Monoclonal antibody against orthopoxvirus and application thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses an anti-positive poxvirus monoclonal antibody and application thereof. The anti-positive poxvirus monoclonal antibody provided by the application is 4H4, 12C3 or 11C9. The anti-positive poxvirus monoclonal antibody provided by the application can specifically recognize and target monkeypox, vaccinia and other positive poxviruses, and shows high virus neutralization capacity, and can be used for preparing a medicine for preventing and / or treating positive poxvirus infection and / or a disease related to positive poxvirus infection, or preparing a detection product for detecting positive poxviruses and diagnosing a disease related to positive poxvirus infection.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a monoclonal antibody against orthopoxvirus and its application. Background Technology

[0002] Monkeypox virus (MPXV) is a zoonotic virus belonging to the genus Orthopoxvirus within the family Poxviridae. Monoclonal antibodies against orthopoxviruses have significant applications in the biomedical field, particularly in the prevention and treatment of diseases caused by orthopoxviruses. With the development of biotechnology, monoclonal antibody technology has become an important tool for developing novel treatment methods and diagnostic tools. Monoclonal antibodies can specifically bind to target antigens, serving not only as highly effective therapeutic agents but also in the preparation of test strips, reagent kits, and other products for detecting related diseases. This has greatly promoted the effective prevention and control of various viral diseases.

[0003] Currently, existing solutions for treating diseases caused by orthopoxvirus typically employ either polyclonal antibodies or traditional antiviral drugs. While polyclonal antibodies possess strong immune response capabilities, their diverse antibody compositions make batch-to-batch variability and stability issues difficult to control. Traditional antiviral drugs, although capable of inhibiting viral replication to some extent, have significant side effects and are prone to drug resistance. Therefore, these methods still have certain limitations in practical applications.

[0004] The use of polyclonal antibodies and traditional antiviral drugs in the current technology has failed to provide antibodies against orthopoxvirus with high consistency and specificity. Furthermore, there is still a large gap in the research on the antiviral mechanism of highly efficient neutralizing monoclonal antibodies and bispecific antibodies against monkeypoxvirus. There is an urgent need for a new, highly efficient and stable monoclonal antibody to make up for the shortcomings of traditional approaches. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this application provides a novel, highly efficient, and stable monoclonal antibody against orthopoxvirus and its application.

[0006] In a first aspect, this application provides a monoclonal antibody against poxvirus, employing the following technical solution:

[0007] A monoclonal antibody against orthopoxvirus, said antibody being 4H4, 12C3, or 11C9;

[0008] The heavy chain variable region of the 4H4 antibody contains three complementarity-determining regions: CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.1~3;

[0009] The light chain variable region of the 4H4 antibody contains three complementarity-determining regions: CDR-L1, CDR-L2, and CDR-L3. The amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO. 4~5; the amino acid sequence of CDR-L2 is KVS.

[0010] The heavy chain variable region of the 12C3 antibody contains three complementarity-determining regions: CDR-H4, CDR-H5, and CDR-H6, ​​the amino acid sequences of which are shown in SEQ ID NO. 8~10.

[0011] The light chain variable region of the 12C3 antibody contains three complementarity-determining regions: CDR-L4, CDR-L5, and CDR-L6. The amino acid sequences of CDR-L4 and CDR-L6 are shown in SEQ ID NO. 11-12; the amino acid sequence of CDR-L2 is YTS.

[0012] The heavy chain variable region of the 11C9 antibody contains three complementarity-determining regions: CDR-H7, CDR-H8, and CDR-H9, and the amino acid sequences of CDR-H7, CDR-H8, and CDR-H9 are shown in SEQ ID NO.15~17;

[0013] The light chain variable region of the 11C9 antibody contains three complementarity-determining regions: CDR-L7, CDR-L8, and CDR-L9. The amino acid sequences of CDR-L7 and CDR-L9 are shown in SEQ ID NO.18~19; the amino acid sequence of CDR-L2 is YAS.

[0014] In this application, the monoclonal antibody comprises at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody; the light chain of the antibody is of the κ type.

[0015] In this application, 4H4, 12C3, and 11C9 can specifically target and bind to monkeypox virus IMV form; 4H4 and 11C9 can target the surface protein A29L of monkeypox virus IMV form, and 12C3 can target the surface protein E8L of monkeypox virus IMV form.

