CN116082499B - Antibody for resisting dengue NS1 protein and application thereof - Google Patents

Antibody for resisting dengue NS1 protein and application thereof Download PDF

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CN116082499B
CN116082499B CN202111315863.4A CN202111315863A CN116082499B CN 116082499 B CN116082499 B CN 116082499B CN 202111315863 A CN202111315863 A CN 202111315863A CN 116082499 B CN116082499 B CN 116082499B
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antibody
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ser
antigen
binding fragment
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CN116082499A (en
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孟媛
钟冬梅
覃文新
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • 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
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    • 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
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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    • C12N2800/00—Nucleic acids vectors
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    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
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    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Abstract

The invention relates to an antibody for resisting dengue NS1 protein, a preparation method and application thereof. The monoclonal antibody of the anti-dengue NS1 protein has high affinity, high reactivity, high sensitivity and specificity to the anti-dengue NS1 protein, and provides an important raw material source for diagnosis of dengue fever.

Description

Antibody for resisting dengue NS1 protein and application thereof
Technical Field
The invention belongs to the technical field of antibodies. More particularly, it relates to an antibody against dengue NS1 protein and uses thereof.
Background
Dengue Fever (DF) is an acute mosquito-borne infectious disease caused by 4 serotypes of viruses (DENV-1, DENV-2, DENV-3, DENV-4), and the transmission of DF mainly through Aedes aegypti and Aedes albopictus is the most widespread, most ill, and more harmful insect-borne viral disease, which is widely prevalent in more than 100 countries and regions in Africa, america, southeast Asia, and Western Pacific regions of the tropics and subtropics worldwide.
Dengue has no specific treatment. Without an adaptive treatment, the mortality rate of dengue hemorrhagic fever may exceed 20% and, through effective supportive care, may be less than 1%. Dengue diagnosis gist: 1) Epidemiological data, including 15 days of activity, whether the patient has passed through a epidemic area, and mosquito bites; 2) Clinical characteristics, sudden onset of disease, fever, "Santong Sanhong", skin diagnosis; 3) Laboratory examination, leucocyte and platelet decrease; detecting positive IgM with serum characteristics; the recovery period IgG is 4 times longer than the acute period; isolating the virus or specific antigen. The detection method for dengue virus clinically comprises virus culture, serological detection, virus nucleic acid detection and the like. The time required for virus isolation is long, and the purpose of rapid diagnosis cannot be achieved, while conventional serological diagnosis is disturbed by the existence of extensive cross reactions. The immunochromatography method marked by the colloidal gold has the characteristics of rapidness, simplicity, convenience, no need of depending on important equipment, capability of realizing on-site detection and the like, and becomes a research hot spot in the current rapid diagnosis of infectious diseases.
The NS1 protein is the only glycoprotein in the non-structural proteins of dengue virus, has extremely strong antigenicity and does not cause the phenomenon of enhancing Antibody-dependent infection (anti-dependent enhancement, ADE), and is used as a target of colloidal gold detection. The colloidal gold detection needs specific monoclonal antibodies aiming at NS1 protein, and the traditional clinical monoclonal antibodies are all murine monoclonal antibodies. The main current monoclonal antibody raw material of the anti-dengue NS1 in the market is imported, the price is high, and the specificity and the sensitivity are all to be improved.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to solve the technical problems of the existing monoclonal antibody against dengue NS1, such as raw material specificity and sensitivity, and the antibody affinity, reactivity, specificity and sensitivity of the anti-dengue NS1 protein prepared by the invention are obviously superior to those of mainstream antibodies in the market.
It is an object of the present invention to provide an antibody or antigen binding fragment comprising the following CDRs:
HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID No. 1;
HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;
HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No.3 or SEQ ID No. 19;
LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID No. 4;
LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 5;
LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 6.
It is another object of the present invention to provide an antibody or antigen binding fragment comprising a heavy chain variable region comprising the above-described HCDR1-3 and a light chain variable region comprising the above-described LCDR1-3.
