WO2023035988A1 - 一种抗登革ns1蛋白的抗体及其应用 - Google Patents

一种抗登革ns1蛋白的抗体及其应用 Download PDF

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WO2023035988A1
WO2023035988A1 PCT/CN2022/115261 CN2022115261W WO2023035988A1 WO 2023035988 A1 WO2023035988 A1 WO 2023035988A1 CN 2022115261 W CN2022115261 W CN 2022115261W WO 2023035988 A1 WO2023035988 A1 WO 2023035988A1
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antibody
seq
antigen
amino acid
acid sequence
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French (fr)
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孟媛
钟冬梅
陈晓倩
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东莞市朋志生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the technical field of antibodies. More specifically, it relates to an antibody against dengue NS1 protein and its application.
  • DF Dengue fever
  • DENV-2 Dexavirus-derived virus
  • DENV-3 Dexavirus-derived virus
  • DENV-4 Dengue fever
  • Aedes aegypti Aedes albopictus
  • DF is an arbovirus disease with the widest distribution, the most incidence, and great harm. It is widely prevalent in more than 100 countries and regions in tropical and subtropical Africa, America, Southeast Asia and the Western Pacific.
  • dengue fever diagnosis include: 1) epidemiological data, activities in the 15 days before the onset, whether you have been to endemic areas, mosquito bite history; 2) clinical features, sudden onset, fever, "three pains and three reds", skin 3) laboratory examination, white blood cells, platelets decreased; detection of serum characteristic IgM positive; convalescent IgG 4 times higher than the acute phase; isolated to virus or specific antigen.
  • Clinically used dengue virus detection methods include virus culture, serological detection, and viral nucleic acid detection.
  • the colloidal gold-labeled immunochromatographic method is fast, simple, does not need to rely on important equipment, and can realize on-site detection. It has become a research hotspot in the rapid diagnosis of infectious diseases.
  • NS1 protein is the only glycoprotein in the non-structural protein of dengue virus (also known as dengue virus), which is highly antigenic and does not cause antibody-dependent infection enhancement (Antibody-dependent enhancement, ADE), so it is used as the target of colloidal gold detection .
  • the detection of colloidal gold requires specific monoclonal antibodies against the NS1 protein, and traditional clinical use is of mouse-derived monoclonal antibodies. Now the mainstream Dengue monoclonal antibody raw materials in the market are imported, the price is high, and the specificity and sensitivity need to be improved.
  • the technical problem to be solved in this disclosure is to overcome the defects of the specificity and sensitivity of the existing Dengue monoclonal antibody raw materials.
  • the antibody affinity, reactivity, specificity and sensitivity of the anti-Dengue NS1 protein prepared by the disclosure are obviously better than the mainstream in the market. Antibody.
  • Heavy chain CDR1 which comprises the amino acid sequence S-G-Y-W-N shown in SEQ ID NO.1, or consists of it;
  • Heavy chain CDR2 which comprises the amino acid sequence Y-I-T-Y-S-G-N-T-Y-Y-N-P-S-I-K-S shown in SEQ ID NO.2, or consists of it;
  • Heavy chain CDR3 which comprises the amino acid sequence Y-R-N-G-N-S-I shown in SEQ ID NO.3, or consists of it;
  • antibody or antigen-binding fragment further comprises:
  • Light chain CDR1 which comprises the amino acid sequence R-A-S-Q-D-I-S-N-Y-L-N shown in SEQ ID NO.4, or consists of it;
  • Light chain CDR2 which comprises the amino acid sequence Y-T-S-R-L-H-S shown in SEQ ID NO.5, or consists of it;
  • Light chain CDR3 which comprises the amino acid sequence Q-Q-G-D-T-I-P shown in SEQ ID NO.6, or consists of it.
  • Another object of the present disclosure is to provide nucleic acids, vectors or cells related to the antibody or antigen-binding fragment.
  • the present disclosure also provides methods of making the antibodies or antigen-binding fragments.
  • the present disclosure also provides an antibody conjugate, and a kit/diagnostic reagent comprising the above-mentioned antibody or antigen-binding fragment or the antibody conjugate.
  • the present disclosure also provides the use of the antibody or antigen-binding fragment or the antibody conjugate in the preparation of a kit or a diagnostic reagent.
  • Fig. 1 is the reducing SDS-PAGE result of DF-8F13R antibody.
  • the present disclosure will be further described below in conjunction with specific examples, but the examples do not limit the present disclosure in any form.
  • the reagents, methods and equipment used in the present disclosure are conventional reagents, methods and equipment in the art.
