CN118666996B - Neutralizing antibody ZJ-11 binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope and its application - Google Patents
Neutralizing antibody ZJ-11 binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope and its application Download PDFInfo
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Abstract
The invention provides a neutralizing antibody ZJ-11 which binds to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope and application thereof. The light chain CDR1 of the neutralizing antibody ZJ-11 comprises an amino acid fragment shown as SEQ ID NO.1, the heavy chain CDR2 comprises an amino acid fragment shown as SEQ ID NO.2 and the heavy chain CDR3 comprises an amino acid fragment shown as SEQ ID NO.3, the light chain CDR1 of the neutralizing antibody ZJ-11 comprises an amino acid fragment shown as SEQ ID NO.4, the light chain CDR2 comprises an amino acid fragment shown as SEQ ID NO.5 and the light chain CDR3 comprises an amino acid fragment shown as SEQ ID NO. 6. The neutralizing antibody ZJ-11 can be specifically combined with conserved epitopes on SARS-CoV-2 and SARS-CoV RBD, so as to prevent the infection of the cell by the sabia virus in a broad spectrum, and finally realize the protection effect. The antibody provided by the invention has strong targeting property and high binding activity, and the antibody prepared by the preparation method provided by the invention has high purity and can be prepared in a large amount.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a neutralizing antibody ZJ-11 combined with coronavirus SARS-CoV-2 and SARS-CoV spike protein (Spikeprotein) RBD epitope, a preparation method and application thereof.
Background
SARS-CoV and SARS-CoV-2 belong to the genus Sa Bei Bingdu subgenera virus (Sarbecovirus) of the genus Beta coronavirus, both are highly pathogenic coronaviruses that infect humans, and the surface of their virions mainly has three structural proteins, spike protein (Spikeprotein), membrane protein (Membraneprotein) and envelope protein (Envelopeprotein). Wherein the S protein is a 180-200kDa transmembrane glycoprotein which is cleaved into S1 and S2 subunits by some proteases on the target cell membrane. The S1 subunit comprises an N-terminal domain (NTD) and a C-terminal Receptor Binding Domain (RBD), which mediates SARS-CoV, SARS-CoV-2 binding to human ACE2 receptor. The S2 subunit consists of fusion peptide (Fusionpeptide, FP), stem helix region (Stem helix, SH), heptad repeats 1 and 2 (Heptadrepeatregion, HR1 and HR 2), transmembrane region and intramembrane region, responsible for mediating the process of viral envelope and target cell membrane fusion, one of the ways in which viruses invade cells.
The neutralizing antibody ZJ-11 therapy is an important means for preventing and treating diseases caused by various pathogen infections, and the development of a novel neutralizing antibody ZJ-11 for Sarbecovirus including SARS-CoV-2 and SARS-CoV is clinically significant in terms of prevention and control of SARS-CoV-2 infection and prevention and control of future similar coronavirus infection.
Disclosure of Invention
Based on this, the object of the present invention consists in providing a neutralizing antibody ZJ-11 binding to the RBD epitopes of the coronaviruses SARS-CoV-2 and SARS-CoV spike proteins, which neutralizing antibody ZJ-11 is able to specifically bind to the conserved epitopes of the RBD domains on the SARS-CoV-2 and SARS-CoV spike proteins, thus preventing the infection of cells by the viruses SARS-CoV-2, SARS-CoV, sha Bei virus (Sarbecovirus) in a broad spectrum, eventually achieving a protective effect.
The aim of the invention can be achieved by the following technical scheme:
The first object of the present invention is to provide a neutralizing antibody ZJ-11 binding to coronaviruses SARS-CoV-2 and SARS-CoV spike protein RBD epitope, the heavy chain CDR1 of said antibody comprising the amino acid sequence as shown in SEQ ID NO.1, the heavy chain CDR2 comprising the amino acid sequence as shown in SEQ ID NO.2 and the heavy chain CDR3 comprising the amino acid sequence as shown in SEQ ID NO. 3;
The light chain CDR1 of the antibody comprises an amino acid sequence shown as SEQ ID NO.4, the light chain CDR2 comprises an amino acid sequence shown as SEQ ID NO.5 and the light chain CDR3 comprises an amino acid sequence shown as SEQ ID NO. 6.
Preferably, the heavy chain variable region of the antibody is shown in SEQ ID NO. 7.
Preferably, the light chain variable region of the antibody is shown in SEQ ID NO. 8.
Preferably, the light chain constant region of the antibody is shown in SEQ ID NO. 9.
Preferably, the heavy chain constant region of the antibody is shown in SEQ ID NO. 10.
