CN111518202A - Novel coronavirus antibody and ELISA detection kit for same - Google Patents

Novel coronavirus antibody and ELISA detection kit for same Download PDF

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CN111518202A
CN111518202A CN202010464260.XA CN202010464260A CN111518202A CN 111518202 A CN111518202 A CN 111518202A CN 202010464260 A CN202010464260 A CN 202010464260A CN 111518202 A CN111518202 A CN 111518202A
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seq
variable region
chain variable
binding protein
antibody
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CN111518202B (en
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张黎
高行素
郑滨洋
孟繁岳
王文娟
郭喜玲
梁祁
朱凤才
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Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health)
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Jiangsu Center For Disease Control And Prevention (jiangsu Institute Of Public Health)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Abstract

The invention discloses a novel coronavirus antibody and an ELISA detection kit for the novel coronavirus antibody, wherein the antibody comprises CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, CDR3 shown in SEQ ID NO.3, CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6 and CDR3 shown in SEQ ID NO. 7. The antibodies of the invention are useful for novel coronavirus detection or novel coronavirus infection diagnosis.

Description

Novel coronavirus antibody and ELISA detection kit for same
Technical Field
The invention belongs to the fields of cellular immunology and molecular biology, and relates to a novel coronavirus antibody and an ELISA (enzyme-Linked immunosorbent assay) detection kit for the novel coronavirus antibody.
Background
The international committee for viral classification named the novel coronavirus SARS-CoV-2 and the world health organization named the pneumonia caused by infection with this virus COVID-19. The virus has strong infectivity and wide transmission path. The virus can adapt to the environment of human body rapidly, has transmission capability in latent period after infection, and reports by some asymptomatic infectors that virus nucleic acid is detected even in various animals. These factors complicate the control of the virus and no effective therapeutic drugs and vaccines are currently on the market.
SARS-CoV-2 belongs to the genus Coronavirus, is a single-stranded positive-strand RNA virus, has a size of about 30kb, has a similarity of 79% to SARS-CoV, and has a similarity of up to about 88% to a Coronavirus (CoV) isolated from Bats. SARS-CoV-2 has typical coronavirus characteristics, and the virus envelope has typical spinous processes, which are shaped like coronages. The Nucleocapsid is of a spiral symmetrical type, the main structural protein is Nucleocapsid Protein (NP), and the total length of the NP is 420 amino acids. The NP has the most content in virus structural protein, is expressed in a large amount in the early stage of host infection, has stronger immunogenicity, and can cause strong immune response of a host. Thus, NP can be used as the main target antigen for serological diagnosis of SARS-CoV-2 infection.
Because specific therapeutic drugs and effective vaccines are not developed successfully, early diagnosis becomes an important measure for preventing and controlling epidemic situations, and early nucleic acid diagnosis and clinical diagnosis become important basis for accurate diagnosis. Although the nucleic acid diagnosis speed is high, the influence of the quality of the sampling is large, false positive and false negative exist, and the implementation of the prevention and control measures is influenced. Nucleic acid detection of part of asymptomatic infected persons is negative in the late stage of the disease process, and missed diagnosis is easy to occur only by nucleic acid detection. Serological diagnosis is to detect the immune response of an organism after pathogen infection, the duration is long, the immune response is stable, and the immune response shows a dynamic change trend along with the progress of the disease course. Serodiagnosis is therefore also an important tool for early diagnosis and assessment of the current state of infection.
Disclosure of Invention
The present invention provides a solution to these and other problems by providing binding proteins (e.g., isolated, recombinant, or synthetic antibodies, or fragments or derivatives thereof) that specifically bind to a novel coronavirus NP protein.
The antibody molecules of the invention may be of any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule. The constant region domains of the antibody (if present) may be selected based on the recommended antibody molecule function. For example, the constant region domain may be a human IgA, IgD, IgE, IgG or IgM domain. In particular, human IgG constant region domains, particularly IgG1, IgG2, IgG3, and IgG4, may be used.
Antibody fragments include, for example, Fab, F (ab)2, Fab ', F (ab ') 2, F (ab ') 3, F (v), Fd, dAb, diabodies, miniantibodies, nanobody fragments, fragments of a single variable domain (e.g., a VH or VL domain), or fragments containing only heavy or light chain domains.
The antibodies of the invention, including fragments and derivatives thereof, against the novel coronavirus NP protein may be monoclonal, polyclonal, murine, chimeric, primatized, humanized or fully human antibodies. The antibodies of the invention may be multimeric, heterodimeric, hemi-dimeric (hemmidimeric), monovalent, bivalent, tetravalent, bispecific, and may include single chain antibodies; and derivatives of these.
In one embodiment of the invention, the antibody against the novel coronavirus NP protein refers to a fully human antibody. Fully human antibodies comprise antibody polypeptides or immunoglobulin variable domains having sequences derived from human immunoglobulins (e.g., from human immunoglobulin coding sequences). The term "human" as applied herein to antibodies or fragments (e.g., variable domains) is free of antibodies from another species (e.g., a mouse) that have been "humanized" by grafting human constant region sequences to the antibody polypeptide (i.e., replacing non-human constant regions with human constant regions) or by grafting human V region framework sequences to immunoglobulin variable domains from a non-human mammal (i.e., replacing the non-human framework regions of the V domains with human framework regions).
The antibodies of the invention comprise synthetic antibodies or recombinant antibodies produced using recombinant DNA techniques, such as antibodies expressed by phage. It should also be construed to include antibodies that have been produced by synthesizing a DNA molecule encoding an antibody that expresses an antibody protein, or determining the amino acid sequence of an antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.
In certain embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and comprises or consists of more than one CDR heavy chain (H) sequence selected from the group consisting of CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and CDR-H3(SEQ ID NO. 3).
In a further embodiment, the binding protein or antibody of the novel coronavirus NP protein comprises at least 2 CDRs selected from the group consisting of CDR-H1(SEQ ID No.1), CDR-H2(SEQ ID No.2) and CDR-H3(SEQ ID No.3), or consists of at least 2 CDRs selected from the group consisting of CDR-H1(SEQ ID No.1), CDR-H2(SEQ ID No.2) and CDR-H3(SEQ ID No. 3).
In a still further embodiment, the binding protein or antibody contains or consists of all 3 of the following CDR H sequences, CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and CDR-H3(SEQ ID NO. 3).
In some embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and contains or consists of one or more CDR light chain (L) sequences selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).
In a further embodiment the novel coronavirus NP protein or antibody comprises at least 2 CDRs selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7), or consists of at least 2 CDRs selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).
In a still further embodiment, the binding protein or antibody of the novel coronavirus NP protein comprises or consists of all 3 CDR L sequences which are CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).
In certain embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and comprises or consists of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7) and/or CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7) and wherein said binding protein further comprises or consists of CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and/or CDR-H3(SEQ ID NO.3) and/or CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and/or CDR-H3(SEQ ID NO. 3).
In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VH sequence of SEQ ID No. 4.
In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VL sequence of SEQ ID No. 8.
In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VL sequence of SEQ ID No.8 and the VH sequence of SEQ ID No. 4.
Binding proteins (e.g., antibodies) of the novel coronavirus NP proteins of the invention can be pegylated. Wherein the antibody may be pegylated on the heavy chain, the light chain, or both chains.
The invention also provides isolated, recombinant and/or synthetic DNA molecules encoding the binding proteins described above.
The DNA sequences of the present invention may comprise, for example, synthetic DNA, cDNA, genomic DNA produced by chemical treatment, or any combination thereof.
In a particular embodiment of the invention, the DNA molecule comprises the amino acid sequences shown in SEQ ID NO.17 and SEQ ID NO. 18.
The invention also provides a vector comprising a DNA molecule as described above.
The invention also provides a host cell comprising a DNA sequence or vector of the invention. In certain aspects, the invention relates to a method for producing a binding protein (e.g., an antibody) as described above, comprising culturing a host cell containing any of the vectors described above under conditions suitable for production of the binding protein by the host cell. In some embodiments, the method comprises recovering the binding protein from the host cell culture.
The host cell may be, for example, a prokaryotic cell, such as E.coli, or other microbial cell, or a eukaryotic cell including, but not limited to, a mammalian cell, such as a human, mouse, monkey, rabbit, goat, hamster, or rat cell, an insect cell, an avian cell, a plant cell, and a lower eukaryotic cell, such as a fungal cell.
In some embodiments of the invention, host cells useful in the practice of the invention can be, for example, (1) bacterial cells such as E.coli, Bacillus crescentus, Streptococcus, Staphylococcus, Streptomyces and Bacillus subtilis cells, and Salmonella typhimurium; (2) fungal Cells and Aspergillus (Aspergillus) Cells, yeast Cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Pichia pastoris (Pichia pastoris), Pichia methanolica (Pichia methanolica), other Pichia species, Kluyveromyces lactis (K.lactis), (3) insect cell lines, such as Cells from Spodoptera frugiperda (Spodoptera frugiperda) (e.g., Sf9 and Sf21 cell lines, and expressSFTM Cells (Protein Sciences Corp., Meriden, CT, USA)), Drosophila S2 Cells, and Trichoplusia ni Hiitrogen (Carlsbad, Calif., USA); (4) mammalian cells, or (5) plant cells.
Typical mammalian cells include COS1 and COS7 cells, Chinese Hamster Ovary (CHO) cells, NSO myeloma cells, NIH3T3 cells, 293 cells, H Ε PG2 cells, HeLa cells, C127, 3T3, BHK, Bowes melanoma cells, L cells, MDCK, HEK293, WI38, murine ES cell lines (e.g., from lines 129/SV, C57/BL6, DBA-1, 129/SVJ), K562, Jurkat cells, and BW 5147.
In certain embodiments, the binding proteins, or nucleic acids, of the invention are labeled with a detectable label, which may be a radioisotope, an enzyme, a dye, or biotin.
In yet other embodiments, the antibodies of the invention are conjugated to an imaging agent, which may be a labeling moiety. The labeling agent may be biotin, a fluorescent or luminescent moiety, a radioactive moiety, a histidine tag, or a peptide tag.
The invention also relates to sequence variants of the binding proteins (e.g., antibodies) described herein, as well as nucleic acid sequences encoding the same. The sequence variants of the invention preferably share at least 90%, 91%, 92%, 93% or 94% identity with the polypeptides of the invention or the nucleic acid sequences encoding them. More preferably, sequence variants share at least 95%, 96%, 97% or 98% identity at the amino acid or nucleic acid level. Most preferably, the sequence variant shares at least 99%, 99.5%, 99.9% or more identity with the polypeptide of the invention or the nucleic acid sequence encoding it.
In certain embodiments, the antibody polypeptides of the invention against the novel coronavirus NP protein are, for example, dabs, fabs, Fab', scFv, Fv, disulfide-bonded fvs, or comprise a single immunoglobulin variable domain, for example a VH or VL domain, that is specific for and monovalent for binding of the novel coronavirus NP protein. Certain binding protein polypeptides of the invention comprise 1, 2 or more CDRs of the invention and an alternative scaffold or universal framework sequence of the invention.
In certain embodiments, the binding protein or antibody of the invention comprises a single variable domain selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL).
The invention also provides compositions comprising the binding proteins of the invention as described above.
The composition can further comprise a second binding protein comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, a light chain variable region CDR 3; wherein the content of the first and second substances,
heavy chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 9;
heavy chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 10;
heavy chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 11;
light chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 13;
light chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 14;
light chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 15;
more preferably, the second binding protein comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO.12, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 16.
An example of such a second binding protein is a monoclonal antibody.
The composition may further comprise additional bioactive or diagnostic agents.
The antibodies of the invention or compositions containing them may be included in a container, package, or dispenser, alone or as part of a kit, together with a label and instructions for administration.
The diagnostic agent includes a detectable substance, examples of which include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for use in positron emission tomography), and non-detectable substancesAs regards the metal ions which can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Pat. No.4,741,900, suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosidase, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin and biotin, suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin, suitable luminescent materials include luminol, suitable bioluminescent materials include luciferase, fluorescein and aequorin, radioisotopes such as, for example, luciferase, luciferin and aequorin125I、131I、111In and90Y、Lu177bismuth, bismuth213Californium252Iridium (III)192And tungsten188Rhenium188、211Astatine, astatine,99Tc。
The terms "pegylated," "polyethylene glycol," or "PEG" include polyalkylene glycol compounds or derivatives thereof, with or without a coupling agent or derivatization with a coupling or activating moiety, such as with a thiol, triflate, tresylate, aziridine, ethylene oxide, or preferably with a maleimide moiety, such as PEG-maleimide. Other suitable polyalkylene glycol compounds include, but are not limited to, maleimide monomethoxy PEG, activated PEG polypropylene glycol, and the following types of charged or neutral polymers: dextrose, polyacetyl neuraminic acid, or other carbohydrate-based polymers, amino acid polymers, and biotin and other affibody derivatives.
The invention also provides methods for producing binding proteins (e.g., antibodies) of the invention, including methods using immortalized cell lines, artificial synthesis, recombinant expression, or phage display techniques. In a particular embodiment, the method of the invention comprises the step of culturing the host cell as described above.
The invention provides an application, which comprises the following applications:
(1) the use of the binding protein as hereinbefore described for the preparation of a novel coronavirus detection product;
(2) use of the binding protein as hereinbefore described for the preparation of a novel diagnostic product for coronavirus infection;
(3) use of a composition as hereinbefore described for the preparation of a test product for a novel coronavirus.
The test product or diagnostic product comprises the kit as described above.
Drawings
FIG. 1 shows a SDS-PAGE pattern of the recombinant SARS-CoV 2NP protein of the present invention;
FIG. 2 is a graph showing the results of detection of antibody titer by indirect ELISA;
FIG. 3 is a graph showing the results of detecting the binding of an antibody to an antigen using WB;
FIG. 4 shows the results of the affinity activity of JS01 detected by SPR;
FIG. 5 shows the results of the affinity activity of JS02 detected by SPR;
FIG. 6 is a graph showing the results of detecting the affinity activity of JS03 using SPR;
FIG. 7 is a graph showing the results of detecting the affinity activity of JS04 using SPR;
FIG. 8 is a graph showing the results of detecting the affinity activity of JS05 using SPR;
FIG. 9 is a graph showing the results of detecting the affinity activity of JS06 using SPR;
FIG. 10 is a graph showing the results of detecting the affinity activity of JS07 using SPR;
FIG. 11 is a graph showing the results of detecting the affinity activity of JS08 using SPR;
FIG. 12 is a graph showing the results of detecting the affinity activity of JS09 using SPR;
FIG. 13 is a graph showing the results of detecting the affinity activity of JS10 using SPR;
FIG. 14 is a graph showing the results of detecting the affinity activity of JS11 using SPR;
FIG. 15 is a graph showing the results of detecting the affinity activity of JS12 using SPR;
FIG. 16 is a graph showing the results of detecting the affinity activity of JS13 using SPR;
FIG. 17 is a graph showing the results of detecting the affinity activity of JS14 using SPR;
FIG. 18 is a graph showing the results of detecting the affinity activity of JS15 using SPR;
FIG. 19 is a graph showing the results of detecting the affinity activity of JS16 using SPR;
FIG. 20 is a graph showing the results of measuring the antibody coating concentration by the double antibody sandwich method;
FIG. 21 is a graph showing the results of detection sensitivity of antibodies by the double antibody sandwich method;
FIG. 22 is a graph showing the detection effect of the antigen detection chromatographic strip of the present invention.
Detailed Description
The invention is further illustrated by the figures and examples. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.
Example 1 antibody screening
Expression of recombinant SARS-CoV2 Nucleoprotein (NP)
1.1 Primary reagents
The SARS-CoV 2NP gene sequence (GenBank sequence number: MT066176.1) and the related primer synthesis and sequencing are all completed by general biological systems (Anhui) limited company; coli DH5 α, BL21(DE3) competent cells were purchased from general biosystems (anhui) ltd; BamHI and NotI endonucleases were purchased from New England Biolabs (NEB); the EXTTaq enzyme was purchased from TaKaRa; HRP-labeled anti-human Fc antibody was purchased from Sigma; other chemical reagents are domestic analytical pure reagents; serum of 2019-nCoV infected patients is collected and stored by the center, and all cases are Jiangsu cases.
1.2 prokaryotic expression plasmid construction
Designing a prokaryotic expression primer of the NP gene, wherein an upstream primer is provided with a BamH I restriction site, and a downstream primer is provided with a Not I restriction site. The primer sequence is as follows: cov2-NP-F: CGGGATCCTCTGATAATGGACCCCAAAATC; cov2-NP-R: ATAAGAATGCGGCCGCAGGCCTGAGTTGAGTCAGCAC. The NP gene was amplified using EX Taq enzyme, and the PCR reaction program was: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 80 s; 10min at 72 ℃. And recovering a 1300 bp target fragment from the PCR product by using glue, performing double enzyme digestion on the PCR product by using BamH I and Not I, connecting the PCR product with a pET28a vector, and transforming E.coli DH5 alpha competent cells. After single colony is selected the next day and sequenced correctly, the quality-improved particles are transformed into prokaryotic expression bacterium E.coli BL21(DE3) competent cells.
1.3NP expression and purification
Culturing NP expressing strain until OD600 is 0.6, adding IPTG with final concentration of 0.5mmol/L, inducing at 16 deg.C for 6h, collecting thallus, ultrasonic crushing, and centrifuging to collect inclusion body. The inclusion bodies were dissolved in 8mol/L urea and then purified by nickel column affinity chromatography. After purification, the urea content is reduced in a gradient manner, the protein is dialyzed and renatured into PBS, and finally the protein expression and purification effects are detected by SDS-PAGE. After the small amount of fermentation is finished, the mixture is put into a 100L fermentation tank for mass fermentation, the fermentation medium is a TB medium (1% glycerol), the fermentation parameter is 280rpm, the aeration ratio is 0.5vvm (15L/min), the pH is controlled to be 6.8-7.2, the tank pressure is 0.06 MPa-0.1 MPa, the fermentation temperature is 16 ℃, and the mixture is cultured for 24 hours.
SDS-PAGE results showed that the total length of NP plus His tag and other additional amino acids in the vector predicted a protein of about 50 × 10 relative molecular mass3Expression bacteria were found to be 50 × 10 after IPTG induction3There is a clear band around, consistent with the expected molecular weight size (FIG. 1A). After the inclusion body is dissolved, the inclusion body is purified by a nickel column, and an obvious elution peak is obtained when the concentration is 150mmol/L imidazole. After the proteins were renatured by dialysis, a single protein band was found to appear at the same position by SDS-PAGE (FIG. 1B). This indicates that the NP was successfully induced and purified to a higher degree. Note: in the figure, M: proteins, Makers; 1: uninduced pET28a-NP expressing bacteria; 2: pET28a-NP recombinant expression bacteria after IPTG induction; 3: and (4) purifying to obtain the recombinant nucleocapsid protein.
Second, phage library construction
1. Collecting peripheral blood of patient with COVID-19 in convalescent period, and separating mononuclear cells (PBMC) from the peripheral blood
In the project, 20ml of peripheral blood of 5 COVID-19 patients before discharge is collected from Jiangsu province, a certain city and after informed consent, on 14 days 2 and 14 months in 2020. 5 patients are in the same transmission chain, one of them is a member of the Wuhan's return, and another 3 patients are infected by the common bath in the hotel bathroom, and the 5 th patient is in a co-workers relationship with one of the 3 patients in the common bath. 5 people are not severe, and are respectively isolated from 2 months, 15 days to 22 days of home-released after treatment. Mononuclear Cells (PBMC) were separated from 20ml of heparin anticoagulated using GE Ficoll-Paque PLUS by density gradient centrifugation.
2. Extraction of RNA and cDNA Synthesis in PBMC
PBMC cell RNA was extracted using the RNeasy Mini Kit from QIAGEN, and then the RNA was reverse-transcribed into cDNA using the First Strand Synthesis Kit from Roche (Transcriptor First Strand cDNA Synthesis Kit, Roche, Cat No.: 04896866001).
3. PCR amplification of VK, VL and VH (EX Taq, Takara, Cat No.: DRR001A)
(1) The amplification VK & VL system is shown in Table 1.
TABLE 1 amplification VK & VL system
Solutions or compositions Volume (μ L)
cDNA 1
EX Buffer(10x) 5
dNTPs(10mM each) 4
P1(10μM) 2
P2(10μM) 2
EX Taq 1U/μl 0.3
dH2O 35.7
(2) The amplified heavy chain Fd fragment system is shown in Table 2.
TABLE 2 amplification of heavy chain Fd segment systems
Figure BDA0002512058060000101
Figure BDA0002512058060000111
(3) The reaction sequence is shown in table 3.
TABLE 3 reaction procedure
Figure BDA0002512058060000112
The PCR product was electrophoresed through 2% agarose gel, and a fragment of about 750bp was recovered.
4. Cloning of the light chain (cloning VK and VL into pComb3H vector)
VK and VL were digested with XbaI and SacI and ligated with pComb3H vector, which was also digested with XbaI and SacI, and the ligation product was recovered and then transfected into XL1-Blue competent cells.
And (3) coating the electric shock bacterium liquid on a 15cm large plate, scraping the bacterium the next day, and obtaining the quality-improved particles, namely the light chain library. The recombinant plasmids were pComb3H-VK and pComb3H-VL at this time.
5. Heavy chain cloning (cloning VH Gene into pComb3H-VK and pComb3H-VL light chain Bank)
The light chain library pComb3-L and Fd fragments are respectively subjected to double enzyme digestion by XhoI and SpeI, are connected with pComb3H-VK and pComb3H-VL which are also subjected to double enzyme digestion by XhoI and SpeI, and are then electrically transformed to obtain the antibody library.
6. Packaging of antibody libraries
(1) Taking out the antibody library from a refrigerator at the temperature of-80 ℃, melting on ice, adding 1ml of the antibody library into 10ml of A + (20 mu g/ml)2YT culture medium, and shaking at the temperature of 37 ℃ and 200rpm for 1 hour;
(2) adding 100ml of A + (100. mu.g/ml), T + (20. mu.g/ml) 2YT medium, and shaking at 200rpm for 1 hour;
(3) plus 1012pfu VCSM13 helper phage, standing at 37 deg.C for 20min, shaking at 200rpm for 2 hr;
(4) adding 70 mu g/ml kanamycin at 30 ℃ and shaking at 200rpm overnight;
(5) centrifuging at 6000rpm for 20min the next day, pouring out the supernatant, adding 4% PEG8000(4g) and 3% NaCl (3g), mixing, and placing on ice for more than 30 min;
(6) and subpackaging in a 50ml centrifuge tube, centrifuging at 9000rpm for 25min, removing supernatant, draining, and resuspending the precipitate with 1ml PBS to obtain the packaged library.
Screening of phage library
1. The recombinant SARS-CoV2 Nucleoprotein (NP) was coated in an immune tube, 3 tubes were coated at 50. mu.g/tube, and left overnight at 4 ℃ with 2% skim milk for the next day to block the immune tube for 1 h.
2. 1.75ml of PBS containing 2% skim milk and 250. mu.l of the phage library were added to the tube, shaken at 37 ℃ for 1h, and then allowed to stand at 37 ℃ for 1 h.
3. The phage library was inverted and washed 20 times with PBST, 5min each.
4. The tube was eluted with 1ml Gly-HCl pH 2.2, left to stand at room temperature for 5min, shaken at 37 ℃ for 5min, then pipetted into a 1.5ml EP tube and neutralized to pH 7 with 57 μ l 2M Tris.
5. The eluate was transferred to a new 50ml centrifuge tube and 10ml of OD 1 fresh XL1-Blue was added immediately, mixed well and incubated at 37 ℃ for 30min, 10ml of 2YT (Amp 100. mu.g/ml, Tet 20. mu.g/ml) was added.
6. Mu.l of the broth was used to determine the volume of the elution pool, and 20ml of the remaining medium was poured into a 500ml Erlenmeyer flask and shaken at 230rpm for 1 hour.
7. 130ml of 2YT (Amp 100ug/ml, Tet 20. mu.g/ml) were added, shaken at 230rpm for 1 h.
8. The helper phage with MOI 20 was added and incubated at 37 ℃ for 30 min.
9. Centrifuge at 3000g for 10min, resuspend pellet into 150ml 2YT (Amp 100. mu.g/ml, Tet 20. mu.g/ml), shake at 37 ℃ at 230rpm for 2 h.
10. 110. mu.l of 70mg/ml kanamycin was added, and 30 ℃ overnight at 230 rpm. Adding 1/5 volume of PEG-NaCl (40ml) the next day, mixing, ice-cooling for at least 1h, centrifuging at 10000g and 4 deg.C for 20min, suspending the precipitate in 2-3ml PBS, centrifuging instantaneously to remove bacteria, and filtering with 0.45 μm filter for the next round of screening.
11. Repeating the screening step for 3 times to achieve the purpose of enriching and screening the phage library.
12. After the third round of enrichment, 2 x 96 clones were picked. After IPTG induction, ELSA detection was performed the next day.
Four, ELISA detection of 2 x 96 clones binding specificity
1.2 pieces of anti-human Fab antibody (1:3000) and 2 pieces of NP protein (2. mu.g/ml) were coated separately and left overnight at 4 ℃.
2. The next day was blocked with 3% skim milk for 1h, then 50. mu.l of induction supernatant and 50. mu.l of skim milk were added, incubated at 37 ℃ for 1h, and washed with PBST.
3. HRP-labeled anti-human Fab antibody (1:3000) was added to each of the 4 plates, incubated at 37 ℃ for 1h, washed with PBST, and then TMB developed.
178 phage antibody fragments which can be specifically combined with NP are obtained through screening, and the fragments are Fab fragments of human origin, including full-length light chain and Fd fragment of heavy chain. 178 single colonies were amplified and sequenced to obtain 159 strains of complete and qualified sequences.
Example 2 expression of full antibodies and related functional validation
Finally selecting 16 antibodies from the 159 antibodies for expression of the whole antibody and relevant function verification, and naming the 16 antibodies as JS01-JS 16.
Wherein the JS12 antibody sequence is shown as follows:
the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO. 1;
the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO. 3;
the amino acid sequence of CDR1 in the variable region of the light chain is shown in SEQ ID NO. 5;
the amino acid sequence of CDR2 in the variable region of the light chain is shown in SEQ ID NO. 6;
the amino acid sequence of CDR3 in the variable region of the light chain is shown in SEQ ID NO. 7;
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.4, and the nucleic acid sequence is shown as SEQ ID NO. 17; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, and the nucleic acid sequence is shown as SEQ ID NO. 18.
The JS08 antibody sequence is shown below:
the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO. 9;
the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO. 10;
the amino acid sequence of CDR3 in the heavy chain variable region is shown in SEQ ID NO. 11;
the amino acid sequence of CDR1 in the variable region of the light chain is shown in SEQ ID NO. 13;
the amino acid sequence of CDR2 in the variable region of the light chain is shown in SEQ ID NO. 14;
the amino acid sequence of CDR3 in the variable region of the light chain is shown in SEQ ID NO. 15.
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region is shown as SEQID NO. 16.
1. Full antibody expression
The 16-strain humanized antibody is constructed into an IgG-type humanized whole molecule antibody, expressed in 293F cells and purified by Protein A for later use.
2. ELISA (enzyme-Linked immuno sorbent assay) for detecting binding specificity of 16-strain antibody and recombinant NP
Recombinant NPs were coated onto ELISA plates with PBS at a concentration of 1. mu.g/ml, all antibody concentrations were diluted to 1mg/ml, then diluted in multiples starting at 1:10000 and incubated at 37 ℃ for 30 min. Then PBST was washed 3 times, HRP-labeled anti-human IgG (1:5000) was added, and after incubation at 37 ℃ for 30min, PBST was washed 3 times, then TMB was developed, and OD450 absorbance values were read after termination.
The dilution titer of the 16 NP antibody was measured by indirect ELISA, and the average OD value of the negative control was 0.119 with a standard deviation of 0.132, so that the cutoff value was defined as
Figure BDA0002512058060000141
The detection titer of the 16-strain antibody was judged to be between 1:80000 and 1:1280000 (FIG. 2).
3. Western Blot results of 16 antibodies and purified NP
Mu.g of the recombinant NP was electrophoresed by SDS-PAGE, transferred to a PVDF membrane, incubated with the above 16 antibodies (0.5. mu.g/ml) at 37 ℃ for 1h, washed 3 times with PBST, then incubated with HRP-labeled anti-human IgG (1:5000) for 30min, washed 3 times with PBST, and then developed on the membrane with DAB.
WB experimental results showed that 16 antibodies were able to specifically bind to recombinantly expressed Nucleoprotein (NP) and a distinct band of color appeared at 50kDa, suggesting that the group of antibodies were all linear epitope antibodies (fig. 3).
4. Antibody affinity activity detection
The antibody affinity determination is completed by a Biacore T200 workstation and is carried out according to the following steps: the CM5 chip was first activated with amino-coupled activators NHS and EDC at 10. mu.l/min for 300s, then the recombinantly expressed SARS-CoV-2NP was diluted to 1ug/mL with 10mM sodium acetate buffer (pH5.5), the Response (RUs) was brought to around 600 by flowing 10. mu.l/min through the chip for 30s, and finally 10. mu.l/min, 420s were set, and the remaining activated sites on the chip surface were blocked with ethanolamine. Serially diluted antibodies were sequentially injected at 25 ℃ at a flow rate of 30. mu.l/min, and after each concentration measurement, CM5 chips were regenerated with glycine-hydrochloric acid (pH2.0), followed by the next concentration measurement. After the experiment was completed, binding affinity was obtained by global fitting of the curve using Biacore T200Evaluation Software.
The experimental results are shown in FIGS. 4-19, JS01-JS16 can efficiently bind to SARS-CoV-2NP protein, and the parameters related to the affinity activity are shown in Table 4.
TABLE 4 antibody affinity parameters
Name of antibody Amount of ligand coupling ka(1/Ms) Kd(1/s) KD(M)
JS01 102RU 2.18E+06 4.79E-04 2.20E-10
JS02 162RU 9.12E+05 5.83E-05 6.39E-11
JS03 102RU 8.97E+05 2.12E-04 2.36E-10
JS04 162RU 7.15E+04 1.91E-04 2.67E-09
JS05 162RU 5.94E+05 2.43E-04 4.09E-10
JS06 136RU 1.61E+05 0.002576 1.61E-08
JS07 110RU 1.44E+06 1.78E-04 1.24E-10
JS08 162RU 3.03E+04 5.77E-06 1.90E-10
JS09 129RU 6.89E+05 4.79E-05 6.94E-11
JS10 136RU 1.47E+06 2.99E-04 2.04E-10
JS11 110RU 1.93E+05 5.62E-05 2.92E-10
JS12 110RU 4.88E+05 7.33E-05 1.50E-10
JS13 110RU 8.05E+05 9.66E-05 1.20E-10
JS14 136RU 1.24E+06 2.21E-04 1.78E-10
JS15 110RU 7.72E+04 1.22E-04 1.58E-09
JS16 136RU 2.68E+05 4.01E-05 1.50E-10
5. Antibody pairing assay
5.1 determination of antibody coating concentration
(1) Mu.l of JS12 antibody was diluted from 5. mu.g/ml to 0.0024. mu.g/ml for 12 dilutions before being coated in ELISA plates. Coating at 4 deg.C overnight, blocking with 1% BSA for 2h, and washing with PBST for 3 times.
(2) 50ng of recombinant NP was added to the first well of each coating concentration, then diluted in multiples to 0.39 ng/well for 8 dilutions, incubated for 1h at 37 ℃ and washed 3 times with PBST.
(3) Adding HRP marked JS08 diluted at 1:1000, incubating for 1h at 37 ℃, PBST washing for 3 times, and reading the OD450nm absorbance value after TMB color development.
As can be seen from the graph in FIG. 20, the amount of the coated antibody has an effect on the detection sensitivity, and the amount of the coating from 5. mu.g/ml to 0.00245. mu.g/ml is not so much affected, and the sensitivity for detecting the NP antigen is less than 3.9 ng/ml. Therefore, in all subsequent pairing experiments, we chose a concentration of 2. mu.g/ml as the antibody coating and 1:4000 as the dilution of the enzyme-labeled antibody.
5.2 double antibody Sandwich method for detecting NP
(1) 16 NP antibodies JS01-JS16 were coated on ELISA plates at 2. mu.g/ml, coated overnight at 4 ℃, blocked with 1% BSA for 2h the next time, and washed with PBST for 3 times.
(2) 0.1. mu.g/ml recombinant NP protein was added, then diluted in multiples to 0.78ng/ml for 8 dilutions, incubated at 37 ℃ for 1h, and washed 3 times with PBST.
(3) HRP-labeled JS08(1:1000) was added, incubated at 37 ℃ for 1h, PBST washed 3 times, TMB developed, and OD450nm absorbance values were read.
As can be seen from FIG. 21, enzyme-labeled JS08 cannot pair with JS06, JS11 and JS08 per se, but can pair with other 13 NP antibodies for double antibody sandwich NP detection. The JS08 and JS16 have the best matching effect, the detection limit can reach below 0.78ng/ml, and the detection limit of other matched antibodies is 12.5-1.56 ng/ml.
6. Sensitivity of double-antibody sandwich immunochromatography for detecting recombinant NP
The anti-JS 08 monoclonal antibody is coated on a nitrocellulose membrane to form a T line, and the anti-human IgG antibody is marked to the C line. After the NP protein is diluted in series, 50 mu L of the NP protein is added into a sample hole, JS01-JS16 antibodies of the labeled colored microspheres on a binding pad under the sample hole and the NP form an immune complex, then the immune complex is migrated to a T line through chromatography, and the T line is combined and fixed with the labeled antibodies to form a colored T line. And the redundant humanized monoclonal antibodies are continuously transferred to the C line and combined with the anti-human antibodies to form the C line. This was used to determine the binding sensitivity to NP.
Respectively matching the JS08 antibody marked by the colored microspheres with the 13 strains of antibodies to prepare the antigen detection chromatographic strip. When the test strip is verified by using 2ng/ml of recombinant NP, all chromatographic strips can see a remarkable detection T line and a quality control C line is also very remarkable (FIG. 22). This indicates that both 13 pairs of antibody combinations can be used to detect nucleoproteins of the novel coronavirus with a limit of detection of less than 2 ng/ml.
Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.
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Claims (10)

1. A binding protein of a novel coronavirus NP protein, which comprises CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2 and CDR3 shown in SEQ ID NO. 3.
2. The binding protein of claim 1, comprising the heavy chain variable region set forth in SEQ ID No. 4.
3. The binding protein of claim 1, further comprising the CDR1 of SEQ ID No.5, the CDR2 of SEQ ID No.6, and the CDR3 of SEQ ID No. 7.
4. The binding protein of claim 3, comprising the light chain variable region set forth in SEQ ID No. 8.
5. An isolated, recombinant or synthetic DNA molecule encoding the binding protein of any one of claims 1-4; preferably, it comprises the sequences shown in SEQ ID NO.17 and 18.
6. A vector comprising the DNA molecule of claim 5.
7. A host cell comprising the vector of claim 6.
8. A method for producing a binding protein for a novel coronavirus NP protein, comprising culturing the host cell of claim 7 under conditions suitable for the host cell to produce the binding protein for the novel coronavirus NP protein.
9. A composition or kit comprising the binding protein of any one of claims 1-4; preferably, the composition or kit further comprises a second binding protein comprising heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, light chain variable region CDR 3; wherein the content of the first and second substances,
heavy chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 9;
heavy chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 10;
heavy chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 11;
light chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 13;
light chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 14;
light chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 15;
more preferably, the second binding protein comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO.12, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 16.
10. A use comprising the use of any one of:
(1) use of a binding protein according to any one of claims 1 to 4 for the preparation of a novel coronavirus detection product;
(2) use of a binding protein according to any one of claims 1 to 4 for the preparation of a novel diagnostic product for coronavirus infection;
(3) use of a composition according to claim 9 for the preparation of a product for the detection of a novel coronavirus.
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CN114456259B (en) * 2020-11-07 2023-06-02 上海科技大学 Anti-novel coronavirus antibody and preparation method and application thereof

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