  • the present disclosure relates to an antibody or antigen-binding fragment comprising the following CDRs:
  • Heavy chain CDR1 which comprises the amino acid sequence S-G-Y-W-N shown in SEQ ID NO.1, or consists of it;
  • Heavy chain CDR2 which comprises the amino acid sequence Y-I-T-Y-S-G-N-T-Y-Y-N-P-S-I-K-S shown in SEQ ID NO.2, or consists of it;
  • Heavy chain CDR3 which comprises the amino acid sequence Y-R-N-G-N-S-I shown in SEQ ID NO.3, or consists of it;
  • antibody or antigen-binding fragment further comprises:
  • Light chain CDR1 which comprises the amino acid sequence R-A-S-Q-D-I-S-N-Y-L-N shown in SEQ ID NO.4, or consists of it;
  • Light chain CDR2 which comprises the amino acid sequence Y-T-S-R-L-H-S shown in SEQ ID NO.5, or consists of it;
  • Light chain CDR3 which comprises the amino acid sequence Q-Q-G-D-T-I-P shown in SEQ ID NO.6, or consists of it.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
  • antigen-binding fragment is a substance comprising part or all of the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but which is still capable of specifically binding to the antigen. Such fragments are biologically active in that they bind to the antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope.
  • Such fragments are selected from Fab (composed of complete light chain and Fd), Fv (composed of VH and VL), scFv (single chain antibody, VH and VL are connected by a linker peptide) or single domain antibody ( Consists of VH only).
  • Such fragments can be produced by recombinant nucleic acid techniques, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • CDRs refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more or even all of the anti-antibody The region of major amino acid residues that contribute to the binding affinity of an antigen-binding fragment thereof to the antigen or epitope it recognizes.
  • CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.
  • CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
  • framework region or "FR” region includes the heavy chain framework region and the light chain framework region, and refers to the region except the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein, heavy Chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; light chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1 , HFR2, HFR3 and HFR4 framework regions.
  • the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs Arrange and connect as follows: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the antibody further comprises framework regions HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region, and framework regions LFR1, LFR2, LFR3, and LFR4 of the light chain variable region, wherein:
  • HFR1 comprises the amino acid sequence shown in SEQ ID NO: 7, or comprises an amino acid sequence with more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 7; or comprises the amino acid sequence shown in SEQ ID NO: 8 Amino acid sequence, or an amino acid sequence comprising more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO:8;
  • HFR2 comprises the amino acid sequence shown in SEQ ID NO: 9, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 9;
  • HFR3 comprises the amino acid sequence shown in SEQ ID NO:10, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO:10;
  • HFR4 comprises the amino acid sequence shown in SEQ ID NO: 11, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 11;
  • LFR1 comprises the amino acid sequence shown in SEQ ID NO: 12, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 12;
  • LFR2 comprises the amino acid sequence shown in SEQ ID NO: 13, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 13;
  • LFR3 comprises the amino acid sequence shown in SEQ ID NO: 14, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 14;
  • LFR4 comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 15.
  • the antibody further comprises framework regions HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region, and framework regions LFR1, LFR2, LFR3, and LFR4 of the light chain variable region, wherein:
  • HFR1 consists of the amino acid sequence shown in SEQ ID NO: 7, or consists of an amino acid sequence with more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 7; or consists of the amino acid sequence shown in SEQ ID NO: 8
  • HFR2 consists of the amino acid sequence shown in SEQ ID NO: 9, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 9;
  • HFR3 consists of the amino acid sequence shown in SEQ ID NO: 10, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 10;
  • HFR4 consists of the amino acid sequence shown in SEQ ID NO: 11, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 11;
  • LFR1 consists of the amino acid sequence shown in SEQ ID NO: 12, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 12;
  • LFR2 consists of the amino acid sequence shown in SEQ ID NO: 13, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 13;
  • LFR3 consists of the amino acid sequence shown in SEQ ID NO: 14, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 14;
  • LFR4 consists of the amino acid sequence shown in SEQ ID NO: 15, or consists of an amino acid sequence having more than 90% identity or homology with the amino acid sequence shown in SEQ ID NO: 15.
  • the antibody further comprises a heavy chain variable region and a light chain variable region: the amino acid sequence of the heavy chain variable region of the antibody consists of SEQ ID NO: 16 or SEQ ID NO: 17;
  • amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof consists of SEQ ID NO: 18.
  • the antibody further comprises a heavy chain constant region and a light chain constant region;
  • the heavy chain constant region is any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM species, the light chain constant region is a ⁇ chain or a ⁇ chain.
  • the species sources of the heavy chain constant region and the light chain constant region are bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, Deer, mink, chicken, duck, goose, turkey, gamecock or human.
  • the amino acid sequence of the heavy chain of the antibody consists of SEQ ID NO: 19 or SEQ ID NO: 20; the amino acid sequence of the light chain of the antibody consists of SEQ ID NO: 21.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , scFv, Fv, Fd, single chain antibody, diabody or domain antibody.
  • the present disclosure also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof.
  • Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA nucleic acid is used when it is ligated into a vector.
  • the present disclosure also relates to vectors containing said nucleic acids.
  • the present disclosure also relates to cells containing said nucleic acid or said vector.
  • the present disclosure also relates to an antibody conjugate comprising the antibody or antigen-binding fragment and a conjugation moiety conjugated thereto;
  • the coupling moiety includes a label selected from purification tags (such as His tags), detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electronic Dense labels such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme , carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling.
