CN116655781A - Antibody FY1 for broad-spectrum recognition of flavivirus E protein - Google Patents
Antibody FY1 for broad-spectrum recognition of flavivirus E protein Download PDFInfo
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- CN116655781A CN116655781A CN202310436563.4A CN202310436563A CN116655781A CN 116655781 A CN116655781 A CN 116655781A CN 202310436563 A CN202310436563 A CN 202310436563A CN 116655781 A CN116655781 A CN 116655781A
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Classifications
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- 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
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—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
-
- 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
Abstract
The invention belongs to the field of biological medicine, and in particular relates to an antibody FY1 for recognizing a flavivirus E protein in a broad spectrum. Specifically, FY1 provided by the invention can identify the flaviviridae viruses including Zika virus, dengue virus type 1, dengue virus type 2 and West Nile virus in a broad spectrum. Accordingly, the invention also provides nucleic acid molecules encoding antibody FY1, vectors expressing antibody FY1, host cells containing antibody FY1.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antibody FY1 for recognizing a flavivirus E protein in a broad spectrum.
Background
The flaviviridae virus is transmitted through mosquito or tick bites, and causes fever, rash, skin mucous membrane bleeding, encephalitis, neonatal deformity and other diseases. Some flaviviridae arboviruses such as YFV, ZIKV are prevalent mainly in africa and south america, but their spread has been expanding in recent years; the prevalence of WNV has expanded from north america to asia, europe since 1999; about 4 million people are annual DENV infected people worldwide, and nearly 7 tens of thousands are JEV infected people.
Arboviruses of the flaviviridae genus include yellow fever virus (yellow fever virus, YFV), zika virus (ZIKV), encephalitis b virus (Japanese encephalitis virus, JEV), dengue virus (DENV), west Nile Virus (WNV), tick-borne encephalitis virus (tick-borne encephalitis virus, TBEV), and the like. It is a class of enveloped single positive-strand RNA viruses, the genome encoding 3 structural proteins: a capsid protein (prM), a pre-membrane protein (E), an envelope protein (E); 7 nonstructural proteins (non-structural protein, NS) were also encoded: NS1, NS2A, NS2, NS3, NS4A, NS4B, and NS5. Structural proteins assemble into virions, whereas non-structural proteins play a role in viral replication and immune escape.
Protein E is a key protein that mediates viral infection and induces neutralizing antibodies. The E protein plays an important role in viral attachment to cells, fusion with endosomal compartments, and modulation of host immune responses. The extracellular domain of the viral E protein is folded into three structurally distinct domains (DI, DII and DII) forming head-to-tail homodimers on the surface of the virion. DI is the central domain that organizes the entire E protein structure. The DII is formed of two extension loops protruding from DI and located in the pocket of the DI and DII interface of adjacent E proteins in the dimer. Located distally of the DII is a glycine-rich hydrophobic sequence called a fusion loop, which contains residues 98-110 and is highly conserved in flaviviruses. This region is involved in a pH dependent type II fusion event; during this process, the area is exposed and redirected outward, making the area available for film contact. DIII forms a seven-chain Ig-like fold, a domain in mature virions that is located at the most distal end of the membrane, and has been thought to be involved in receptor binding.
Disclosure of Invention
The invention aims to provide an antibody FY1 for recognizing a flavivirus E protein in a broad spectrum and application thereof. Specifically, FY1 provided by the invention can identify the flaviviridae viruses including Zika virus, dengue virus type 1, dengue virus type 2 and West Nile virus in a broad spectrum.
In a first aspect of the invention there is provided an antibody that recognizes the E protein of a flavivirus, the antibody comprising:
the amino acid sequences of CDR1, CDR2 and CDR3 are variable heavy chains shown in SEQ ID NO.1-3 respectively; and
the amino acid sequences of CDR1, CDR2 and CDR3 are the variable light chains shown in SEQ ID Nos. 4-6, respectively.