[0016] Optionally, the amino acid sequence of the heavy chain variable region of the 4H4 antibody includes:

[0017] (a1) The amino acid sequence shown in SEQ ID NO. 6; or

[0018] (a2) An amino acid sequence of SEQ ID NO. 6 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 6; or

[0019] (a3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.6, and has the same amino acid sequence as the protein shown in SEQ ID NO.6;

[0020] The amino acid sequence of the light chain variable region of the 4H4 antibody includes:

[0021] (a4) The amino acid sequence shown in SEQ ID NO.7; or

[0022] (a5) An amino acid sequence of SEQ ID NO. 7 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 7; or

[0023] (a6) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.7, and has the same amino acid sequence as the protein shown in SEQ ID NO.7;

[0024] The amino acid sequence of the heavy chain variable region of the 12C3 antibody includes:

[0025] (b1) The amino acid sequence shown in SEQ ID NO.13; or

[0026] (b2) An amino acid sequence of SEQ ID NO. 13 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO. 13; or

[0027] (b3) Has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.13, and has the same amino acid sequence as the protein shown in SEQ ID NO.13;

[0028] The amino acid sequence of the light chain variable region of the 12C3 antibody includes:

[0029] (b4) The amino acid sequence shown in SEQ ID NO.14; or

[0030] (b5) An amino acid sequence of SEQ ID NO. 14 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO. 14; or

[0031] (b6) Has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.14, and has the same amino acid sequence as the protein shown in SEQ ID NO.14;

[0032] The amino acid sequence of the heavy chain variable region of the 11C9 antibody includes:

[0033] (c1) The amino acid sequence shown in SEQ ID NO.20; or

[0034] (c2) An amino acid sequence of SEQ ID NO. 20 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 20; or

[0035] (c3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.20, and has the same amino acid sequence as the protein shown in SEQ ID NO.20;

[0036] The amino acid sequence of the light chain variable region of the 11C9 antibody includes:

[0037] (c4) The amino acid sequence shown in SEQ ID NO.21; or

[0038] (c5) An amino acid sequence of SEQ ID NO. 21 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO. 21; or

[0039] (c6) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO.21 and has the same amino acid sequence as the protein shown in SEQ ID NO.21.

[0040] Secondly, this application provides a recombinant protein comprising the aforementioned anti-poxvirus monoclonal antibody and a tag sequence for assisting expression and / or purification.

[0041] In this application, the tag sequence is selected from at least one of His tags, GGGS sequences, and FLAG tags.

[0042] Thirdly, this application provides a biomaterial comprising at least one of d1-d16;

[0043] (d1) A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1 to 2 or the recombinant protein of claim 3;

[0044] (d2) An expression cassette containing the nucleic acid molecule described in (d1);

[0045] (d3) A carrier containing the nucleic acid molecule described in (d1);

[0046] (d4) A carrier containing the expression box described in (d2);

[0047] (d5) A transgenic cell line containing the nucleic acid molecules described in (d1);

[0048] (d6) A transgenic cell line containing the expression cassette described in (d2);

[0049] (d7) A transgenic cell line containing the vector described in (d3);

[0050] (d8) A transgenic cell line containing the vector described in (d4);

[0051] (d9) Microorganisms containing the nucleic acid molecules described in (d1);

[0052] (d10) Microorganisms containing the expression cassette described in (d2);

[0053] (d11) Microorganisms containing the carrier described in (d3);

[0054] (d13) Microorganisms containing the carrier described in (d4);

[0055] (d14) A virus containing the nucleic acid molecule described in (d1);

[0056] (d15) A virus containing the expression cassette described in (d2);

[0057] (d16) A virus containing the vector described in (d3);

[0058] (d17) is a virus containing the vector described in (d4).

[0059] In this application, the transgenic cell line does not contain propagation material.

[0060] Fourthly, this application provides a conjugate comprising the monoclonal antibody or the recombinant protein, and a conjugation portion;

[0061] The coupling portion comprises at least one of the following: a detectable marker, a drug, a toxin, an electron-dense marker, a biotin / avidin protein, a spin marker, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, and a viral capsid protein.

[0062] Optionally, the detectable marker is a fluorescent or luminescent marker.