  • purification tags such as His tags
  • detectable labels such as colloidal gold, radioactive labels, luminescent substances, colored substances
  • enzymes such as fluorescent labels, chromophore labels, electronic Dense labels such as radioisotop
  • the present disclosure also relates to a kit or diagnostic reagent comprising said antibody or antigen-binding fragment or said antibody conjugate, wherein:
  • the kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to dengue NS1 protein.
  • the kit or diagnostic reagent is used to diagnose dengue fever or dengue virus infection, or to detect dengue virus.
  • the present disclosure also relates to the use of the antibodies or antigen-binding fragments or antibody conjugates described herein for diagnosing dengue or dengue virus infection, or for detecting dengue virus.
  • the present disclosure also relates to methods of diagnosing dengue or dengue virus infection in a subject, comprising:
  • the presence of said immune complex therein is indicative of dengue or dengue virus infection.
  • the present disclosure also relates to a method of detecting dengue virus or dengue virus NS1 protein in a test sample comprising:
  • the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment.
  • the immune complex further includes a second antibody, and the second antibody binds to the dengue virus or dengue virus NS1 protein antigen.
  • restriction enzymes and rTaq DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • Embodiment 1 The preparation of the antibody of anti-dengue NS1 protein
  • the mRNA was extracted from the hybridoma cell line secreting Anti-DF 8F13 monoclonal antibody, and the DNA product was obtained by RT-PCR.
  • the product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transformed into DH5 ⁇ In the competent cells, after the growth of colonies, four clones of the heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were taken and sent to the gene sequencing company for sequencing.
  • VNL gene sequence is 321bp, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 354bp, which belongs to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.
  • pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has been introduced with HindIII, BamHI, EcoRI and other polyclonal restriction sites, and named as pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T
  • the VL and VH gene-specific primers of the Anti-DF 8F13 antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and 0.73KB was amplified by PCR amplification The Light Chain gene fragment and the 1.4kb Heavy Chain gene fragment.
  • the Heavy Chain and Light Chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were connected to the 3.4A expression vector to obtain the Heavy Chain gene. Recombinant expression plasmids for Chain and Light Chain.
  • Step 2 Dilute the plasmid prepared in step (2) to 40 ⁇ g/100 ⁇ L with ultrapure water, adjust the CHO cells to 1.43 ⁇ 10 7 cells/mL in a centrifuge tube, mix 100 ⁇ L of the above plasmid with 700 ⁇ L of cells, transfer to the electroporation cup, electroporation , count the next day; 25 ⁇ mol/L MSX 96-well pressurized culture for about 25 days.
  • the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/mL. The expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.
  • Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Fed-batch feeding Start feeding daily after culturing in shake flasks for 72 hours.
  • HyClone TM Cell Boost TM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. , has been supplemented until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day.
  • Affinity purification was performed with a proteinA affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g of foreign control antibody as a control.
  • the electropherogram is shown in Figure 1. Two bands were revealed after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain).
  • amino acid sequences of HCDR1-3 of DF-8F13R are shown in SEQ ID NO.1-3 respectively; the amino acid sequences of LCDR1-3 are shown in SEQ ID NO.4-6 respectively .
  • amino acid sequences of the heavy chain variable region, the light chain variable region, the heavy chain and the light chain are respectively shown in SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.21.
  • DF-8F13RMb1 Perform a series of single point mutations on DF-8F13R, repeat steps 1-(3) to 3-(2), and screen to obtain DF-8F13RMb1 through affinity analysis and activity identification.
  • amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain and light chain of the DF-8F13RMb1 antibody are SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.20 and SEQ ID NO.20 and SEQ ID NO.20, respectively. ID NO.21 is shown.
  • the purified antibody was diluted to 10 ⁇ g/mL with PBST, and the Dengue type I antigen (purchased from Fapon Biotech) was also serially diluted with PBST;
  • Kd represents the equilibrium dissociation constant, that is, affinity
  • Kon represents the association rate constant
  • Kdis represents the dissociation rate constant
  • the monoclonal antibody prepared in Example 1 was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples were taken for state observation at 7 days, 14 days, and 21 days, and The activity test was carried out on the samples left for 21 days (the activity of the samples was assessed by the OD results of enzyme immunoassay).
  • Embodiment 4 performance evaluation
  • the antibody prepared in the above Example 1 and the control antibody were used as labeled antibodies, respectively, and used together with another anti-NS1 antibody (obtained from Feipeng Biotechnology, which can also be obtained by immunization with NS1 immunogen) as a coating antibody, to detect Dengue I Type, II and III antigens (purchased from Faipeng Biology), compare the performance difference between the antibody prepared in Example 1 and the control antibody on the colloidal gold platform, the antibody prepared in Example 1 is in the immunoassay on the gold standard In terms of color development, it shows a superior performance level than the control antibody. See the table below for details:
  • the color development of gold standard is composed of C plus numbers. The smaller the number after C, the stronger the color development and the higher the activity; the higher the number after C, the weaker the color development and lower activity; the number is followed by "+” It is slightly stronger than without color by 0.5-1C, and the number with "-" is slightly lower than without color by 0.5-1C. B means no or very weak color development.
