CN116836271A - Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus - Google Patents

Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus Download PDF

Info

Publication number
CN116836271A
CN116836271A CN202210289992.9A CN202210289992A CN116836271A CN 116836271 A CN116836271 A CN 116836271A CN 202210289992 A CN202210289992 A CN 202210289992A CN 116836271 A CN116836271 A CN 116836271A
Authority
CN
China
Prior art keywords
thr
ser
val
gly
pro
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.)
Granted
Application number
CN202210289992.9A
Other languages
Chinese (zh)
Other versions
CN116836271B (en
Inventor
孟媛
钟冬梅
唐丽娜
曹慧方
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.)
Dongguan Pengzhi Biotechnology Co Ltd
Original Assignee
Dongguan Pengzhi 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 Dongguan Pengzhi Biotechnology Co Ltd filed Critical Dongguan Pengzhi Biotechnology Co Ltd
Priority to CN202210289992.9A priority Critical patent/CN116836271B/en
Publication of CN116836271A publication Critical patent/CN116836271A/en
Application granted granted Critical
Publication of CN116836271B publication Critical patent/CN116836271B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Landscapes

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

Abstract

The application discloses an antibody for resisting type-B streptococcus, a reagent and a kit for detecting type-B streptococcus, and relates to the technical field of antibodies. The anti-type B streptococcus antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for the detection of the streptococcus B.

