CN116444657B - Antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses - Google Patents

Antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses Download PDF

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CN116444657B
CN116444657B CN202210022971.0A CN202210022971A CN116444657B CN 116444657 B CN116444657 B CN 116444657B CN 202210022971 A CN202210022971 A CN 202210022971A CN 116444657 B CN116444657 B CN 116444657B
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CN116444657A (en
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孟媛
李蔚芝
钟冬梅
游辉
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Dongguan Pengzhi Biotechnology Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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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
    • 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
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    • 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]
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • 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

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Abstract

The application discloses an antibody for resisting novel coronavirus, a reagent for detecting novel coronavirus and a kit, and relates to the technical field of antibodies. The antibodies against the novel coronaviruses disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has better affinity to N protein of the novel coronavirus, and the novel coronavirus detected by using the antibody has better sensitivity and specificity. The application provides more excellent antibody selection for the detection of novel coronaviruses.

Description

Antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses
Technical Field
The application relates to the technical field of antibodies, in particular to an antibody for resisting novel coronaviruses, a novel coronavirus detection reagent and a kit.
Background
Since the occurrence of the epidemic situation of the novel coronavirus 2019-nCoV pneumonia, the epidemic situation is spread worldwide, and the life safety and the physical health of human beings are seriously threatened. Respiratory droplets and intimate contact transmission are the primary transmission pathways for new coronavirus pneumonia, with the possibility of transmission through aerosols in the case of prolonged exposure to high concentration aerosols in a relatively closed environment. 2019-nCoV is very contagious, and most patients develop clinical symptoms after infection, but some patients are asymptomatic infected patients. Common signs of a person infected with coronavirus are respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. Although no specific treatment method is available for the diseases caused by the novel coronaviruses at present, the cure rate of the patients with mild symptoms or asymptomatic diseases can be greatly improved by treatment, and the treatment time is shortened. Thus, detection or identification of patients becomes particularly important.
The novel coronavirus mainly uses nucleic acid detection and viral gene sequencing as etiology definitive evidence at present, and false negative results can appear in the nucleic acid detection due to the influence of various factors such as sampling, operation, reagents and the like. The positive detection rate of virus nucleic acid of a highly suspected 2019 novel coronavirus (2019-nCoV) infected patient is only 30-50%. In addition, the nucleic acid detection has high requirements on instruments, detection sites and environmental conditions, has the defects of long detection time, low flux and the like, and is not convenient for large-scale detection of people under the current epidemic situation. Compared with the nucleic acid detection method, the antibody detection sample is serum or plasma, is less influenced by sample sampling, is favorable for early diagnosis and suspicious case elimination, and is rapid and convenient to detect and suitable for large-scale screening. Thus, antibody detection kits become more important.
Therefore, it is highly desirable to develop a rapid and convenient detection kit for clinical detection.
Structural proteins of the novel coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), which proteins comprise a plurality of epitopes. N protein and viral genome RNA are intertwined to form a viral nucleocapsid, which plays an important role in the synthesis process of viral RNA. Meanwhile, N protein is relatively conserved, the proportion of the N protein in structural proteins of viruses is the largest, and the organism can generate high-level antibodies against the N protein in early infection. Finally, the N protein is an important marker protein of the novel coronavirus, and the antigen can be detected through the N protein monoclonal antibody by utilizing the principle of specific binding of the antigen and the antibody, so that the sample is directly proved to contain the novel coronavirus, and the detection of the novel coronavirus is realized.
At present, 2019-nCoV N protein monoclonal antibody products are few, and the performances are different.
In view of this, the present application has been made.
Disclosure of Invention
The application aims to provide an antibody for resisting a novel coronavirus, a reagent and a kit for detecting the novel coronavirus, wherein the antibody can specifically bind to N protein of the novel coronavirus, has higher affinity to the N protein, and has better sensitivity and specificity for detecting the novel coronavirus by using the antibody. The application provides a richer antibody selection for the detection of novel coronaviruses.
In order to achieve the above object, according to one aspect of the present application, there is provided an antibody or a functional fragment thereof against a novel coronavirus or an N protein thereof, which comprises HCDR1, HCDR2, HCDR3 having amino acid sequences shown in SEQ ID NOS: 1-3, and LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NOS: 4-6.
Further, the antibody or functional fragment thereof also has HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID NOS.7-10, and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID NOS.11-14, or amino acid sequences having at least 80% homology with each of the sequences.
In order to achieve the above object, according to a second aspect of the present application, there is provided an antibody against a novel coronavirus or an N protein thereof or a functional fragment thereof, the antibody or the functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, HFR1, LFR2, LFR4 is the amino acid sequence of HCDR1, HCDR2, LCDR3, LFR4.
In order to achieve the above object, according to a third aspect of the present application, there is provided an antibody against a novel coronavirus or an N protein thereof, comprising a heavy chain and/or a light chain, the heavy chain comprising the heavy chain variable region as described above and the heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In order to achieve the above object, according to a fourth aspect of the present application, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present application, there is provided a reagent or kit for detecting a novel coronavirus or an N protein thereof, the reagent or kit comprising the above antibody or a functional fragment thereof or the above antibody conjugate.
In order to achieve the above object, the present application also provides a vector, a recombinant cell and a method for preparing the above antibody or a functional fragment thereof.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of reducing SDS-PAGE of the anti-novel coronavirus antibody of example 1.
Detailed Description
