonly used ELISA detection methods include a double-antibody sandwich method, an indirect method, a competition method, an immunosuppression method and the like, and the basic principles are antigen-antibody binding reactions. The ELISA kit based on the method has wide application in inspection and quarantine and other occasions. The affinity and stability of the antibody in the partial inspection and quarantine method are very strict, so that the used antibody has very high affinity and very good stability with the antigen specifically bound with the antibody, and the application and storage environment of the antibody is more extensive so as to be more beneficial to the popularization of the detection method. Traditional monoclonal antibodies perform well in terms of antigen affinity, and although the stability of the monoclonal antibodies can be improved by adding a protective agent or changing a buffer solution, the storage conditions of the monoclonal antibodies are still relatively strict, and the monoclonal antibodies are generally transported or stored at low temperature. The single-domain antibody is more excellent in affinity and stability, the affinity of the single-domain antibody with an antigen can reach a pM level before affinity maturation is carried out on the single-domain antibody, the room temperature stability of the single-domain antibody is good, and the transportation and storage cost can be reduced. In addition, the single-domain antibody can be expressed in a large amount of solubility in a prokaryotic expression system, the production cost is reduced, and the stability of antibody batches is improved.
The growth stimulation expression gene 2 protein (ST 2) is a specific cell marker, belongs to interleukin 1 receptor (IL-1R) family, is related to inflammatory bowel disease, diabetes, heart failure, coronary heart disease and the like, and is involved in inflammatory reaction and promotion of mast cell activation. The main types of ST2 are soluble ST2 (sST 2) and transmembrane ST2 (ST 2L), both of which are associated with the cardiovascular system. sST2 is a decoy receptor, and blocks IL-33/ST2 signal transduction after being combined with interleukin 33 (IL-33), thereby inhibiting activation of nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK), weakening myocardial protection effect, and promoting heart injury and remodeling. sST2 and heart failure: according to statistics, the incidence rate of heart failure in China is 0.7-0.9 per thousand, and about 50 ten thousand of new heart failure patients occur every year. The clinical symptoms of heart failure are atypical, the condition changes frequently, and the diagnosis is susceptible to the experience of doctors and the like. Currently, there is no independent diagnostic test for heart failure, the primary diagnostic basis being patient history and signs. As the incidence of heart failure increases, biomarkers are increasingly being used for early diagnosis, treatment and prognosis evaluation of heart failure. The role of sST2 as a novel biomarker of Heart failure in inflammatory responses, fibrosis and cardiac stress has attracted widespread attention as GAGGIN H K et al, Circ Heart Fail, 2013, 6 (6): 1206-1213. DOI: 10.1161/CIRCHARTFAILURE.113.00045. WU et al, found that sST2 has a low biological variability in healthy subjects and is therefore more suitable for monitoring changes in the condition of heart failure than other biomarkers (e.g.amino-terminal pro-brain natriuretic peptide, NT-proBNP). The results of a study involving 167 patients with chronic systolic heart failure with grade I-III cardiac function and a left ventricular ejection fraction < 45% showed that the level of sST2 positively correlated with cardiac function stratification, worsening chronic heart failure hospitalization, mortality, and the like. sST2 and Acute Myocardial Infarction (AMI): in patients with AMI, the concentration of sST2 was elevated prematurely and was associated with poor cardiac remodeling. Weir et al found that sST2 correlated negatively with Left Ventricular (LV) ejection fraction and positively with baseline and infarct volume index 24 weeks after AMI. The Pascal-Figal et al study also supported a relationship between sST2 and post-AMI adverse remodeling in an experimental model of AMI, where myocardial expression of sST2 rose rapidly within the first 4 weeks and was associated with ongoing fibrotic and inflammatory processes. In long-term follow-up, higher concentrations of sST2 are more likely to have HF. Elevated plasma levels of sST2 are also significantly associated with impaired cardiac structural, functional and hemodynamic parameters in patients with acute HF dyspnea. sST2 is also associated with inflammation: in acute HF patients, Rehman et al studies showed a correlation between sST2 and inflammation, such as temperature, white blood cell count, and C-reactive protein (CRP). In addition, HF patients have higher levels of sST2 with inflammatory lung disease. Indeed, sST2 was identified as involved in autoimmune and inflammatory diseases by modulating T helper type 2 responses. Thus, an increase in sST2 in reports of acute heart disease patients represents an adverse reaction that abrogates the LL-33/ST2L system interaction and leads to an adverse phenotype associated with myocardial stretch, fibrosis, adverse remodeling, inflammation and worse hemodynamic impairment. Based on many clinical studies published in recent years, sST2 has important prognostic value in several acute and chronic cardiovascular diseases. Prediction of mortality in HF patients the serum sST2 concentration strongly predicts mortality within 1 year in patients with acute heart failure and patients without heart failure. The concentration of sST2 in HF patients was significantly higher than in non-HF dyspnea patients, but the diagnosis of acute HF sST2 was clearly lower than NT-proBNP. In the study of patients with acute heart failure, sST2 again became the first choice for predicting 1 month and 1 year mortality; and exhibit a higher predictive value than other biomarkers, such as NT-proBNP, BNP, CRP or troponin. Detection of sST2 concentrations improved mortality, risk stratification for cardiovascular death and HF hospitalization, and increased prognostic information for other biomarkers. Furthermore, patients with elevated sST2 values are at a much higher risk of adverse outcome than patients with reduced sST 2. In acute HF, ST2 levels are not correlated with factors such as age, weight, etc., and their predictive value is complementary to natriuretic peptides; the combination of ST2 and NTproBNP significantly improved risk stratification. sST2 also predicts cardiovascular death and heart failure for 30 days and 1 year in ACS patients with elevated non-ST segments. In stable coronary artery disease, sST2 also predicts mortality and cardiovascular death. The ACCF/AHA guideline of 2013 has incorporated sST2 as a marker of myocardial fibrosis and suggested its use as class iib for risk stratification in patients with acute decompensated HF (evidence level a) or chronic HF (evidence level B).
Currently, most of the detection methods for sST2 are double-antibody sandwich methods, and the solid-phase antibody used is a monoclonal antibody. The monoclonal antibody has the problems of long preparation period and high cost, and the cost of the sST2 detection kit is undoubtedly increased.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide a single domain antibody aiming at sST2, a derivative protein and application thereof, the single domain antibody can be obtained through prokaryotic expression or yeast expression after obtaining the high-specificity single domain antibody, the yield and the purification efficiency are high, the batch quality is stable, the affinity of the single domain antibody has certain advantages compared with the conventional antibody, the antigen capture capacity and the detection effect can be improved, the detection sensitivity is improved, in addition, the single domain antibody belongs to a monoclonal antibody, the unicity and the controllability of a detection reagent can be ensured, and the detection standard of a detection kit can be better controlled.
The single domain antibody is a monoclonal antibody, has generally higher affinity and better stability than the traditional monoclonal antibody, and also has the advantages of the traditional monoclonal antibody. Therefore, the invention utilizes the advantages to obtain single-domain antibodies aiming at sST2, and screens 4 pairs of single-domain antibodies (2B 4-1A7, 1A9-1A7, 2B4-2B10 and 1A9-2B 10) without competitive inhibition by competitive ELISA, wherein each pair of single-domain antibodies is respectively used for coating a solid phase carrier and preparing an enzyme conjugate in an sST2 detection kit. The double-site sandwich method has high specificity, and can carry out heat preservation reaction on a detected sample and an enzyme-labeled antibody together to carry out one-step detection, thereby shortening the detection time.
In a first aspect of the invention, there is provided a single domain antibody to sST2, the single domain antibody consisting of a heavy chain comprising heavy chain CDR1 as set forth in any one of SEQ ID NOs 41-59, CDR2 as set forth in any one of SEQ ID NOs 60-79 and CDR3 as set forth in any one of SEQ ID NOs 80-99.