Preferably, the sequence of the constant region of the antibody is the sequence of an IgG1 constant region.
Preferably, the species source of the constant region of the antibody is human.
The heavy chain variable region shown in SEQ ID No.7 and the light chain variable region shown in SEQ ID No.8 further comprise a Framework Region (FR), and the amino acid sequences of 4 FRs do not directly participate in the binding reaction.
The antibodies provided by the invention are capable of specifically binding to SARS-CoV-2 and SARS-CoV spike protein RBD, which binding epitopes are highly conserved in SARS-CoV-2 and SARS-CoV.
A second object of the present invention is to provide a recombinant protein comprising one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region and the light chain variable region defined in the first object.
A third object of the present invention is to provide a detection reagent, a detection kit or a medicament comprising the neutralizing antibody ZJ-11 provided in the first object or the recombinant protein provided in the second object.
The fourth object of the present invention is to provide a nucleic acid comprising a nucleic acid sequence encoding the neutralizing antibody ZJ-11 binding to SARS-CoV-2 and SARS-CoV spike protein RBD epitopes of coronavirus.
A fifth object of the present invention is to provide a recombinant expression vector comprising the above-mentioned nucleic acid.
Preferably, the recombinant expression vector is an antibody expression vector.
Preferably, the recombinant expression vector is AbVec2.0-IGHG1 or AbVec1.1-IGKC.
The sixth object of the present invention is to provide a host cell comprising the above specific antibody, or the above nucleic acid, or the above recombinant expression vector.
Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.
The seventh object of the present invention is to provide a method for preparing the neutralizing antibody ZJ-11 binding to the SARS-CoV-2 and SARS-CoV spike protein RBD epitopes of coronaviruses, comprising constructing the above-mentioned host cell, culturing, and collecting the neutralizing antibody ZJ-11 binding to the SARS-CoV-2 and SARS-CoV spike protein RBD epitopes of coronaviruses.
Preferably, the host cell is a Freestyle 293-F cell or an Expi293F cell.
Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects:
The neutralizing antibody ZJ-11 provided by the application contains CDRs of specific sequences, and can specifically and conservatively epitope on RBD structural domains of SARS-CoV-2 and SARS-CoV spike protein, thereby preventing the infection of the cell by the sabia virus (Sarbecovirus) and finally realizing the protective effect. The neutralizing antibody ZJ-11 provided by the application has broad spectrum, and can be suitable for preventing and controlling the infection of representative viruses in various SARS-CoV-2 mutant strains and other Sha Bei virus viruses (Sarbecovirus). Meanwhile, the neutralizing antibody ZJ-11 provided by the application has higher neutralizing activity. Overall, the present application provides a new candidate for the prevention and treatment of sabal virus (Sarbecovirus) infection.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of detection of the binding activity of the monoclonal neutralizing antibody ZJ-11 in example 2 of the present invention
FIG. 2 is a graph showing the results of neutralization test of SARS-CoV-2 mutant strain pseudovirus by monoclonal neutralizing antibody ZJ-11 of example 2 of the present invention;
FIG. 3 is a graph showing the results of the test for neutralizing pseudovirus of a representative strain of sabia virus (Sarbecovirus) with the monoclonal neutralizing antibody ZJ-11 of example 2 of the present invention;
Detailed Description
The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the invention and are not intended to limit the scope of the invention in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the invention, and equivalents thereof are also intended to fall within the scope of the invention. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention, it being understood that the invention may be practiced without one or more of these details.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.
Terminology
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).
The term "plural", and the like in the present invention refers to, unless otherwise specified, a number of 2 or more. For example, "one or more" means one kind or two or more kinds.
As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present invention can be implemented, the technical problem of the present invention is solved, and the technical effect expected by the present invention is achieved.
Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the invention. If there are multiple "preferences" in a solution, if there is no particular description and there is no conflict or constraint, then each "preference" is independent of the others.
In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.
In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
In the present invention, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody [ see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991) ]. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be classified into different classes, mainly class 5 immunoglobulins: igA, igD, igE, igG and IgM, some of which can be further classified into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2, depending on the amino acid sequence of their heavy chain constant region. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies. Chimeric antibodies are a molecule in which different portions are derived from different animal species, e.g., chimeric antibodies having variable regions from murine monoclonal antibodies, and constant regions from human immunoglobulins (see, e.g., U.S. patent 481757 and U.S. patent 4816397, which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. patent 5585089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably produced by amino acid substitution.
The following are some specific examples.
Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
In some embodiments of the invention, the host cell is a Freestole 293-F cell or an Expi293F cell.