It is another object of the invention to provide nucleic acids, vectors or cells related to said antibodies or antigen binding fragments.
The invention also provides methods of making the antibodies or antigen binding fragments.
The invention also provides an antibody conjugate and a kit/diagnostic reagent comprising the antibody or antigen binding fragment or the antibody conjugate.
The invention also provides the use of the antibody or antigen binding fragment or the antibody conjugate in the preparation of a kit or diagnostic reagent.
Drawings
FIG. 1 shows the result of SDS-PAGE of DF-5M32RMb1 antibodies.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The present invention relates to an antibody or antigen binding fragment comprising the following CDRs:
HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID No. 1;
HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;
HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No.3 or SEQ ID No. 19;
LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID No. 4;
LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 5;
LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 6.
In the present invention, 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. The term "antigen binding fragment" is a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab (consisting of intact light chain and Fd), fv (consisting of VH and VL), scFv (single chain antibody, with a linker peptide between VH and VL) or single domain antibody (consisting of VH only). Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.
In the present invention, 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 contributing to the binding affinity of an antibody or antigen binding fragment to the antigen or epitope it recognizes. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present invention, 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 invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In the present invention, 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 can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions.
In the present invention, 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 some embodiments, 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 or consists of any one of the amino acid sequences SEQ ID NO 7 or SEQ ID NO 20;
HFR2-4 comprises the amino acid sequence of SEQ ID NO 8-10 in sequence, or consists of the amino acid sequence of SEQ ID NO 8-10 in sequence;
LFR1-4 comprises the amino acid sequences of SEQ ID NO. 11-14 in sequence, or consists of the amino acid sequences of SEQ ID NO. 11-14 in sequence.
In other embodiments, each of the framework region amino acid sequences of an antibody or antigen binding fragment 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 some embodiments, the antibody or antigen binding fragment of the binding affinity K D ≤6.36*10 -8 M。
In a second aspect, the invention relates to an antibody or antigen binding fragment comprising a heavy chain variable region comprising the above-described HCDR1-3 and the above-described HFR1-4 and/or a light chain variable region comprising the above-described LCDR1-3 and the above-described LFR1-4.
In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs 15, 21-23; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 16.
In some embodiments, the amino acid sequence of the heavy chain variable region consists of the amino acid sequence set forth in any one of SEQ ID NOs 15, 21-23; the light chain variable region consists of an amino acid sequence shown in SEQ ID NO. 16.
In some embodiments, the antibody further comprises a constant region;
in some embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region;
in some embodiments, 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 kappa-type or lambda-type light chain constant regions.
In some embodiments, the species source of the heavy and light chain constant regions is cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock, or human.
In some embodiments, the species source of the heavy chain constant region and the light chain constant region is mouse.
In some embodiments, 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 more than 80% of homology with SEQ ID NO. 25.
In other embodiments, the constant region amino acid sequence of an antibody or antigen binding fragment 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 constant region (SEQ ID NO:24, 25) described above.
In some embodiments, the 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.
In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2, scfv, fv, fd, single chain antibody, diabody, or domain antibody.
The invention also relates to nucleic acids encoding the antibodies or antigen binding fragments.
Nucleic acids are typically RNA or DNA, and nucleic acid molecules may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are used when they are incorporated into vectors.
The invention also relates to a vector containing said nucleic acid.
The invention also relates to a cell containing said nucleic acid or said vector.
The invention also relates to an antibody conjugate comprising the antibody or antigen binding fragment and a coupling moiety coupled thereto;
in some embodiments, the coupling moiety comprises a detectable label selected from the group consisting of a purification tag (e.g., his tag), such as colloidal gold, a radiolabel, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, saccharide oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin-labeling.
In some embodiments, the coupling moiety is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
The invention also relates to a kit or diagnostic reagent comprising said antibody or antigen binding fragment or said antibody conjugate, wherein:
optionally, the 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 NS 1.