Specifically, the heavy chain includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR 3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of the following (1) to (20):
(1) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:60, CDR3 shown in SEQ ID NO: 80;
(2) CDR1 shown in SEQ ID NO. 42, CDR2 shown in SEQ ID NO. 61, CDR3 shown in SEQ ID NO. 81;
(3) CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 62, CDR3 shown in SEQ ID NO. 82;
(4) CDR1 shown in SEQ ID NO. 44, CDR2 shown in SEQ ID NO. 63, CDR3 shown in SEQ ID NO. 83;
(5) CDR1 shown in SEQ ID NO. 45, CDR2 shown in SEQ ID NO. 64, CDR3 shown in SEQ ID NO. 84;
(6) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 65, CDR3 shown in SEQ ID NO. 85;
(7) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 66, CDR3 shown in SEQ ID NO. 86;
(8) CDR1 shown in SEQ ID NO. 48, CDR2 shown in SEQ ID NO. 67, CDR3 shown in SEQ ID NO. 87;
(9) CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 68, CDR3 shown in SEQ ID NO. 88;
(10) CDR1 shown in SEQ ID NO. 50, CDR2 shown in SEQ ID NO. 69, CDR3 shown in SEQ ID NO. 89;
(11) CDR1 shown in SEQ ID NO. 51, CDR2 shown in SEQ ID NO. 70, CDR3 shown in SEQ ID NO. 90;
(12) CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 71, CDR3 shown in SEQ ID NO. 91;
(13) CDR1 shown in SEQ ID NO. 53, CDR2 shown in SEQ ID NO. 72, CDR3 shown in SEQ ID NO. 92;
(14) CDR1 shown in SEQ ID NO. 54, CDR2 shown in SEQ ID NO. 73, CDR3 shown in SEQ ID NO. 93;
(15) CDR1 shown in SEQ ID NO. 55, CDR2 shown in SEQ ID NO. 74, CDR3 shown in SEQ ID NO. 94;
(16) CDR1 shown in SEQ ID NO. 56, CDR2 shown in SEQ ID NO. 75, CDR3 shown in SEQ ID NO. 95;
(17) CDR1 shown in SEQ ID NO. 57, CDR2 shown in SEQ ID NO. 76, CDR3 shown in SEQ ID NO. 96;
(18) CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 77, CDR3 shown in SEQ ID NO. 97;
(19) CDR1 shown in SEQ ID NO. 54, CDR2 shown in SEQ ID NO. 78, CDR3 shown in SEQ ID NO. 98;
(20) CDR1 shown in SEQ ID NO. 59, CDR2 shown in SEQ ID NO. 79 and CDR3 shown in SEQ ID NO. 99.
The CDR combinations of the above (1) to (20) correspond to SEQ ID NO.1 to 20 in order.
All the above sequences may be replaced with a sequence having "at least 80% homology" with the sequence or a sequence having only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".
In a preferred embodiment, the sequence of the single domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4;
the amino acid sequences of the FR regions of the framework regions are:
a variant of FR1 or FR1 as represented by any one of SEQ ID NO 100-109, said variant of FR1 comprising a substitution of at most 3 amino acids in said FR 1;
110-126, said variant of FR2 comprising a substitution of up to 3 amino acids in said FR 2;
a variant of FR3 or FR3 as set forth in any one of SEQ ID NO:127-146, said variant of FR3 comprising a substitution of up to 3 amino acids in said FR 3;
FR4 or FR4 variant as set forth in any one of SEQ ID NO:147-152, said FR4 variant comprising a substitution of at most 3 amino acids in said FR 4.
In one embodiment, the single domain antibody to sST2 binds to a polypeptide selected from SEQ ID NOs: 1-20 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding to sST2 protein.
In another preferred embodiment, the single domain antibody to sST2 is identical to a sequence selected from SEQ ID NOs: 1-20 has at least 95% sequence homology and is capable of specifically binding to sST2 protein.
A second aspect of the invention provides single domain antibodies against sST2, said single domain antibodies being as set forth in any one of SEQ ID nos. 1-20, respectively, or said single domain antibodies having at least 95% sequence homology with the amino acid sequence set forth in any one of SEQ ID nos. 1-20.
In one embodiment, the nucleic acid molecule encoding the single domain antibody to sST2 is identical to a sequence selected from SEQ ID NOs: 21-40 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and encodes a single domain antibody against sST2 that is capable of specifically binding to sST2 protein.
Preferably, the coding sequence of the single domain antibody is as shown in any one of SEQ ID No.21-40, or has at least 95% sequence homology with any one of SEQ ID No.21-40, respectively.
A third aspect of the invention is to provide the aforementioned Fc fusion antibody against a single domain antibody of sST 2.
In a fourth aspect of the invention, nucleotide molecules are provided which encode the aforementioned single domain antibodies against sST2, and the nucleotide sequences are set forth in SEQ ID NOs: 21-40 or at least 95% sequence homology with any one of SEQ ID nos. 21-40.
A fifth aspect of the present invention provides an expression vector comprising a nucleotide molecule encoding the aforementioned single domain antibody or the aforementioned Fc fusion antibody or the aforementioned nucleotide molecule.
A sixth aspect of the invention provides a host cell or non-human organism which can express the aforementioned single domain antibody against sST2, or which comprises the aforementioned expression vector.
The present invention also provides a method of producing a single domain antibody or Fc fusion antibody thereof against sST2, comprising the steps of: (a) culturing the aforementioned host cell under conditions suitable for the production of a single domain antibody or Fc fusion antibody thereof, thereby obtaining a culture comprising the single domain antibody or Fc fusion antibody thereof against sST 2; (b) isolating or recovering said single domain antibody to sST2 or an Fc fusion antibody thereof from said culture; and (c) optionally purifying and/or modifying the single domain antibody against sST2 or an Fc fusion antibody thereof obtained in step (b).
A seventh aspect of the invention provides an sST2-ELISA detection kit comprising the aforementioned single domain antibody to sST 2.
An eighth aspect of the present invention provides the use of the aforementioned single domain antibody against sST2 in the preparation of an sST2-ELISA test kit, or the use of the aforementioned single domain antibody against sST2 in the preparation of a kit for detecting sST2 protein in a human (or a method for detecting sST2 protein in a human using the single domain antibody against sST 2), or the use of the aforementioned single domain antibody against sST2 in the isolation and purification of a sST2 protein fusion protein.
The single domain antibody is a VHH comprising only antibody heavy chains and no antibody light chains.
Compared with the prior art, the invention has the beneficial effects that: the single domain antibody as the sST2 protein detection antibody has higher affinity with the traditional monoclonal antibody, and compared with the polyclonal antibody in the existing detection kit, the single domain antibody has more single component, better batch stability and easier implementation of quality control. Therefore, the reliability of the actual detection result of the sST2 protein content is higher.
In the subsequent research, researchers can also fuse the target protein and the sST2 tag protein by a genetic engineering method, then the fusion protein is expressed or secreted in cells, the sST2 fusion protein is detected by using the single-domain antibody specific to the sST2 protein, and the target protein in the cells or the culture medium is further purified, so that the high-purity sST2 fusion protein is obtained.
Drawings
FIG. 1 is an electrophoresis diagram of PCR products for detecting the insertion rate of a target fragment in a constructed single domain antibody phage display library, wherein the number M is a DNA molecule marker, and the numbers 1 to 15 are PCR products of different clones randomly picked from the constructed single domain antibody library against the sST2 protein;
FIG. 2 is a detailed result of biopanning the constructed single domain antibody library, where P/N = number of monoclonal bacteria grown after phage eluted from positive well in biopanning infected with TG1 bacteria/number of monoclonal bacteria grown after phage eluted from positive well infected with TG1 bacteria, which parameter is gradually increased after enrichment occurs; I/E = total number of phage added to positive wells per round of biopanning/total number of phage eluted from positive wells per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred;
FIG. 3 is an SDS-PAGE electrophoresis of crude protein solutions purified by nickel column resin affinity chromatography of expressed single domain antibodies against sST2 protein;
FIG. 4 is an SDS-PAGE electrophoresis of human Fc fusion sST2 single domain antibody expressed in mammalian expression system after Protein A affinity chromatography purification, with the number M being Protein marker;
FIG. 5 shows the results of ELISA experiments with single domain antibodies against sST2 protein expressed in prokaryotic expression system, all of which were expressed and purified according to example 4;
fig. 6 is the detection of competition concentration (EC 80) against sST2 protein by a competition ELISA.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
Single domain antibodies (sdabs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which do not naturally contain a light chain), single domain antibodies derived from conventional antibodies, and engineered antibodies.
Single domain antibodies may be derived from any species, including mouse, human, camel, llama, goat, rabbit and cow. For example, naturally occurring VHH molecules may be derived from antibodies provided by species in the family camelidae (e.g. camel, dromedary, llama and guanaco). Like intact antibodies, single domain antibodies are capable of selectively binding to a particular antigen. Single domain antibodies may contain only the variable domains of immunoglobulin chains, with CDR1, CDR2 and CDR3, and the framework regions.
As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% homology.
In the present invention, a nanobody against sST2 can be obtained from a sequence having high sequence homology to CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" to the sequences in (1) - (20), or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" may accomplish the purpose of the invention (i.e., to derive a protein).
