WO2023078452A1 - Anticorps contre la protéine ns1 de la dengue et utilisation associée - Google Patents

Anticorps contre la protéine ns1 de la dengue et utilisation associée Download PDF

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WO2023078452A1
WO2023078452A1 PCT/CN2022/130485 CN2022130485W WO2023078452A1 WO 2023078452 A1 WO2023078452 A1 WO 2023078452A1 CN 2022130485 W CN2022130485 W CN 2022130485W WO 2023078452 A1 WO2023078452 A1 WO 2023078452A1
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antibody
antigen
amino acid
acid sequence
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孟媛
钟冬梅
覃文新
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东莞市朋志生物科技有限公司
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    • G01N33/56983—Viruses
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    • 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
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C12N2800/00—Nucleic acids vectors
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    • G01N2333/08—RNA viruses
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    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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    • G01N2469/10—Detection of antigens from microorganism in sample from host

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.
  • Dengue fever is an acute mosquito-borne infectious disease caused by four serotypes of viruses (DENV-1, DENV-2, DENV-3, DENV-4), mainly transmitted by Aedes aegypti and 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 1) epidemiological data, activities within 15 days after onset, whether you have been to endemic areas, history of mosquito bites; 2) clinical features, sudden onset, fever, "three pains and three reds", skin diagnosis ; 3) Laboratory tests showed a decrease in white blood cells and platelets; the serum characteristic IgM was positive; IgG in the convalescent phase was 4 times higher than that in the acute phase; viruses or specific antigens were isolated.
  • 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. It has strong antigenicity and does not cause antibody-dependent infection enhancement (Antibody-dependent enhancement, ADE), so it is used as the target of colloidal gold detection.
  • ADE antibody-dependent enhancement
  • 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 anti-dengue NS1 monoclonal antibody raw materials in the market are imported, the price is high, and the specificity and sensitivity need to be improved.
  • the present disclosure provides an antibody or an antigen-binding fragment, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1-HCDR3 include or are any of SEQ ID NO: 15, SEQ ID NO: 21-23 The amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region is shown; the LCDR1-LCDR3 includes or is an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.
  • the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system.
  • the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.
  • the present disclosure provides an antibody or antigen-binding fragment comprising the following CDRs:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO.2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19, or consists of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO.5, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.
  • the antibody or antigen-binding fragment 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, the HFR1 Comprising any amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:20, having more than 90% homology with SEQ ID NO:7 or having more than 80% homology with SEQ ID NO:20; HFR2 includes SEQ ID NO:8 or an amino acid sequence having more than 80% homology with SEQ ID NO:8; HFR3 comprises SEQ ID NO:9 or an amino acid sequence having more than 80% homology with SEQ ID NO:9; HFR4 comprises SEQ ID NO:10 or an amino acid sequence having more than 80% homology with SEQ ID NO:10; and LFR1 comprising SEQ ID NO:11 or an amino acid sequence having more than 80% homology with SEQ ID NO:11; LFR2 Comprising SEQ ID NO: 12 or an amino acid sequence having
  • the present disclosure provides an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1-3 and the above-mentioned HFR1-4, the light chain variable region comprises the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.
  • the heavy chain variable region comprises an amino acid sequence selected from any of SEQ ID NO: 15 and SEQ ID NO: 21-23, or consists of it.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or consists of it.
  • the antibody or antigen-binding fragment further comprises a constant region.
  • the constant region comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from 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 a lambda-type light chain constant region.
  • the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man.
  • the species source of the constant region is mouse.
  • the heavy chain constant region is SEQ ID NO: 24 or an amino acid sequence having more than 80% homology with SEQ ID NO: 24;
  • the light chain constant region is SEQ ID NO: 25 or an amino acid sequence with SEQ ID NO: 24 NO:25 amino acid sequence with more than 80% homology.
  • the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • the present disclosure also provides an antibody or an antigen-binding fragment, comprising a heavy chain and/or a light chain, the heavy chain comprising the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain comprising the above-mentioned light chain chain variable region and the aforementioned light chain constant region.
  • amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 17, 26, 27, 28; the amino acid sequence of the light chain is shown in SEQ ID NO: 18.
  • the disclosure also provides a nucleic acid encoding the antibody or antigen-binding fragment of any of the above.
  • the present disclosure also provides a cell comprising the above-mentioned nucleic acid.
  • the present disclosure also provides a method for preparing the antibody or antigen-binding fragment described in any one of the above, the method comprising culturing the above-mentioned cells.
  • the present disclosure also provides an antibody conjugate comprising the antibody or antigen-binding fragment of any one of the above and a conjugation moiety conjugated thereto.
  • the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromophore label, an electron-dense label , such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, sugar One or more of oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label.
  • a detectable label such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromophore label, an electron-dense label , such as radioiso
  • the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • the present disclosure also provides a kit or a diagnostic reagent, which comprises the antibody or antigen-binding fragment described in any one of the above or the antibody conjugate described above.