EXAMPLE 1 construction, expression and purification of expression vectors for anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11
The anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibodies ZJ-11 prepared in this example have heavy chain variable regions comprising VHCDR1 as shown in SEQ ID NO.1, VHCDR2 as shown in SEQ ID NO.2 and VHCDR3 as shown in SEQ ID NO.3, and light chain variable regions comprising VLCDR1 as shown in SEQ ID NO.4, VLCDR2 as shown in SEQ ID NO.5 and VLCDR3 as shown in SEQ ID NO. 6.
Specifically, the heavy chain variable region is shown as SEQ ID NO.7, the light chain variable region is shown as SEQ ID NO.8, the light chain constant region is shown as SEQ ID NO.9, and the heavy chain constant region is shown as SEQ ID NO. 10.
SEQ ID NO.1:SYGMS,
SEQ ID NO.2:TINSGGRSTYYPDSVQG,
SEQ ID NO.3:HSGNYVDEAMDY,
SEQ ID NO.4:SASSSVSVMH,
SEQ ID NO.5:TTSNLAS,
SEQ ID NO.6:HQWSSWT,
SEQ ID NO.7:
EVQLVESGGDLVKPGESLKLSCVASGFTFSSYGMSWVRQTPDKRLDWVATINSGGRST YYPDSVQGRFTISRDNAKNTLYLQMSSLKSDDTAMFYCARHSGNYVDEAMDYWGQGTSV TVSS,
SEQ ID NO.8:
QLVLTQSPAIISASLGEEITLTCSASSSVSVMHWYQQKSGTSPKLLIFTTSNLASGVPSRF SGSGSGTFYSLTISSVEAEDAADYYCHQWSSWTFGGGTKVEIK,
SEQ ID NO.9:
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,
SEQ ID NO.10:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
The preparation method of the anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11 comprises the following steps:
1. Nucleotide sequence fragments containing the heavy chain variable region (SEQ ID NO. 7) and the light chain variable region (SEQ ID NO. 8) encoding anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibodies ZJ-11 were incorporated into AbVec2.0-IGHG1 and AbVec1.1-IGKC, respectively, containing the heavy chain constant region sequence fragments of human IgG1 antibody (reference Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.Tiller T,Meffre E,Yurasov S,Tsuiji M,Nussenzweig MC,Wardemann H.J Immunol Methods.2008Jan 1;329(1-2):112-24.Epub 2007Oct 31.10.1016/j.jim.2007.09.017PubMed 17996249) for vector use) to give recombinant expression vectors capable of expressing the heavy chain and light chain of the antibody of interest, respectively.
2. Transfection of cells, expression and purification of anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11
1. Transfection of cells and expression of monoclonal neutralizing antibody ZJ-11
Transfection was performed using the Gibco Expi293F expression system and following the instructions, the steps were outlined as follows:
Mixing 30 mug of two recombinant expression vector DNA respectively expressing the heavy chain and the light chain of the monoclonal neutralizing antibody ZJ-11 with a transfection reagent ExpiFectamineTM, and standing at room temperature for 20 minutes to form a stable complex;
then added to 25.5mL of an Expi 293F cell culture broth that had been adjusted to a concentration of 2.9X10 6 cells/mL;
Culturing in a shaker at 37℃and 8% (v/v) CO 2 at 125rpm for 20 hours;
Adding a transfection enhancer 1 and a transfection enhancer 2 carried by an Expi293F expression system;
The culture was continued for 4 days at 37℃in a shaker at 8% (v/v) CO 2 at 125 rpm.
2. Purification
The culture prepared in step 1 was collected, and the cell culture supernatant was collected by centrifugation at 3000rpm for 15 minutes, and antibody purification was performed using ProteinA magnetic beads from Genscript. The purification steps are briefly described as follows:
mixing ProteinA magnetic beads with cell culture supernatant, and combining with a shaking table at room temperature for 4 hours;
The magnetic rack adsorbs the magnetic beads, the cell supernatant is discarded, and the magnetic beads are washed 5 times with 1 XPBS of pH7.00.1% (v/v) Tween 20;
elution with Elutionbuffer at pH2.00.1M glycine;
Equilibrated with pH8.51M Tris buffer;
The equilibrated monoclonal neutralizing antibody ZJ-11 was desalted and subjected to DPBS solvent substitution.
The purified neutralizing antibody ZJ-11 was stored in a-80℃refrigerator.
