CN116063466A - anti-SARS-CoV-2 virus N protein binding antibody and application thereof - Google Patents

anti-SARS-CoV-2 virus N protein binding antibody and application thereof Download PDF

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CN116063466A
CN116063466A CN202210954692.8A CN202210954692A CN116063466A CN 116063466 A CN116063466 A CN 116063466A CN 202210954692 A CN202210954692 A CN 202210954692A CN 116063466 A CN116063466 A CN 116063466A
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monoclonal antibody
antibody
amino acid
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李林
郭垚霖
张伯寒
李天一
刘永健
李韩平
韩婧婉
贾磊
王晓林
李敬云
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Academy of Military Medical Sciences AMMS of PLA
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    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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

Abstract

The invention discloses a binding antibody for resisting SARS-CoV-2 virus N protein and application thereof. The monoclonal antibody N2E5 heavy chain of the invention is variableThe amino acid of the region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table; the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 is shown as SEQ ID NO.5 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.6 in the sequence table. Kinetic constants K of N2E5 and N8C6 and N proteins D 1.42×10 respectively ‑8 M and 1.31X10 ‑8 M. The antibody has high affinity with the N protein of the novel coronavirus, and can be widely used in detection kits of the novel coronavirus, clinical treatment and the like.

Description

anti-SARS-CoV-2 virus N protein binding antibody and application thereof
Technical Field
The invention belongs to the field of biotechnology, and in particular relates to a binding antibody for resisting SARS-CoV-2 virus N protein and application thereof.
Background
The new type of pneumonia (now called covd-19) is caused by the coronavirus SARS-CoV-2. Covd-19 patients often exhibit mild or moderate symptoms such as fever, coughing, and severe patients may develop acute respiratory distress syndrome, acute cardiac injury, multiple organ failure, and secondary infections. The healed patient may also have irreversible pulmonary fibrosis and potential damage to the reproductive and hematopoietic systems may occur.
SARS-CoV-2 belongs to the genus Sarbecovirus of the subfamily β of the subfamily Coronaviridae and belongs to a different subgenera of the same genus as severe acute respiratory syndrome coronavirus (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV) and middle east respiratory syndrome coronavirus (Middle East Respiratory Syndrome Coronavirus, MERS-CoV). The SARS-CoV-2 single-stranded ribonucleic acid genome is about 29.9kb in size and consists of 11 genes, the genome being arranged in the order of 5 '-replicase (orf 1/ab) -structural protein [ spike protein (S) -envelope protein (E) -membrane protein (M) -nucleocapsid protein (N) ] -3'.
The envelope anchored S protein can be attached to a host in the infection process, the extracellular domain of the S protein has two subunits of S1 and S2, the S1 subunit is combined with a receptor on the surface of a host cell, and the fusion of a virus membrane and the host membrane is mediated by the S2 subunit. The M protein can bind to the nucleocapsid and play a role in the formation and assembly of the envelope. The E protein is mainly responsible for the assembly and release of the virus. N protein is nucleocapsid protein and is combined with viral RNA genome.
Disclosure of Invention
The technical problem to be solved by the invention is how to identify the N protein of the novel coronavirus and/or how to obtain a binding antibody against the N protein of the novel coronavirus.
In order to solve the technical problems, the invention firstly provides an antibody for resisting novel coronavirus N protein. The antibody is A or/and B. The a may be monoclonal antibody N2E5 or an antigen binding portion thereof and/or monoclonal antibody N8C6 or an antigen binding portion thereof. The monoclonal antibody N2E5 or antigen binding portion thereof contains a polypeptide having the designation N2E5-V H Heavy chain variable region of (2) and designated N2E5-V L Light chain variable region of (a). The N2E5-V H And N2E5-V L Each consisting of a determinant complementary region and a framework region. The N2E5-V H And said N2E5-V L The determinant complementary regions of (a) may each consist of CDR1, CDR2 and CDR 3.
The N2E5-V H The amino acid sequence of CDR1 of (1) may be positions 26-33 of SEQ ID NO.1 of the sequence Listing.
The N2E5-V H The amino acid sequence of CDR2 of (1) may be positions 51-57 of SEQ ID NO.1 of the sequence Listing.
The N2E5-V H The amino acid sequence of CDR3 of (1) may be positions 96-110 of SEQ ID NO.1 of the sequence Listing.
The N2E5-V L The amino acid sequence of CDR1 of (2) may be positions 26-31 of SEQ ID NO.2 of the sequence Listing.