In some embodiments, the nucleic acid sequence identical to SEQ ID NO: sequences 1-20 that are substituted for only one or a few amino acids compared to sequences containing, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions may also achieve the objects of the invention. Indeed, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2, and CDR3 combination in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution, which may generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, for example conservative amino acid substitutions are those in which one or a few amino acids within the following groups (a) - (d) are replaced by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: asp, Asn, Glu, Gln; (b) polar positively charged residues: his, Arg, Lys; (c) aromatic residue: phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val and Cys. Particularly preferred conservative amino acid substitutions are as follows: asp substituted by Glu; asn is replaced by Gln or His; glu is substituted with Asp; gln is substituted by Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys substituted by Arg, Gln; phe is replaced by Met, Leu, Tyr; trp is substituted by Tyr; tyr is substituted by Phe, Trp; ala substituted by Gly or Ser; ser substituted by Thr; thr is substituted by Ser; gly by Ala or Pro; met is substituted by Leu, Tyr or Ile; leu is substituted by Ile or Val; ile is substituted by Leu or Val; val is substituted by Ile or Leu; cys is substituted with Ser. In addition, the skilled person knows that the creativity of single domain antibodies is found in the CDR1-3 region, whereas the framework region sequence FR1-4 is not unalterable and the sequence of FR1-4 may take the form of conservative sequence variants of the sequences disclosed in the present invention.
Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
The invention adopts sST2 protein with the purity of 95% purchased from Novoprotein company to immunize Alabar bactrian camel, extracts the bactrian camel peripheral blood lymphocytes after 7 times of immunization and establishes a single domain antibody library of the sST2 protein. In the antibody screening process, sST2 polypeptide is coupled with Biotin (sST 2-Biotin), neutral avidin protein is coupled on an enzyme label plate, sST2 polypeptide is indirectly displayed on the surface of the enzyme label plate by utilizing the characteristic of combination of Biotin and neutral avidin, so that the epitope of the sST2 polypeptide is exposed, then a single domain antibody gene library (camel heavy chain antibody phage display gene library) after sST2 protein immunization is screened by utilizing a phage display technology, and a single domain antibody strain which can be efficiently expressed in escherichia coli is obtained.
And (3) expressing the single domain antibody obtained by screening in escherichia coli, purifying through agarose coupled with nickel ions, and analyzing the purified nano antibody.
The invention will be further illustrated with reference to the following specific examples.
Example 1: construction of a single domain antibody library against the sST2 protein:
(1) mixing 1mg of sST2 antigen and Freund's adjuvant in equal volume, immunizing a Xinjiang bactrian camel once a week for 7 times continuously, and stimulating B cells to express specific nano-antibodies in the immune process; (2) after the immunization is finished, extracting 100ml of camel peripheral blood lymphocytes and extracting total RNA; (3) synthesizing cDNA and amplifying VHH by using nested PCR; (4) restriction enzymes Pst I and Not I were used to digest 20. mu.g of pMECS phage display vector and 10μ g VHH and joining the two fragments; (5) the ligation products were transformed into electroporation competent cells TG1, and an sST2 protein phage display library was constructed and the library volume was determined, the size of the library volume being approximately 2X 109(ii) a Meanwhile, the correct insertion rate of the target fragment in the created library is detected through colony PCR, the result of colony PCR is shown in figure 1, 34 clones are randomly selected to be used as colony PCR, and the result shows that the insertion rate reaches 90%.
Example 2: screening for single domain antibodies against sST2 protein:
(1) culturing 200 μ L of recombinant TG1 cells in 2 × TY culture medium, adding 40 μ L of helper phage VCSM13 to infect TG1 cells, culturing overnight to amplify phage, precipitating phage with PEG/NaCl the next day, centrifuging, and collecting amplified phage; (2) NaHCO diluted at 100mM pH 8.33500ug of the neutral avidin protein is coupled on an enzyme label plate, is placed at 4 ℃ overnight, and is provided with a negative control hole at the same time; (3) the next day 100. mu.L of biotinylated sST2 protein (sST 2-Biotin) was added, incubated for 2 hours at room temperature, and 100. mu.L of PBS was added to negative control wells; (4) after 2 hours, 100 mu L of 3% skim milk is added, and the mixture is sealed for 2 hours at room temperature; (5) after the end of blocking, 100. mu.l of the amplified phage library (approx.2X 10) was added11Individual phage particles), and reacting for 1h at room temperature; (6) after 1 hour of action, wash 5 times with PBS +0.05% Tween-20 to wash away unbound phage; (7) the phage specifically bound to the sST2 protein was dissociated with trypsin at a final concentration of 25mg/mL and infected with E.coli TG1 cells in logarithmic growth phase, cultured at 37 ℃ for 1h, phage was generated and collected for the next round of screening, and the same screening process was repeated for 3 rounds to obtain enrichment step by step, the enrichment effect is shown in FIG. 2, the P/N value was about 10000 from 2.2 to the third round of the first round, and the I/E quality was about 13 from 13605 to the third round of the first round, demonstrating that the library has a very significant enrichment against sST 2.
Example 3: screening of specific positive clones by phage enzyme-linked immunosorbent assay (ELISA):
(1) 3 rounds of screening are carried out on the sST2 protein according to the single-domain antibody screening method, after the screening is finished, the phage enrichment factor aiming at the sST2 protein reaches more than 10 (about 10000), and the protein is screenedSelecting 400 single colonies from the obtained positive clones, respectively inoculating the single colonies into a 96-deep-well plate containing a TB culture medium containing 100 mu g/mL ampicillin, setting a blank control, culturing at 37 ℃ until the logarithmic phase, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, and culturing at 28 ℃ overnight; (2) obtaining a crude antibody by using a permeation cracking method; neutral avidin protein was diluted to 100mM NaHCO, pH 8.33Neutralizing, coating 100 mu g of neutral avidin protein in an enzyme label plate at 4 ℃ overnight, and adding 100ug of ST2-Biotin protein into the enzyme label plate the next day; (3) taking 100uL of the crude antibody extract obtained in the step, transferring the crude antibody extract to an ELISA plate added with an antigen, and incubating for 1h at room temperature; (4) unbound antibody was washed away with PBST, 100ul of Mouse anti-HA tag antibody (Mouse anti-HA antibody, Thermo Fisher) diluted at 1:2000 was added, and incubated at room temperature for 1 h; (5) unbound antibody was washed away with PBST, 100ul of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted 1:20000 was added, and incubated at room temperature for 1 h; (6) washing away unbound antibodies by PBST, adding horseradish peroxidase developing solution, reacting at 37 ℃ for 15min, adding a stop solution, and reading an absorption value at a wavelength of 450nm on an enzyme-labeling instrument; (7) when the OD value of the sample hole is more than 5 times of that of the control hole, judging the sample hole as a positive cloning hole; (8) the bacteria of the positive cloning wells were shaken in LB medium containing 100. mu.g/ul ampicillin to extract plasmids and sequenced.
Gene sequences of each clone strain are analyzed according to Vector NTI (sequence alignment software), strains with the same CDR1, CDR2 and CDR3 sequences are regarded as the same clone strain, strains with different sequences are regarded as different clone strains, and finally, single domain antibodies (SEQ ID NO. 1-20) specific to sST2 protein are obtained. The amino acid sequence of the antibody is in a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the whole VHH is formed. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally single domain antibody protein is obtained.
The CDR and FR sequences of the 20 single domain antibodies are shown in tables 1-6.
FR4 sequences of Table 120 Single Domain antibodies
Figure 852398DEST_PATH_IMAGE001
CDR1 sequences of the 220 single domain antibodies in Table
Figure 35118DEST_PATH_IMAGE002
CDR2 sequences of the 320 single domain antibodies in Table
Figure 408330DEST_PATH_IMAGE003
CDR3 sequences of Table 420 Single Domain antibodies
Figure 6802DEST_PATH_IMAGE005
FR1 sequences of the Table 520 Single Domain antibodies
Figure 165382DEST_PATH_IMAGE006
FR2 sequences of the Single Domain antibodies of Table 620
Figure 456686DEST_PATH_IMAGE007
The FR3 sequence (SEQ ID NO. 127-146) is in one-to-one correspondence with single domain antibodies 1A10, 1A7, 1A9, 1B2, 1B5, 2A2, 2B10, 2B4, 2C2, 2E8, 2F1, 2F4, 2F9, 3A10, 3B1, 3B7, 3C5, 3C8, 4A1 and 4D2 in sequence.