EXAMPLE 2 functional analysis of anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11
1. Detection of the binding Activity of human anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11 prepared in example 1 with antigen
ELISA was used to determine the ability of monoclonal neutralizing antibody ZJ-11 to bind to spike protein S of spike protein S, SARS-CoV of SARS-CoV-2 and RBD domains of SARS-CoV-2 and SARS-CoV.
The steps are briefly described as follows:
(1) The ELISA plate is coated with 25ng of SARS-CoV-2 Wild strain (Wild Type, WT) spike protein S (trimer S6P), SARS-CoV spike protein S (trimer S2P) and SARS-CoV-2, SARS-CoV RBD protein per well, and 0.1M Na 2 HPO4 (pH 9.0) is used as coating liquid at 4 ℃ overnight;
(2) DPBS of 10% (v/v) calf serum is used as a blocking solution, and is blocked for 2 hours at 37 ℃, and the neutralizing antibody ZJ-11 to be detected prepared in example 1 after serial gradient dilution is added for incubation for 2 hours at 37 ℃;
(3) HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) diluted 1:10000 was added as secondary antibody and incubated at 37℃for 1 hour;
(4) After color development with TMB single-component color development solution, the reaction was stopped with Solarbio ELISA stop solution, and the absorbance at 450nm was measured with an ELISA reader.
Results:
As shown in FIG. 1, the results of the detection of the binding activity of the monoclonal neutralizing antibody ZJ-11 to the antigen are shown in FIG. 1, wherein the binding activity of the monoclonal neutralizing antibody ZJ-11 to SARS-CoV-2S protein is EC 50 =0.0016. Mu.g/mL, the binding activity to SARS-CoV S protein is EC 50 =0.003. Mu.g/mL, and the binding activity to SARS-CoV-2 and SARS-CoV RBD protein EC 50 are 0.0043. Mu.g/mL and 0.0140. Mu.g/mL, respectively.
2. Detection of the neutralizing Activity of human anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11 against SARS-CoV-2 mutant pseudo-virus prepared in example 1
The SARS-CoV-2 pseudovirus neutralization assay was performed using SARS-CoV-2 pseudovirus wild strain (WT), mutant strains Beta, delta, omicron BA.1, BA.5, XBB, EG.5, JN.1.
The pCDNA3.1-SARS-CoV-2 (WT) -S plasmid, pCDNA3.1-Beta-S plasmid, pCDNA3.1-Delta-S plasmid, pCDNA3.1-Omicron BA.1-S plasmid were each 20. Mu.g transfected into 293T cells, the 293T cells were infected with VSV DELTA G (with luciferase reporter) for 24 hours, and the cell supernatants were collected for 48 hours to prepare pseudo-viruses corresponding to SARS-CoV-2WT, mutants Beta, delta, omicron BA.1, BA.5, XBB, and EG.5, JN.1 pseudo-viruses were purchased from Darui technologies;
Mixing pseudovirus and serial gradient diluted monoclonal neutralizing antibody ZJ-11, incubating at 37 ℃ for 1 hour, adding into digested 293T-ACE2 cells, and culturing at 96 Kong Baiban at 37 ℃ for 24 hours;
After the cell plate is discarded, adding a chromogenic substrate (Steady-Glo Luciferase asssy) of luciferase, incubating for 3 minutes at room temperature, and detecting a fluorescence value;
inhibition was calculated by comparison with read from wells with pseudovirus alone and pseudovirus neutralization activity (indicated by IC 50) was calculated.
Results:
The test results of the pseudo virus neutralization test of the monoclonal neutralizing antibody ZJ-11 are shown in FIG. 2, and the monoclonal neutralizing antibody ZJ-11 can neutralize various SARS-CoV-2 mutant pseudo viruses in a broad spectrum and has better neutralization activity as shown in FIG. 2 (WT,0.0287μg/mL;Beta,0.0931μg/mL;Delta,0.2331μg/mL;Omicron BA.1,0.2984μg/mL;Omicron BA.5,1.7889μg/mL;Omicron XBB,3.1735μg/mL;Omicron EG.5,3.1282μg/mL;Omicron JN.1,4.3129μg/mL).
3. Detection of the neutralizing Activity of human anti-coronavirus SARS-CoV-2 and SARS-CoV neutralizing antibody ZJ-11 against a pseudovirus representative strain of Sha Bei virus subgenera virus (Sarbecovirus) prepared in example 1
Antibody neutralization activity was detected using a representative strain of the sabia virus (Sarbecovirus) SARS-CoV, bat-CoVWIV1, bat-CoVSHC014, bat-CoVRaTG.