  • the disclosure provides an antibody against dengue NS1 protein, which has high antibody affinity, reactivity, specificity and sensitivity, and can be used for the diagnosis of dengue fever or dengue virus.

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Abstract

一种抗登革NS1蛋白的抗体及其制备方法和用途。制备的抗登革NS1蛋白的单克隆抗体对抗登革NS1蛋白具有高亲和性、高反应活性、高灵敏度和特异性，为登革热疾病的诊断提供了重要的原料来源。

Description

一种抗登革NS1蛋白的抗体及其应用
相关申请的交叉引用
本申请要求于2021年09月10日提交中国专利局的申请号为202111059913.7、名称为“一种抗登革NS1蛋白的抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开属于抗体技术领域。更具体地，涉及一种抗登革NS1蛋白的抗体及其应用。
背景技术
登革热(dengue fever，DF)是由4个血清型病毒(DENV-1、DENV-2、DENV-3、DENV-4)引起的急性蚊媒传染病，主要通过埃及伊蚊和白纹伊蚊传播。DF是分布最广，发病最多，危害较大的一种虫媒病毒性疾病，广泛流行于全球热带和亚热带的非洲、美洲、东南亚和西太平洋地区的100多个国家和地区。
登革热没有特效的治疗方法。如果没有适应的治疗，登革出血热的病死率可超过20％，经过有效的支持疗法，病死率可低于1％。登革热诊断要点包括：1)流行病学资料，发病前15天的活动情况，有否去过流行区，蚊虫叮咬历；2)临床特征，突然起病，发热，“三痛三红”，皮诊；3)实验室检查，白细胞、血小板下降；检测血清特性IgM阳性；恢复期IgG比急性期有4倍增长；分离到病毒或特异性抗原。临床上用于登革病毒的检测方法有病毒培养、血清学检测、病毒核酸检测等。病毒分离所需时间较长，达不到快速诊断的目的，而常规的血清学诊断又因存在广泛的交叉反应而受到干扰。胶体金标记的免疫层析方法具有快速、简便、不需要依赖重要装备、能够实现现场检测等特点，成为目前传染病快速诊断中研究的热点。
NS1蛋白是登革病毒(也称登革热病毒)非结构蛋白中唯一的糖蛋白，抗原性极强且不引发抗体信赖性感染增强现象(Antibody-dependent enhancement，ADE)，所以作为胶体金检测的靶标。而胶体金检测需要针对NS1蛋白的特异性单克隆抗体，传统临床用的都是鼠源性的单克隆抗体。现在市场主流Dengue单克隆抗体原料来源于进口，价格高，特异性、灵敏度都有待提升。
发明内容
本公开要解决的技术问题是克服现有Dengue单克隆抗体原料特异性及灵敏度的缺陷 问题，本公开制备的抗登革NS1蛋白的抗体亲和力、反应活性、特异性及灵敏度方面明显优于市场主流抗体。
本公开的目的是提供一种抗体或抗原结合片段，所述抗体或抗原结合片段含有以下CDRs：
重链CDR1，其包含SEQ ID NO.1所示的氨基酸序列S-G-Y-W-N，或由其组成；
重链CDR2，其包含SEQ ID NO.2所示的氨基酸序列Y-I-T-Y-S-G-N-T-Y-Y-N-P-S-I-K-S，或由其组成；和
重链CDR3，其包含SEQ ID NO.3所示的氨基酸序列Y-R-N-G-N-S-I，或由其组成；
并且所述抗体或抗原结合片段还包含：
轻链CDR1，其包含SEQ ID NO.4所示的氨基酸序列R-A-S-Q-D-I-S-N-Y-L-N，或由其组成；
轻链CDR2，其包含SEQ ID NO.5所示的氨基酸序列Y-T-S-R-L-H-S，或由其组成；和
轻链CDR3，其包含SEQ ID NO.6所示的氨基酸序列Q-Q-G-D-T-I-P，或由其组成。
本公开的另一目的是提供所述抗体或抗原结合片段相关的核酸、载体或细胞。
本公开还提供了制备所述抗体或抗原结合片段的方法。
本公开还提供了一种抗体偶联物，以及包含上述抗体或抗原结合片段或所述抗体偶联物的试剂盒/诊断试剂。
本公开还提供了所述抗体或抗原结合片段或所述抗体偶联物在制备试剂盒或诊断试剂中的用途。
附图说明
图1是DF-8F13R抗体的还原性SDS-PAGE结果。
具体实施方式
以下结合具体实施例来进一步说明本公开，但实施例并不对本公开做任何形式的限定。除非特别说明，本公开采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本公开涉及一种抗体或抗原结合片段，所述抗体或抗原结合片段包含以下CDRs：
重链CDR1，其包含SEQ ID NO.1所示的氨基酸序列S-G-Y-W-N，或由其组成；
重链CDR2，其包含SEQ ID NO.2所示的氨基酸序列Y-I-T-Y-S-G-N-T-Y-Y-N-P-S-I-K-S，或由其组成；和
重链CDR3，其包含SEQ ID NO.3所示的氨基酸序列Y-R-N-G-N-S-I，或由其组成；
并且所述抗体或抗原结合片段还包含：
轻链CDR1，其包含SEQ ID NO.4所示的氨基酸序列R-A-S-Q-D-I-S-N-Y-L-N，或由其组成；
轻链CDR2，其包含SEQ ID NO.5所示的氨基酸序列Y-T-S-R-L-H-S，或由其组成；和
轻链CDR3，其包含SEQ ID NO.6所示的氨基酸序列Q-Q-G-D-T-I-P，或由其组成。
在本公开中，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，双特异性或多特异性抗体，以及嵌合抗体，只要它们展示所需的生物学活性。术语“抗原结合片段”是包含抗体CDR的一部分或全部的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab(由完整的轻链和Fd构成)，Fv(由VH和VL构成)，scFv(单链抗体，VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。此类片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
在本公开中，术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区，指包含一种或多种或者甚至全部的对抗体或其抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在本公开具体实施方式中，CDRs是指所述抗体的重链和轻链的高度可变区。