Description

Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus
Technical Field
The application relates to the technical field of antibodies, in particular to an antibody for resisting type-B streptococcus, a reagent for detecting type-B streptococcus and a kit.
Background
Streptococcus B (group B streptococci, GBS), also known as Streptococcus agalactiae, is a gram positive bacterium that is commonly colonized in the gastrointestinal tract, perineum and vagina, and is found in the intestinal tract or vagina of approximately 10-35% of healthy women. In most cases it is harmless, but when pregnant women infect GBS they will be transmitted to the neonate at the time of delivery, which is one of the major causes of neonatal infection and death, with mortality rates of up to 4-6% for infected neonates. GBS causes severe pulmonary infections and is susceptible to systemic sepsis due to immature neonatal immunity, and is associated with premature delivery, low weight, early water loss, onset within one week of neonatal birth, and rapid exacerbation of symptoms within 48 hours to death. Bacterial meningitis can be caused if the brain is infected, and even if the brain is cured, neurological sequelae such as cerebral palsy, epilepsy, intelligent disorder and the like can be caused. It is recommended by the American society of obstetrics and gynecology that a check of Streptococcus B be made, preferably at 35 to 37 weeks. GBS also causes invasive infections in the elderly and in adults with susceptibility factors (e.g., pregnancy, diabetes, and low immunity).
Immunological detection methods are a means for detecting antibodies and antigens based on specific reactions, and are often used for detecting micro-bioactive substances such as microorganisms, proteins, and hormones because the detected signals can be amplified and displayed by isotopes, enzymes, chemiluminescent substances, and the like. Currently, the GBS detection method mainly includes a colloidal gold immunochromatography method, which is an immunological detection method that uses colloidal gold to amplify and display a detected signal. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunoassay, chemiluminescent method, etc. The immunological detection methods described above all require antibodies directed against GBS. Thus, there is a strong need in the art for antibodies that are effective and bind to GBS and detect it.
In view of this, the present application has been made.
Disclosure of Invention
Aiming at the problem of less sources of the existing anti-B-type streptococcus antibodies, the application provides an anti-B-type streptococcus antibody with improved activity, and provides an important raw material source for detecting the B-type streptococcus.
In order to achieve the above object, according to one aspect of the present application, there is provided an antibody or a functional fragment thereof comprising HCDR1 having an amino acid sequence shown in SEQ ID NO. 1, HCDR2 having an amino acid sequence shown in SEQ ID NO. 2 or 15, HCDR3 having an amino acid sequence shown in SEQ ID NO. 3, and LCDR1, LCDR2, LCDR3 having an amino acid sequence shown in SEQ ID NO. 4 to SEQ ID NO. 6.
In order to achieve the above object, according to a second aspect of the present application, there is provided an anti-type B streptococcus antibody or a functional fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present application, there is provided an anti-type B streptococcus antibody comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present application, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present application, there is provided a reagent or kit for detecting streptococcus B, the reagent or kit comprising the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.
In order to achieve the above object, according to a sixth aspect of the present application there is provided the use of an antibody as defined above or a functional fragment thereof, an antibody conjugate as defined above or a reagent or kit as defined above in the detection of streptococcus B.
In order to achieve the above object, the present application also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of reducing SDS-PAGE of Anti-GBS-4G7mut1 to Anti-GBS-4G7mut4.
Detailed Description
The application provides an antibody or a functional fragment thereof, which comprises HCDR1 with an amino acid sequence shown as SEQ ID NO. 1, HCDR2 with an amino acid sequence shown as SEQ ID NO. 2 or 15, HCDR3 with an amino acid sequence shown as SEQ ID NO. 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4 to SEQ ID NO. 6. The antibodies have improved affinity and activity.
In the present application, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.
In the present application, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present application, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, and the 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The application adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the application.
In the present application, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present application, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, HFR3 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 16.
In an alternative embodiment, LFR3 of said antibody or functional fragment thereof is shown in SEQ ID NO. 17.
In another aspect, embodiments of the present application provide an antibody or a functional fragment thereof against streptococcus B, the antibody or the functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, HFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR4, or the amino acid sequence of HCDR1, HCDR 3.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 18 to 21.
In an alternative embodiment, the light chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 22 to 23.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is derived from rabbit species.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 24 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 25.
In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the above-described constant region (SEQ ID NO:24 or 25).
In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the application provides an antibody against streptococcus B comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs 26 to 29.
In an alternative embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs 30 to 31.
In another aspect, the application provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the application.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (chlorophyll), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a nitrocellulose membrane.
In another aspect, the application provides a reagent or kit for detecting streptococcus B, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.
In another aspect, the application provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate or a reagent or kit as described above in the detection of streptococcus B.
In another aspect, the application provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the application provides a vector comprising the nucleic acid molecule described above.
In another aspect, the application provides a cell comprising the vector described above.
In another aspect, the application provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
On the basis of the present application, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present application to prepare the antibody or the functional fragment thereof by any technique.
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present application will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present application are described in further detail below in connection with the examples.
Example 1 preparation of Anti-GBS-4G7 monoclonal antibodies
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMARTTMRACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting anti-B streptococcus monoclonal antibodies, DNA products are obtained through an RT-PCR method, rTaq DNA polymerase is used for carrying out an A adding reaction on the products, the products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain gene clone and the Light Chain gene clone are respectively taken, and 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of Anti-GBS-4G7 antibody variable region genes
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 336bp, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 342bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene family.
(3) Construction of recombinant antibody expression plasmids