The amino acid sequence SEQ ID NO.1-20 of the single domain antibody corresponds one to one in sequence to single domain antibodies 1A10, 1A7, 1A9, 1B2, 1B5, 2A2, 2B10, 2B4, 2C2, 2E8, 2F1, 2F4, 2F9, 3A10, 3B1, 3B7, 3C5, 3C8, 4A1 and 4D 2.
Example 4: purification and expression of specific single-domain antibody of sST2 protein in host bacterium escherichia coli
(1) Sequencing analysis obtained from the abovePlasmids of different clones (pMECS-VHH) were electrically transformed into E.coli HB2151 and plated on LB + amp + glucose plates containing ampicillin and glucose, and cultured overnight at 37 ℃; (2) selecting a single colony to be inoculated in 5mL LB culture solution containing shore penicillin, and carrying out shake culture at 37 ℃ overnight; (3) inoculating 1mL of overnight cultured strain to 330mL of TB culture medium, shake culturing at 37 deg.C, and culturing to OD600nmAdding 1M IPTG when the value reaches 0.6-0.9, and carrying out shake culture at 28 ℃ overnight; (4) centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by use of osmotic bursting method; (5) the antibody was purified by nickel column affinity chromatography, and the purified single domain antibody was shown in FIG. 3. In fig. 3, VHH1-8 inside 3-1 corresponds to single domain antibodies 1a10, 1a7, 1a9, 1B2, 1B5, 2a2, 2B10, 2B4, respectively; 3-2 internal VHHs 9-17 correspond to single domain antibodies 2C2, 2E8, 2F1, 2F4, 2F9, 3A10, 3B1, 3B7 and 3C5 respectively; VHH18-20 inside 3-3 corresponded to single domain antibodies 3C8, 4A1, 4D2, respectively.
Example 5: construction of Fc fusion antibody eukaryotic expression vector of specific single domain antibody of sST2 protein
The target sequence obtained in example 3 was subcloned into eukaryotic expression vectors: (1) the antibody screened out in the example 3 is subjected to Sanger sequencing to obtain a nucleotide sequence; (2) the nucleotide sequence (SEQ ID NO. 21-40) after codon optimization is synthesized into the carrier RJK-V4-hFC designed and modified by the company by a sequence synthesis mode, and the preparation process of the carrier RJK-V4-hFC is described in example 14; (3) transforming a recombinant eukaryotic expression vector constructed by a company into DH5 alpha escherichia coli, culturing, carrying out plasmid macro-extraction, and removing endotoxin; (4) carrying out sequencing identification on the greatly extracted plasmid; (5) and preparing the recombinant vector which is determined to be error-free for subsequent eukaryotic cell transfection expression.
Example 6: fc fusion antibody of specific single domain antibody of sST2 protein is expressed in suspension ExpicHO-S cells
(1) 3 days before transfection at 2.5X 105(ii) passaging and expanding ExpCHO-S-cells in/ml cells, the calculated required cell volume being transferred to a cell containing fresh, pre-warmed 120ml (final volume) of ExpICHO.in a 500ml shake flask of an expression medium; to achieve a cell concentration of about 4X 106 -6×106Viable cells/mL; (2) one day prior to transfection, ExpicHO-S-chamber cells were diluted to a concentration of 3.5X 106Viable cells/mL, cells were cultured overnight; (3) on the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 7X 106 -10×106Viable cells/mL; (4) cells were diluted to 6X 10 with fresh ExpiCHO ™ expression medium preheated to 37 ℃6Viable cells/mL. The calculated required cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) ExpicHO ™ expression medium; (5) gently inverting and mixing the Expifectamine-antibody CHO reagent, diluting the Expifectamine-antibody CHO reagent with 3.7ml of OptiPRO-antibody medium, and swirling or mixing; (6) diluting plasmid DNA with refrigerated 4ml OptiPRO ™ culture medium, and mixing; (7) incubating the Expifactamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding the Expifactamine CHO/plasmid DNA complex into the prepared cell suspension, and gently swirling the shake flask in the adding process; (8) shake culturing cells at 37 ℃, 8% CO 2, humidified air; (9) on day 1 (18-22 h post transfection), 600ul Expifeacamine ™ CHO Enhancer and 24ml ExpicHO feed were added. (10) Supernatants were collected approximately 8 days after transfection (cell viability below 70%).
Example 7: expression of Fc fusion antibodies of specific single domain antibodies of the sST2 protein in 293F cells in suspension
Recombinant single domain antibody expression experimental protocol (taking 500ml shake flask as an example):
(1) 3 days before transfection at 2.5X 105The 293F cells were passaged and expanded in culture and the calculated required cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 120ml (final volume) OPM-293 CD05 Medium. The cell concentration is about 2X 106-3×106Viable cells/mL.
(2) On the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 2X 106-3×106Viable cells/mL.
(3) By usingPre-warmed OPM-293 CD05 Medium cells were diluted to 1X 106Viable cells/mL. The required cell volume was calculated and transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) of medium.
(4) Diluting PEI (1 mg/ml) reagent with 4ml of Opti-MEM medium, and swirling or blowing to mix evenly; the plasmid DNA was diluted with 4ml Opt-MEM medium, vortexed, mixed well, and filtered through a 0.22um filter tip. Incubate at room temperature for 5 min.
(5) Diluted PEI reagent was added to the diluted DNA and mixed by inversion. The PEI/plasmid DNA complex was incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, with gentle swirling of the flask during the addition.
(6) Cells were incubated at 37 ℃ in 5% CO2And shake culturing at 120 rpm.
(7) 5ml OPM-CHO PFF05 feed was added at 24h, 72h post transfection.
(8) Supernatants were collected approximately 7 days after transfection (cell viability below 70%).
Example 8: purification of human Fc recombinant Single Domain antibodies
(1) Filtering the protein expression supernatant obtained in example 6 or 7 with a 0.45 μm disposable filter to remove insoluble impurities;
(2) performing affinity chromatography purification on the filtrate by using a Protein purifier, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human-derived Fc and Protein A;
(3) passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is bound to the packing;
washing the impurity protein bound on the column by low-salt and high-salt buffer solutions;
(4) performing a system of target proteins bound to the column with a low pH buffer;
(5) adding the eluent into Tris-HCl solution with pH9.0 rapidly for neutralization;
(6) after the neutralized protein solution was dialyzed, SDS-PAGE analysis was performed to confirm that the protein was at a purity of 95% or more and at a concentration of 0.5mg/mL or more, and then the protein was stored at a low temperature for future use, as shown in FIG. 4. In fig. 4, 1-14 inside 4-1 correspond to single domain antibodies 1a10, 1a7, 1a9, 1B2, 1B5, 2a2, 2B10, 2B4, 2C2, 2E8, 2F1, 2F4, 2F9, 3a10, respectively; 4-2, 1-6, respectively, correspond to single domain antibodies 3B1, 3B7, 3C5, 3C8, 4a1, 4D 2.
Example 9: primary detection of binding capacity of prokaryotic expression nano antibody and sST2
Carrying out primary detection on the affinity of the single-domain antibody purified in the example 4 to sST 2; the first step of determining the affinity of the recombinant single domain antibody to sST2 using a quantitative standard is as follows;
(1) 100 ng/100. mu.L of standard sST2 sample was coated onto ELISA plates;
(2) sealing the coated plate by using skimmed milk powder;
(3) adding the single domain antibody against sST2 obtained in example 4;
(4) adding a monoclonal antibody of a mouse anti-HA label marked by HRP;
(5) adding a chromogenic substrate TMB;
(6) adding a stop solution to terminate the reaction;
(7) measurement of OD450 values P/N values were calculated and as a result, as shown in fig. 5, it can be seen that all 20 single domain antibodies against sST2 have strong affinity to sST 2.
Example 10: competitive concentration (EC 80) detection of sST2 protein by competitive ELISA
(1) The single-domain antibody of 6-strain sST2 with the P/N value >12 in example 9 was coated on an ELISA plate at 100 ng/100. mu.L;
(2) sealing the coated plate by using Casein;
(3) add an isocratic dilution of standard sST2 sample (with HA-tag);
(4) adding a monoclonal antibody of a mouse anti-HA label marked by HRP;
(5) adding a chromogenic substrate TMB;
(6) adding a stop solution to terminate the reaction;
(7) OD450 values were measured and EC80 results were calculated as shown in FIG. 6;
example 11: specific single domain antibody against sST2 protein in a double antibody sandwich-ELISA assay for competitive inhibition detection
(1) Coating the single-domain antibody of 6 strains of sST2 with a P/N value of >12 and Nb _ sST2 (negative control) on an ELISA plate at 100ng/100 mu L;
(2) sealing the coated plate by using 5% Milk Vertex for 10 min;
(4) the sST2 protein was added to the resulting mixture to a final concentration of EC80 for each sST2 single domain antibody, and the mixture was mixed in equal proportions with the antibody of example 8 (the antibody of example 7 purified from example 8) in double concentration and used without further incubation.