在本公开中，重链互补决定区用HCDR表示，其包括HCDR1、HCDR2和HCDR3；轻链互补决定区用LCDR表示，其包括LCDR1、LCDR2和LCDR3。本领域常用的CDR标示方法包括：Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本公开采用Kabat注释标准标示CDR区，但其他方法标示的CDR区也属于本公开的保护范围。
在本公开中，“框架区”或“FR”区包括重链框架区和轻链框架区，是指抗体重链可变区和轻链可变区中除CDR之外的区域；其中，重链框架区可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4框架区；轻链框架区可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4框架区。
在本公开中，重链可变区由以下编号的CDR与FR按如下组合排列连接获得：HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4；轻链可变区由以下编号的CDR与FR 按如下组合排列连接获得：LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。
在一些实施方式中，所述抗体还包含重链可变区的框架区HFR1、HFR2、HFR3和HFR4，和轻链可变区的框架区LFR1、LFR2、LFR3和LFR4，其中：
HFR1包含SEQ ID NO:7所示的氨基酸序列，或包含与SEQ ID NO:7所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；或包含SEQ ID NO:8所示的氨基酸序列，或包含与SEQ ID NO:8所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
HFR2包含SEQ ID NO:9所示的氨基酸序列，或包含与SEQ ID NO:9所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
HFR3包含SEQ ID NO:10所示的氨基酸序列，或包含与SEQ ID NO:10所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
HFR4包含SEQ ID NO:11所示的氨基酸序列，或包含与SEQ ID NO:11所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；和
LFR1包含SEQ ID NO:12所示的氨基酸序列，或包含与SEQ ID NO:12所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
LFR2包含SEQ ID NO:13所示的氨基酸序列，或包含与SEQ ID NO:13所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
LFR3包含SEQ ID NO:14所示的氨基酸序列，或包含与SEQ ID NO:14所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列；
LFR4包含SEQ ID NO:15所示的氨基酸序列，或包含与SEQ ID NO:15所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列。
在一些实施方式中，所述抗体还包含重链可变区的框架区HFR1、HFR2、HFR3和HFR4，和轻链可变区的框架区LFR1、LFR2、LFR3和LFR4，其中：
HFR1由SEQ ID NO:7所示的氨基酸序列组成，或由与SEQ ID NO:7所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；或由SEQ ID NO:8所示的氨基酸序列组成，或由与SEQ ID NO:8所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
HFR2由SEQ ID NO:9所示的氨基酸序列组成，或由与SEQ ID NO:9所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
HFR3由SEQ ID NO:10所示的氨基酸序列组成，或由与SEQ ID NO:10所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
HFR4由SEQ ID NO:11所示的氨基酸序列组成，或由与SEQ ID NO:11所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；和
LFR1由SEQ ID NO:12所示的氨基酸序列组成，或由与SEQ ID NO:12所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
LFR2由SEQ ID NO:13所示的氨基酸序列组成，或由与SEQ ID NO:13所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
LFR3由SEQ ID NO:14所示的氨基酸序列组成，或由与SEQ ID NO:14所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成；
LFR4由SEQ ID NO:15所示的氨基酸序列组成，或由与SEQ ID NO:15所示的氨基酸序列具有90％以上同一性或同源性的氨基酸序列组成。
在一些实施方式中，所述抗体还包含重链可变区和轻链可变区：所述抗体的重链可变区的氨基酸序列由SEQ ID NO:16或SEQ ID NO:17组成；
所述抗体或其抗原结合片段的轻链可变区的氨基酸序列由SEQ ID NO:18组成。
在一些实施方式中，所述抗体还包含重链恒定区和轻链恒定区；所述重链恒定区为IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种，所述轻链恒定区为κ链或λ链。
在一些实施方式中，所述重链恒定区和轻链恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一些实施方式中，所述抗体的重链的氨基酸序列由SEQ ID NO:19或SEQ ID NO:20组成；所述抗体的轻链的氨基酸序列由SEQ ID NO:21组成。