The application provides an antibody or a functional fragment thereof for resisting novel coronavirus or N protein thereof, which comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1-3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4-6.
In the present application, 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.
In the present application, 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 application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present application, 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 application adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the application.
In the present application, 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 may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present application, 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 alternative embodiments, the antibody or functional fragment thereof further has HFR1, HFR2, HFR3, HFR4 having an amino acid sequence shown in SEQ ID NOS.7-10, and LFR1, LFR2, LFR3, and LFR4 having an amino acid sequence shown in SEQ ID NOS.11-14, or an amino acid sequence having at least 80% homology to each of the sequences.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof 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 an alternative embodiment, the antibody or functional fragment thereof is in K D ≤10 -9 The affinity of M binds to the novel coronavirus or its N protein.
In an alternative embodiment, the antibody or functional fragment thereof is in K D ≤10 -10 The affinity of M binds to a novel coronavirus or N protein thereof;
K D reference is made to the method in the embodiment of the application.
In another aspect, embodiments of the present application provide an antibody or a functional fragment thereof against a novel coronavirus or an N protein thereof, the antibody or the functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR3, HFR4, HFR1, LFR2, r3, LFR4 is the amino acid sequence of HFR1, HCDR2, HCDR3, LFR4.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in SEQ ID NO. 15;
in an alternative embodiment, the light chain variable region amino acid sequence is set forth in SEQ ID NO. 16.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 17 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 18.
In other embodiments, the constant region sequence 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 above-described constant region (SEQ ID NO:17 or 18).
In alternative embodiments, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the application provides an antibody against a novel coronavirus or an N protein thereof, comprising a heavy chain and/or a light chain, said heavy chain comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 19; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.
In another aspect, the application provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the application.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (chlorophyll), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a magnetic microsphere.
In another aspect, the application provides a reagent or kit for detecting a novel coronavirus or an N protein thereof, the reagent or kit comprising an antibody or functional fragment thereof or an antibody conjugate as described above.
In another aspect, the application provides the use of the above-described reagent or kit in the detection of novel coronaviruses or N proteins thereof.
In another aspect, the application provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the application provides a vector comprising the nucleic acid molecule described above.
In another aspect, the present application provides recombinant cells comprising the above vector.
In another aspect, the application provides a method of making an antibody or functional fragment thereof comprising: the recombinant cells as described above were cultured.
On the basis of the present application, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present application to prepare the antibody or the functional fragment thereof by any technique.
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present application will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present application are described in further detail below in connection with the examples.
Example 1 preparation of antibody 9B54
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased fromTOYOBO Co. BD SMART TM RACE cDNA Amplification 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.
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting antibodies against novel coronavirus N proteins, DNA products are obtained through an RT-PCR method, the products are inserted into a pMD-18T vector after an A adding reaction by rTaq DNA polymerase, the products are transformed into DH5 alpha competent cells, after colony growth, the Heavy Chain gene clone and the Light Chain gene clone are respectively taken, and 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of 9B54 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 321bp, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 369bp, belongs to the VH1 gene family, and the front of the gene fragment is 57bp leader peptide sequence (the sequences of the Heavy and light chains of 9B54 are respectively shown as SEQ ID NO:19 and 20).
(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 antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.72kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment 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
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
Diluting 2019-nCoV N protein antigen to 1 mug/ml by using the coating solution, wherein 100 mug/ml of the coating solution is used for each well, and the temperature is 4 ℃ overnight; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50. Mu.L/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient plasmid transformation were active against 2019-nCoV N protein antigen.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion 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
Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were regulated to 1.43X10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 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; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 6 Performing seed preservation by using cells/ml and 2 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 inoculating density, and calculating the expanding culture volume according to production requirements, 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. 2. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, 2. Mu.g of an external control antibody was used as a control, and the electrophoresis pattern was shown in FIG. 1 below, showing two bands after reducing SDS-PAGE, 1 Mr was 50KD and the other Mr was 28KD.
Example 2
Performance detection of antibodies
(1) Activity detection
Coating liquid (main component NaHCO) 3 ) Diluting 2019-nCoV N protein antigen to 3 mug/ml for coating a microplate, wherein each well is 100 mug, and the temperature is 4 ℃ overnight; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody in the example 1, 100 μl/well, 37 ℃ for 30-60 min; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 2 below.
TABLE 2 Activity data
(2) Affinity detection
Purified antibodies were diluted to 10. Mu.g/mL with PBST using an AMC sensor, and 2019-nCoV N protein antigen 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. K (K) D Representing equilibrium dissociation constant, i.e. affinity; kon represents the binding rate; kdis represents the dissociation rate. The results are shown in Table 3 below.
Table 3 affinity assay data
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control 2.43E-08 4.52E+03 1.10E-04
9B54 1.02E-10 1.11E+06 1.13E-04
(3) Stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the enzyme-free activity assay OD for 21 days.
TABLE 4 Table 4
Sample concentration (ng/ml) 12.5 1.5625 0
4 ℃,21 days sample 2.122 0.691 0.047
Sample at-80℃for 21 days 2.154 0.647 0.051
37 ℃ and 21 days of sample 2.133 0.666 0.057
(4) Evaluation of Performance
The above 9B54 was used as a labeled antibody in combination with another novel coronavirus (commercially available from Phpeng) to detect a novel coronavirus.
1. Marking
(1) Preparing colloidal gold: firstly heating chloroauric acid solution to boiling by adopting a traditional sodium citrate reduction method, rapidly adding a certain proportion of trisodium citrate solution, uniformly stirring, stopping heating when the color of the solution is changed into wine red and is not changed any more, and cooling to room temperature to obtain a colloidal gold solution with the concentration of four parts per million;
(2) Marking: adding 0.2M K2CO3 solution into the colloidal gold solution to adjust the pH value to 6.0-7.5;
(3) And (3) centrifuging: adding a labeled antibody into the colloidal gold solution with the pH adjusted, uniformly mixing, adding a sealing agent, stopping labeling, centrifuging at 10000rpm/7min/4 ℃, and removing the supernatant;
(4) And (3) re-dissolving: re-suspending to 100uL, and performing ultrasonic treatment for 2-3 times;
(5) Gold paving: the concentrated gold obtained by the re-suspension is diluted and spread on a glass cellulose membrane, and then is put into a freeze dryer for freeze drying (1-2 h) or is put into a drying room at 37 ℃ for drying overnight.
2. Coating
(1) Assembling the nitrocellulose membrane and the colloidal gold PVC base plate for standby;
(2) Diluting the coated antibody to 1.0-2.0mg/mL, uniformly drawing lines on an NC film by using a metal spraying film drawing instrument, and then placing the coated antibody into a 37 ℃ incubator for drying for at least 45 min. Assembling and cutting strips, and adding samples for detection.
3. Detection of
(1) Quality control product: vaccine, 2019-nCoV N antigen, negative nasal swab, pharyngeal swab, and saliva.
The antibody is used as a labeled antibody and is matched with another new crown antibody, the performance difference of the antibody is compared on a colloidal gold platform, and the antibody can achieve a better performance level than a control. The specific table is as follows:
TABLE 5
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
The partial amino acid sequence related to the application is as follows:
sequence numbering Sequence fragments
SEQ ID NO：1 DYNMG
SEQ ID NO：2 AIIPNNGGTIYNQKFKG
SEQ ID NO：3 EAYRDYDVKTWF
SEQ ID NO：4 SASQGIRNYLN
SEQ ID NO：5 YTSTLHS
SEQ ID NO：6 QQYSKFP
SEQ ID NO：7 EVLLQQSGPELVNPGASVKIPCKASGYTFT
SEQ ID NO：8 WVKQSHGKSLEWIG
SEQ ID NO：9 KATLTVDESSSTAYMELRSLTSEDTAVYYCAR
SEQ ID NO：10 AYWGQGTLVTVSA
SEQ ID NO：11 DIQMTQTTSSLSASLGDRVTISC
SEQ ID NO：12 WYQQKPDGTVKLLIY
SEQ ID NO：13 GVPSRFSGSGSGTDYSLTISNLEPEDLATYFC
SEQ ID NO：14 YTFGGGTKLEIK
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> antibody against novel coronavirus, reagent and kit for detecting novel coronavirus
<130> P2021105CN01
<160> 20
<170> PatentIn version 3.3
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Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
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Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
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Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Phe Cys
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Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
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Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
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Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
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Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
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Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr
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Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
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Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Lys Phe Pro Tyr
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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
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Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
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Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
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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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Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
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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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Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
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Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
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Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
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Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
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Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
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Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
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Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
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Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
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Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
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Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
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Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
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Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
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Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
115 120 125
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
130 135 140
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
180 185 190
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
195 200 205
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
210 215 220
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
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Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
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Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu
260 265 270
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln
275 280 285
Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
290 295 300
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
305 310 315 320
Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro
340 345 350
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met
355 360 365
Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
370 375 380
Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
385 390 395 400
Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn
405 410 415
Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu
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His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
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Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr
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Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Lys Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210

Claims (31)

1. An antibody or functional fragment thereof against a novel coronavirus or an N protein thereof, wherein the antibody or functional fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs 1-3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs 4-6.
2. The antibody or functional fragment thereof according to claim 1, further having amino acid sequences of SEQ ID NOs 7 to 10 or 80% homologous thereto HFR1, HFR2, HFR3, HFR4 and amino acid sequences of SEQ ID NOs 11 to 14 or 80% homologous thereto LFR1, LFR2, LFR3 and LFR4.
3. The antibody or functional fragment thereof according to claim 2, wherein the antibody or functional fragment thereof has a KD of 1 x 10 or less -9 The affinity of M binds to the novel coronavirus or its N protein.
4. An antibody or a functional fragment thereof against a novel coronavirus or an N protein thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 15;
the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
5. The antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the antibody or functional fragment thereof further comprises a constant region.
6. The antibody or functional 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 functional 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 functional fragment thereof according to claim 5, wherein the constant region is derived from bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
9. The antibody or functional fragment thereof according to claim 8, wherein the constant region is of murine origin.
10. The antibody or functional fragment thereof of claim 6 wherein the heavy chain constant region sequence is as shown in SEQ ID No. 17 or has at least 80% homology thereto and the light chain constant region sequence is as shown in SEQ ID No. 18 or has at least 80% homology thereto.
11. The antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
12. An antibody or a functional fragment thereof against a novel coronavirus or an N protein thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 19; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.
13. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1-12.
14. The antibody conjugate of claim 13, wherein the antibody or functional fragment thereof is labeled with a label.
15. The antibody conjugate of claim 14, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
16. The antibody conjugate of claim 15, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes, rhodamine-based dyes, cy-based dyes, alexa-based dyes, and protein-based dyes.
17. The antibody conjugate of claim 15, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
18. The antibody conjugate of claim 15, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、1 53 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
19. the antibody conjugate of claim 15, wherein the chemiluminescent reagent is selected from the group consisting of luminol, luciferin, crustacean fluorescein, ruthenium bipyridine, acridinium esters, dioxane, lomustine and peroxyoxalate.
20. The antibody conjugate of claim 15, wherein the nanoparticle-based label is selected from the group consisting of a colloid, an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
21. The antibody conjugate of claim 20, wherein the colloid is selected from the group consisting of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microsphere.
22. The antibody conjugate of claim 21, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
23. The antibody conjugate of claim 13, wherein the antibody or functional fragment thereof is coated onto a solid phase.
24. The antibody conjugate of claim 23, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
25. The antibody conjugate of claim 23, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
26. The antibody conjugate of claim 23, wherein the solid phase is a magnetic microsphere.
27. A reagent or kit for detecting a novel coronavirus or an N protein thereof, comprising the antibody or functional fragment thereof according to any one of claims 1-12 or the antibody conjugate according to any one of claims 13-26.
28. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1-12.
29. A vector comprising the nucleic acid of claim 28.
30. A recombinant cell comprising the vector of claim 29.
31. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 12, comprising: culturing the recombinant cell of claim 30.
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CN113150136A (en) * 2021-04-30 2021-07-23 杭州贤至生物科技有限公司 Preparation of novel coronavirus N protein monoclonal antibody
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