[0063] Fifthly, the application of the monoclonal antibodies, recombinant proteins, biomaterials, and conjugates provided in this application in the preparation of drugs, reagents, detection plates, kits, or detection chips.

[0064] Optionally, the drug is a drug for the prevention and / or treatment of orthopoxvirus infection and / or diseases associated with orthopoxvirus infection.

[0065] In this application, the orthopoxvirus includes monkeypoxvirus and / or viruses highly homologous to monkeypoxvirus.

[0066] Optionally, the diseases associated with vaccinia virus infection include: skin lesions, pneumonia, corneal infection, sepsis, encephalitis, etc.

[0067] Optionally, the virus with high homology to monkeypox virus includes vaccinia virus; high homology means homology ≥90%.

[0068] Optionally, the drug has at least one of the functions e1-e2:

[0069] e1 for the prevention and / or treatment of orthopoxvirus infection,

[0070] e2 for the prevention and / or treatment of diseases associated with orchiopeptidosis virus;

[0071] The reagent, detection plate, kit, or detection chip has at least one function of f1-f2:

[0072] F1 test for vaccinia virus,

[0073] f2 is used to diagnose diseases associated with orthopoxvirus infection.

[0074] In a sixth aspect, this application provides a pharmaceutical composition comprising the monoclonal antibody, the recombinant protein, the biological material or the conjugate, and a pharmacologically acceptable carrier.

[0075] The pharmaceutical composition has at least one of the functions e1-e2:

[0076] e1 for the prevention and / or treatment of orthopoxvirus infection,

[0077] e2 is used for the prevention and / or treatment of diseases associated with orthopoxvirus.

[0078] In a seventh aspect, this application provides a detection product comprising the monoclonal antibody, the recombinant protein, the biological material, or the conjugate.

[0079] The test product has at least one of the functions f1-f2:

[0080] F1 test for vaccinia virus,

[0081] f2 is used to diagnose diseases associated with orthopoxvirus infection.

[0082] Optionally, the detection product is a reagent, detection plate, detection chip, or kit.

[0083] In summary, this application has the following beneficial effects:

[0084] This application provides a monoclonal antibody against orthopoxvirus, 4H4, 12C3, or 11C9. This anti-orthopoxvirus monoclonal antibody can specifically recognize and target orthopoxviruses such as monkeypox and vaccinia, and exhibits highly efficient viral neutralization capabilities, demonstrating significant clinical application value in the prevention and treatment of orthopoxvirus infection. Therefore, the anti-orthopoxvirus monoclonal antibody provided in this application can be used to prepare drugs for the prevention and / or treatment of orthopoxvirus infection and / or diseases related to orthopoxvirus infection, or to prepare diagnostic products for detecting orthopoxvirus and diagnosing diseases related to orthopoxvirus infection. Attached Figure Description

[0085] Figure 1 The purified monkeypox virus protein E8L ( Figure 1 A) and A29L ( Figure 1 B) SDS-PAGE results;

[0086] Figure 2 SDS-PAGE results of purified monoclonal antibodies 4H4, 12C3, and 11C9;

[0087] Figure 3A represents the ELISA binding activity of monoclonal antibody 4H4 to monkeypox virus protein A29L;

[0088] Figure 3 B represents the ELISA binding activity of monoclonal antibody 11C9 to monkeypox virus protein A29L;

[0089] Figure 3 C represents the ELISA binding activity of monoclonal antibody 12C3 to monkeypox virus protein E8L;

[0090] Figure 3 E represents the binding activity of monoclonal antibodies 4H4 and 11C9 to inactivated vaccinia virus (Tiantan strain);

[0091] Figure 3 F represents the binding activity of monoclonal antibody 12C3 to inactivated vaccinia virus (Tian Tan strain);

[0092] Figure 4 A represents the results of an in vitro neutralization experiment of monoclonal antibodies 4H4 and 11C9 against vaccinia virus (Tiantan strain).

[0093] Figure 4 B represents the results of the in vitro neutralization experiment of monoclonal antibody 12C3 against vaccinia virus (Tian Tan strain);

[0094] Figure 4 C represents the results of in vitro neutralization experiments of monoclonal antibodies 4H4, 11C9, and 12C3 against monkeypox virus. Detailed Implementation

[0095] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0096] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, medical microbiology, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Crystallizable fragments (Fc fragments) correspond to the CH2 and CH3 functional regions of an antibody. They can crystallize and have no antigen-binding activity, but possess functions such as complement fixation and binding to Fc receptors. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into highly denatured regions called complementarity-determining regions (CDRs), interspersed with more conserved framework regions (FRs). The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The term "antibody" is not limited to any specific method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0098] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are commonly obtained using hybridoma techniques first reported by Kohler et al., but can also be obtained using recombinant DNA techniques.