Preferably, the antibody comprises Fab, fab', F (ab) 2, fv or scFv.
In particular, the Fab (Fragment of antigen binding, antigen binding fragment) refers to a portion of an antibody molecule comprising a variable region and a constant region of a light chain, a variable region and a constant region of a heavy chain, which are disulfide-bonded. The Fab 'refers to the dimer of Fab'. Fv refers to the smallest fragment of an antibody that binds to the complete antigen binding site. An Fv fragment comprises the variable region of a light chain bound to the variable region of a heavy chain. The scFv (Single-Chain Fragment Variable, single-chain variable region fragment, single-chain antibody) refers to an engineering antibody formed by directly connecting a light chain variable region and a heavy chain variable region or connecting the light chain variable region and the heavy chain variable region through a peptide chain.
More preferably, the variable heavy chain sequence of the antibody is shown in SEQ ID NO. 7.
More preferably, the variable light chain sequence of the antibody is shown in SEQ ID NO. 8.
In particular, the flaviviridae viruses include viruses well known in the art; more specifically, zika virus, dengue virus type 1, dengue virus type 2 and West Nile virus are included.
In another aspect of the invention there is provided an isolated nucleic acid molecule comprising one or both of:
1) Nucleic acid sequences encoding a variable heavy chain of an antibody, said variable heavy chain comprising CDR1, CDR2 and CDR3 of amino acid sequences SEQ ID No.1-3, respectively.
2) Nucleic acid sequences encoding a variable light chain of an antibody, said variable light chain comprising CDR1, CDR2 and CDR3 of amino acid sequences SEQ ID nos. 4-6, respectively.
Preferably, the nucleic acid sequence encoding the variable heavy chain of the antibody is shown in SEQ ID NO. 9.
Preferably, the nucleic acid sequence encoding the variable light chain of the antibody is shown in SEQ ID NO. 10.
The term "CDR" refers to the Complementarity Determining Regions (CDRs) of which three constitute the binding characteristics CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region and three constitute the binding characteristics CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region. Defining CDR boundaries and lengths varies in different classification and numbering systems, whereby CDRs may be defined by Kabat, chothia, contact or any other boundaries.
In a further aspect the invention provides a vector comprising the aforementioned isolated nucleic acid molecule or expressing the aforementioned antibody.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
In a further aspect the invention provides a host cell in which an antibody according to the invention is expressed or which comprises an isolated nucleic acid molecule or vector as hereinbefore described.
In another preferred embodiment, the host cell comprises: coli, yeast cells, mammalian cells, phage, or combinations thereof.
In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, CHO cell, BHK cell, NSO cell or COS cell.
In yet another aspect of the invention, a conjugate is provided, which may be chemically or genetically labeled to provide a detectable antibody; detectable moieties include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, and nonradioactive paramagnetic metal ions.
In another aspect the invention provides a kit comprising said antibody or conjugate.
Preferably, the kit further comprises a detection reagent; in a preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particle and imaging agent.
In another aspect the invention provides the use of any one or more of the aforementioned antibodies, isolated nucleic acid molecules, vectors or host cells for the preparation of a product that binds to a flavivirus.
Preferably, the product that binds to a flavivirus is a product that detects a flavivirus.
Preferably, the antibodies of the invention in the product are bound to a solid phase (solid support).
In particular, the term "solid support" according to the present invention may be a plate, a tube or polystyrene beads, as is well known in radioimmunoassays and enzyme immunoassays. In addition, various porous materials such as nylon, nitrocellulose, cellulose acetate, glass fibers and other porous polymers may be used as the solid support.
More preferably, the antibody is attached to the solid phase by its constant or variable region.
In another aspect, the invention provides a method of producing antibodies that broadly recognize the E protein of a flavivirus, comprising culturing the aforementioned host cell.