The use of said antibody or antigen binding fragment or said antibody conjugate in the preparation of a kit or diagnostic reagent is also within the scope of the present invention.
Embodiments of the present invention will be described in detail below with reference to examples.
In the following examples, restriction enzymes, rTaq DNA polymerase, were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMARTTMRACE cDNAAmplification 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.
Example 1 preparation of antibodies against dengue NS1 protein
1. Construction of expression plasmid
(1) Preparation of Anti-DF 5M32 antibody Gene
mRNA is extracted from hybridoma cell strains secreting Anti-DF 5M32 monoclonal antibodies, DNA products are obtained through an RT-PCR method, rTaq DNA polymerase is used for carrying out an A adding reaction, then the products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colony growth, heavy Chain (heavychain) and Light Chain (Light Chain) gene clones are respectively taken for 4 clone gene sequencing companies to sequence.
(2) Sequence analysis of Anti-DF 5M32 antibody variable region Gene
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 339bp, and the front of the VL gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 351bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene sequence.
(3) Construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector 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 gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific F.primers of the Anti-DF 5M32 antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a Light Chain gene fragment of 0.74KB and a Heavy Chain gene fragment of 1.4KB 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
Diluting the plasmid prepared in the step 1- (3) to 40 mug/100 mug by ultrapure water, regulating CHO cells to 1.43 multiplied by 107cells/mL in a centrifuge tube, mixing 100 mug of the plasmid with 700 mug of the cell, transferring the mixture into an electrorotating cup, electrorotating, sampling and counting on days 3, 5 and 7, and collecting and detecting on day 7.
Coating the micro-porous plate by diluting sheep anti-mouse IgG 1ug/ml with coating liquid (main component NaHCO 3), wherein each hole is 100uL and the temperature is 4 ℃ overnight; the next day, the washing solution (main component Na2 HPO4+Nacl) is washed for 2 times and is patted dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted cell supernatant at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing at 37deg.C for 1 hr; removing liquid in the plate, beating, adding diluted DENV positive serum, 100uL per well, and 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; adding a DENV monoclonal antibody marked with HRP, 100uL per well, and at 37 ℃ for 30min; adding a developing solution A (50 uL/hole, main component citric acid+sodium acetate+acetanilide+carbamide peroxide), and adding a developing solution B (50 uL/hole, main component citric acid+EDTA.2Na+TMB+concentrated HCL) for 10min; adding stop solution (EDTA.2Na+ concentrated H2SO 4) to obtain 50 uL/hole; OD was read on the microplate reader at 450nm (reference 630 nm).
The results showed that the reaction OD was still greater than 1.0 after 1000-fold dilution of the cell supernatant, and less than 0.1 without cell supernatant wells, indicating that antibodies generated after transient plasmid conversion were active against DENV antigen.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: 50. Mu.L of Buffer, 100. Mu.g/tube of plasmid prepared in step 1- (3), 10. Mu.L of PvuI enzyme, 500. Mu.L of sterile water, and performing water bath enzyme digestion at 37 ℃ 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
Diluting the plasmid prepared in the step 2- (2) to 40 mug/100 mug by ultrapure water, regulating CHO cells to 1.43 multiplied by 107cells/mL in a centrifuge tube, mixing 100 mug of the plasmid with 700 mug of the cell, transferring the mixture into an electrorotating cup, electrorotating, and counting the next day; 25 mu mol/L MSX 96 wells were incubated under pressure for approximately 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 strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; 3 days later, seed preservation and batch culture are carried out, the cell density is adjusted to be 0.5 multiplied by 106cells/mL, batch culture is carried out by 2.2mL, and seed preservation is carried out by 2mL, wherein the cell density is 0.3 multiplied by 106 cells/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, and calculating the expanded culture volume according to the production requirement, 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. 6.6. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
The amino acid sequences of HCDR1-3 of DF-5M32RMb1 obtained by the steps are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of LCDR1-3 are shown in SEQ ID NO.4-6, respectively. The amino acid sequences of the heavy chain variable region, the light chain variable region, the heavy chain and the light chain are respectively shown as SEQ ID NO. 15-18.