(5) Incubation with anti-HA antibody (HRP labeled);
(6) adding a chromogenic substrate TMB;
(7) adding a stop solution to terminate the reaction;
(8) OD450 values were measured and antibody 4 pairs were identified that were free of competitive inhibition, successfully paired, and could be used in the sST2-ELISA detection kit: 2B4-1A7, 1A9-1A7, 2B4-2B10, and 1A9-2B10, as shown in Table 7 below.
TABLE 7 detection of competitive inhibition of specific single domain antibodies against the sST2 protein in a two-antibody sandwich-ELISA assay
Figure 989298DEST_PATH_IMAGE008
Example 12: application of specific single-domain antibody aiming at sST2 protein in sST2-ELISA kit (double-antibody sandwich method)
(1) Detecting the content of sST2 in a target sample by a double-antibody sandwich method by using the single-domain antibodies obtained in example 8 and example 4, wherein two different single-domain antibodies are used;
(2) firstly, a standard substance is used for measuring and drawing a standard curve of the content of sST2 and OD450, and the steps are as follows;
(3) coating the single-domain antibody obtained by identification and purification in the embodiment 8 on an ELISA plate as a capture antibody;
(4) sealing the coated plate by using skimmed milk powder;
(5) adding a standard sample for incubation;
(6) adding the single-domain antibody obtained in example 4 as a detection antibody (with an HA tag), and incubating;
(7) incubation with anti-HA antibody (HRP labeled);
(8) adding a chromogenic substrate TMB;
(9) adding a stop solution to terminate the reaction;
(10) measuring an OD450 value, and drawing a standard curve;
(11) and then detecting the sample to be detected based on the standard curve obtained in the step.
Example 13: application of single-domain antibody aiming at sST2 protein in separation and purification of sST2 protein fusion protein
Purification of sST2 protein was performed by coupling single domain antibodies to sST2 protein to hydrogen bromide activated agarose resin.
Example 14: construction of eukaryotic expression vector RJK-V4-hFc
The target vector RJK-V4-hFC for single domain antibody is modified by fusing Fc segment in heavy chain coding sequence of human IgG (NCBI Accession No.: AB 776838.1) based on Invitrogen commercial vector pCDNA3.4 (vector data link: https:// Assets. thermofisher. com/TFS-Assets/LSG/manual/pcdna 3-4 _ topo _ ta _ cloning _ kit _ man. pdf), i.e., the vector contains IgG heavy chain Hinge region (Hinge) CH2 and CH3 regions. The specific modification scheme is as follows:
(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;
(2) introducing a Multiple Cloning Site (MCS) and a 6 XHis tag at the 5 'end and the 3' end of the Fc fragment coding sequence respectively by means of overlapping PCR;
(3) amplifying the fragment by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively in a PCR mode;
(4) the recombinant DNA fragments in pcDNA3.4 and (3) are digested with restriction enzymes XbaI and AgeI respectively;
(5) and (3) connecting the vector and the insert after enzyme digestion under the action of T4 ligase, then transforming the connection product into escherichia coli, amplifying, sequencing and verifying to obtain the recombinant plasmid.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Sequence listing
<110> Nanjing Congjiekang Biotech Co., Ltd
<120> Single domain antibody against sST2, and derived protein and application thereof
<160> 152
<170> SIPOSequenceListing 1.0
<210> 1
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Ala Thr Phe Ile Asn Trp Ser Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala Ala Ile Glu Ser
35 40 45
Gly Gly Ser Pro Thr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Leu
50 55 60
Ser Thr Asp Val Ala Lys Asn Ile Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Ala Asp Thr Ala Met Tyr Tyr Cys Ala Ala Lys Lys Val Trp
85 90 95
Ala Arg Glu Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
100 105 110
Ser
<210> 2
<211> 123
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Ile Tyr Ser Arg Asn Cys Val Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile Ala Ala Leu Tyr Thr
35 40 45
Ala Gly Gly Tyr Thr Tyr Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Arg Pro
85 90 95
Ser Asn Ser Leu Thr Cys Gly Ile Ala Thr Ile Thr Thr Asp Asp Val
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 3
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Thr Tyr Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val Ala Gly Ile Asp Ser
35 40 45
Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asp Thr Leu Tyr Leu Gln Met Asp Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Leu Ser Pro Arg
85 90 95
Cys Asp Phe Asp Arg Ser Ser Arg Val Thr Asn Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 4
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Thr Ser Gly Val Thr Tyr Arg Arg Thr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
35 40 45
Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Gly Ile Tyr Tyr Cys Ala Ala Glu Ala Gly
85 90 95
Asp Glu Cys Ser Ala Ser Ser Val Phe Arg Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 5
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Gly Ile Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
35 40 45
Asn Gly Glu Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Arg Asn
85 90 95
Thr Cys Gly Leu Arg Gly Asp Phe Tyr Asn Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 7
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Gly Arg Arg Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Thr Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Ser
35 40 45
Gly Asp Ile Thr Phe Tyr Gly Asp Ser Val Arg Gly Arg Phe Thr Ile
50 55 60
Ser Arg Asp Asp Ala Lys Asn Ala Val Tyr Leu Gln Met Asp Arg Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Gly Asp
85 90 95
Tyr Cys Ser Thr Arg Pro Phe Ala Phe Asn Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 7
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Val Ser Gly Tyr Thr Ser Arg His Cys Met Gly Trp Phe Arg
20 25 30
Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu Tyr Pro Ala
35 40 45
Thr Gly Ser Ala Phe Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Asp Asp Thr Ala Leu Tyr Tyr Cys Ala Val Glu Ser Thr Ser
85 90 95
Leu Cys Ala Ala Arg Thr Val Pro Ser Phe Lys Phe Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 8
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Ala Asp Met Gly Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser Ser Leu Gly Asp
35 40 45
Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Arg Leu Trp
85 90 95
Thr Ala Ser Thr Val Val Pro Asp Thr Pro Cys Leu Asp Glu Ile Glu
100 105 110
Asp Phe Gly Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Val Ser Gly Asn Met Tyr Arg Arg Asn Cys Ile Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
35 40 45
Ala Gly Gly Tyr Ser Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Pro Lys Asn Thr Val Tyr Leu Gln Met Asn Asn
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Asp Gly Arg
85 90 95
Asp Gly Asp Leu Cys Ala Ala Arg Ser Asp Phe Arg Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 10
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr His Asp Tyr Ser Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala Ser Met Tyr Thr
35 40 45
Gly Gly Ala Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ala Gln Asp Asn Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Thr Arg
85 90 95
Pro Ile Val Asp Arg Trp Cys Gly Leu Pro Trp Pro Leu Tyr Thr Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 11
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Phe Arg Arg Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val Leu Tyr Thr
35 40 45
Gly Val Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Lys Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Asp Ser Gly
85 90 95
Pro Leu Cys Gly Leu Gly Ile Gln Phe Gly Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 12
<211> 116
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Ile Asn Tyr Arg Met Gly Trp Phe Arg
20 25 30
Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Met Ala Ser Ala Ile
35 40 45
Ser Glu Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Arg
50 55 60
Asp Ser Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro
65 70 75 80
Glu Asp Thr Gly Met Tyr Tyr Cys Ala Ala Gly Arg Val Trp Gly Arg
85 90 95
Leu Trp Asn Pro Asp Asp Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
<210> 13
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Ile Thr Tyr Ser Arg Asn Cys Leu Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Thr Ile Tyr Thr
35 40 45
Ser Thr Gly Thr Thr Tyr Phe Asp Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Val Ser
85 90 95
Gly Trp Ser Val Cys Ser Arg Ser Thr Lys Tyr Asn Tyr Trp Gly His
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 14
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Leu Asp Ser
35 40 45
Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Leu Thr Cys
85 90 95
Tyr Cys Ser Asp Gly Tyr Cys Tyr Asn Gly Asp Glu Tyr Asn Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 15
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Ala Ser Gly Val Thr Phe Ser Arg Ser Cys Ile Gly Trp Phe
20 25 30
Arg Gln Ile Pro Gly Lys Glu Arg Glu Gly Val Ala Val Val Tyr Thr
35 40 45
Gly Gly Arg Ser Ile Asp Tyr Ser Ala Tyr Val Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser His Asp Asn Ala Trp Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala
85 90 95
Asp Phe Leu Leu Gln Glu Pro Cys Thr Val Leu Ser Ser Arg Gly Phe
100 105 110
Lys Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Glu Tyr Ala Tyr Ile Asn Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Val Ile Cys Ser
35 40 45