More specifically, the production method comprises the operations of culturing the host cell and obtaining an antibody against the flavivirus E protein from the culture;
alternatively, the method comprises the steps of introducing the vector into a host cell, culturing the host cell, and obtaining antibodies against the flavivirus E protein from the culture.
Preferably, the manipulation of obtaining antibodies against the E protein of the flaviviridae virus can be accomplished by methods commonly used in the art, such as, for example, centrifugation of the medium and host cells, high pressure homogenization of the disrupted cells, centrifugation to remove cell debris, and affinity chromatography to purify the antibodies.
In another aspect of the invention there is provided a method for detecting a flavivirus E protein for non-diagnostic purposes comprising the step of contacting a sample to be tested with an antibody of the invention.
More specifically, the method further comprises the step of detecting a reaction of the sample to be tested after binding to the antibody.
Preferably, the sample to be measured may be an environmental sample, for example, air, water, soil, cultivation equipment (cultivation environment), or the like.
Preferably, the sample to be tested may be an animal or human body from a non-human being.
Preferably, the sample to be tested may be a physiological fluid such as blood, serum, plasma, saliva, ocular secretions, cerebrospinal fluid, pus, exudates, milk, sweat, tears, ear exudates, sputum, lymph, urine, faeces, oronasal secretions; tissues such as lung, spleen and kidney.
Preferably, the sample to be detected may be subjected to pretreatment, such as extraction, addition, separation, dilution, concentration, filtration, distillation, dialysis, etc., and the above pretreatment may improve the accuracy and sensitivity of the detection.
Preferably, the method further comprises detecting the virus (antigen) by means of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography or the like.
Preferably, the method may be based on a competition method or a sandwich method.
The term "competition method" as used herein is a method of comparing the relationship between the amount of antigen in a sample and the amount of a known amount of labeled antigen competing for binding to an antibody. The step of performing a competition-based immunological assay comprises: a sample containing an unknown amount of the antigen of interest (i.e., a flavivirus in the present invention) is added to a solid support that has been coated with antibodies, while a predetermined amount of the labeled antigen of interest is added to react. After incubation, the solid support is rinsed and the amount of antigen of interest is quantified by detecting the activity of the label bound to the support.
In the term "sandwich method" according to the present invention, the antigen of interest in the sample is sandwiched between the coating mab and the labeling mab, and then a substrate for a marker such as an enzyme is added, and the presence of the antigen is detected and judged by the change in the color of the substrate. For example, a sample containing an unknown amount of the antigen of interest is first added to a solid support that has been physically or chemically pre-coated with the monoclonal antibodies of the invention for reaction. Then, the labeled monoclonal antibody of the present invention is added for reaction. After incubation, the support is rinsed and the activity of the label bound to the support is detected.