pcDNATM3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.72kb Light Chain gene fragment and a 1.39kb Heavy Chain gene fragment are amplified by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
2 stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube at cells/mlAnd (3) turning into an electric rotating cup, carrying out electric rotation, sampling and counting on days 3, 5 and 7, and collecting samples and detecting on day 7.
The GBS cultures were diluted 1000-fold with coating solution (NaHCO 3 as main ingredient) 100uL per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-rabbit IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50. Mu.L/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0 and that the reaction OD without cell supernatant wells was less than 0.1, indicating that antibodies produced after transient plasmid transformation were active on GBS cultures.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strain with high antibody concentration and relative concentration, and transferring to 24 holes for about 3 daysRight turn 6 holes; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 6 Performing seed preservation by using cells/ml and 2 ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.
Example 2 Activity optimization
The Anti-GBS-4G7 monoclonal antibody obtained in example 1 was not sufficiently active on GBS cultures, but was not sufficiently active, and thus, the applicant had performed directed mutation on the light chain CDRs and the heavy chain CDRs of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. The monoclonal antibody with remarkably improved antibody activity is obtained through screening and is named as: anti-GBS-4G7mut1 to Anti-GBS-4G7mut4. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:
TABLE 1 antibody sequences
Sample name Heavy chain sequence number Light chain sequence number
Anti-GBS-4G7mut1 SEQ ID NO:26 SEQ ID NO:30
Anti-GBS-4G7mut2 SEQ ID NO:27 SEQ ID NO:31
Anti-GBS-4G7mut3 SEQ ID NO:28 SEQ ID NO:31
Anti-GBS-4G7mut4 SEQ ID NO:29 SEQ ID NO:31
Example 3 detection of Performance of antibodies
1 Activity assay
The GBS cultures were diluted 1000-fold with coating solution (NaHCO 3 as main ingredient) 100uL per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding goat anti-rabbit IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in the following table.
TABLE 2 Activity data
Sample concentration (ng/ml) 60 30 15 7.5 3.75 0
Control 1.332 0.791 0.462 0.285 0.182 0.031
Anti-GBS-4G7mut1 1.564 1.015 0.755 0.431 0.209 0.043
Anti-GBS-4G7mut2 1.868 1.488 0.982 0.517 0.302 0.021
Anti-GBS-4G7mut3 1.669 1.175 0.767 0.443 0.217 0.052
Anti-GBS-4G7mut4 1.96 1.549 0.976 0.602 0.347 0.057
2 evaluation of Performance
The antibody is used as a coating antibody, and is matched with another Anti-GBS labeled end monoclonal antibody respectively, the performance difference of the antibody and a control (market mainstream antibody) is compared on a colloidal gold platform, and the prepared antibody can achieve better performance level than the control. The specific properties are shown in the following table:
table 3 performance evaluation data
3 stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. The following table shows the OD results of the enzyme-free activity assay for 21 days of antibody assessment.
Table 4 stability data
Sample concentration (ng/ml) 30 7.5 0
4 ℃,21 days sample 1.616 0.705 0.009
Sample at-80℃for 21 days 1.623 0.698 0.011
37 ℃ and 21 days of sample 1.661 0.701 0.016
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
The partial amino acid sequence related to the application is as follows:
sequence numbering Sequence fragments
SEQ ID NO:1 RNAMS
SEQ ID NO:2 LIAIGGATFYPNWAKG
SEQ ID NO:3 EMAGSF
SEQ ID NO:4 QSSQSVLDNKNLA
SEQ ID NO:5 KASSPAS
SEQ ID NO:6 AGEYYSSGYY
SEQ ID NO:15 IIAIGGATFYPNWAKG
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> antibodies against Streptococcus type B, reagents and kits for detecting Streptococcus type B
<130> P2022031CN01
<160> 31
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 1
Arg Asn Ala Met Ser
1 5
<210> 2
<211> 16
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 2
Leu Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
1 5 10 15
<210> 3
<211> 6
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 3
Glu Met Ala Gly Ser Phe
1 5
<210> 4
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 4
Gln Ser Ser Gln Ser Val Leu Asp Asn Lys Asn Leu Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 5
Lys Ala Ser Ser Pro Ala Ser
1 5
<210> 6
<211> 10
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 6
Ala Gly Glu Tyr Tyr Ser Ser Gly Tyr Tyr
1 5 10
<210> 7
<211> 29
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 7
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser
20 25
<210> 8
<211> 14
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 8
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
1 5 10
<210> 9
<211> 30
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 9
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Leu Thr
1 5 10 15
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
20 25 30
<210> 10
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 10
Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 11
<211> 22
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 11
Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly
1 5 10 15
Thr Val Thr Ile Asn Cys
20
<210> 12
<211> 15
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 12
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile Tyr
1 5 10 15
<210> 13
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 13
Gly Val Pro Ser Arg Phe Ser Gly Asn Gly Ser Gly Thr Gln Phe Thr
1 5 10 15
Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
20 25 30
<210> 14
<211> 12
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 14
Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys
1 5 10
<210> 15
<211> 16
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 15
Ile Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
1 5 10 15
<210> 16
<211> 30
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 16
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Ile Thr
1 5 10 15
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
20 25 30
<210> 17
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 17
Gly Val Pro Ser Arg Phe Ser Gly Asp Gly Ser Gly Thr Gln Phe Thr
1 5 10 15
Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
20 25 30
<210> 18
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 18
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Leu Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Leu Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 19
<211> 113
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 19
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Ile Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 20
<211> 113
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 20
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Leu Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 21
<211> 113
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 21
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Ile Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Leu Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 22
<211> 111
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 22
Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly
1 5 10 15
Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Asp Asn Lys
20 25 30
Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu
35 40 45
Ile Tyr Lys Ala Ser Ser Pro Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Asn Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln
65 70 75 80
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Glu Tyr Tyr Ser Ser
85 90 95
Gly Tyr Tyr Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 110
<210> 23
<211> 111
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 23
Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly
1 5 10 15
Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Asp Asn Lys
20 25 30
Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu
35 40 45
Ile Tyr Lys Ala Ser Ser Pro Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Asp Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln
65 70 75 80
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Glu Tyr Tyr Ser Ser
85 90 95
Gly Tyr Tyr Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 110
<210> 24
<211> 323
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 24
Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly
1 5 10 15
Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn
35 40 45
Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys
65 70 75 80
Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala
85 90 95
Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly
100 105 110
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
115 120 125
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
130 135 140
Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val
145 150 155 160
Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile
165 170 175
Arg Val Val Ser Thr Leu Pro Ile Thr His Gln Asp Trp Leu Arg Gly
180 185 190
Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile
195 200 205
Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val
210 215 220
Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser
225 230 235 240
Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu
245 250 255
Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala
260 265 270
Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser Val
275 280 285
Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met
290 295 300
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser
305 310 315 320
Pro Gly Lys
<210> 25
<211> 104
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 25
Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp
1 5 10 15
Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr
20 25 30
Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr
35 40 45
Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr
50 55 60
Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser
65 70 75 80
His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val
85 90 95
Gln Ser Phe Asn Arg Gly Asp Cys
100
<210> 26
<211> 436
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 26
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Leu Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Leu Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys
115 120 125
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly
130 135 140
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr
145 150 155 160
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr
180 185 190
Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
195 200 205
Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu
210 215 220
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
260 265 270
Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr
275 280 285
Ile Arg Val Val Ser Thr Leu Pro Ile Thr His Gln Asp Trp Leu Arg
290 295 300
Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
325 330 335
Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val
340 345 350
Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val
355 360 365
Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro
370 375 380
Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser
385 390 395 400
Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg
420 425 430
Ser Pro Gly Lys
435
<210> 27
<211> 436
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 27
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Ile Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys
115 120 125
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly
130 135 140
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr
145 150 155 160
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr
180 185 190
Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
195 200 205
Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu
210 215 220
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
260 265 270
Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr
275 280 285
Ile Arg Val Val Ser Thr Leu Pro Ile Thr His Gln Asp Trp Leu Arg
290 295 300
Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
325 330 335
Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val
340 345 350
Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val
355 360 365
Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro
370 375 380
Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser
385 390 395 400
Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg
420 425 430
Ser Pro Gly Lys
435
<210> 28
<211> 436
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 28
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Leu Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys
115 120 125
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly
130 135 140
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr
145 150 155 160
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr
180 185 190
Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
195 200 205
Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu
210 215 220
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
260 265 270
Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr
275 280 285
Ile Arg Val Val Ser Thr Leu Pro Ile Thr His Gln Asp Trp Leu Arg
290 295 300
Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
325 330 335
Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val
340 345 350
Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val
355 360 365
Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro
370 375 380
Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser
385 390 395 400
Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg
420 425 430
Ser Pro Gly Lys
435
<210> 29
<211> 436
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 29
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Asn Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Ser Ile Gly
35 40 45
Ile Ile Ala Ile Gly Gly Ala Thr Phe Tyr Pro Asn Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Gly Thr Ser Thr Thr Val Asp Leu Arg Leu Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Met
85 90 95
Ala Gly Ser Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys
115 120 125
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly
130 135 140
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr
145 150 155 160
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr
180 185 190
Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
195 200 205
Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu
210 215 220
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
260 265 270
Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr
275 280 285
Ile Arg Val Val Ser Thr Leu Pro Ile Thr His Gln Asp Trp Leu Arg
290 295 300
Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
325 330 335
Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val
340 345 350
Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val
355 360 365
Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro
370 375 380
Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser
385 390 395 400
Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg
420 425 430
Ser Pro Gly Lys
435
<210> 30
<211> 215
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 30
Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly
1 5 10 15
Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Asp Asn Lys
20 25 30
Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu
35 40 45
Ile Tyr Lys Ala Ser Ser Pro Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Asn Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln
65 70 75 80
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Glu Tyr Tyr Ser Ser
85 90 95
Gly Tyr Tyr Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly
100 105 110
Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln
115 120 125
Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe
130 135 140
Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr
145 150 155 160
Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr
165 170 175
Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His
180 185 190
Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln
195 200 205
Ser Phe Asn Arg Gly Asp Cys
210 215
<210> 31
<211> 215
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 31
Gln Val Leu Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly
1 5 10 15
Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Leu Asp Asn Lys
20 25 30
Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu
35 40 45
Ile Tyr Lys Ala Ser Ser Pro Ala Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Asp Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Gln
65 70 75 80
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Glu Tyr Tyr Ser Ser
85 90 95
Gly Tyr Tyr Tyr Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly
100 105 110
Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln
115 120 125
Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe
130 135 140
Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr
145 150 155 160
Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr
165 170 175
Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His
180 185 190
Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln
195 200 205
Ser Phe Asn Arg Gly Asp Cys
210 215