在一些实施方式中，所述抗原结合片段选自Fab，Fab'，F(ab') 2，scFv，Fv，Fd，单链抗体，双价抗体或结构域抗体。
本公开还涉及核酸，所述核酸编码所述抗体或其抗原结合片段。
核酸通常是RNA或DNA，核酸分子可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时采用DNA核酸。
本公开还涉及载体，所述载体含有所述核酸。
本公开还涉及细胞，所述细胞含有所述核酸或所述载体。
本公开还涉及一种抗体偶联物，包含所述抗体或抗原结合片段以及与其偶联的偶联部分；
可选地，所述偶联部分包括选自纯化标签(如His标签)，可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱 性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记。
本公开还涉及一种试剂盒或诊断试剂，其包含所述抗体或抗原结合片段或所述抗体偶联物，其中：
可选地，所述试剂盒或诊断试剂还包含结合除登革NS1蛋白以外的抗体或抗原结合片段、抗体偶联物，或融合蛋白。
所述抗体或其抗原结合片段或所述抗体偶联物在制备试剂盒或诊断试剂中的用途，同样在本公开的保护范围之内。
在一些实施方式中，所述试剂盒或诊断试剂用于诊断登革热或登革热病毒感染，或检测登革热病毒。
本公开还涉及本文所述的抗体或抗原结合片段或抗体偶联物用于诊断登革热或登革热病毒感染，或检测登革热病毒的用途。
本公开还涉及诊断受试者中登革热或登革热病毒感染的方法，包括：
将本文所述的抗体或抗原结合片段或本文所述的抗体偶联物与所述受试者的样品在能够与登革热病毒NS1蛋白产生免疫反应的条件下接触，以及
检测是否存在抗体或抗原结合片段或抗体偶联物与登革热病毒NS1蛋白的免疫复合物，
其中存在所述免疫复合物指示登革热或登革热病毒感染。
本公开还涉及检测测试样品中的登革热病毒或登革热病毒NS1蛋白的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的登革热病毒或登革热病毒NS1蛋白抗原与本文所述的抗体或抗原结合片段、偶联物或试剂盒接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述抗原的存在。
在一些实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述抗体或抗原结合片段结合。
在一些实施方式中，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述登革热病毒或登革热病毒NS1蛋白抗原结合。
下面将结合实施例对本公开的实施方案进行详细描述。
以下实施例中，限制性内切酶、rTaq DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART TM RACE cDNA Amplification Kit试剂盒购自 Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
实施例1 抗登革NS1蛋白的抗体的制备
1、表达质粒构建
(1)Anti-DF 8F13抗体基因制备
从分泌Anti-DF 8F13单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取重链(Heavy Chain)及轻链(Light Chain)基因克隆各4个克隆送基因测序公司进行测序。
(2)Anti-DF 8F13抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为321bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为354bp，属于VH1基因家族，其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2022115261-appb-000001
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计Anti-DF 8F13抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.73KB的Light Chain基因片段和1.4kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因连接3.4A表达载体中，得到Heavy Chain和Light Chain的重组表达质粒。
2、稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
将步骤1-(3)步骤制备得到的质粒用超纯水稀释至40μg/100μL，于离心管中准备1.43×10 7cells/mL的CHO细胞。将100μL上述质粒与700μL上述CHO细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
用包被液(主要成分NaHCO 3)稀释羊抗鼠IgG 1ug/ml，进行微孔板包被，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的细胞上清，100uL/孔，37℃，60min；甩掉板内液体，拍干，加入20％鼠阴性血封闭，每孔120ul，37℃， 1h；甩掉板内液体，拍干，加入稀释的Dengue阳性血清，每孔100uL，37℃，40min；洗涤液清洗5次，拍干；加入标记HRP的Dengue单克隆抗体(与纯化抗体能配对，购自菲鹏)，每孔100uL，37℃，30min；加入显色液A液(50uL/孔，主要成份柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50uL/孔，主要成份柠檬酸+EDTA·2Na+TMB+浓HCL)，10min；加入终止液(EDTA·2Na+浓H 2SO 4)，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对Dengue抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂：Buffer 50μL、步骤1-(3)步骤制备得到的质粒100μg/管、PvuⅠ酶10μL、无菌水补至500μL，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株