The N2E5-V L The amino acid sequence of CDR2 of (2) may be positions 49-51 of SEQ ID NO.2 of the sequence Listing.
The N2E5-V L The amino acid sequence of CDR3 of (2) may be SEQ ID N of the sequence ListingO.2, positions 88-98.
The B may be monoclonal antibody N8C6 or an antigen binding portion thereof. The monoclonal antibody N8C6 or antigen binding portion thereof may contain a polypeptide having the designation N8C6-V H Heavy chain variable region of (C) and having the designation N8C6-V L Light chain variable region of (a). The N8C6-V H And N8C6-V L Each consisting of a determinant complementary region and a framework region. The N8C6-V H And said N8C6-V L Is composed of CDR1, CDR2 and CDR 3.
The N8C6-V H The amino acid sequence of CDR1 of (2) may be positions 26-33 of SEQ ID NO.5 of the sequence Listing;
the N8C6-V H The amino acid sequence of CDR2 of (2) may be positions 51-58 of SEQ ID NO.5 of the sequence Listing;
the N8C6-V H The amino acid sequence of CDR3 of (2) may be positions 97-107 of SEQ ID NO.5 of the sequence Listing;
the N8C6-V L The amino acid sequence of CDR1 of (2) may be positions 26-34 of SEQ ID NO.6 of the sequence Listing;
the N8C6-V L The amino acid sequence of CDR2 of (2) may be positions 52-54 of SEQ ID NO.6 of the sequence Listing;
the N8C6-V L The amino acid sequence of CDR3 of (2) may be positions 91-101 of SEQ ID NO.6 of the sequence Listing.
In the above antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 may be SEQ ID NO.1 of the sequence Listing or have at least 80% identity with SEQ ID NO. 1. The amino acid sequence of the light chain variable region of the monoclonal antibody N2E5 can be SEQ ID NO.2 or have at least 80% identity with SEQ ID NO.2 in a sequence table. Wherein the amino acid sequence inconsistencies may be in the Framework Regions (FR).
The amino acid sequence of the heavy chain variable region of the monoclonal antibody N8C6 may be SEQ ID No.5 of the sequence Listing or have at least 80% identity with SEQ ID No. 5. The amino acid sequence of the light chain variable region of the monoclonal antibody N8C6 can be SEQ ID NO.6 of the sequence Listing or have at least 80% identity with SEQ ID NO.6. Wherein the amino acid sequence inconsistencies may be in the Framework Regions (FR).
The at least 80% identity may be at least 80%, 85% or 95% identity.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
Herein, the at least 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Variants of the antibodies of the invention having improved affinity and/or potency may be obtained by employing methods known in the art and are included within the scope of the invention. For example, amino acid substitutions may be used to obtain antibodies with further improved avidity. Alternatively, codon optimization of the nucleotide sequence may also be used to improve translational efficiency in expression systems used to produce antibodies. In addition, polynucleotides comprising sequences that optimize antibody specificity or neutralizing activity by applying directed evolution to any of the nucleic acid sequences of the present invention are also within the scope of the present invention.
In the above antibody, the monoclonal antibody may be any of the following:
a) A single chain antibody;
b) A fusion antibody comprising a) said single chain antibody;
c) Fab fragments;
d) Fv fragments;
the term "Fab fragment" is a heterodimer formed by the disulfide bond between the heavy chain Fd and the intact light chain, containing only one antigen binding site. After the coding genes of the heavy chain Fd and the complete light chain are connected and the bacterial protein signal peptide genes are fused, fab antibodies (Fab fragments) can be expressed in the E.coli endocrine, and the complete three-dimensional folding and intra-chain and inter-chain disulfide bonds are realized. The heavy chain Fd refers to about 1/2 of the H chain portion (about 225 amino acid residues, including VH, CH1 and part of the hinge region) of the Fab.
The term "Fv fragment" refers to a functional Fv antibody that can be assembled by separately constructing vectors containing the VH and VL genes, co-transfecting the cells, and separately expressing them; a termination codon may be placed between the VH and VL in the vector to express two small molecule protein fragments, respectively, which are then non-covalently bound to form an Fv antibody (Fv fragment).
The term "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ') 2 A molecule.
The term "F (ab') 2 The fragment "comprises two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab') 2 Fragments consist of two Fab' fragments held together by disulfide bonds between the two heavy chains.