[0099] As used in this article, the term "single-chain antibody" refers to a single-chain variable region fragment, which is a genetically engineered fusion protein. It is a small molecule composed of the variable regions of the antibody heavy chain and the variable regions of the light chain linked by a peptide chain.

[0100] As used in this article, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence of a target pathogen.

[0101] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0102] As used herein, the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.

[0103] As used in this article, the term "homology" refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules. It reflects the relationship between branches that originate from the same ancestor during evolution, exhibiting an essential similarity due to a common evolutionary or ontological origin, but their functions are not necessarily the same.

[0104] As used herein, the term "Escherichia coli expression system" refers to an expression system consisting of Escherichia coli (strain) and a vector, wherein the Escherichia coli (strain) is derived from commercially available strains, such as, but not limited to: GI698, ER2566, BL21(DE3), B834(DE3), BLR(DE3).

[0105] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0106] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0107] As used in this article, the term "vaccinia virus (Tiantan strain)" refers to an attenuated strain obtained from smallpox patients in my country after inoculation and passage in animals such as monkeys, rabbits, and cattle.

[0108] As used in this article, the term "TCID" 50 "50% tissue culture infection dose" refers to the amount of virus required to induce cytopathic effects or death in half of the cells within a culture plate or test tube, and is used to characterize the viral titer.

[0109] As used in this article, the term "ELISA" stands for Enzyme-Linked Immunosorbent Assay, which is an immunoassay method that uses the specific binding of antigens or antibodies to the surface of a solid-phase carrier and the colorimetric reaction catalyzed by enzymes labeled on the antibody or antigen to detect the target analyte.

[0110] As used in this article, the term "IC" 50 "" refers to the half-maximal inhibitory concentration of the antagonist being measured. In this paper, it represents the antibody concentration at which the antibody neutralizes the virus to 50% of its maximum effect in in vitro neutralization experiments.

[0111] To obtain protective neutralizing antibodies, the inventors of this application first used recombinant monkeypox virus proteins A29L (GenBank: QJQ40281.1) and E8L (GenBank: QJQ40248.1) as antigens, and directly immunized BALB / c mice subcutaneously (50ug / mouse) using a sequential immunization strategy, with each immunization occurring two weeks apart. Three days after the initial immunization, mouse spleen cells were fused with mouse myeloma cells using cell fusion technology, and high-titer monoclonal hybridoma cells were screened using recombinant A29L and E8L proteins as antigens. Then, RT-PCR was performed on the selected monoclonal hybridoma cells to obtain the sequence encoding the variable region of the antibody. Furthermore, the monoclonal hybridoma cells were mass-produced in mammalian ascites fluid, and the antibodies were captured using a Protein A / G prefabricated gravity column. A series of functional tests were performed on antibodies 4H4, 11C9, and 12C3. The results showed that antibodies 4H4, 11C9, and 12C3 could specifically bind to orthopoxvirus and neutralize vaccinia virus in vitro.

[0112] The raw materials, reagents, solvents, etc. used in the embodiments of this application can all be obtained commercially. Example 1

[0113] Expression and purification of recombinant laryngopharynx virus proteins A29L and E8L

[0114] Using EcoRI and XhoI enzymes, DNA fragments encoding monkeypox virus proteins A29L (amino acid sequence shown in SEQ ID NO. 22) and E8L (amino acid sequence shown in SEQ ID NO. 23) were ligated into the pET25b vector. The ligation product was transformed into BL21 *E. coli* competent cells, and single colonies were picked and inoculated into 10 mL of LB medium at 37°C for 6–8 hours; then transferred to 2 L of LB medium and incubated at 37°C until OD600 = 0.4–0.6. Subsequently, IPTG was added to the culture to a final concentration of 0.5 mM, and the culture was continued at 16°C for 16–20 hours. After incubation, the bacterial pellet was harvested by high-speed centrifugation. The pellet was washed twice with 1×PBS and then sonicated in an ice-water bath. Afterward, the pellet was discarded by high-speed centrifugation, and the protein in the supernatant was purified by Ni-NTA affinity chromatography. The purity of the target protein was identified by SDS-PAGE, and the results are shown below. Figure 1 As shown.