  • the kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to an antigen fragment other than dengue NS1 protein.
  • the present disclosure also provides the use of any one of the antibodies or antigen-binding fragments or antibody conjugates described above in the preparation of kits or diagnostic reagents.
  • the reagent or kit is used for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or diseases related to dengue virus infection.
  • the present disclosure also provides the antibody or antigen-binding fragment or the antibody conjugate described in any one of the above or the kit or diagnostic reagent for detecting dengue virus or dengue NS1 protein and diagnosing dengue Use in virus infection or diseases related to dengue virus infection.
  • the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
  • the present disclosure also provides a method for diagnosing a subject infected with dengue virus or a disease related to dengue virus infection, comprising:
  • the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
  • the present disclosure also provides a method of detecting dengue virus or dengue NS1 protein in a test sample, comprising:
  • the dengue virus or dengue NS1 protein antigen in the test sample is combined with the antibody or its functional fragment described in any one of the above, or the described
  • the antibody conjugate, or said reagent or kit is contacted to form an immune complex;
  • 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 that binds to the dengue virus or dengue NS1 protein antigen.
  • Fig. 1 is the reducing SDS-PAGE result of DF-5M32RMb1 antibody.
  • Some embodiments of the present disclosure provide an antibody or an antigen-binding fragment, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include any of SEQ ID NO: 15, SEQ ID NO: 21-23 An amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region shown; said LCDR1-LCDR3 includes an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.
  • the HCDR1-HCDR3 are amino acid sequences consistent with the HCDR1-HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO: 15 and SEQ ID NO: 21-23; the LCDR1-HCDR3 LCDR3 is an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.
  • the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system.
  • the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO.2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19, or consists of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO.5, or consists of it;
  • LCDR3 which comprises the amino acid sequence 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 antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment 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 denoted by "HCDR", which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is denoted 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.
  • a "framework region” or "FR” region includes a heavy chain framework region and a light chain framework region and refers to an antibody heavy chain variable region (which may be denoted as VH) and a light chain variable region (which may be denoted as VL) Regions other than the CDRs in ; where the heavy chain framework region is denoted by "HFR” and can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework The regions are indicated by "LFR” and can be further subdivided into contiguous regions separated by CDRs, comprising the LFR1, LFR2, LFR3 and LFR4 framework regions.
  • the heavy chain variable region is obtained by joining the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the terms "subject” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • the antibody further comprises the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region, wherein:
  • HFR1 comprises any amino acid sequence of SEQ ID NO:7 or SEQ ID NO:20, or consists of it;
  • HFR2-4 comprises the amino acid sequence of SEQ ID NO:8-10 successively, or consists of the amino acid sequence of SEQ ID NO:8-10 successively;
  • LFR1-4 comprises the amino acid sequence of SEQ ID NO:11-14 successively, or consists of the amino acid sequence of SEQ ID NO:11-14 successively.
  • each framework region of the antibody or antigen-binding fragment provided by the present disclosure may be identical to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14) 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.
  • the binding affinity K D of the antibody or antigen-binding fragment to Dengue NS1 is ⁇ 6.36 ⁇ 10 -8 M.
  • Some embodiments of the present disclosure also provide an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1 -3 and the aforementioned HFR1-4, the light chain variable region comprising the aforementioned LCDR1-3 and the aforementioned LFR1-4.
  • the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 15, SEQ ID NO: 21-23; the light chain variable region comprises SEQ ID NO : the amino acid sequence shown in 16.
  • the amino acid sequence of the heavy chain variable region consists of any amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 21-23; the light chain variable region consists of SEQ ID NO: Amino acid sequence composition shown in ID NO:16.
  • the antibody further comprises a constant region
  • the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region” can be represented as CH; a "light chain constant region” can be represented as CL .
  • the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region.
  • the species sources of the heavy chain constant region and the light chain constant region are cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, Deer, mink, chicken, duck, goose, turkey, gamecock or human.
  • the species origin of the heavy chain constant region and the light chain constant region is mouse.
  • the heavy chain constant region is SEQ ID NO:24 or an amino acid sequence having more than 80% homology with SEQ ID NO:24; the light chain constant region is SEQ ID NO:25 or an amino acid sequence with SEQ ID NO:24 NO:25 amino acid sequence with more than 80% homology.
  • the amino acid sequence of the constant region of the antibody or antigen-binding fragment provided by the present disclosure may have at least 80%, 81%, or 82% of the above-mentioned corresponding constant region (SEQ ID NO: 24, 25). %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • amino acid sequence of the heavy chain of the antibody consists of SEQ ID NO: 17; the amino acid sequence of the light chain of the antibody consists of SEQ ID NO: 18.
  • the above 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 the antibodies or antigen-binding fragments described above.
  • 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.
  • Some embodiments of the present disclosure also relate to vectors containing the above-mentioned nucleic acids.
  • Some embodiments of the present disclosure also relate to cells containing the nucleic acids or vectors described above.
  • Some embodiments of the present disclosure also relate to an antibody conjugate, comprising the above-mentioned antibody or antigen-binding fragment and a coupling moiety coupled 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 , electron-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-labeled.
  • 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 , electron-dense labels, such as
  • the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • kits or diagnostic reagent comprising the above-mentioned antibody or antigen-binding fragment or antibody conjugate, wherein:
  • the above kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to a protein other than dengue NS1.
  • Some embodiments of the present disclosure also provide the use of the above antibodies or antigen-binding fragments or antibody conjugates in the preparation of kits or diagnostic reagents.
  • the above reagent or kit is used for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or diseases related to dengue virus infection.
  • Some embodiments of the present disclosure also provide the above-mentioned antibodies or antigen-binding fragments, antibody conjugates or kits or diagnostic reagents for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or related to dengue virus infection use in disease.
  • the above-mentioned diseases related to dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
  • Some embodiments of the present disclosure also provide a method for diagnosing a subject infected with dengue virus or a disease associated with dengue virus infection, comprising:
  • the above-mentioned diseases related to dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
  • Some embodiments of the present disclosure also provide a method for detecting dengue virus or dengue NS1 protein in a test sample, comprising:
  • the dengue virus or dengue NS1 protein antigen in the test sample is contacted with the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit to form immune complexes;
  • the immune complex further includes a second antibody, which binds to the above-mentioned antibody or antigen-binding fragment;
  • the immune complex further includes a second antibody, which binds to the above dengue virus or dengue 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.
  • the disclosure provides an anti-dengue NS1 antibody, a reagent and a kit for detecting dengue virus, the antibody can specifically bind to the dengue NS1 protein, and has a high affinity for it, and the antibody can be used to detect dengue virus or dengue NS1 protein has better sensitivity or specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of dengue virus.
  • Embodiment 1 The preparation of the antibody of anti-dengue NS1 protein
  • the mRNA was extracted from the hybridoma cell line secreting Anti-DF 5M32 monoclonal antibody prepared in our laboratory, and the DNA product was obtained by RT-PCR method, and the product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase , transformed into DH5 ⁇ competent cells, and after the colonies grew out, four heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were respectively taken and sent to a gene sequencing company for sequencing.
  • VNL gene sequence is 339bp, 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 351bp, 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 5M32 antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and 0.74KB 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 respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were respectively connected to the 3.4A expression vectors, respectively. Recombinant expression plasmids for the heavy and light chains were obtained.
  • Step 2-(2) Dilute the plasmid prepared in step 2 to 40 ⁇ g/100 ⁇ L with ultrapure water, adjust 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, and transfer to the electroporation cup, Electroporation, counting the next day; 25 ⁇ mol/LMSX 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 amount of cells 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 Feed feeding starts every day when the shake flask is cultured for 72 hours.
  • HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day.
  • Affinity purification was performed with a proteinA affinity chromatography column. Take 6.6 ⁇ g of the purified antibody for reducing SDS-PAGE, and the electropherogram is shown in the figure.
  • DF-5M32RMb1 Two bands were shown 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-5M32RMb1 obtained through the above steps 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.15-18.
  • DF-5M32RMb1 Analyzes the structure of DF-5M32RMb1, design mutation primers, repeat steps 1-(3) to 3-(2), and obtain DF-8F13RMb2-4 through activity identification and screening.
  • the heavy chain variable region of DF-8F13RMb2-4 is shown in SEQ ID NO.21-23
  • the heavy chain constant region is shown in SEQ ID NO.24
  • the light chain is the same as DF-5M32RMb1.
  • the purified antibody was diluted to 10ug/ml with PBST, and the DENV-IV antigen (an NS1 protein, purchased from Fapon) was serially diluted with PBST;
  • KD means the equilibrium dissociation constant, that is, affinity
  • Kon means the association rate constant
  • Kd means 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 which had been placed for 21 days.
  • the activity test method refer to Example 2 (the activity of the sample was evaluated by the OD result 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, type II, type III and type IV 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 shows a higher than control antibody A better level of performance.
  • another anti-NS1 antibody obtained from Feipeng Biotechnology, which can also be obtained by immunization with NS1 immunogen
  • Dengue I Type, type II, type III and type IV antigens purchased from Faipeng biology
  • the anti-dengue virus antibody provided by the disclosure has good affinity to the NS1 protein of the dengue virus, and the detection of the dengue virus by using the antibody has good sensitivity and specificity.
  • This disclosure provides a more excellent antibody selection for the detection of dengue virus, therefore, the anti-dengue virus antibody, dengue virus detection reagent and kit provided by the disclosure have excellent practical performance and broad market application prospects .

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Abstract

L'invention concerne un anticorps contre une protéine NS1 de la dengue et une méthode de préparation associée. L'anticorps fournit une source de matière première pour le diagnostic de la maladie de la dengue.
PCT/CN2022/130485 2021-11-08 2022-11-08 Anticorps contre la protéine ns1 de la dengue et utilisation associée WO2023078452A1 (fr)

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