Gly Gly Gly Ala Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Gly Ala Lys Tyr Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Arg Arg
85 90 95
Pro Tyr Tyr Ser Gly Ala Tyr Phe Arg Thr Ala Asp Tyr Asn Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 17
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Ser Gly Tyr Phe Tyr Ser Arg Leu Cys Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Thr Gly Arg Glu Ala Val Ala Thr Ser
35 40 45
Tyr Pro Gly Ile Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp
85 90 95
Ala Thr Trp Asn Thr Cys Gly Ile Thr Lys Thr Thr Tyr Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 18
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Arg Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Thr Pro Gly Lys Gly Arg Glu Gly Val Ala Ser Ile Asn Ser
35 40 45
Asp Gly Thr Thr Ile Tyr Gly Asn Phe Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Glu Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Val Asp Ser
85 90 95
Thr Cys Ala Pro Met Pro Ser Trp Phe Thr Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 19
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Val Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Asp Ser
35 40 45
Arg Gly Ser Thr Thr Tyr Ala Asp Ser Leu Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Lys Tyr Tyr Cys Ala Ala Gly Asp Gly Tyr
85 90 95
Gly Leu Glu His Ile Cys Ile Pro His Tyr Tyr Ala Met Asn Tyr Trp
100 105 110
Gly Glu Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 20
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Phe Thr Tyr Arg Arg Ala Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
35 40 45
Ser Gly Gly Met Thr Phe Tyr Asp Ala Ser Val Arg Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Ala Gly Gly Gly
85 90 95
Asp Tyr Cys Ala Thr Asn Ala Arg Phe Phe Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 21
<211> 339
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gagagcggcg gcggcagcgt gcagcccggc ggcagcctga ggctgagctg cgccgccagc 60
ggcgccacct tcatcaactg gagcatgggc tggttcaggc aggcccccgg caaggagagg 120
gaggtggtgg ccgccatcga gagcggcggc agccccacct acgccgacag cgtgaaggac 180
aggttcaccc tgagcaccga cgtggccaag aacatcctgt acctgcagat gaacagcctg 240
aagcccgccg acaccgccat gtactactgc gccgccaaga aggtgtgggc cagggagttc 300
gcctactggg gccagggcac ccaggtgacc gtgagcagc 339
<210> 22
<211> 369
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacatct acagcaggaa ctgcgtgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcatcg ccgccctgta caccgccggc ggctacacct actacagcgc cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaggcccag caacagcctg 300
acctgcggca tcgccaccat caccaccgac gacgtgtggg gccagggcac ccaggtgacc 360
gtgagcagc 369
<210> 23
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcaccta ctgcatggcc tggttcaggc aggccctggg caaggagagg 120
gagggcgtgg ccggcatcga cagcgacggc agcaccacct acgccgacag cgtgaagggc 180
aggttcacca tcagcaagga caacgccaag gacaccctgt acctgcagat ggacagcctg 240
aagcccgagg acaccgccat gtactactgc gccgccctga gccccaggtg cgacttcgac 300
aggagcagca gggtgaccaa ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 24
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccaccagc 60
ggcgtgacct acaggaggac ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatcta caccagcggc ggcttcacct tctacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccc tgtacctgca gatgaacacc 240
ctgaagcccg aggacaccgg catctactac tgcgccgccg aggccggcga cgagtgcagc 300
gccagcagcg tgttcaggag ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 25
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcggcat ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatcta caccaacggc gagtacacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg acaggaacac ctgcggcctg 300
aggggcgact tctacaacta ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 26
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacaccg gcaggaggaa ctgcatgggc tggttcaggc aggccaccgg caaggagagg 120
gagggcgtgg ccgccatcta cagcggcgac atcaccttct acggcgacag cgtgaggggc 180
aggttcacca tcagcaggga cgacgccaag aacgccgtgt acctgcagat ggacaggctg 240
aagcccgagg acaccgccat gtactactgc gccgccggcc ccggcgacta ctgcagcacc 300
aggcccttcg ccttcaacta ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 27
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgtggtgagc 60
ggctacacca gcaggcactg catgggctgg ttcaggcagg cccccggcaa ggagagggag 120
ggcgtggccg ccctgtaccc cgccaccggc agcgccttct acggcgacag cgtgaagggc 180
aggttcacca tcagccagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgacg acaccgccct gtactactgc gccgtggaga gcaccagcct gtgcgccgcc 300
aggaccgtgc ccagcttcaa gttcaggggc cagggcaccc aggtgaccgt gagcagc 357
<210> 28
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg caccgccagc 60
ggcttcacct tcggcgacgc cgacatgggc tggtacaggc aggcccccgg caacgagtgc 120
gagctggtga gcagcctggg cgacaacggc agcacctact acgccgacag cgtgaagggc 180
aggttcacca tcagccagga caacgccaag aacaccgtgt acctgcagat gaacagcctg 240
aagcccgagg acaccgccgt gtactactgc gccgccgaga ggctgtggac cgccagcacc 300
gtggtgcccg acaccccctg cctggacgag atcgaggact tcggctactg gggcaggggc 360
acccaggtga ccgtgagcag c 381
<210> 29
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgtggtgagc 60
ggcaacatgt acaggaggaa ctgcatcggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatcta caccgccggc ggctacagct actacggcga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacccc aagaacaccg tgtacctgca gatgaacaac 240
ctgaagcccg aggacaccgc catgtactac tgcgccaccg acggcaggga cggcgacctg 300
tgcgccgcca ggagcgactt caggtactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 30
<211> 363
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacaccc acgactacag ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagagggtgg ccagcatgta caccggcggc gccgccacct actacgccga cagcgtgaag 180
ggcaggttca ccatcgccca ggacaacgcc aagaacaccc tgttcctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg acaccaggcc catcgtggac 300
aggtggtgcg gcctgccctg gcccctgtac accggccagg gcacccaggt gaccgtgagc 360
agc 363
<210> 31
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gagagcggcg gcggcagcgt gcagagcggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct tcaggaggta ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgtgctgta caccggcgtg gacatcacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaagaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc cctgtactac tgcgccgccg acagcggccc cctgtgcggc 300
ctgggcatcc agttcggcta ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 32
<211> 348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacca tcaactacag gatgggctgg ttcaggcagg cccccggcaa ggagagggag 120
ggcgtggcca tggccagcgc catcagcgag gactacgccg acagcgtgaa gggcaggttc 180
agcatcagca gggacagcgc caagaacacc ctgtacctgc agatgaacag cctgaagccc 240
gaggacaccg gcatgtacta ctgcgccgcc ggcagggtgt ggggcaggct gtggaacccc 300
gacgactaca acagctgggg ccagggcacc caggtgaccg tgagcagc 348
<210> 33
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg caccgccagc 60
ggcatcacct acagcaggaa ctgcctgggc tggttcaggc aggcccccgg caaggagagg 120
gagtgggtgg ccaccatcta caccagcacc ggcaccacct acttcgacga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccgtgagcgg ctggagcgtg 300
tgcagcagga gcaccaagta caactactgg ggccacggca cccaggtgac cgtgagcagc 360
<210> 34
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gagagcggcg gcggcagcgt gcagaccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcagcta ctgcatggcc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccctgga cagcgacggc agcaccacct acgccgacag cgtgaagggc 180
aggttcaccg tgagcaagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acaccgccat gtactactgc gccgccgacc tgacctgcta ctgcagcgac 300
ggctactgct acaacggcga cgagtacaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 35
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gaaagcggcg gcggcagcgt gcaggcgggc ggcagcctgc gcctgagctg cgtggcgagc 60
ggcgtgacct ttagccgcag ctgcattggc tggtttcgcc agattccggg caaagaacgc 120
gaaggcgtgg cggtggtgta taccggcggc cgcagcattg attatagcgc gtatgtggat 180
agcgtgaaag gccgctttac cattagccat gataacgcgt ggaacaccgt gtatctgcag 240
atgaacagcc tgaaaccgga agataccgcg atgtattatt gcgcggcgga ttttctgctg 300
caggaaccgt gcaccgtgct gagcagccgc ggctttaaat tttggggcca gggcacccag 360
gtgaccgtga gcagc 375
<210> 36
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
gagtacgcct acatcaacta ctgcatgggc tggttcaggc aggtgcccgg caaggagagg 120
gagggcgtgg ccgtgatctg cagcggcggc ggcgccacct actacaccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacggcgcc aagtacaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcaggaggcc ctactacagc 300
ggcgcctact tcaggaccgc cgactacaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 37
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gagagcggcg gcggcagcgc ccaggccggc ggcagcctga ggctgagctg caccgccagc 60
ggcagcggct acttctacag caggctgtgc atgggctggt tcaggcaggc ccccggcacc 120
ggcagggagg ccgtggccac cagctacccc ggcatcggca gcacctacta cgccgacagc 180
gtgaagggca ggttcaccat cagccaggac aacgccaaga acaccatgta cctgcagatg 240
aacagcctga agcccgagga caccgccatg tactactgcg ccgccgacgc cacctggaac 300
acctgcggca tcaccaagac cacctacgcc tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 38
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcaggta ctgcatgggc tggttcaggc agacccccgg caagggcagg 120
gagggcgtgg ccagcatcaa cagcgacggc accaccatct acggcaactt cgtgaagggc 180
aggttcacca tcagcgagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acaccgccat gtactactgc gccgccaggg tggacagcac ctgcgccccc 300
atgcccagct ggttcaccta ctggggccag ggcacccagg tgaccgtgag cagc 354
<210> 39
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga gggtgagctg cgccgccagc 60
ggctacacct acagcagcta ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccagcatcga cagcaggggc agcaccacct acgccgacag cctgaagggc 180
aggttcacca tcagcaagga caacgccaag aacaccctgt acctgcagat gaacaacctg 240
aagcccgagg acaccgccaa gtactactgc gccgccggcg acggctacgg cctggagcac 300