[0115] Figure 1 The results showed that Example 1 yielded high-purity A29L and E8L proteins with sizes of 11 kDa and 30 kDa, respectively. Example 2

[0116] Preparation of monoclonal antibodies 4H4, 12C3, and 11C9

[0117] Using recombinant monkeypox virus proteins A29L (GenBank: QJQ40281.1) and E8L (GenBank: QJQ40248.1) as antigens, BALB / c mice were directly immunized subcutaneously (50 μg / mouse) using a sequential immunization strategy, with each immunization occurring two weeks apart. Three days after the initial immunization, mouse spleen cells were fused with mouse myeloma cells using cell fusion technology, and high-titer monoclonal hybridoma cells were screened using recombinant A29L or E8L protein coating as antigens. Then, RT-PCR was performed on the selected monoclonal hybridoma cells to obtain the sequences encoding the variable regions of antibodies (see Table 1 below). Furthermore, the monoclonal hybridoma cells were mass-produced in mammalian ascites fluid, and antibodies were captured using a Protein A / G prefabricated gravity column.

[0118] After extensive experimental research, two neutralizing monoclonal antibodies targeting A29L (4H4, 11C9) and one neutralizing monoclonal antibody targeting E8L (12C3) were discovered. Figure 2 The SDS-PAGE results showed that three high-purity monoclonal antibodies were obtained, with heavy and light chain molecular weights of approximately 50 kDa and 25 kDa, respectively.

[0119] Table 1. Sequence information encoding the antibody variable region

[0120]

[0121] Example 3

[0122] Investigating the ability of antibodies 4H4, 12C3, and 11C9 to specifically bind to pea sprout virus (Tiantan strain).

[0123] (a) Detection of the binding ability between monoclonal antibodies and antigen proteins using ELISA:

[0124] Monkeypox protein A29L and E8L were coated onto ELISA plates at a concentration of 1 μg / ml; subsequently, 96-well plates were blocked with 5% skim milk. The monoclonal antibodies 4H4, 12C3, and 11C9 to be tested were serially diluted and added to each well of the ELISA plate at a volume of 100 μL. An irrelevant IgG control was used. The plates were incubated at 37°C for 30 minutes. The plates were washed three times with ELISA wash buffer (PBST), followed by the addition of 100 μL of diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody, and incubated at 37°C for 30 minutes. After washing the plates three times with PBST, the chromogenic reagent was added, and the reaction was terminated after 15 minutes of development. The absorbance at 450 nm was then read on a microplate reader. The results are shown below. Figure 3 As shown in A~3C.

[0125] Depend on Figure 3 As shown in A~3C, antibodies 4H4 and 11C9 can specifically target and bind to monkeypox virus protein A29L, and antibody 12C3 can specifically target and bind to monkeypox virus protein E8L, while the PBS control does not show specific binding activity.

[0126] (ii) Detection of the binding affinity between monoclonal antibodies and vaccinia virus (Tian Tan strain) using ELISA:

[0127] BHK-21 cells infected with vaccinia virus (Tian Tan strain) for 48 h were degassed, and the supernatant was removed. An appropriate amount of 1×PBS was added, and the cells were subjected to three freeze-thaw cycles at -80°C. The precipitate was discarded by high-speed centrifugation, and the supernatant virus solution was obtained. The virus solution was inactivated at 56°C for 30 min and then stored at -80°C for long-term preservation. The inactivated virus solution prepared above was diluted 20-fold and coated onto an ELISA plate. Subsequent procedures were the same as before. Results are as follows. Figure 3 As shown in E~3F.

[0128] Depend on Figure 3 From E to 3F, it can be seen that antibodies 4H4, 11C9, and 12C3 can specifically target and bind to vaccinia virus, among which 12C3 has the strongest ability to bind to inactivated virus.