Claims (12)

1. An antibody or functional fragment thereof against streptococcus B, wherein said antibody or functional fragment thereof comprises HCDR1 having the amino acid sequence shown in SEQ ID No. 1, HCDR2 having the amino acid sequence shown in SEQ ID No. 2 or 15, HCDR3 having the amino acid sequence shown in SEQ ID No. 3, and LCDR1, LCDR2, LCDR3 having the amino acid sequence shown in SEQ ID No. 4 to SEQ ID No. 6.
2. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10 and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14; or an amino acid sequence having at least 80% homology to each of the above sequences;
optionally, the amino acid sequence of HFR3 of the antibody or functional fragment thereof is shown in SEQ ID NO. 16;
alternatively, the amino acid sequence of LFR3 of said antibody or functional fragment thereof is shown as SEQ ID NO. 17.
3. An anti-streptococcus B antibody or a functional fragment thereof, wherein said antibody or functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, LFR1, LFR2, LFR3, LFR4 of the amino acid sequence of HFR1, HFR2, HFR3, LCDR3, LFR4 of claim 1, HFR2, HFR3, LFR4;
alternatively, the heavy chain variable region amino acid sequence is as shown in any one of SEQ ID NOs 18 to 21;
alternatively, the light chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 22 to 23.
4. The antibody or functional fragment thereof according to any one of claims 1 to 3, wherein the antibody or functional fragment thereof further comprises a constant region;
optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;
alternatively, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD; the light chain constant region is selected from a kappa-type or lambda-type light chain constant region;
alternatively, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin;
optionally, the constant region is derived from a species of rabbit;
alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 24 or has at least 80% homology thereto;
alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 25 or has at least 80% homology thereto.
5. The antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
6. An anti-streptococcus B antibody comprising a heavy chain and/or a light chain, wherein the heavy chain comprises the heavy chain variable region of claim 3 and the heavy chain constant region of claim 4; the light chain comprises the light chain variable region of claim 3 and the light chain constant region of claim 4;
alternatively, the heavy chain has an amino acid sequence as set forth in any one of SEQ ID NOs 26 to 29;
alternatively, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs 30 to 31.
7. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1 to 6;
optionally, the antibody or functional fragment thereof is labeled with a label;
optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;
optionally, the fluorescent dye is selected from fluorescein dye and its derivative, rhodamine dye and its derivative, cy series dye and its derivative, alexa series dye and its derivative, and protein dye and its derivative;
alternatively, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase;
optionally, the radioisotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110 msin, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F;
optionally, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, clorine and its derivatives, and peroxyoxalate and its derivatives;
optionally, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;
optionally, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex;
optionally, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver, and colloidal selenium;
optionally, the antibody or functional fragment thereof is coated to a solid phase;
alternatively, the solid phase is selected from the group consisting of microspheres, plates, and membranes;
alternatively, the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.
8. A reagent or kit for detecting streptococcus B, comprising the antibody or functional fragment thereof according to any one of claims 1 to 6 or the antibody conjugate according to claim 7.
9. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1 to 6.
10. A vector comprising the nucleic acid of claim 9.
11. A cell comprising the nucleic acid of claim 9 or the vector of claim 10.
12. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 6, comprising: culturing the cell of claim 11.
CN202210289992.9A 2022-03-23 2022-03-23 Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus Active CN116836271B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210289992.9A CN116836271B (en) 2022-03-23 2022-03-23 Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210289992.9A CN116836271B (en) 2022-03-23 2022-03-23 Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus

Publications (2)

Publication Number Publication Date
CN116836271A true CN116836271A (en) 2023-10-03
CN116836271B CN116836271B (en) 2024-04-05

Family

ID=88160379

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210289992.9A Active CN116836271B (en) 2022-03-23 2022-03-23 Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus

Country Status (1)

Country Link
CN (1) CN116836271B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106932574A (en) * 2017-03-04 2017-07-07 天津市宝坻区人民医院 The detection method of Type B streptococcal infection

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106932574A (en) * 2017-03-04 2017-07-07 天津市宝坻区人民医院 The detection method of Type B streptococcal infection

Also Published As

Publication number Publication date
CN116836271B (en) 2024-04-05

Similar Documents

Publication Publication Date Title
CN116693676B (en) Anti-mycoplasma pneumoniae antibody, reagent for detecting mycoplasma pneumoniae and kit
CN116554315A (en) Antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses
CN117088982A (en) Anti-methamphetamine antibodies, reagents and kits for detecting methamphetamine
CN116836271B (en) Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus
CN117088983B (en) Anti-amphetamine antibodies, reagents and kits for detecting amphetamine
CN117285638B (en) Anti-buprenorphine antibodies, reagents and kits for the detection of buprenorphine
CN116836279B (en) Anti-tetraiodothyroxine antibody, reagent and kit for detecting tetraiodothyroxine
CN116693678B (en) Anti-mycoplasma pneumoniae antibody, reagent for detecting mycoplasma pneumoniae and kit
CN116836273B (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN116693683B (en) Anti-testosterone antibody, reagent for detecting testosterone and kit
CN116444656B (en) Antibody, reagent and method for identifying novel crown mutant antigen
CN116496401B (en) Anti-abnormal prothrombin antibody, reagent for detecting abnormal prothrombin and kit
CN117285639B (en) Anti-ethyl glucuronide antibody, and reagent and kit for detecting ethyl glucuronide
CN116693677B (en) Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis
CN117088984B (en) Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine
CN116836278B (en) Anti-progesterone antibody, kit for detecting progesterone
CN116854812A (en) Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis
CN116925216A (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN116836274A (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN117088975A (en) Anti-albumin antibody, reagent and kit for detecting albumin
CN117402239B (en) Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit
CN117229408A (en) Anti-barbital compound antibody, reagent for detecting barbital drugs and kit
CN116903734A (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN116496394A (en) Antibodies against S100 protein, reagents and kits for detecting S100 protein
CN117327177A (en) Anti-adiponectin antibody, reagent for detecting adiponectin and kit

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