The term "single chain antibody (ScFv)" refers to a single polypeptide chain, called a single chain antibody (ScFv), in which the light and heavy chain variable region genes are linked by an appropriate oligonucleotide linker (linker). Polypeptide chains spontaneously fold into their natural conception, preserving Fv specificity and affinity.
The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antigen binding fragments retain at least some of the binding specificity of the parent antibody. Typically, the antigen binding fragment retains at least 10% of the parent binding activity when activity is expressed on a molar basis. In particular, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
The term "nanobody (single domain antibody)" means that the antibody heavy chain V region is expressed by genetic engineering methods to obtain an antibody containing only VH fragments. The ability of single domain antibodies to bind to antigen and their stability are essentially identical to those of full antibodies.
The term "bispecific antibody" refers to a bispecific antibody that is produced in large quantities, with high uniformity and purity, by introducing two sets of light and heavy chain genes into myeloma cells, and selecting the appropriate antibody constant regions and Ig types. In addition, bispecific antibodies can also be obtained by chemical cross-linking techniques or hybrid-hybridoma techniques.
The term "Minimal Recognition Unit (MRU)" refers to a single CDR structure comprising only the variable domain, and has a molecular mass of only about 1% of that of an intact antibody, and can bind to the corresponding antigen.
The antibodies of the invention may be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, then host cells are transfected, the transfected host cells are cultured under specific conditions, and the antibodies of the invention are expressed.
The antigen binding fragments may be antigen binding fragments that can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody, as is well known to those of skill in the art.
In order to solve the above technical problems, the present invention also provides a nucleic acid molecule encoding the monoclonal antibody N2E5 or antigen binding portion thereof described above and/or a nucleic acid molecule encoding the monoclonal antibody N8C6 or antigen binding portion thereof described above.
The nucleic acid molecule may be a DNA molecule as described in c 1) or c 2) or c 3) below:
c1 A DNA molecule encoding a monoclonal antibody, wherein the heavy chain variable region encoding sequence of the monoclonal antibody can be a DNA molecule with a sequence shown as SEQ ID No.3, and the light chain variable region encoding sequence of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID No. 4.
c2 A DNA molecule encoding a monoclonal antibody; the heavy chain variable region coding sequence of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO.7, and the light chain variable region coding sequence of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO. 8.
c3 A DNA having more than 90% identity with the DNA molecule defined in c 1) or c 2) and encoding said monoclonal antibody or antigen binding portion thereof.
Herein, the at least 90% identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In order to solve the technical problems, the invention also provides a biological material. The biological material may be an expression cassette, a recombinant vector, a recombinant microorganism and/or a recombinant animal cell line comprising the nucleic acid molecules described above.
Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), viral vectors (e.g., baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (e.g., herpes simplex viruses), etc. In one embodiment of the invention, the vector may specifically be a pADSCFV-S vector or a pcDNA3.1 (+) vector.
The microorganism described herein may be a yeast, a bacterium or a fungus. Wherein the bacteria may be derived from Escherichia (Escherichia), erwinia (Erwinia), agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), bacillus (Bacillus), etc.; the yeast may be Pichia (P.pastoris).
The cell line (host cell) refers to a cell that can be used to introduce a vector, including but not limited to: eukaryotic cells (e.g., yeast cells, aspergillus), animal cells (e.g., mammalian cells, insect cells), or prokaryotic cells. In one embodiment of the invention, the cell line may be in particular HEK293-F cells.
The terms "cell" and "cell line" are used interchangeably and all such designations include progeny thereof.
The use of the antibodies described above for detecting novel coronavirus N proteins is also within the scope of the present invention.
In order to solve the technical problems, the invention also provides application of the antibody and/or the biological material in preparation of products for detecting or diagnosing novel coronaviruses.
In order to solve the technical problems, the invention also provides application of the antibody and/or the biological material in preparation or development of a novel coronavirus clinical treatment product.
In order to solve the technical problems, the invention also provides application of the antibody and/or the biological material in preparing or developing a novel medicine for treating diseases caused by coronaviruses.
In order to solve the technical problems, the invention also provides application of the antibody and/or the biological material in preparation of products for detecting or diagnosing diseases caused by novel coronaviruses.