步骤2-(2)步骤制备得到的质粒用超纯水稀释至40μg/100μL，调节CHO细胞1.43×10 7cells/mL于离心管中，100μL上述质粒与700μL细胞混合，转入电转杯，电转，次日计数；25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔，并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6cells/mL，2.2mL进行批培养，细胞密度0.3×10 6cells/mL，2mL进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3、重组抗体生产
(1)细胞扩培
细胞复苏之后先在125mL规格的摇瓶中培养，接种体积为30mL，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万cells/mL接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万cells/mL左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyClone TM Cell Boost TM Feed 7a每天流加初始培养体积的3％，Feed 7b 每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化。取4μg纯化的抗体进行还原性SDS-PAGE，4μg外来对照抗体作为对照，电泳图如图1所示。在还原性SDS-PAGE后显示两条带，一条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
对纯化后的抗体测序及Kabat分析，得到DF-8F13R的HCDR1-3的氨基酸序列分别如SEQ ID NO.1-3所示；LCDR1-3的氨基酸序列分别如SEQ ID NO.4-6所示。重链可变区、轻链可变区、重链及轻链的氨基酸序列分别如SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.19及SEQ ID NO.21所示。
对DF-8F13R进行一系列的单点突变，重复步骤1-(3)至3-(2)，经亲和力分析及活性鉴定，筛选得到DF-8F13RMb1。经测序分析，DF-8F13RMb1抗体的重链可变区、轻链可变区、重链及轻链的氨基酸序列分别如SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.20及SEQ ID NO.21所示。
实施例2 亲和力分析及活性鉴定
1、亲和力分析
利用AMC传感器，纯化后的抗体用PBST稀释到10μg/mL，Dengue I型抗原(购自菲鹏生物)同样以PBST进行梯度稀释；
运行流程：缓冲液1(PBST；主要成分为Na 2HPO 4、NaCl和吐温20)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH1.69的GLY溶液及缓冲液3进行传感器再生，输出数据。
表1
样品名称 Kd(M) Kon(1/Ms) Kdis(1/s)
对照 7.06E-07 1.84E+03 1.30E-03
DF-8F13R 5.49E-08 3.15E+04 1.73E-03
DF-8F13RMb1 4.88E-08 3.64E+04 1.78E-03
注：Kd表示平衡解离常数即亲和力；Kon表示结合速率常数；Kdis表示解离速率常数。
2、活性鉴定
用包被液(主要成分NaHCO3)稀释羊抗鼠IgG 1ug/ml，进行微孔板包被，每孔100uL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120uL，37℃，1h，拍干；加入稀释后的纯化抗体，100uL/孔，37℃，60min；甩掉板内液体，拍干，加入20％鼠阴性血封闭，每孔120ul，37℃，1h；甩掉板内液体，拍干，加入稀释的Dengue阳性血清每孔100uL，37℃，40min；洗涤 液清洗5次，拍干；加入标记HRP的Dengue单克隆抗体(与纯化抗体能配对的，购自菲鹏)，每孔100uL，37℃，30min；加入显色液A液(50uL/孔)，加入显色液B液(50uL/孔)，10min；加入终止液，50uL/孔；酶标仪上450nm(参考630nm)处读OD值。
表2
样品浓度(ng/ml) 500 125 62.5 31.25 7.8125 0
对照 1.958 1.563 1.001 0.544 0.143 0.068
DF-8F13R 2.253 1.725 1.261 0.758 0.249 0.042
DF-8F13RMb1 2.295 1.748 1.266 0.758 0.309 0.038
实施例3 稳定性考核
将实施例1制备得到的单克隆抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对放置21天的样品进行活性检测(利用酶免检测OD结果考核样品的活性)。
DF-8F13R稳定性测试结果如表3所示，结果显示，DF-8F13R及DF-8F13RMb1三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明本公开制备得到的自产抗体稳定性好。
表3
样品浓度(ng/mL) 500 62.5 0
4℃，21天样品 2.315 1.276 0.094
-80℃，21天样品 2.307 1.239 0.083
37℃，21天样品 2.353 1.254 0.067
实施例4 性能评估
将上述实施例1中制备的抗体与对照抗体作为标记抗体，分别与另一株作为包被抗体的抗NS1抗体(获自菲鹏生物，也可用NS1免疫原免疫得到)配套使用，检测Dengue I型、II型和III型抗原(购自菲鹏生物)，在胶体金平台上比较实施例1中制备的抗体与对照抗体的性能差异，实施例1中制备的抗体在免疫测定中在金标显色方面显示出比对照抗体更优的性能水平。具体见下表：
表4
Figure PCTCN2022115261-appb-000002
备注：金标显色以C加数字组成，C后面的数字越小表示显色越强，活性越高；C后面的数字越高表示显色越弱，活性越低；数字后带“+”比不带显色略强0.5-1C，数字后带“-”比不带显色略低0.5-1C。B表示不显色或者显色很弱。