The steps are briefly described as follows:
The pCDNA3.1-SARS-CoV-S plasmid, pCDNA3.1-Bat-CoVWIV1-S plasmid and pCDNA3.1-Bat-CoVSHC014-S plasmid and pCDNA3.1-Bat-CoVRaTG-S plasmid were each 20. Mu.g transfected into 293T cells, and 293T cells were infected with VSVDeltaG (with luciferasereporter) for 24 hours, and the cell supernatants were collected for 48 hours to prepare pseudo-viruses corresponding to SARS-CoV, bat-CoVWIV1, bat-CoVSHC014 and Bat-CoVRaTG 13;
Mixing pseudovirus and serial gradient diluted monoclonal neutralizing antibody ZJ-11, incubating at 37 ℃ for 1 hour, adding into digested 293T-ACE2 cells, and culturing at 96 Kong Baiban at 37 ℃ for 24 hours;
After the cell plate is discarded, adding a chromogenic substrate (Steady-GloLuciferaseasssy) of luciferase, incubating for 3 minutes at room temperature, and detecting a fluorescence value;
inhibition was calculated by comparison with read from wells with pseudovirus alone and pseudovirus neutralization activity (indicated by IC 50) was calculated.
Results:
As can be seen from FIG. 3, the neutralizing antibody ZJ-11 can neutralize a wide range of sabia viruses (Sarbecovirus) and has better neutralizing activity (SARS-CoV, 0.0546. Mu.g/mL; bat-CoVWIV1, 0.0235. Mu.g/mL; bat-CoVSHC014, 0.0179. Mu.g/mL; bat-CoVRaTG, 0.0087. Mu.g/mL) as well as the neutralizing activity of the monoclonal neutralizing antibody ZJ-11 against the pseudovirus strain Sha Bei virus (Sarbecovirus) in FIG. 3.
TABLE 1
The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (12)
1. A neutralizing antibody ZJ-11 binding to the epitope of coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD, characterized by comprising a heavy chain and a light chain,
The amino acid sequence of the heavy chain CDR1 of the antibody is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 3;
The amino acid sequence of the light chain CDR1 of the antibody is shown as SEQ ID NO.4, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 6.
2. Neutralizing antibody ZJ-11 binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitopes according to claim 1, wherein said neutralizing antibody ZJ-11 fulfils one or more of the following conditions:
1) The heavy chain variable region of the antibody is shown as SEQ ID NO. 7;
2) The light chain variable region of the antibody is shown as SEQ ID NO. 8.
3. The neutralizing antibody ZJ-11 binding to the RBD epitopes of coronaviruses SARS-CoV-2 and SARS-CoV spike protein as claimed in claim 1, wherein the sequence of the constant region of said antibody is the sequence of the IgG1 constant region, or/and the species of the constant region of said antibody is of human origin.
4. The neutralizing antibody ZJ-11 binding to the RBD epitopes of coronaviruses SARS-CoV-2 and SARS-CoV spike protein as defined in claim 1 wherein the light chain constant region of said antibody is as defined in SEQ ID NO.9 or/and the heavy chain constant region of said antibody is as defined in SEQ ID NO. 10.
5. Use of a neutralizing antibody ZJ-11 binding to coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitopes as claimed in any of claims 1 to 4 for the preparation of a SARS-CoV or SARS-CoV-2 assay product or for the preparation of an anti-SARS-CoV or SARS-CoV-2 medicament.
6. A detection reagent, a detection kit or a medicament, characterized by comprising the neutralizing antibody ZJ-11 binding to the coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope as claimed in any one of claims 1 to 4.
7. A nucleic acid, characterized in that it is a nucleic acid sequence encoding the neutralizing antibody ZJ-11 binding to the coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitopes according to any one of claims 1 to 4.
8. A recombinant expression vector comprising the nucleic acid of claim 7, wherein the recombinant expression vector is an antibody expression vector.
9. The recombinant expression vector of claim 8, wherein the recombinant expression vector is an abvec2.0-IGHG1 and/or an abvec1.1-IGKC expression vector.
10. A host cell comprising the neutralizing antibody ZJ-11 of any one of claims 1 to 4, or the nucleic acid of claim 7, or the recombinant expression vector of claim 8.
11. The host cell of claim 10, wherein the host cell is a Freestyle 293-F cell or an Expi293F cell.
12. A method for producing a neutralizing antibody ZJ-11 binding to the coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope as defined in any one of claims 1 to 4, comprising constructing the host cell as defined in claim 9, culturing, and collecting the neutralizing antibody ZJ-11 binding to the coronavirus SARS-CoV-2 and SARS-CoV spike protein RBD epitope.
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