The screening of the present invention resulted in 2 novel binding antibodies against novel coronavirus Nucleocapsid proteins (nucleoapsid, N protein, SARS-CoV-2N protein), the monoclonal antibodies N2E5 and N8C6 being capable of recognizing novel coronavirus N proteins, the single chain antibodies comprising a heavy chain variable region and a light chain variable region. The N2E5 heavy chain variable region has three complementarity determining regions having amino acid sequences shown as positions 26-33, 51-57 and 96-110 of SEQ ID NO.1; the N2E5 light chain variable region has three complementarity determining regions having amino acid sequences shown in positions 26-31, 49-51 and 88-98 of SEQ ID NO. 2. The N8C6 heavy chain variable region has three complementarity determining regions having amino acid sequences shown as positions 26-33, 51-58 and 97-107 of SEQ ID NO. 5; the N8C6 light chain variable region has three complementarity determining regions having amino acid sequences shown in positions 26-34, 52-54 and 91-101 of SEQ ID NO.6.
The invention has the beneficial effects that:
the single-chain antibody of the invention can specifically recognize the N protein of SARS-COV-2 virus, the N2E5 and N8C6Kinetic constant K of monoclonal antibody and N protein D 1.42×10 respectively -8 M and 1.31X10 -8 M。
The antibody has high affinity with N protein of novel coronavirus, and can be widely used for detection, clinical treatment and the like of the novel coronavirus.
Drawings
FIG. 1 is a diagram showing the identification of SDS-PAGE proteins of monoclonal antibodies N2E5 and N8C6. A is the detection result of the monoclonal antibody N2E 5; b is the detection result of the monoclonal antibody N8C6. M is protein Marker,1 is cell culture supernatant, 2 is Flow Through,3 is purified product No.1, 4 is purified product No.2, 5 is purified product No.3, 6 is purified product No.4, and 7 is purified product No. 5.
FIG. 2 is a Western Blot identification of N2E5 antibodies and N8C6 antibodies. A is the identification result of the monoclonal antibody N2E 5; b is the identification result of the monoclonal antibody N8C6. M is protein Marker,1 is 5 mug SARS-CoV-2N protein, 2 is 10 mug SARS-CoV-2N protein, 3 is 15 mug SARS-CoV-2N protein, 4 is 20 mug SARS-CoV-2N protein.
FIG. 3 is a graph showing the affinity of the N2E5 antibody and the N8C6 antibody for the N protein of SARS-CoV-2 virus.
Detailed Description
Animal virus: the biological material is available to the public from the applicant in accordance with the national biosafety regulations, and is used only for repeated experiments related to the present invention, and is not used for other purposes.
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The sources of reagents and carriers in the examples of the present invention are as follows:
the heavy chain expression vector Igγ1 and the light chain expression vector Iglambda are provided by the central infectious disease prevention and control national center for preventing and controlling HIV infection in China (related documents: hu Yuanyuan, kang Jia, etc.. A group of monoclonal antibodies targeting HIV membrane proteins are isolated and functionally identified [ J ] J. Chinese experiment and J. 2. Of clinical virology, 2021, 35, vol. 35: 141-147;Tiller T,Meffre E,Yurasov S,Tsuiji M, nussenzweig MC, wardemann H.efficiency generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.J. Immunomethods.20088 Jan 1;329 (1-2): 112-24).
FreeStyle TM 293 expression medium: gibco, cat No. 12338026.
293F cells: the cells are provided by the central infectious disease prevention and control laboratory of the central AIDS prevention and control center for Chinese disease prevention and control (related documents: hu Yuanyuan, kang Jia, etc.. A group of monoclonal antibodies targeting HIV membrane proteins are isolated and functionally identified [ J ] J J.China experiment and J.4.35, 2 nd: 141-147 of J.2021, 4 th month).
SARS-CoV-2N protein (as a mixture of His-tagged and untagged N proteins): shanghai Biyun biotechnology Co., ltd., product number P2328-1mg.
Example 1 discovery of antibodies
B cells of a patient suffering from the COVID-19 are infected by using EB virus, and through the steps of subcloning screening, antibody light and heavy chain variable region amplification, expression vector construction, antibody expression and purification, antibody affinity identification and the like, 2 humanized IgG antibodies capable of specifically recognizing SARS-CoV-2 virus Nucleocapsid protein (Nucleocapid, N protein, SARS-CoV-2N protein) are obtained, which are respectively named monoclonal antibody N2E5 and monoclonal antibody N8C6.