Analyzing the structure of DF-5M32RMb1, designing mutation primer, repeating steps 1- (3) to 3- (2), and screening to obtain DF-8F13RMb2-4 after activity identification. Through sequencing analysis, the heavy chain variable regions of DF-8F13RMb2-4 are shown as SEQ ID NO.21-23, the heavy chain constant regions are shown as SEQ ID NO.24, and the light chain is identical to DF-5M32RMb 1.
Example 2 affinity assay and Activity characterization
1. Affinity analysis
The purified antibody was diluted to 10ug/ml with PBST using AMC sensor, and DENV-IV antigen (an NS1 protein, available from Figpeng) was gradient diluted with PBST;
the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output.
TABLE 1
Sample name KD(M) Kon(1/Ms) Kdis(1/s)
Control antibodies 8.11E-07 2.34E+03 1.90E-03
DF-5M32RMb1 5.96E-08 2.14E+04 1.28E-03
DF-5M32RMb2 5.78E-08 3.19E+04 1.84E-03
DF-5M32RMb3 6.36E-08 2.93E+04 1.86E-03
DF-5M32RMb4 5.29E-08 3.32E+04 1.76E-03
Note that: KD represents equilibrium dissociation constant, i.e. affinity; kon represents the binding rate constant; kdis represents the dissociation rate constant.
2. Activity assay
Coating the micro-porous plate by diluting sheep anti-mouse IgG 1ug/ml with coating liquid (main component NaHCO 3), wherein each hole is 100uL and the temperature is 4 ℃ overnight; the next day, the washing liquid (main component Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted purified antibody at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing at 37deg.C for 1 hr; removing liquid in the plate, beating, adding diluted DENV positive serum, 100uL per well, and 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; the HRP-labeled DENV monoclonal antibody (available from Fipeng, paired with purified antibody) was added at 100uL per well, 37℃for 30min; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; OD was read on the microplate reader at 450nm (reference 630 nm).
TABLE 2
Sample concentration (ng/ml) 12.5 6.25 3.125 1.5625 0.78125 0
Control antibodies 1.537 0.996 0.692 0.480 0.317 0.045
DF-5M32RMb1 2.098 1.520 0.963 0.654 0.489 0.039
DF-5M32RMb2 2.042 1.474 0.960 0.603 0.471 0.031
DF-5M32RMb3 1.965 1.496 0.952 0.622 0.479 0.043
DF-5M32RMb4 2.013 1.487 0.923 0.631 0.482 0.032
Example 3 stability assessment
The monoclonal antibody prepared in example 1 was placed at 4 ℃, -80 ℃, -37 ℃ (refrigerator) for 21 days, samples were taken for 7 days, 14 days and 21 days for status observation, and activity detection was performed on the samples placed for 21 days (activity of the samples was checked using the results of enzyme-free detection OD).
The stability test results of DF-5M32RMb1 are shown in Table 3, and the results show that the DF-5M32RMb1 to DF-5M32RMb4 have no obvious protein state change after the antibodies are placed for 21 days under three examination conditions, and the activity of the antibodies has no descending trend along with the increase of the examination temperature, so that the self-produced antibodies prepared by the invention have good stability.
TABLE 3 Table 3
Sample concentration (ng/mL) 6.25 3.125 0
4 ℃,21 days sample 1.421 0.897 0.041
Sample at-80℃for 21 days 1.469 0.811 0.033
37 ℃ and 21 days of sample 1.395 0.803 0.039
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> an antibody against dengue NS1 protein and use thereof
<130> P2021055CN01
<160> 25
<170> PatentIn version 3.5
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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val
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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
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Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
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Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
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Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val
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Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
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Claims (25)

1. An antibody or antigen-binding fragment thereof against dengue virus or NS1 protein thereof, wherein the antibody or antigen-binding fragment thereof comprises the following CDRs:
HCDR1, its amino acid sequence is shown in SEQ ID NO. 1;
HCDR2, its amino acid sequence is shown in SEQ ID NO. 2;
HCDR3, its amino acid sequence is shown in SEQ ID NO.3 or SEQ ID NO. 19;
LCDR1, the amino acid sequence of which is shown in SEQ ID NO. 4;
LCDR2, its amino acid sequence is shown in SEQ ID NO. 5;
LCDR3 has the amino acid sequence shown in SEQ ID NO. 6.