atctgcatcc cccactacta cgccatgaac tactggggcg agggcaccca ggtgaccgtg 360
agcagc 366
<210> 40
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggcttcacct acaggagggc ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatcta caccagcggc ggcatgacct tctacgacgc cagcgtgagg 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc cacctactac tgcgccgccg gcggcggcga ctactgcgcc 300
accaacgcca ggttcttcaa ctactggggc cagggcaccc aggtgaccgt gagcagc 357
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Ala Thr Phe Ile Asn Trp Ser
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Gly Tyr Ile Tyr Ser Arg Asn Cys
1 5
<210> 43
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gly Tyr Thr Tyr Ser Thr Tyr Cys
1 5
<210> 44
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Gly Val Thr Tyr Arg Arg Thr Cys
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Gly Tyr Thr Tyr Ser Gly Ile Cys
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gly Tyr Thr Gly Arg Arg Asn Cys
1 5
<210> 47
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gly Tyr Thr Ser Arg His Cys
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gly Phe Thr Phe Gly Asp Ala Asp
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Gly Asn Met Tyr Arg Arg Asn Cys
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Gly Tyr Thr His Asp Tyr Ser Cys
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gly Tyr Thr Phe Arg Arg Tyr Cys
1 5
<210> 52
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Gly Tyr Thr Ile Asn Tyr Arg
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Gly Ile Thr Tyr Ser Arg Asn Cys
1 5
<210> 54
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Gly Tyr Thr Tyr Ser Ser Tyr Cys
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Gly Val Thr Phe Ser Arg Ser Cys
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Glu Tyr Ala Tyr Ile Asn Tyr Cys
1 5
<210> 57
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Gly Ser Gly Tyr Phe Tyr Ser Arg Leu Cys
1 5 10
<210> 58
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Gly Tyr Thr Tyr Ser Arg Tyr Cys
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Gly Phe Thr Tyr Arg Arg Ala Cys
1 5
<210> 60
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Ile Glu Ser Gly Gly Ser Pro
1 5
<210> 61
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Leu Tyr Thr Ala Gly Gly Tyr Thr
1 5
<210> 62
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Ile Asp Ser Asp Gly Ser Thr
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Ile Tyr Thr Ser Gly Gly Phe Thr
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Ile Tyr Thr Asn Gly Glu Tyr Thr
1 5
<210> 65
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Ile Tyr Ser Gly Asp Ile Thr
1 5
<210> 66
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Leu Tyr Pro Ala Thr Gly Ser Ala
1 5
<210> 67
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Leu Gly Asp Asn Gly Ser Thr
1 5
<210> 68
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Ile Tyr Thr Ala Gly Gly Tyr Ser
1 5
<210> 69
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Met Tyr Thr Gly Gly Ala Ala Thr
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Leu Tyr Thr Gly Val Asp Ile Thr
1 5
<210> 71
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Ala Ser Ala Ile Ser Glu
1 5
<210> 72
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Ile Tyr Thr Ser Thr Gly Thr Thr
1 5
<210> 73
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Leu Asp Ser Asp Gly Ser Thr
1 5
<210> 74
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Val Tyr Thr Gly Gly Arg Ser Ile Asp Tyr Ser
1 5 10
<210> 75
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Ile Cys Ser Gly Gly Gly Ala Thr
1 5
<210> 76
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Ser Tyr Pro Gly Ile Gly Ser Thr
1 5
<210> 77
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Ile Asn Ser Asp Gly Thr Thr
1 5
<210> 78
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Ile Asp Ser Arg Gly Ser Thr
1 5
<210> 79
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Ile Tyr Thr Ser Gly Gly Met Thr
1 5
<210> 80
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Ala Ala Lys Lys Val Trp Ala Arg Glu Phe Ala Tyr
1 5 10
<210> 81
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Ala Ala Ala Arg Pro Ser Asn Ser Leu Thr Cys Gly Ile Ala Thr Ile
1 5 10 15
Thr Thr Asp Asp Val
20
<210> 82
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Ala Ala Leu Ser Pro Arg Cys Asp Phe Asp Arg Ser Ser Arg Val Thr
1 5 10 15
Asn
<210> 83
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Ala Ala Glu Ala Gly Asp Glu Cys Ser Ala Ser Ser Val Phe Arg Ser
1 5 10 15
<210> 84
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Ala Ala Asp Arg Asn Thr Cys Gly Leu Arg Gly Asp Phe Tyr Asn Tyr
1 5 10 15
<210> 85
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Ala Ala Gly Pro Gly Asp Tyr Cys Ser Thr Arg Pro Phe Ala Phe Asn
1 5 10 15
Tyr
<210> 86
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Ala Val Glu Ser Thr Ser Leu Cys Ala Ala Arg Thr Val Pro Ser Phe
1 5 10 15
Lys Phe
<210> 87
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Ala Ala Glu Arg Leu Trp Thr Ala Ser Thr Val Val Pro Asp Thr Pro
1 5 10 15
Cys Leu Asp Glu Ile Glu Asp Phe Gly Tyr
20 25
<210> 88
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Ala Thr Asp Gly Arg Asp Gly Asp Leu Cys Ala Ala Arg Ser Asp Phe
1 5 10 15
Arg Tyr
<210> 89
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Ala Ala Asp Thr Arg Pro Ile Val Asp Arg Trp Cys Gly Leu Pro Trp
1 5 10 15
Pro Leu Tyr
<210> 90
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Ala Ala Asp Ser Gly Pro Leu Cys Gly Leu Gly Ile Gln Phe Gly Tyr
1 5 10 15
<210> 91
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Ala Ala Gly Arg Val Trp Gly Arg Leu Trp Asn Pro Asp Asp Tyr Asn
1 5 10 15
Ser
<210> 92
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Ala Ala Ala Val Ser Gly Trp Ser Val Cys Ser Arg Ser Thr Lys Tyr
1 5 10 15
Asn Tyr
<210> 93
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Ala Ala Asp Leu Thr Cys Tyr Cys Ser Asp Gly Tyr Cys Tyr Asn Gly
1 5 10 15
Asp Glu Tyr Asn Tyr
20
<210> 94
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Ala Ala Asp Phe Leu Leu Gln Glu Pro Cys Thr Val Leu Ser Ser Arg
1 5 10 15
Gly Phe Lys Phe
20
<210> 95
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Ala Ala Gly Arg Arg Pro Tyr Tyr Ser Gly Ala Tyr Phe Arg Thr Ala
1 5 10 15
Asp Tyr Asn Tyr
20
<210> 96
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Ala Ala Asp Ala Thr Trp Asn Thr Cys Gly Ile Thr Lys Thr Thr Tyr
1 5 10 15
Ala Tyr
<210> 97
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Ala Ala Arg Val Asp Ser Thr Cys Ala Pro Met Pro Ser Trp Phe Thr
1 5 10 15
Tyr
<210> 98
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Ala Ala Gly Asp Gly Tyr Gly Leu Glu His Ile Cys Ile Pro His Tyr
1 5 10 15
Tyr Ala Met Asn Tyr
20
<210> 99
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Ala Ala Gly Gly Gly Asp Tyr Cys Ala Thr Asn Ala Arg Phe Phe Asn
1 5 10 15
Tyr
<210> 100
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser
20
<210> 101
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 102
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Thr Ser
20
<210> 103
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser
20
<210> 104
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Ala Ser
20
<210> 105
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Val Ser
20
<210> 106
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Val Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 107
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 108
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 109
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 110
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val Ala
1 5 10 15
Ala
<210> 111
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile Ala
1 5 10 15
Ala
<210> 112
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 112
Met Ala Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Gly
<210> 113
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 113
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 114
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 114
Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 115
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 115
Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser
1 5 10 15
Ser
<210> 116
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 116
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 117
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 117
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala
1 5 10 15
Ser
<210> 118
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 118
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Val
<210> 119
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 119
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Met
<210> 120
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala
1 5 10 15
Thr
<210> 121
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 121
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 122
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 122
Ile Gly Trp Phe Arg Gln Ile Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Val
<210> 123
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 123
Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Val
<210> 124
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 124
Met Gly Trp Phe Arg Gln Ala Pro Gly Thr Gly Arg Glu Ala Val Ala
1 5 10 15
Thr
<210> 125
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 125
Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Gly Arg Glu Gly Val Ala
1 5 10 15
Ser
<210> 126
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 126