Monoclonal antibody N2E5: the heavy chain variable region and the light chain variable region of the monoclonal antibody N2E5 are shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 is shown in SEQ ID NO. 2. The monoclonal antibody N2E5 heavy chain variable region is provided with three Complementarity Determining Regions (CDRs) which are respectively a heavy chain CDR1 (the amino acid sequence is the 26 th-33 th positions of SEQ ID NO.1 in a sequence table), a heavy chain CDR2 (the amino acid sequence is the 51 th-57 th positions of SEQ ID NO.1 in the sequence table) and a heavy chain CDR3 (the amino acid sequence is the 96 th-110 th positions of SEQ ID NO.1 in the sequence table); the monoclonal antibody N2E5 light chain variable region has three Complementarity Determining Regions (CDRs) which are respectively light chain CDR1 (the amino acid sequence is the 26 th-31 th positions of SEQ ID NO.2 in the sequence table), light chain CDR2 (the amino acid sequence is the 49 th-51 th positions of SEQ ID NO.2 in the sequence table) and light chain CDR3 (the amino acid sequence is the 88 th-98 th positions of SEQ ID NO.2 in the sequence table).
Monoclonal antibody N8C6: the monoclonal antibody N8C6 heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 5; the amino acid sequence of the monoclonal antibody N8C6 light chain variable region is shown as SEQ ID NO.6. The heavy chain variable region of the monoclonal antibody N8C6 has three Complementarity Determining Regions (CDRs) which are respectively a heavy chain CDR1 (the amino acid sequences are the 26 th to 33 th positions of SEQ ID NO.5 in the sequence table, a heavy chain CDR2 (the amino acid sequences are the 51 th to 58 th positions of SEQ ID NO.5 in the sequence table), and a heavy chain CDR3 (the amino acid sequences are the 97 th to 107 th positions of SEQ ID NO.5 in the sequence table), and the light chain variable region of the monoclonal antibody N2E5 has three Complementarity Determining Regions (CDRs) which are respectively a light chain CDR1 (the amino acid sequences are the 26 th to 34 th positions of SEQ ID NO.6 in the sequence table), a light chain CDR2 (the amino acid sequences are the 52 th to 54 th positions of SEQ ID NO.6 in the sequence table), and a light chain CDR3 (the amino acid sequences are the 91 th to 101 th positions of SEQ ID NO.6 in the sequence table) (Table 1).
TABLE 1 amino acid sequences of the CDR regions of monoclonal antibodies N2E5 and N8C6
Figure BDA0003790816300000071
Note that: the naming system for the above CDRs is Kabat.
Example 2 preparation of antibodies
1. The variable regions of the antibodies are amplified by using specific primers (Table 2) with enzyme cutting sites by respectively connecting a DNA molecule of a heavy chain variable region of a monoclonal antibody N2E5 (nucleotide sequence is SEQ ID NO.3 in a sequence table), a DNA molecule of a heavy chain variable region of a monoclonal antibody N8C6 (nucleotide sequence is SEQ ID NO.4 in a sequence table), a DNA molecule of a light chain variable region of a monoclonal antibody N2E5 (nucleotide sequence is SEQ ID NO.7 in a sequence table) and a DNA molecule of a light chain variable region of a monoclonal antibody N8C6 (nucleotide sequence is SEQ ID NO.8 in a sequence table) to a T cloning vector to obtain 4 recombinant T vectors containing different variable region DNA molecules, and respectively using the 4 recombinant T vectors as templates. In Table 2, primer pair N2E5H5'AgeI/N2E5H3' SalI was used to amplify the monoclonal antibody N2E5 heavy chain variable region, primer pair N2E5L5'AgeI/N2E5L3' XhoI was used to amplify the monoclonal antibody N2E5 light chain variable region, primer pair N8C6H5'AgeI/N8C6H3' SalI was used to amplify the monoclonal antibody N8C6 heavy chain variable region, and primer pair N8C6L5'AgeI/N8C6L3' XhoI was used to amplify the monoclonal antibody N8C6 light chain variable region. The PCR reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.
TABLE 2 antibody variable region amplification primers incorporating cleavage sites
Figure BDA0003790816300000081
Note that: underlined indicates the cleavage site
TABLE 3 introduction of cleavage sites into PCR reaction System
Figure BDA0003790816300000082
TABLE 4 restriction site introduction PCR reaction procedure
Figure BDA0003790816300000091
And (3) carrying out electrophoresis identification on all PCR products, performing gel cutting purification, and simultaneously performing concentration measurement.