2. The antibody or antigen-binding fragment thereof of claim 1, further comprising 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 antibody or antigen-binding fragment thereof
HFR1 comprises 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 more than 80% homology with SEQ ID NO. 20;
HFR2 comprises 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 comprises SEQ ID NO. 11 or an amino acid sequence having more than 80% homology with SEQ ID NO. 11;
LFR2 comprises SEQ ID NO. 12 or an amino acid sequence having more than 80% homology with SEQ ID NO. 12;
LFR3 comprises SEQ ID NO. 13 or an amino acid sequence having more than 80% homology with SEQ ID NO. 13;
LFR4 comprises SEQ ID NO. 14 or an amino acid sequence having more than 80% homology with SEQ ID NO. 14.
3. An antibody or antigen-binding fragment thereof against dengue virus or NS1 protein thereof, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1-3 of claim 1 and HFR1-4 of claim 2 and a light chain variable region comprising LCDR1-3 of claim 1 and LFR1-4 of claim 2.
4. An antibody or antigen-binding fragment thereof against dengue virus or NS1 protein thereof, characterized in that the heavy chain variable region comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs 15, 21-23;
the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16.
5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, further comprising a constant region.
6. The antibody or antigen-binding fragment thereof of claim 5, wherein the constant region comprises a heavy chain constant region and/or a light chain constant region.
7. The antibody or antigen-binding fragment thereof of claim 6, wherein 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 kappa-type or lambda-type light chain constant regions.
8. The antibody or antigen binding fragment thereof according to claim 5, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.
9. The antibody or antigen-binding fragment thereof of claim 8, wherein the cow is a dairy cow.
10. The antibody or antigen-binding fragment thereof of claim 8, wherein the chicken is turkey or cocktail.
11. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant region is of murine species origin.
12. The antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain constant region is SEQ ID No.24 or an amino acid sequence having 80% or more homology with SEQ ID No. 24; the light chain constant region is SEQ ID NO. 25 or an amino acid sequence with more than 80% of homology with SEQ ID NO. 25.
13. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.
14. A cell comprising the nucleic acid of claim 13.
15. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, comprising culturing the cell of claim 14.
16. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 and a conjugate moiety conjugated thereto.
17. The antibody conjugate of claim 16, wherein the conjugate moiety is selected from a purification tag or a detectable label.
18. The antibody conjugate of claim 17, wherein the conjugate moiety is selected from one or more of colloidal gold, a radiolabel, a luminescent substance, a colored substance, and an enzyme.
19. The antibody conjugate of claim 17, wherein the coupling moiety is selected from one or more of a fluorescent label, a chromophore label, and an electron dense label.
20. The antibody conjugate of claim 17, wherein the conjugate moiety is selected from one or more of a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, biotin, avidin, and spin-labeling.
21. The antibody conjugate of claim 20, wherein the conjugate moiety is selected from one or more of glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.
22. The antibody conjugate of claim 16, wherein the conjugate moiety is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
23. A kit or diagnostic reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the antibody conjugate according to any one of claims 16 to 22.
24. The kit or diagnostic reagent of claim 23, further comprising an antibody or antigen binding fragment thereof, an antibody conjugate, or a fusion protein that binds to an antigen fragment other than dengue NS1 protein.
25. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or an antibody conjugate according to any one of claims 16 to 22 in the preparation of a kit or diagnostic reagent.
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