Specifically, as is well known in the art, the flaviviridae genus includes the following viruses: apoli virus, aroa virus (including the following types: the viruses Bussula quara virus, paullinia cucullata virus Iguape virus, naranjal virus, bagaza virus) Ban Ji virus Banzi virus, bouboui virus, bucara Sha Bianfu virus Bukalasa bat virus, caxipanai virus Capipacore virus, karil island virus Carey Island virus, cell fusion factor virus Cell fusing agent virus, bovine bone mountain virus Cowbone Ridge virus, dakar bat virus, dengue virus Denguee virus (including the following types: dengue virus type 1 Dengue virus type 1, dengue virus type 2 Dengue virus type 2, dengue virus type 3 Dengue virus type 3, dengue virus type 4 Dengue virus type 4), edge Hill virus, enrober bat virus Entebbe bat virus (including the following types: sokoluk virus, caldazole virus, kluka virus, kluyveromyces virus Gadgets Gully virus, ilheus virus (including the following types: luo Xiao virus Rocio virus), saccharum turkey meningitis virus Israel turkey meningoencephalomyelitis virus, japanese encephalitis virus Japanese encephalitis virus, zhu Gela virus Jugra virus, hu Diya Pa virus, kadamm virus, kadougo Du Gu virus Kedougo virus, kekokober virus (including the following types: stratford virus), koutang virus, kotanogo virus, kognus forest virus Kyasanur Forest disease virus, langat virus Lango virus, st.Louis encephalitis virus Louis encephalitis virus, sheep jumping virus Louping ill virus, sheep jumping virus type Louping ill virus British subtype, the sheep jumping disease virus type Louping ill virus Irish subtype, sheep jumping disease virus spanish subtype Louping ill virus Spanish subtype, sheep jumping disease virus turkish subtype Louping ill virus Turkish subtype) milban virus Meaban virus, moromi virus Modoc virus, monda hepialus encephalitis virus Montana myotis leukoencephalitis virus, ink Lei Gunao inflammatory virus Murray Valley encephalitis virus (including the following types: alfuy virus), entaya virus, emblica hemorrhagic fever virus Omsk hemorrhagic fever virus, gold-edged bat virus Phnom Penh bat virus (including the following: black wind tunnel virus, batu Cave virus), powassan virus, rikura Wo Bingdu Rio Bravo virus, royal Farm virus (including the following types: karshi virus), saboya virus, wo Ya virus (including the following types: the Botikum virus Potiskum virus), saint Louis encephalitis virus, the Sal Vieja virus, the St.Pacific virus San Perlita virus, the Somarz reef virus Saumarez Reef virus, the Sepik virus, the Tamana bat virus, the Tembusu virus, the Tick-borne encephalitis virus Tick-borne encephalitis virus (including the following types: european Tick-borne encephalitis virus Tick-borne encephalitis virus European subtype, far east Tick-borne encephalitis virus Tick-borne encephalitis virus Far Eastern subtype, siberian Tick-borne encephalitis virus Tick-borne encephalitis virus Siberian subtype), qiu Lieni virus Tyuleniy virus, uganda S virus, ustutu virus, wessel Brownian virus Wesselsbron virus, west Nile virus (including the following types: kunjin virus), ya Wen De virus Yaounde virus, yellow fever virus Yellow fever virus, jokule virus, zika virus (including the following types: the stond Wen Ni virus Spondweni virus).
In particular, the invention in the specific examples demonstrated that the antibodies of the invention have specific binding activity against Zika virus, dengue virus type 1 Dengue virus type, dengue virus type 2 Dengue virus type 2, west Nile virus, a representative binding activity of the antibodies of the invention against Flaviviridae.
Drawings
FIG. 1 is a diagram showing the results of SDS-PAGE for detecting an antibody of the present invention.
FIG. 2 is a graph showing the results of an ELISA method for detecting specific binding of the antibody of the present invention to the Zika virus E protein and the dengue virus E protein type 2.
FIG. 3 is a graph showing the results of a flow cytometry assay for the specific binding of an antibody of the present invention to dengue virus type 1E protein.
FIG. 4 is a graph of experimental results of flow cytometry for detecting specific binding of antibodies of the present invention to the E protein of West Nile Virus.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
Example 1 preparation of antibodies
1. Experimental procedure(1) The structures of the heavy chain variable region and the light chain variable region of the antibody provided by the invention are shown in table 1 and table 2, the heavy chain variable region and the light chain variable region of the antibody are synthesized by a gene synthesis method, and fragments are cloned into a vector by a molecular cloning method.