And (3) enzyme cutting: the purified PCR products and antibody expression vector IgGamma 1 and antibody light chain expression vector Iglambda were set up as in the systems of Table 5 and Table 6 on an ultra-low temperature ice box.
TABLE 5 cleavage reaction System
Figure BDA0003790816300000092
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TABLE 6 cleavage reaction procedure
Figure BDA0003790816300000093
After the cleavage efficiency was detected by agarose gel electrophoresis, the target fragment was ligated to the antibody expression vector by incubating with T4 DNA ligase at 37℃for 3 hours, and the ligation system is shown in Table 7.
TABLE 7 ligation reaction procedure
Figure BDA0003790816300000094
Conversion: the ligation product was transformed into DH 5. Alpha. E.coli competence, and the monoclonal colonies were grown up and the strain was preserved. Extracting plasmids of bacterial liquid, carrying out enzyme digestion identification, and carrying out sequencing identification on the identified plasmids to obtain recombinant heavy chain expression vectors Iggamma 1-N2E5 and Iggamma 1-N8C6, recombinant light chain expression vectors Iglambda-N2E 5 and Iglambda-N8C 6 which are successfully constructed.
2. Construction of recombinant cells
Co-transfecting a recombinant heavy chain expression vector (Ig gamma 1-N2E 5) and a recombinant light chain expression vector (Ig lambda-N2E 5) with FreeStyle TM 293 expression medium to obtain the recombinant cell 293F/gamma 1HC-N2E5 expressing monoclonal antibody N2E5.
Co-transfecting a recombinant heavy chain expression vector (Ig gamma 1-N8C 6) and a recombinant light chain expression vector (Ig lambda-N8C 6) of a monoclonal antibody N8C6 into FreeStyle TM 293F cells cultured in 293 expression medium to obtain recombinant cells 293F/gamma 1HC-N8C6 expressing monoclonal antibody N8C6.
The co-transfection procedure was as follows:
preparing cells: 28mL fresh, pre-warmed serum-free 293 was used TM Dilution of expression Medium 3X 10 7 The 293F cells were shake-bottled to 125mL of sterile cells.
Preparation of lipid-DNA complexes: at the position of
Figure BDA0003790816300000101
In I, 30 μg of plasmid is diluted, and the concentration ratio of the recombinant light and heavy chain expression vector of the antibody is 1:1. 60 μl of 293fetin was diluted in Opti-MEM serum free medium TM Transfection reagent, make serum culture medium total amount 1mL, incubating for 5min at room temperature. Adding diluted plasmid into diluted reagent, mixing gently, incubating for 20-30 min at room temperature to form DNA-reagent complex.
2mL of the above complex was added to each cell suspension bottle. 5% CO at 125r/min 37 DEG C 2 The cells are cultured in a cell culture tank.
3. Preparation of antibodies
3.1 collecting supernatant
After culturing the recombinant cells obtained in step 2 for 48 hours, a culture expressing monoclonal antibody N2E5 (designated as 293FN2E5 culture) and a culture expressing monoclonal antibody N8C6 (293F/N8C 6 culture) were obtained, respectively, and the cultures were transferred into a centrifuge tube, and the supernatant was collected by centrifugation at 400g for 10min and filtered through a 0.22 μm filter.
3.2 antibody purification
The supernatant obtained by filtration was purified according to instructions using the protein A/G antibody purification kit of Thermo Fisher Scientific (NO. 89980) to obtain a purified product of monoclonal antibody N2E5 and a purified product of monoclonal antibody N8C6.
The purification steps are as follows:
the filtered supernatant was diluted with an equal volume of binding buffer. And loosening the top cover on the rotary column, and pulling the bottom seal open. The column was placed in a 15mL collection tube, 400g centrifuged for 1min and the stock was discarded. The column was equilibrated with 2mL of binding buffer. Centrifuge 1000g for 1min, discard filtrate. This step was repeated 1 time. The diluted sample was applied to the column and the Flow water was collected and designated "Flow Through".
The column was washed with 15mL of binding buffer, placed in a clean 15mL collection tube, centrifuged at 1000g for 1min, and the filtrate was discarded. 100. Mu.L of the neutralization buffer was added to 5 1.5mL Ep tubes, 1mL of elution buffer was added to the column, and 1000g was centrifuged for 1min. The filtrate was transferred to an Ep tube containing a neutralization buffer and the collected solution was stored as "purified product-1". This step was repeated 4 times to obtain 5 gradients of purified product, which was stored at 4 ℃.