上述实施例为本公开较佳的实施方式，但本公开的实施方式并不受上述实施例的限制，其他的任何未背离本公开的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本公开的保护范围之内。
工业实用性
本公开提供一种抗登革NS1蛋白的抗体，具有高的抗体亲和力、反应活性、特异性及灵敏度，可用于对登革热或登革热病毒的诊断。

Claims (14)

  1. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含以下CDRs：
    重链CDR1，其包含SEQ ID NO.1所示的氨基酸序列S-G-Y-W-N，或由其组成；
    重链CDR2，其包含SEQ ID NO.2所示的氨基酸序列Y-I-T-Y-S-G-N-T-Y-Y-N-P-S-I-K-S，或由其组成；和
    重链CDR3，其包含SEQ ID NO.3所示的氨基酸序列Y-R-N-G-N-S-I，或由其组成；
    并且所述抗体或抗原结合片段还包含：
    轻链CDR1，其包含SEQ ID NO.4所示的氨基酸序列R-A-S-Q-D-I-S-N-Y-L-N，或由其组成；
    轻链CDR2，其包含SEQ ID NO.5所示的氨基酸序列Y-T-S-R-L-H-S，或由其组成；和
    轻链CDR3，其包含SEQ ID NO.6所示的氨基酸序列Q-Q-G-D-T-I-P，或由其组成。
  2. 根据权利要求1所述的抗体或抗原结合片段，还包含重链可变区的框架区
    HFR1、HFR2、HFR3和HFR4，和轻链可变区的框架区LFR1、LFR2、LFR3和LFR4，所述
    HFR1包含选自SEQ ID NO:7、SEQ ID NO:8、与SEQ ID NO:7具有90％以上同源性、与SEQ ID NO:8具有90％以上同源性的氨基酸序列；
    HFR2包含SEQ ID NO:9或与SEQ ID NO:9具有90％以上同源性的氨基酸序列；
    HFR3包含SEQ ID NO:10或与SEQ ID NO:10具有90％以上同源性的氨基酸序列；
    HFR4包含SEQ ID NO:11或与SEQ ID NO:11具有90％以上同源性的氨基酸序列；和
    LFR1包含SEQ ID NO:12或与SEQ ID NO:12具有90％以上同源性的氨基酸序列；
    LFR2包含SEQ ID NO:13或与SEQ ID NO:13具有90％以上同源性的氨基酸序列；
    LFR3包含SEQ ID NO:14或与SEQ ID NO:14具有90％以上同源性的氨基酸序列；
    LFR4包含SEQ ID NO:15或与SEQ ID NO:15具有90％以上同源性的氨基酸序列。
  3. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链可变区和轻链可变区，
    所述重链可变区包含SEQ ID NO:16或SEQ ID NO:17所示的氨基酸序列，或由其组成；
    所述轻链可变区包含SEQ ID NO:18所示的氨基酸序列，或由其组成。
  4. 根据权利要求1或3所述的抗体或抗原结合片段，还包含重链恒定区和/或轻链 恒定区：所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种的重链恒定区；所述轻链恒定区为κ链或λ链；
    可选地，所述重链恒定区和轻链恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
  5. 一种核酸，其特征在于，所述核酸编码权利要求1～4任一所述的抗体或抗原结合片段。
  6. 一种细胞，其特征在于，所述细胞包含权利要求5所述的核酸。
  7. 一种制备权利要求1～4任一所述的抗体或抗原结合片段的方法，其特征在于，所述方法包括培养权利要求6所述的细胞。
  8. 一种抗体偶联物，其特征在于，所述抗体偶联物包含权利要求1～4任一所述的抗体或抗原结合片段以及与其偶联的偶联部分；
    优选地，所述偶联部分选自纯化标签或可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记的一种或多种。
  9. 一种试剂盒或诊断试剂，其特征在于，所述试剂盒或诊断试剂包含权利要求1～4任一所述的抗体或抗原结合片段或权利要求8所述的抗体偶联物；
    优选地，所述试剂盒或诊断试剂还包含结合除登革NS1蛋白以外的抗原片段的抗体或其抗原结合片段、抗体偶联物，或融合蛋白。
  10. 权利要求1～4任一所述的抗体或抗原结合片段或权利要求8所述的抗体偶联物在制备试剂盒或诊断试剂中的用途。
  11. 根据权利要求10所述的用途，其特征在于，所述试剂盒或诊断试剂用于诊断登革热或登革热病毒感染，或检测登革热病毒。
  12. 权利要求1～4任一所述的抗体或抗原结合片段或权利要求8所述的抗体偶联物用于诊断登革热或登革热病毒感染，或检测登革热病毒的用途。
  13. 诊断受试者中登革热或登革热病毒感染的方法，包括：
    将权利要求1～4任一所述的抗体或抗原结合片段或权利要求8所述的抗体偶联物与受试者的样品在能够与登革热病毒NS1蛋白产生免疫反应的条件下接触，以及
    检测是否存在抗体或抗原结合片段或抗体偶联物与登革热病毒NS1蛋白的免疫复合物，
    其中存在所述免疫复合物指示登革热或登革热病毒感染。
  14. 一种检测测试样品中的登革热病毒或登革热病毒NS1蛋白抗原的方法，其包括：
    a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的登革热病毒或登革热病毒NS1蛋白抗原与权利要求1～4任一所述的抗体或抗原结合片段、权利要求8所述的抗体偶联物或权利要求9所述的试剂盒接触以形成免疫复合物；和
    b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述抗原的存在；
    优选的，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述抗体或抗原结合片段结合；
    优选的，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述登革热病毒或登革热病毒NS1蛋白抗原结合。
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