TABLE 1 Structure of the heavy chain variable region of the antibodies of the invention
TABLE 2 Structure of the light chain variable region of the antibodies of the invention
| Sequence(s) | Position of | Length of | SEQ ID NO | |
| LFR1 | QSVLTQPPSLSGTPGQRVTISC | 1-22 | 22 | - |
| CDR-L1 | SGGQSNIGSNFVY | 23-35 | 13 | 4 |
| LFR2 | WYQHPPGTAPKLLIF | 36-50 | 15 | - |
| CDR-L2 | RNNQRPS | 51-57 | 7 | 5 |
| LFR3 | GVPDRFSGSKSGTSASLAVSGLRTEDEADYFC | 58-89 | 32 | - |
| CDR-L3 | AVWDNTLNVWV | 90-100 | 11 | 6 |
| LFR4 | FGGGTKLTVL | 101-110 | 10 | - |
(2) The recombinant vector for expressing the antibody constructed in the step 1 is transfected into 293T cells in the logarithmic growth phase, fresh culture medium is changed for 6 to 8 hours after transfection, and the cells are cultured in an 8% CO2 incubator at 37 ℃ for 96 hours. The transfection supernatant was collected, centrifuged at 4000rpm for 1 hour and purified by Protein A affinity chromatography. The expression and purification of the antibodies were examined by SDS-PAGE.
2. Results
The results are shown in FIG. 1, and the relatively pure protein is obtained, and the light chain and the heavy chain of the antibody after melting can be observed, which indicates that the antibody preparation is successful.
Example 2 binding Activity of ELISA detection antibodies to Zika Virus E protein and dengue virus type 2E protein
The coating solution was diluted to 1. Mu.g/mL with Zika virus E protein and dengue virus type 2E protein, 100. Mu.L per well was added to the ELISA plate and placed in a wet box overnight at 4 ℃. The plate washer was cleaned 3 times, 1.5% casein blocked, 200. Mu.L per well, and the wet box blocked at 37℃for 1h. The antibodies were diluted to different concentrations with 1xPBS and added to the ELISA plate at 100. Mu.L per well, reacted at 37℃for 1h in a wet box, the ELISA plate was washed 3 times, goat anti-human (Fab') 2-HRP secondary antibody was added, reacted at room temperature for 45min, the ELISA plate was washed 5 times and developed with 100. Mu.L TMB substrate, reacted for 3min and the reaction was stopped with 100. Mu.L 2N H2SO4, and read at 450nm by ELISA. And drawing an antibody-antigen binding curve with the antibody concentration as an abscissa and the OD value as an ordinate.
As shown in FIG. 2, the antibody prepared by the invention can specifically recognize the Zika virus E protein and the dengue virus type 2E protein on the solid carrier, and has good dose dependency of the combination between the antigen and the antibody along with the increase of the concentration of the antibody.
Example 3 binding Activity of flow-detection antibody FY1 to West Nile Virus and dengue virus type 1E protein
According to Lipo3000 transfection reagent instructions, west Nile virus and dengue virus type 1E protein expression plasmid are respectively transfected into BHK21 cells, cells are collected after 48hr, fixed at room temperature for 20min, the cells are washed 3 times by using cell permeabilization liquid, antibody FY1 (5 mug/mL) is added for incubation, room temperature for 20min, the cell permeabilization liquid is washed 3 times, secondary antibody (anti-human IgG-FITC) is added for marking, light shielding at 4 ℃ is carried out for 30min, and after the cell permeabilization liquid is washed 3 times, the cells are resuspended in 1% paraformaldehyde for flow cytometry.
The results of the assay are shown in FIGS. 3-4, where FIG. 3 shows the binding to dengue virus type 1E protein and FIG. 4 shows the binding to West Nile virus E protein. The results show that the antibody prepared by the invention can specifically recognize the E protein of the West Nile virus and the dengue virus type 1.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (10)
1. An antibody that broad-spectrum recognizes a flavivirus E protein, the antibody comprising:
the amino acid sequences of CDR1, CDR2 and CDR3 are variable heavy chains shown in SEQ ID NO.1-3 respectively; and, a step of, in the first embodiment,
the amino acid sequences of CDR1, CDR2 and CDR3 are the variable light chains shown in SEQ ID Nos. 4-6, respectively.