3.3 detection of purified product
The purified products were detected using human total IgG content ELISA kit (NO. BDEL-0254-96T) from Beijing Boolong immune technology Co., ltd, and the human total IgG concentrations of monoclonal antibody N2E5 purified products 1-5 were 6608.04ng/mL, 6222.81ng/mL, 4632.42ng/mL, 4594.73ng/mL, 3421.90ng/mL, respectively. The total human IgG concentration of the purified products 1 to 5 of the monoclonal antibody N8C6 was 6695.55ng/mL, 6731.94ng/mL, 5577.87ng/mL, 5547.51ng/mL, 3991.43ng/mL, respectively.
3.4 analysis of antibody purity
The culture supernatant obtained in step 3.1, the "Flow Through" produced during the purification in step 3.2 and the resulting purified antibody product were subjected to SDS-PAGE protein electrophoresis. As a result, as shown in FIG. 1, culture supernatants (293F/N2E 5 culture and 293F/N8C6 culture supernatants, FIG. 1, lane 1 of A and FIG. 1, lane 1 of B) and "Flow Through" (FIG. 1, lane 2 of A and FIG. 1, lane 2 of B) had bands of hetero proteins, whereas the antibody purified products had bands of interest at only 55kDa (antibody heavy chain size) and 25kDa (antibody light chain size).
3.5Western Blot detection
SARS-CoV-2N protein (about 47kDa, including His-tagged SARS-CoV-2N protein and non-His-tagged SARS-CoV-2N protein) was loaded on a gradient of 5. Mu.g, 10. Mu.g, 15. Mu.g, 20. Mu.g, subjected to SDS-PAGE protein electrophoresis, transferred to PVDF membrane and then subjected to a reaction of 1: step 3.2 purification of 1000-fold dilution the resulting monoclonal antibody N2E5 or monoclonal antibody N8C6 was incubated. As shown in FIG. 2, the Western Blot detection results show that the monoclonal antibodies N2E5 and N8C6 can bind to SARS-CoV-2N protein, and that there are two bands specific at about 47kDa, namely His-tagged SARS-CoV-2N protein and non-His-tagged SARS-CoV-2N protein.
Example 3 affinity detection of antibodies to SARS-CoV-2 Virus N protein
Using biofilm layer interference techniques (Biolayer interferometry, BLI): the Gator biofilm layer interferometer performs affinity detection on the purified monoclonal antibody N2E5 and the monoclonal antibody N8C6 obtained in example 2.
The detection results are shown in FIG. 3. Kinetic constants K of monoclonal antibody N2E5 and monoclonal antibody N8C6 and N protein D 1.42×10 respectively -8 M and 1.31X10 -8 M, indicates that the affinity of the two monoclonal antibodies is higher.
In conclusion, the monoclonal antibodies N2E5 and N8C6 obtained by screening can specifically identify and bind SARS-COV-2 virus N protein, have high affinity with N protein, and can be used for preparing a novel coronavirus detection kit and developing novel products for coronavirus clinical treatment.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. An antibody against novel coronavirus N protein, characterized in that: the antibody is A or/and B, wherein A is monoclonal antibody N2E5 or antigen binding portion thereof and/or monoclonal antibody N8C6 or antigen binding portion thereof; the monoclonal antibody N2E5 or antigen binding portion thereof contains a polypeptide designated N2E5-V H Heavy chain variable region of (2) and designated N2E5-V L Light chain variable region of (2), said N2E5-V H And N2E5-V L Are composed of a determinant complementary region and a framework region; the N2E5-V H And said N2E5-V L Is composed of CDR1, CDR2 and CDR 3;
the N2E5-V H The amino acid sequence of CDR1 of (1) is SEQ ID NO.1 in a sequence table;
the N2E5-V H The amino acid sequence of CDR2 of (1) is 51-57 of SEQ ID NO.1 in the sequence table;
the N2E5-V H The amino acid sequence of CDR3 of (1) is 96-110 th site of SEQ ID NO.1 in the sequence table;
the N2E5-V L The amino acid sequence of CDR1 of (2) is 26-31 of SEQ ID NO.2 in the sequence table;
the N2E5-V L The amino acid sequence of CDR2 of (2) is the 49 th-51 th position of SEQ ID NO.2 in the sequence table;
the N2E5-V L The amino acid sequence of CDR3 of (2) is 88-98 of SEQ ID NO.2 in the sequence table;
the B is a monoclonal antibody N8C6 or an antigen binding portion thereof, and the monoclonal antibody N8C6 or the antigen binding portion thereof contains a polypeptide named N8C6-V H Heavy chain variable region of (C) and having the designation N8C6-V L Light chain variable region of (C) N8C6-V H And N8C6-V L Are composed of a determinant complementary region and a framework region; the N8C6-V H And said N8C6-V L Is composed of CDR1, CDR2 and CDR 3;
the N8C6-V H The amino acid sequence of CDR1 of (2) is the 26 th-33 th position of SEQ ID NO.5 in the sequence table;
the N8C6-V H The amino acid sequence of CDR2 of (1) is 51-58 of SEQ ID NO.5 in the sequence table;
the N8C6-V H The amino acid sequence of CDR3 of (2) is 97 th-107 th position of SEQ ID NO.5 in the sequence table;
the N8C6-V L The amino acid sequence of CDR1 of (1) is the 26 th-34 th position of SEQ ID NO.6 in the sequence table;
the N8C6-V L The amino acid sequence of CDR2 of (2) is 52-54 th site of SEQ ID NO.6 in the sequence table;
the N8C6-V L The amino acid sequence of CDR3 of (2) is the 91 st-101 st position of SEQ ID NO.6 in the sequence table.