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ser
<210> 127
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 127
Thr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Leu Ser Thr Asp Val
1 5 10 15
Ala Lys Asn Ile Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Ala Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 128
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 128
Tyr Tyr Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 129
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 129
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ala Lys Asp Thr Leu Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 130
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 130
Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp
20 25 30
Thr Gly Ile Tyr Tyr Cys
35
<210> 131
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 131
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 132
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 132
Phe Tyr Gly Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp
1 5 10 15
Ala Lys Asn Ala Val Tyr Leu Gln Met Asp Arg Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 133
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 133
Phe Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp
20 25 30
Thr Ala Leu Tyr Tyr Cys
35
<210> 134
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 134
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 135
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 135
Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Pro Lys Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 136
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 136
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ala Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 137
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 137
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Leu Tyr Tyr Cys
35
<210> 138
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 138
Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Ser
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 139
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 139
Tyr Phe Asp Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 140
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 140
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Lys Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 141
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 141
Ala Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser His Asp Asn
1 5 10 15
Ala Trp Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 142
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 142
Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Gly
1 5 10 15
Ala Lys Tyr Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 143
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 143
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 144
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 144
Ile Tyr Gly Asn Phe Val Lys Gly Arg Phe Thr Ile Ser Glu Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 145
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 145
Thr Tyr Ala Asp Ser Leu Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp
20 25 30
Thr Ala Lys Tyr Tyr Cys
35
<210> 146
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 146
Phe Tyr Asp Ala Ser Val Arg Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Thr Tyr Tyr Cys
35
<210> 147
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 147
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 148
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 148
Thr Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 149
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 149
Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 150
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 150
Trp Gly His Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 151
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 151
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 152
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 152
Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
1 5 10

Claims (8)

1. Single domain antibody against sST2, characterized in that: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR 3;
the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are as follows (1) or (2):
(1) CDR1 shown in SEQ ID NO. 48, CDR2 shown in SEQ ID NO. 67, CDR3 shown in SEQ ID NO. 87;
(2) CDR1 shown in SEQ ID NO. 42, CDR2 shown in SEQ ID NO. 61, and CDR3 shown in SEQ ID NO. 81.
2. The single domain antibody against sST2 according to claim 1, wherein: the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 8 or SEQ ID NO. 2.
3. Fc fusion antibody according to claim 1 or 2 against a single-domain antibody to sST 2.
4. A nucleotide molecule encoding the single domain antibody to sST2 according to claim 1 or 2, wherein: the nucleotide sequences are respectively shown as SEQ ID NO: 28. SEQ ID NO: 22, respectively.
5. An expression vector comprising a nucleotide molecule encoding the single domain antibody of claim 1 or 2 or the Fc fusion antibody of claim 3 or the nucleotide molecule of claim 4.
6. A host cell capable of expressing the single domain antibody against sST2 of claim 1 or 2, or comprising the expression vector of claim 5.
7. sST2-ELISA detection kit comprising the single domain antibody of claim 1 or 2.
8. Use of the single domain antibody against sST2 according to claim 1 or 2 in the preparation of a sST2-ELISA test kit, or use of the single domain antibody against sST2 according to claim 1 or 2 in the preparation of a kit for detecting sST2 protein in a human, or use of the single domain antibody against sST2 according to claim 1 or 2 in the isolation and purification of a sST2 protein fusion protein.
CN202111491678.0A 2021-12-08 2021-12-08 Single-domain antibody aiming at sST2, and derivative protein and application thereof Active CN113912729B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111491678.0A CN113912729B (en) 2021-12-08 2021-12-08 Single-domain antibody aiming at sST2, and derivative protein and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111491678.0A CN113912729B (en) 2021-12-08 2021-12-08 Single-domain antibody aiming at sST2, and derivative protein and application thereof

Publications (2)

Publication Number Publication Date
CN113912729A CN113912729A (en) 2022-01-11
CN113912729B true CN113912729B (en) 2022-03-04

Family

ID=79248903

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111491678.0A Active CN113912729B (en) 2021-12-08 2021-12-08 Single-domain antibody aiming at sST2, and derivative protein and application thereof

Country Status (1)

Country Link
CN (1) CN113912729B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103154027A (en) * 2010-04-09 2013-06-12 重症监护诊断股份有限公司 Soluble human st-2 antibodies and assays
CN112920275A (en) * 2019-12-06 2021-06-08 菲鹏生物股份有限公司 Binding proteins, reagents and kits that specifically bind to sST2

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103154027A (en) * 2010-04-09 2013-06-12 重症监护诊断股份有限公司 Soluble human st-2 antibodies and assays
CN112920275A (en) * 2019-12-06 2021-06-08 菲鹏生物股份有限公司 Binding proteins, reagents and kits that specifically bind to sST2

Also Published As

Publication number Publication date
CN113912729A (en) 2022-01-11

Similar Documents

Publication Publication Date Title
CN111018985B (en) Application of single-domain antibody aiming at bovine serum albumin BSA
CN111057148B (en) Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof
CN109970858B (en) CD22 single domain antibody, nucleotide sequence and kit
CN113150136B (en) Preparation of novel coronavirus N protein monoclonal antibody
CN110003335B (en) CD47 single domain antibody, nucleic acid and kit
CN110003336B (en) PD-1 single domain antibody, nucleotide sequence and kit
CN111793132A (en) Monoclonal antibody of human procalcitonin and preparation method and application thereof
CN111138533B (en) Single domain antibody against hepatitis A virus and derived protein thereof
CN112321708A (en) Peanut allergen Arah3 specific nano antibody and application thereof
CN114276445B (en) Rotavirus recombinant protein specific antibody, plasmid vector and method
CN110423277B (en) Nano antibody of PD-1 and clinical application thereof
CN111138532B (en) Use of single domain antibodies against hepatitis a virus
CN105218677A (en) Anti-triazophos single-chain antibody and application thereof
CN113912729B (en) Single-domain antibody aiming at sST2, and derivative protein and application thereof
CN109734804B (en) Nano antibody aiming at H3K64Ac/H3K64 fragment and application thereof
CN112010969A (en) High-affinity enhanced green fluorescent protein nano antibody and screening method of coding gene thereof
CN114805559B (en) Fully human anti-new coronavirus receptor binding domain single-chain antibody No4 and its application
CN110903359B (en) Preparation of campylobacter jejuni recombinant protein and monoclonal antibody thereof
CN114213543A (en) Anti-canine pancreatic lipase specific single-chain antibody, plasmid vector and method
CN116987194B (en) Anti-idiotype nano antibody of mimic epitope peptide of human ST2 antigen and application thereof
CN112390880A (en) Lupin allergen Lupan1 specific nano antibody and application thereof
CN110003334B (en) Polypeptide, CD19 single domain antibody, preparation method thereof, nucleotide sequence and kit
CN117866100B (en) Single-domain antibodies or antigen-binding fragments thereof directed against D-dimer and related biomaterials and uses thereof
CN110066337B (en) anti-TNF-alpha antibody
CN111499739B (en) Antibody and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant