CN117534750A - Antibody for resisting novel coronavirus nucleocapsid protein or antigen binding fragment thereof and application thereof - Google Patents
Antibody for resisting novel coronavirus nucleocapsid protein or antigen binding fragment thereof and application thereof Download PDFInfo
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- CN117534750A CN117534750A CN202311341345.9A CN202311341345A CN117534750A CN 117534750 A CN117534750 A CN 117534750A CN 202311341345 A CN202311341345 A CN 202311341345A CN 117534750 A CN117534750 A CN 117534750A
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Classifications
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- 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
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/14—Antivirals for RNA viruses
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
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- Molecular Biology (AREA)
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Abstract
The invention belongs to the technical field of antibodies, and discloses an antibody for resisting novel coronavirus nucleocapsid protein or an antigen binding fragment thereof and application thereof. The antibody or antigen binding fragment thereof has better specificity on the novel coronavirus and/or nucleocapsid protein of the novel coronavirus, and can be used for detecting the existence or the level of the novel coronavirus nucleocapsid protein in a sample, detecting the novel coronavirus and diagnosing diseases related to the novel coronavirus infection.
Description
Technical Field
The invention belongs to the technical field of antibodies, and particularly relates to an antibody for resisting novel coronavirus nucleocapsid protein or an antigen binding fragment thereof and application thereof.
Background
Diseases caused by the novel coronavirus (SARS-CoV-2) infection are prevalent to varying degrees worldwide. At present, no specific medicine aiming at novel coronavirus infection exists, and quick screening and blocking of a transmission path of an infected person are the most effective methods for controlling the transmission of the infected person, so that a detection method which is convenient, quick and accurate to study is particularly important for epidemic prevention and control.
SARS-CoV-2 is a membranous single-stranded RNA virus, 4 structural proteins are spike (S) protein, membrane (M) protein, envelope (E) protein, nucleocapsid (N) protein, N protein is an important component of SARS-CoV-2, is an alkaline protein composed of 419 amino acids, has a molecular weight of 45.62kDa, is highly conserved in coronavirus genus, has 90% homology with N protein of SARS coronavirus, can recognize and bind RNA, participates in viral genome RNA packaging, regulates cell metabolism and cell cycle, N protein is a main immunogen in host immune response, induces strong antibody reaction, and can be used as diagnostic antigen and immunogen.
Currently, detection means for novel coronaviruses include nucleic acid detection, antibody detection, and antigen detection. Although nucleic acid detection is the main method for detecting novel coronaviruses, the method has high sensitivity, specificity and accuracy, and the disadvantage is that the detection requires high conditions and takes a long time. The antibody detection is that the antibody can be detected only after a certain time of virus infection, the antibody can not be detected in early stage of virus infection, in addition, the vaccinated population can also detect the new coronavirus antibody, and false positive exists, so that the antigen detection is of great significance. Antigen detection is generally used in the acute infection phase, i.e. sample detection within 7 days of symptoms of the suspected population, and antigen positive results can be used for early diversion and rapid management of the suspected population. The method is simple and convenient to operate, the detection result can be obtained within 15 to 20 minutes, and residents can finish sampling and self-testing at home. Compared with the gold standard for detecting nucleic acid, although the detection has a certain detection omission probability, the antigen detection is an effective detection means and can early discover new coronavirus infection relative to the conditions of high risk, high epidemic, aggregation infection and the like in the detection process.
The common novel coronavirus antigen rapid detection method comprises a colloidal gold method, a latex method and a fluorescence immune layer analysis method, and is applicable to large-scale rapid detection aiming at common screening, wherein the colloidal gold detection method has very low requirements on personnel, sites and equipment. The method does not need special treatment specimens, can qualitatively detect the novel coronavirus in the nasal swab sample in vitro, obtains detection results by naked eye observation within 15min, can automatically finish detection, avoids cross infection between doctors and patients, breaks through the limitation of detection technology on personnel and places, and has the advantages of strong specificity, high sensitivity, simplicity, convenience, rapidness, naked eye interpretation, high stability and the like. In order to realize the rapid detection of the novel coronavirus antigen, research and acquisition of the monoclonal antibody of the N protein of the novel coronavirus are of great significance.
Disclosure of Invention
The object of the first aspect of the present invention is to provide an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein.
The object of the second aspect of the present invention is to provide a recombinant protein.
The object of a third aspect of the present invention is to provide a biomaterial related to the antibody against a novel coronavirus nucleocapsid protein of the first aspect of the present invention or an antigen binding fragment thereof or the recombinant protein of the second aspect.
The object of the fourth aspect of the present invention is to provide a conjugate.
The object of the fifth aspect of the present invention is to provide a solid support.
The object of a sixth aspect of the present invention is to provide the use of an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein according to the first aspect of the present invention, a recombinant protein according to the second aspect, a biomaterial according to the third aspect, a conjugate according to the fourth aspect, and/or a solid support according to the fifth aspect for the preparation of a product.
A seventh aspect of the invention is directed to a product.
An object of the eighth aspect of the present invention is to provide the method for producing the antibody against a novel coronavirus nucleocapsid protein of the first aspect of the present invention or an antigen-binding fragment thereof or the recombinant protein of the second aspect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein (N protein), said antibody or antigen binding fragment thereof comprising a heavy chain and a light chain:
the heavy chain of the antibody or antigen binding fragment thereof comprises:
a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);
the light chain of the antibody or antigen binding fragment thereof comprises:
a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence of SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).
Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L3 of said antibody or antigen binding fragment thereof are set forth in SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 16. SEQ ID NO:17, the amino acid sequence of CDR-L2 is: LVS, the CDRs are defined in IMGT definition scheme.
Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 of said antibody or antigen binding fragment thereof are as shown in SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO:17, said CDRs are defined according to the Kabat definition scheme.
Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 of said antibody or antigen binding fragment thereof are as shown in SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 10. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO:17, said CDRs are defined in Chothia definition scheme.
Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 of said antibody or antigen binding fragment thereof are as shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO:22, the CDR is defined in a Contact definition scheme.
Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a1 SEQ ID NO:2; or (b)
a2 SEQ ID NO:2 via one or several amino acid substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:2, the protein shown in the formula 2 has the same functional amino acid sequence; or (b)
a3 With SEQ ID NO:2 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:2, the protein shown in the formula 2 has the same functional amino acid sequence;
the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
b1 SEQ ID NO:4, a step of; or (b)
b2 SEQ ID NO:4 via one or several amino acid substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:4, wherein the protein shown in the formula 4 has the same functional amino acid sequence; or (b)
b3 With SEQ ID NO:4 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:4, and the protein has the same functional amino acid sequence.
Preferably, the antibody or antigen binding fragment thereof comprises at least one of a full length antibody, fab ', F (ab') 2, fv, scFv, bispecific antibody, multispecific antibody.
Preferably, the heavy chain of the antibody or antigen binding fragment thereof further comprises a heavy chain constant region; and/or
The light chain of the antibody or antigen binding fragment thereof further comprises a light chain constant region.
In a second aspect of the invention, there is provided a recombinant protein comprising: the antibody or antigen-binding fragment thereof against the novel coronavirus nucleocapsid protein of the first aspect of the present invention; and
optionally a tag sequence to assist expression and/or purification.
Preferably, the tag sequence is selected from at least one of the following group: his tag, GGGS sequence, FLAG tag.
In a third aspect of the invention there is provided a biomaterial associated with an antibody or antigen-binding fragment thereof against a novel coronavirus nucleocapsid protein of the first aspect of the invention, or a recombinant protein of the second aspect of the invention, said biomaterial comprising at least one of h 1) to h 8):
h1 A nucleic acid molecule encoding an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein of the first aspect of the invention, or a recombinant protein of the second aspect;
h2 An expression cassette comprising h 1) the nucleic acid molecule;
h3 A vector comprising h 1) said nucleic acid molecule;
h4 A vector comprising h 2) the expression cassette;
h5 A transgenic cell line comprising h 1) said nucleic acid molecule;
h6 A transgenic cell line comprising h 2) said expression cassette;
h7 A transgenic cell line comprising h 3) the vector;
h8 A transgenic cell line comprising h 4) said vector.
Preferably, the transgenic cell line does not comprise propagation material.
Preferably, the nucleic acid molecule encoding an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein of the first aspect of the invention comprises a nucleic acid molecule encoding a heavy chain variable region of an anti-novel coronavirus nucleocapsid protein of the first aspect of the invention and a nucleic acid molecule encoding a light chain variable region of an anti-novel coronavirus nucleocapsid protein of the first aspect of the invention.
Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the anti-novel coronavirus nucleocapsid protein of the first aspect of the present invention comprises:
a211 As shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b)
a212 SEQ ID NO:1 by substitution and/or deletion and/or addition of one or several nucleotides and which correspond to SEQ ID NO:1, and the nucleic acid molecule has the nucleotide sequence with the same function; or (b)
a213 With SEQ ID NO:1 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:1, and the nucleic acid molecule has the nucleotide sequence with the same function;
the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the anti-novel coronavirus nucleocapsid protein of the first aspect of the present invention comprises:
a221 As shown in SEQ ID NO:3, a nucleotide sequence shown in figure 3; or (b)
a222 SEQ ID NO:3 via one or several nucleotide substitutions and/or deletions and/or additions, and which correspond to SEQ ID NO:3, the nucleic acid molecules shown in the formula I have nucleotide sequences with the same functions; or (b)
a223 With SEQ ID NO:3 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:3, and the nucleic acid molecules shown in the formula I have nucleotide sequences with the same functions.
In a fourth aspect of the invention, there is provided a conjugate comprising: at least one of an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein of the first aspect of the invention and a recombinant protein of the second aspect of the invention;
and a coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron dense label, biotin/avidin, a spin label, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein.
Preferably, the detectable label is a fluorescent or luminescent label.
Preferably, the detectable label is selected from any one of acridinium ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.
Preferably, the radionuclide is selected from at least one of Tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, and Re-188.
Preferably, the medicament is other medicament (e.g. antiviral medicament: fepima Weirui darcy, or interferon, etc.) for the prevention (prophylaxis or treatment) of a novel coronavirus infection and/or a disease associated with a novel coronavirus infection.
Preferably, the disease associated with a novel coronavirus infection comprises a novel coronavirus infection.
In a fifth aspect of the invention there is provided a solid support having coupled to its surface an antibody or antigen binding fragment thereof against the novel coronavirus nucleocapsid protein of the first aspect of the invention and/or the recombinant protein of the second aspect.
In a sixth aspect, the invention provides the use of an antibody or antigen binding fragment thereof of the first aspect of the invention against a novel coronavirus nucleocapsid protein, a recombinant protein of the second aspect, a biological material of the third aspect, a conjugate of the fourth aspect, and/or a solid support of the fifth aspect for the preparation of a product.
Preferably, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip.
Preferably, the medicament has at least one of the functions i 1) to i 2):
i1 Prevention and treatment of novel coronavirus infection;
i2 Preventing and treating diseases related to novel coronavirus infection.
Preferably, the reagent, assay plate, assay chip or kit has at least one of the functions j 1) to j 3):
j1 Detecting the presence or level of a novel coronavirus nucleocapsid protein in a sample;
j2 Detecting a novel coronavirus;
j3 Diagnosing a disease associated with a novel coronavirus infection.
Preferably, the disease associated with a novel coronavirus infection comprises a novel coronavirus infection.
In a seventh aspect of the invention, there is provided a product comprising at least one of k 1) to k 4):
k1 The antibody or antigen binding fragment thereof against the novel coronavirus nucleocapsid protein of the first aspect of the present invention;
k2 A recombinant protein according to the second aspect of the invention;
k3 A conjugate according to the fourth aspect of the invention;
k4 A solid support according to the fifth aspect of the present invention.
Preferably, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip.
Preferably, the medicament has at least one of the functions i 1) to i 2):
i1 Prevention and treatment of novel coronavirus infection;
i2 Preventing and treating diseases related to novel coronavirus infection.
Preferably, the reagent, assay plate, assay chip or kit has at least one of the functions j 1) to j 3):
j1 Detecting the presence or level of a novel coronavirus nucleocapsid protein in a sample;
j2 Detecting a novel coronavirus;
j3 Diagnosing a disease associated with a novel coronavirus infection.
Preferably, the disease associated with a novel coronavirus infection comprises a novel coronavirus infection.
In an eighth aspect of the invention there is provided a method of preparing an antibody or antigen binding fragment thereof against a novel coronavirus nucleocapsid protein according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention by culturing a transgenic cell line according to the third aspect of the invention.
The beneficial effects of the invention are as follows:
the invention provides an antibody or antigen binding fragment thereof for resisting a novel coronavirus nucleocapsid protein, which has better specificity for the novel coronavirus and/or the nucleocapsid protein of the novel coronavirus, and can be used for detecting the existence or the level of the novel coronavirus nucleocapsid protein in a sample, detecting the novel coronavirus and diagnosing diseases related to the novel coronavirus infection.
Drawings
FIG. 1 is a graph showing SDS-PAGE of eluted samples of imidazole from example 1 at different collection times: wherein M is a protein molecular weight standard (Marker), 1 is a negative control, 2 is a positive control, and 3-13 are eluted samples of different collection times at a concentration of 250mM imidazole.
FIG. 2 is a graph showing the results of serum titer detection of mice after immunization in example 2.
FIG. 3 is a graph showing the results of ascites titer tests in mice after injection of the cell line 23187N in example 3.
FIG. 4 is a diagram showing SDS-PAGE of the purified novel coronavirus N protein mab 23187N of example 3: wherein M is a protein molecular weight standard (Marker), and 1 and 2 are purified novel coronavirus N protein monoclonal antibody 23187N.
FIG. 5 is a graph showing the results of specific detection of novel coronavirus N protein mab 23187N in example 3.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
EXAMPLE 1 obtaining novel coronavirus N recombinant proteins
1. Construction of recombinant expression vectors
The cDNA sequence of SARS-CoV-2 is used as template, primer is designed and synthesized according to NCBI sequence number (No. MT123290.1), SARS-CoV-2N gene sequence (the amplified product is 28277-29536bp of MT 123290.1), the product is identified by agarose gel electrophoresis, the carrier pMAL-c6T and PCR amplified product are digested with NotI and BamHI, the target gene and carrier large fragment are recovered by agarose gel electrophoresis back gel recovery kit, connected according to conventional mode, the competent cell of colibacillus BL21 (DE 3) is transformed, monoclonal is selected, amplified and cultured, and the expression carrier pMAL-c6T N is obtained by PCR amplification, enzyme digestion and sequencing identification.
Expression and purification of SARS-CoV-2N protein
The positive BL21 (DE 3) clone containing the recombinant expression vector pMAL-c6T N is identified to be correct, plasmid is extracted after amplification culture, the plasmid is transformed in a conventional mode, single colony is picked up to be amplified and cultured in LB culture medium containing ampicillin resistance, when the concentration of bacterial liquid reaches 0.5, the bacterial liquid is subjected to induced expression 18h under the conditions of 0.1mM IPTG, 200rpm and 16 ℃, the bacterial liquid is collected by centrifugation at 4000rpm for 20min at 4 ℃, the bacterial liquid is subjected to resuspension washing by 20mL PBS, binding buffer ice bath is added for ultrasonic disruption for 3min, the ultrasonic power is 30W, ultrasonic treatment is repeated for 2s at intervals until the bacterial liquid is relatively clear, and the crushed supernatant is collected by centrifugation at 12000rpm for 20min at 4 ℃ for purification.
Purifying the target protein (N recombinant protein) by adopting nickel column affinity chromatography: loading 50% NI-NTA into column, washing the column with 4mL deionized water, adding 5mL Binding Buffer balance column, loading the split supernatant containing N recombinant protein into column, collecting penetrating liquid, loading again, washing unbound impurity protein with Binding Buffer, adding 20mL Washing Buffer to wash impurity protein, eluting protein with 250mM imidazole Buffer, and performing SDS-PAGE on the eluted samples at different collection times to detect sample purity, wherein the results are shown in FIG. 1: the purity of the N protein obtained by the affinity chromatography is higher. The BCA method determines the concentration of the purified N recombinant protein.
EXAMPLE 2 establishment of novel coronavirus N protein monoclonal antibody cell lines
1. Immunization of mice
Balb/c mice were immunized with the N recombinant protein obtained in example 1 as an immunogen. First immunization (first immunization is carried out on day 1) is emulsified by Freund's complete adjuvant and immunogen according to the volume ratio of 1:1, the dose is 100 mug/mouse, and 5 immune spots are injected subcutaneously on the back and abdomen; secondary immunization was performed on day 15 using Freund's incomplete adjuvant to immunogen in a volume ratio of 1:1 emulsifying, wherein the dosage is 50 mug/mouse, and the immunization method is the same as the previous method; three immunizations were performed on day 29, with the immunization method being the same as two-immunization; on day 36, trace amounts of tail blood were collected for ELISA assay with antibody titers greater than 1:10000, followed by antigen impact immunization and spleen cell fusion. If the antibody titer is less than 1:10000, four immunizations are performed on day 43 in a three-way manner; abdominal impact immunization was performed 3 days prior to cell fusion, and 100. Mu.g of immunogen was directly injected intraperitoneally without adjuvant.
2. Mouse serum titer detection
(1) Antigen coating: the N recombinant protein obtained in example 1 was adjusted to 1. Mu.g/mL as a coating antigen in a coating buffer, 100. Mu.L was added to each well of an ELISA plate, and the temperature was 4℃overnight.
(2) Washing: the next day the well liquid was discarded, the wells were patted dry and washed 2 times with PBST in a plate washer.
(3) Closing: 200. Mu.L of blocking solution was added to each well and incubated at 37℃for 1h.
(4) Serum (primary antibody) was prepared: mice were bled by tail-breaking, and serum from non-immunized mice was used as a negative control.
(5) Adding an antibody: the serum sample to be tested is diluted by a sealing liquid in a multiple ratio respectively, the serum of the non-immunized mice is diluted by 1:2500 to be used as a negative control, the sealing liquid is used as a blank control, the sealing liquid acts for 1h at 37 ℃, the liquid in the hole is discarded, the mixture is beaten dry, PBST is washed for 2 times, and the mixture is beaten dry.
(6) Adding enzyme-labeled secondary antibodies: the enzyme-labeled secondary antibody (HRP-goat anti-mouse) was diluted 5000-fold with blocking solution, incubated at 37℃for 1h, the liquid in the wells was discarded, and the wells were dried.
(7) Color development and color comparison: adding 50 mu L/hole of TMB color development liquid, developing color at 37 ℃ for 15min in dark, adding 100 mu L/hole of 2M sulfuric acid to terminate the reaction, and measuring the A value at 450nm by an enzyme-labeling instrument. And (3) calculating: the highest dilution factor of the antiserum when the ratio P/N of the A value of the hole to be detected to the A value of the negative control hole is more than or equal to 2.1 is the titer/titer of the serum. Mice exceeding 1:l00000 were ready for the next fusion. The results are shown in FIG. 2, and the indirect ELISA method is used for identifying the reaction of the recombinant N protein and immune serum, and the titer of the mouse serum is 1:60000.
3. Cell fusion
The spleen cells of immunized mice and SP2/0 mouse myeloma cells are mixed in a 50mL centrifuge tube according to the proportion of 5:1 in a sterile way, the supernatant is washed twice by a culture medium, 0.9mL of preheated PEG-1500 is slowly added in 50s, so that the cells are uniformly dispersed in the PEG as much as possible, and the mixture is kept stand for 1min. Slowly dripping 20mL of preheated serum-free DMEM medium at 37 ℃, adding 2mL in the first two minutes, adding 18mL in the last two minutes, completely adding in 4min, standing for 3min, centrifuging at 800rpm for 5min, discarding the supernatant, adding preheated FBS and HAT medium, gently blowing, mixing, transferring into 96-well culture plates, transferring 200 mu L per well, and culturing in an incubator.
4. Screening of positive hybridoma cells
And after the tenth day of cell fusion, the fused cells are paved with more than 50 percent of holes, and the hybridoma cells are screened by adopting an indirect ELISA method.
5. Subcloning of Positive hybridoma cells
Subcloning the positive holes obtained by screening by adopting a limiting dilution method, observing the number and the positions of cell clusters in the positive holes under an inverted microscope, sucking the cell clusters by using a 200 mu L suction head in a super clean bench, and diluting the cell number to 1-2 cells in 100 mu L. Removing prepared feeder cells, adding diluted cells into 96-well plate, labeling 100 μl each, adding 5% CO 2 Culturing for 9d at 37 ℃ in an incubator. Through 3 subcloning until 1 cell per hole, the titer of cell supernatant is detected by an indirect ELISA method, the positive rate reaches 100%, the cell strain 23187N is amplified and cultured and stored.
EXAMPLE 3 preparation and identification of novel coronavirus N protein monoclonal antibodies
3.1 preparation of novel coronavirus N protein monoclonal antibody ascites and determination of ascites titer
By means of 12-16 week old BALB/C female mice were intraperitoneally injected with 0.5mL of sterilized liquid paraffin, and after 10 days each mice was injected with 0.5mL of the cell suspension of 23187N (5X 10) of the cell line preserved in example 2 5 Individual/individual, cell suspension injected day 1 d); and collecting ascites after the 7d mice obviously swell the abdominal cavity, centrifuging at 3000rpm for 20min, removing adipose tissues, sucking the supernatant, and freezing at-20 ℃ for later use. The indirect ELISA method is used for determining the titer of ascites, and the result is shown in figure 3: ascites antibody titer is 1:1280000.
3.2 purification of monoclonal antibodies
Collecting ascites, purifying novel coronavirus N protein monoclonal antibody 23187N by octanoic acid-ammonium sulfate precipitation method, and specifically comprises the following steps: taking ascites, centrifuging at 12000rpm for 5min at 4 ℃, taking supernatant, adding 2 times of 0.06M acetic acid buffer solution (pH 4.0), and adjusting the pH value to 4.5; adding 33 mu L of n-octanoic acid into each mL of ascites, stirring at room temperature for 30min, standing at 4 ℃ for 1h to enable the hybrid protein to be fully precipitated, centrifuging at 4 ℃ for 30min, adding 0.277g of ammonium sulfate powder into each mL of liquid in the supernatant, placing a beaker on a magnetic stirrer, stirring for 1h, centrifuging at 4 ℃ for 20min at 10000g, discarding the supernatant, dissolving the precipitate by PBS, and identifying the purity of the monoclonal antibody by SDS-PAGE, wherein the result is shown in FIG. 4: the novel coronavirus N protein monoclonal antibody 23187N with high purity is obtained.
3.3 type and subclass identification of monoclonal antibodies
The experimental operation is carried out by adopting the instruction of the mouse subtype identification kit, and the type and the subclass of the novel coronavirus N protein monoclonal antibody 23187N are IgG2a and kappa light chains.
Meanwhile, monoclonal antibody 23187N (i.e., novel coronavirus N protein mab 23187N purified in 3.2 of example 3) produced by cell line 23187N stored in example 2 was sequenced by the company Ai Ji biotechnology limited, and the results were as follows: the nucleotide sequence of the heavy chain variable region of the novel coronavirus N protein mab 23187N is: CAGGTCCAA CTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGTTACACGTTCACCAGCTACTGGATGAACTGGATTAAGCAGAGGCCTGAGCAAGGCCTTGAGTGGATTGGAAGGATTAATCCTTACGATAGTGAAACTCACTACAATCAAAAGTTCAAGGACAAGGCCATATTGACTGTAGACAAGTCCTCCAACACAGCCTACATGCAATTCAACAGCCTGACATCTGGGGACTCTGCGGTCTATTACTGTGCATGTTCCTGGTTTCCTGACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 1), which corresponds to the amino acid sequence: QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWIKQRPEQGLEWIGRI NPYDSETHYNQKFKDKAILTVDKSSNTAYMQFNSLTSGDSAVYYCACSWFPDWGQGTLVT VSA (SEQ ID NO: 2); the nucleotide sequence of the light chain variable region is: GATGTCGTGATGACCCAGACTCC ACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAAATAGAGATGGAAAGACATATTTGAACTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCTTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTGCTGGCAAGGTACACATTTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 3), which corresponds to the amino acid sequence: DVVMTQTPLTLSVTIG QPASISCKSSQSLLNRDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTL KISRVEAEDLGIYYCWQGTHLWTFGGGTKLEIK (SEQ ID NO: 4).
The CDR-H1, CD R-H2 and CDR-H3 of the heavy chain variable region of the novel coronavirus N protein mab 23187N defined by different definition schemes are shown in table 1.
TABLE 1 CDR-H1, CDR-H2, CDR-H3, defined by different definition schemes for the heavy chain variable region
The CDR-L1, CD R-L2 and CDR-L3 of the light chain variable region of the novel coronavirus N protein mab 23187N defined by different definition schemes are shown in Table 2.
TABLE 2 CDR-L1, CDR-L2, CDR-L3, defined by different definition schemes for the light chain variable region
3.4 specific detection of monoclonal antibodies
Inactivated 2019-nCoV, human coronavirus OC43 (HCoV-OC 43), human coronavirus 229E (HCoV-229E), H1N1 influenza A virus, H3N2 influenza A virus, influenza B virus (Victoria), influenza B virus (Yamagata) are used as antigens, respectively (the inactivated viruses tested are all references obtained from Chinese food and drug assay institute or Chinese disease prevention control center); the ELISA plate was coated under the same conditions, the monoclonal antibody 23187N purified in 3.2 of example 3 was used as a primary antibody, the goat anti-mouse IgG marked with HRP was used as a secondary antibody, the A450mn value was read on the ELISA plate, the average value was taken from 3 wells of each sample, the specificity of the monoclonal antibody was detected by the indirect ELISA method (see example 2), and as shown in FIG. 5, the monoclonal antibody 23187N produced by the cell line 23187N preserved in example 2 (i.e., the novel coronavirus N protein monoclonal antibody 23187N purified in 3.2 of example 3) had better specificity and could be used for detecting novel coronaviruses and/or diseases caused by novel coronaviruses.
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.
Claims (10)
1. An antibody or antigen-binding fragment thereof against a novel coronavirus nucleocapsid protein, said antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain:
the heavy chain of the antibody or antigen binding fragment thereof comprises:
a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);
the light chain of the antibody or antigen binding fragment thereof comprises:
a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence of SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).
2. The antibody or antigen-binding fragment thereof of claim 1, wherein:
the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 16. SEQ ID NO:17, the amino acid sequence of CDR-L2 is: LVS, the CDRs being defined by an IMGT definition scheme; or (b)
The amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2 and the CDR-L3 of the antibody or the antigen binding fragment thereof are shown in SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO:17, said CDRs being defined by the Kabat definition scheme; or (b)
The amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2 and the CDR-L3 of the antibody or the antigen binding fragment thereof are shown in SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 10. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO:17, said CDRs being defined by a Chothia definition scheme; or (b)
The amino acid sequences of the CDR-H1, the CDR-H2, the CDR-H3, the CDR-L1, the CDR-L2 and the CDR-L3 of the antibody or the antigen binding fragment thereof are shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO:22, said CDR being defined in a Contact definition scheme;
preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises:
a1 SEQ ID NO:2; or (b)
a2 SEQ ID NO:2 via one or several amino acid substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:2, the protein shown in the formula 2 has the same functional amino acid sequence; or (b)
a3 With SEQ ID NO:2 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:2, the protein shown in the formula 2 has the same functional amino acid sequence;
the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof comprises:
b1 SEQ ID NO:4, a step of; or (b)
b2 SEQ ID NO:4 via one or several amino acid substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:4, wherein the protein shown in the formula 4 has the same functional amino acid sequence; or (b)
b3 With SEQ ID NO:4 has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology to SEQ ID NO:4, wherein the protein shown in the formula 4 has the same functional amino acid sequence;
preferably, the antibody or antigen binding fragment thereof comprises at least one of a full length antibody, fab ', F (ab') 2, fv, scFv, bispecific antibody, multispecific antibody;
preferably, the heavy chain of the antibody or antigen binding fragment thereof further comprises a heavy chain constant region; and/or
The light chain of the antibody or antigen binding fragment thereof further comprises a light chain constant region.
3. A recombinant protein comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-2; and
optionally a tag sequence to assist expression and/or purification.
4. A biological material associated with the antibody or antigen-binding fragment thereof of any one of claims 1-2, or the recombinant protein of claim 3, the biological material comprising at least one of h 1) to h 8):
h1 A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-2, or the recombinant protein of claim 3;
h2 An expression cassette comprising h 1) the nucleic acid molecule;
h3 A vector comprising h 1) said nucleic acid molecule;
h4 A vector comprising h 2) the expression cassette;
h5 A transgenic cell line comprising h 1) said nucleic acid molecule;
h6 A transgenic cell line comprising h 2) said expression cassette;
h7 A transgenic cell line comprising h 3) the vector;
h8 A transgenic cell line comprising h 4) said vector.
5. A conjugate, comprising: at least one of the antibody or antigen binding fragment thereof of any one of claims 1-2 and the recombinant protein of claim 3;
and a coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron dense label, biotin/avidin, a spin label, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein;
preferably, the detectable label is a fluorescent or luminescent label;
preferably, the detectable label is selected from any one of acridinium ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase;
preferably, the radioisotope is selected from at least one of Tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177 and Re-188.
6. A solid support having coupled to its surface the antibody or antigen-binding fragment thereof according to any one of claims 1-2, and/or the recombinant protein according to claim 3.
7. (1) The use of at least one of (5) in the preparation of a product;
(1) The antibody or antigen-binding fragment thereof of any one of claims 1-2;
(2) The recombinant protein of claim 3;
(3) The biomaterial of claim 4;
(4) The conjugate of claim 5;
(5) The solid support of claim 6;
preferably, the product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip;
preferably, the medicament has at least one of the functions i 1) to i 2):
i1 Prevention and treatment of novel coronavirus infection;
i2 Prevention and treatment of diseases associated with novel coronavirus infection;
preferably, the reagent, assay plate, assay chip or kit has at least one of the functions j 1) to j 3):
j1 Detecting the presence or level of a novel coronavirus nucleocapsid protein in a sample;
j2 Detecting a novel coronavirus;
j3 Diagnosing a disease associated with a novel coronavirus infection.
8. A product comprising at least one of k 1) to k 4):
k1 An antibody or antigen-binding fragment thereof according to any one of claims 1-2;
k2 A recombinant protein according to claim 3;
k3 A conjugate according to claim 5;
k4 A solid support according to claim 6.
9. The product according to claim 8, wherein:
the product comprises at least one of a medicine, a reagent, a detection plate, a kit and a detection chip;
preferably, the medicament has at least one of the functions i 1) to i 2):
i1 Prevention and treatment of novel coronavirus infection;
i2 Prevention and treatment of diseases associated with novel coronavirus infection;
preferably, the reagent, assay plate, assay chip or kit has at least one of the functions j 1) to j 3):
j1 Detecting the presence or level of a novel coronavirus nucleocapsid protein in a sample;
j2 Detecting a novel coronavirus;
j3 Diagnosing a disease associated with a novel coronavirus infection.
10. The method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or the recombinant protein according to claim 3, which is obtained by culturing the transgenic cell line according to claim 4.
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Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070103548A (en) * | 2006-04-19 | 2007-10-24 | 대한민국(관리부서 질병관리본부장) | Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof |
| US20100240872A1 (en) * | 2007-07-04 | 2010-09-23 | Forerunner Pharma Research Co., Ltd. | ANTI-Muc 17 ANTIBODY |
| US20140127236A1 (en) * | 2011-06-01 | 2014-05-08 | Antitope Limited | Humanised anti-cd52 antibodies |
| CN112679605A (en) * | 2021-03-15 | 2021-04-20 | 安源医药科技（上海）有限公司 | Antibodies or antigen binding fragments thereof against novel coronavirus nucleocapsid proteins and uses thereof |
| CN112961222A (en) * | 2020-02-04 | 2021-06-15 | 中国科学院微生物研究所 | 2019 novel coronavirus N protein linear epitope peptide, monoclonal antibody and application |
| US20210311054A1 (en) * | 2020-04-01 | 2021-10-07 | Institut Pasteur | Severe acute respiratory syndrome (sars) - associated coronavirus diagnostics |
| CN113583116A (en) * | 2020-04-30 | 2021-11-02 | 养生堂有限公司 | Antibodies against SARS-CoV-1 or SARS-CoV-2 and uses thereof |
| US20210341478A1 (en) * | 2020-04-29 | 2021-11-04 | Antaimmu Biomed Co., Ltd. | Recombinant antibodies, kits comprising the same, and uses thereof |
| US20210340189A1 (en) * | 2020-04-30 | 2021-11-04 | Regents Of The University Of Minnesota | Sars-cov-2 n protein and methods of use |
| CN113603769A (en) * | 2021-07-20 | 2021-11-05 | 贵州省人民医院 | Hybridoma cell strain capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody and establishment method and application thereof |
| CN113999303A (en) * | 2021-12-30 | 2022-02-01 | 北京健乃喜生物技术有限公司 | Novel coronavirus nucleocapsid protein antibodies for in vitro diagnosis |
| WO2022036337A1 (en) * | 2020-08-13 | 2022-02-17 | Diaclone Llc | Compositions and methods for recombinant polypeptide mimicking sars-cov-2 nucleocapsid protein (np) |
| US20220056112A1 (en) * | 2020-08-11 | 2022-02-24 | Yurogen Biosystems Llc | MONOCLONAL ANTIBODIES AGAINST SARS-CoV-2 NUCLEOCAPSID PROTEIN AND USES THEREOF |
| KR20220055423A (en) * | 2020-10-26 | 2022-05-03 | 한국기초과학지원연구원 | Detection method of SARS-CoV-2 using novel SARS-CoV-2 specific antibody |
| WO2023038477A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Antibody specific to coronavirus nucleocapsid protein and use thereof |
| CN115806611A (en) * | 2022-11-16 | 2023-03-17 | 杭州华葵金配生物科技有限公司 | Antibody against novel coronavirus N protein and application thereof |
| KR20230039786A (en) * | 2021-09-10 | 2023-03-21 | 한국생명공학연구원 | An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof |
| CN116425870A (en) * | 2023-05-24 | 2023-07-14 | 陕西师范大学 | Novel coronavirus N protein resistant monoclonal antibody 31A8, and product and application thereof |
| WO2023154824A1 (en) * | 2022-02-10 | 2023-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies that broadly target coronaviruses |
| CN116655779A (en) * | 2022-11-16 | 2023-08-29 | 杭州华葵金配生物科技有限公司 | Novel coronavirus antibodies and uses thereof |
-
2023
- 2023-10-16 CN CN202311341345.9A patent/CN117534750A/en active Pending
Patent Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070103548A (en) * | 2006-04-19 | 2007-10-24 | 대한민국(관리부서 질병관리본부장) | Monoclonal antibody against nucleocapsid protein of sars coronavirus and the use thereof |
| US20100240872A1 (en) * | 2007-07-04 | 2010-09-23 | Forerunner Pharma Research Co., Ltd. | ANTI-Muc 17 ANTIBODY |
| US20140127236A1 (en) * | 2011-06-01 | 2014-05-08 | Antitope Limited | Humanised anti-cd52 antibodies |
| CN112961222A (en) * | 2020-02-04 | 2021-06-15 | 中国科学院微生物研究所 | 2019 novel coronavirus N protein linear epitope peptide, monoclonal antibody and application |
| US20210311054A1 (en) * | 2020-04-01 | 2021-10-07 | Institut Pasteur | Severe acute respiratory syndrome (sars) - associated coronavirus diagnostics |
| US20210341478A1 (en) * | 2020-04-29 | 2021-11-04 | Antaimmu Biomed Co., Ltd. | Recombinant antibodies, kits comprising the same, and uses thereof |
| CN113583116A (en) * | 2020-04-30 | 2021-11-02 | 养生堂有限公司 | Antibodies against SARS-CoV-1 or SARS-CoV-2 and uses thereof |
| US20210340189A1 (en) * | 2020-04-30 | 2021-11-04 | Regents Of The University Of Minnesota | Sars-cov-2 n protein and methods of use |
| US20220056112A1 (en) * | 2020-08-11 | 2022-02-24 | Yurogen Biosystems Llc | MONOCLONAL ANTIBODIES AGAINST SARS-CoV-2 NUCLEOCAPSID PROTEIN AND USES THEREOF |
| WO2022036337A1 (en) * | 2020-08-13 | 2022-02-17 | Diaclone Llc | Compositions and methods for recombinant polypeptide mimicking sars-cov-2 nucleocapsid protein (np) |
| KR20220055423A (en) * | 2020-10-26 | 2022-05-03 | 한국기초과학지원연구원 | Detection method of SARS-CoV-2 using novel SARS-CoV-2 specific antibody |
| CN112679605A (en) * | 2021-03-15 | 2021-04-20 | 安源医药科技（上海）有限公司 | Antibodies or antigen binding fragments thereof against novel coronavirus nucleocapsid proteins and uses thereof |
| CN113603769A (en) * | 2021-07-20 | 2021-11-05 | 贵州省人民医院 | Hybridoma cell strain capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody and establishment method and application thereof |
| KR20230039786A (en) * | 2021-09-10 | 2023-03-21 | 한국생명공학연구원 | An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof |
| WO2023038477A1 (en) * | 2021-09-10 | 2023-03-16 | 한국생명공학연구원 | Antibody specific to coronavirus nucleocapsid protein and use thereof |
| CN113999303A (en) * | 2021-12-30 | 2022-02-01 | 北京健乃喜生物技术有限公司 | Novel coronavirus nucleocapsid protein antibodies for in vitro diagnosis |
| WO2023154824A1 (en) * | 2022-02-10 | 2023-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies that broadly target coronaviruses |
| CN115806611A (en) * | 2022-11-16 | 2023-03-17 | 杭州华葵金配生物科技有限公司 | Antibody against novel coronavirus N protein and application thereof |
| CN116655779A (en) * | 2022-11-16 | 2023-08-29 | 杭州华葵金配生物科技有限公司 | Novel coronavirus antibodies and uses thereof |
| CN116425870A (en) * | 2023-05-24 | 2023-07-14 | 陕西师范大学 | Novel coronavirus N protein resistant monoclonal antibody 31A8, and product and application thereof |
Non-Patent Citations (9)
| Title |
|---|
| CURTIS D. HODGE等: "Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein", 《MABS》, vol. 13, no. 01, 12 April 2021 (2021-04-12), pages 1905978 * |
| GENBANK: "7WP8_E: Chain E, 2B4 heavy chain", 《GENBANK》, 13 September 2023 (2023-09-13) * |
| GENBANK: "ALA14854.1: ALA14854.1", 《GENBANK》, 25 August 2015 (2015-08-25) * |
| JAMES S TERRY等: "Development of a SARS-CoV-2 nucleocapsid specific monoclonal antibody", 《VIROLOGY》, vol. 558, 1 February 2021 (2021-02-01), pages 28 - 37, XP086530250, DOI: 10.1016/j.virol.2021.01.003 * |
| 丘立文等: "人冠状病毒SARS-CoV、229E和OC43核衣壳(N)蛋白单克隆抗体的制备及3株人冠状病毒N蛋白的抗原相关性观察", 《南方医科大学学报》, vol. 26, no. 03, 28 March 2006 (2006-03-28), pages 290 - 293 * |
| 同重湘等: "基于金标免疫层析技术SARS-CoV-2抗体检测COVID-19的临床应用价值分析", 《兰州大学学报(医学版)》, vol. 46, no. 02, 8 April 2020 (2020-04-08), pages 19 - 24 * |
| 祝岸凯: "新型冠状病毒N蛋白单克隆抗体的制备、表征及其应用", 《中国优秀硕士学位论文全文数据库》, no. 2023, 15 February 2023 (2023-02-15) * |
| 鞠守勇: "新型冠状病毒(SARS-CoV-2)核衣壳蛋白(N蛋白)的表达研究", 《武汉职业技术学院学报》, vol. 19, no. 02, 15 April 2020 (2020-04-15), pages 104 - 107 * |
| 高原、陈川、王晶: "2019新型冠状病毒的抗原抗体检测", 《计量学报》, vol. 41, no. 05, 28 May 2020 (2020-05-28), pages 513 - 517 * |
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