2. The antibody of claim 1, comprising Fab, fab', F (ab) 2, fv, or scFv;
preferably, the sequence of the variable heavy chain of the antibody is shown in SEQ ID NO. 7;
preferably, the sequence of the variable light chain of the antibody is shown in SEQ ID NO. 8.
3. An isolated nucleic acid molecule comprising one or both of:
1) A nucleic acid sequence encoding the variable heavy chain of the antibody of claim 1; preferably, the nucleic acid sequence encoding the variable heavy chain of the antibody is shown in SEQ ID NO. 9;
2) A nucleic acid sequence encoding a variable light chain of the antibody of claim 1; preferably, the nucleic acid sequence encoding the variable light chain of the antibody is shown in SEQ ID NO. 10;
preferably, the nucleic acid molecule encodes the antibody of claim 1.
4. A vector comprising the isolated nucleic acid molecule of claim 3 or expressing the antibody of claim 1;
preferably, the vector is an expression vector.
5. A host cell expressing the antibody of claim 1, or comprising the isolated nucleic acid molecule of claim 3 or the vector of claim 4;
preferably, the host cell comprises: coli, yeast cells, mammalian cells, phage, or combinations thereof;
preferably, the host cell is preferably a mammalian cell;
more preferably, the host cell is a HEK293 cell, CHO cell, BHK cell, NSO cell or COS cell.
6. A conjugate, said conjugate being an antibody having a detectable moiety-tag;
preferably, the detectable moiety comprises an enzyme, prosthetic group, fluorescent material, luminescent material, radioactive material, positron emitting metal, and non-radioactive paramagnetic metal ion.
7. A kit comprising the conjugate of claim 6 or the antibody of claim 1.
8. A method of producing an antibody that recognizes the E protein of a flavivirus, the method comprising culturing the host cell of claim 5; or alternatively, the process may be performed,
introducing the isolated nucleic acid molecule of claim 3 or the vector of claim 4 into a cell for cell culture;
more specifically, the production method comprises the operations of culturing cells and obtaining antibodies against the flavivirus E protein from the culture;
preferably, said obtaining antibodies against the flavivirus E protein from the culture is performed by any one or more of the following methods: the medium and host cells are centrifuged, the cells are broken up by high pressure homogenization, the cell debris is removed by centrifugation or the antibody is purified by affinity chromatography.
9. A method for detecting the E protein of a flaviviridae virus for non-diagnostic purposes, said method comprising the step of contacting a sample to be tested with an antibody according to claim 1;
preferably, the antibody of claim 1 is an antibody with a detectable moiety label;
more specifically, the method further comprises the step of detecting a reaction after the sample to be tested is bound to the antibody;
preferably, the sample to be tested comprises an animal body or a human from a non-human;
preferably, the sample to be tested comprises physiological fluids such as blood, serum, plasma, saliva, ocular secretions, cerebrospinal fluid, pus, exudates, milk, sweat, tears, ear exudates, sputum, lymph, urine, faeces, oronasal secretions; tissues such as lung, spleen and kidney;
preferably, the sample to be tested may be pretreated;
preferably, the step of pre-treatment comprises extraction, addition, separation, dilution, concentration, filtration, distillation, dialysis;
preferably, the method further comprises detecting the flavivirus E protein by means of enzyme-linked immunosorbent assay, enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography or the like;
preferably, the method is based on a competition method or a sandwich method;
preferably, the antibodies in the product are bound to a solid phase;
preferably, the solid phase comprises a plate, a test tube, polystyrene beads, nylon, nitrocellulose, cellulose acetate, glass fiber;
more preferably, the antibody is attached to the solid phase by its constant or variable region.
10. Use of any one or more of the antibody of claim 1, the isolated nucleic acid molecule of claim 3, the vector of claim 4 or the host cell of claim 5, the conjugate of claim 6 and the kit of claim 7 for the preparation of a product for detecting a flavivirus;
preferably, the flavivirus is Zika virus, dengue virus type 1, dengue virus type 2, west Nile virus.
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