2. The antibody of claim 1, wherein: the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 is SEQ ID NO.1 in the sequence table, and the amino acid sequence of the light chain variable region of the monoclonal antibody N2E5 is SEQ ID NO.2 in the sequence table;
the amino acid sequence of the heavy chain variable region of the monoclonal antibody N8C6 is SEQ ID NO.5 in the sequence table, and the amino acid sequence of the light chain variable region of the monoclonal antibody N8C6 is SEQ ID NO.6 in the sequence table.
3. The antibody of claim 1 or 2, wherein: the monoclonal antibody is any one of the following:
a) A single chain antibody;
b) A fusion antibody comprising a) said single chain antibody;
c) Fab fragments;
d) Fv fragments.
4. A nucleic acid molecule encoding the monoclonal antibody N2E5 or antigen binding portion thereof according to claim 1 or 2 and/or a nucleic acid molecule encoding the monoclonal antibody N8C6 or antigen binding portion thereof according to claim 1 or 2;
the nucleic acid molecule is a DNA molecule as described in c 1) or c 2) or c 3) below:
c1 A DNA molecule of the coding sequence of a heavy chain variable region of the monoclonal antibody, wherein the coding sequence of the heavy chain variable region of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO.3, and the coding sequence of the light chain variable region of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO. 4;
c2 A DNA molecule encoding a monoclonal antibody; the heavy chain variable region coding sequence of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO.7, and the light chain variable region coding sequence of the monoclonal antibody is a DNA molecule with a sequence shown as SEQ ID NO. 8;
c3 A DNA having more than 90% identity with the DNA molecule defined in c 1) or c 2) and encoding said monoclonal antibody or antigen binding portion thereof.
5. A biomaterial characterized in that: the biological material is an expression cassette, a recombinant vector, a recombinant microorganism and/or a recombinant animal cell line comprising the nucleic acid molecule of claim 4.
6. Use of an antibody according to any one of claims 1-3 for detecting novel coronavirus N proteins.
7. Use of an antibody according to any one of claims 1-3 and/or a biomaterial according to claim 5 for the preparation of a product for the detection or diagnosis of a novel coronavirus.
8. Use of an antibody according to any one of claims 1-3 and/or a biomaterial according to claim 5 for the preparation or development of a product for the clinical treatment of a novel coronavirus.
9. Use of an antibody according to any one of claims 1-3 and/or a biomaterial according to claim 5 for the preparation or development of a medicament for the treatment of a disease caused by a novel coronavirus.
10. Use of an antibody according to any one of claims 1-3 and/or a biomaterial according to claim 5 for the manufacture of a product for the detection or diagnosis of a novel coronavirus-caused disease.
CN202210954692.8A 2022-08-10 2022-08-10 anti-SARS-CoV-2 virus N protein binding antibody and application thereof Pending CN116063466A (en)

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Publication number Priority date Publication date Assignee Title
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116675765A (en) * 2023-06-02 2023-09-01 保定国兰生物技术有限公司 Coronavirus N protein monoclonal antibody and application thereof
CN116675765B (en) * 2023-06-02 2023-11-24 保定国兰生物技术有限公司 Coronavirus N protein monoclonal antibody and application thereof

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