[0129] Example 4

[0130] Evaluation of the in vitro neutralization capabilities of monoclonal antibodies 4H4, 12C3, and 11C9 against vaccinia virus and laryngophobia virus

[0131] (a) Neutralization test of vaccinia virus (Tiantan strain):

[0132] The neutralizing activity of the antibody was determined using a micro-neutralization assay (MRNT). Monoclonal antibodies were serially diluted to 4H4, 11C9, and 12C3, and then incubated with an equal volume of 100 TCID50 of virus at 37°C for 1 hour. After incubation, the virus was added to 96-well plates pre-inoculated with BHK-21 cells and incubated at 37°C in a 5% CO2 incubator for 1 hour to allow adsorption. The supernatant was then removed. After washing, maintenance medium (containing 2% fetal bovine serum) was added, and the plates were cultured for another 4 days. Cytopathic effect (CPE) was observed after 4 days, and the neutralizing titer (half-inhibitory concentration, IC50) of the antibody was calculated. 50 ), the result is as follows Figure 4 As shown in A and 4B.

[0133] From 4A and 4B, we know that 4H4, 11C9, and 12C3 have an IC50 value against vaccinia virus (Tiantan strain). 50 The concentrations were 7.78 μg / mL, 29.26 μg / mL, and 13.80 μg / mL, respectively, indicating that monoclonal antibodies 4H4, 11C9, and 12C3 have different levels of neutralizing ability against vaccinia virus, with 4H4 exhibiting the strongest neutralizing activity, followed by 12C3.

[0134] (ii) Neutralization test of monkeypox virus:

[0135] The focal reduction neutralization assay (FRNT) performed in a biosafety level 3 laboratory determined the neutralizing activity of the antibody. The antibody was serially diluted and incubated with an equal volume of monkeypox virus at 37°C for 1 hour. The mixture (containing 200 infectious units of live virus) was then transferred to 96-well plates pre-inoculated with Vero E6 cells and incubated at 37°C, 5% CO2 for 1 hour before removal. After washing, maintenance medium (containing 1.6% carboxymethyl cellulose and 2% fetal bovine serum) was added, and the plates were incubated at 37°C, 5% CO2 for 24 hours. After 24 hours, the supernatant was removed, and cells were fixed with 4% paraformaldehyde, infiltrated with 0.1% Triton X-100, and then incubated with the corresponding HRP-bound monoclonal antibody. Reaction development was performed using KPL TrueBlue peroxidase substrate. The number of monkeypox virus infection focals was calculated using ELISpot, and the results are shown below. Figure 4 As shown in C.

[0136] Depend on Figure 4 As shown in C, the IC50 values ​​of 4H4, 11C9, and 12C3 against monkeypox virus are 8.19 μg / mL, 22.81 μg / mL, and 13.86 μg / mL, respectively, indicating that the monoclonal antibodies 4H4, 11C9, and 12C3 have different degrees of neutralizing ability against monkeypox virus, with 4H4 exhibiting the strongest neutralizing activity, followed by 12C3.

[0137] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A monoclonal antibody against orthopoxvirus, characterized in that, The antibody is 4H4; The heavy chain variable region of the 4H4 antibody contains three complementarity-determining regions: CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.1~3, respectively. The light chain variable region of the 4H4 antibody contains three complementarity-determining regions: CDR-L1, CDR-L2, and CDR-L3. The amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO. 4~5, respectively. The amino acid sequence of CDR-L2 is KVS. The 4H4 antibody can bind to monkeypox virus protein A29L and vaccinia virus.

2. The monoclonal antibody against orthopoxvirus according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the 4H4 antibody includes: (a1) The amino acid sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain variable region of the 4H4 antibody includes: (a4) The amino acid sequence shown in SEQ ID NO.

7.

3. A recombinant protein, characterized in that, It consists of a monoclonal antibody against orthopoxvirus as described in claim 1 or 2 and a tag sequence that assists in expression and / or purification.

4. A biomaterial, characterized in that, The biomaterial is at least one of d1-d16; (d1) Encoding the monoclonal antibody nucleic acid molecule according to any one of claims 1 to 2; (d2) An expression cassette containing the nucleic acid molecule described in (d1); (d3) A carrier containing the nucleic acid molecule described in (d1); (d4) A carrier containing the expression box described in (d2); (d5) A transgenic cell line containing the nucleic acid molecules described in (d1); (d6) A transgenic cell line containing the expression cassette described in (d2); (d7) A transgenic cell line containing the vector described in (d3); (d8) A transgenic cell line containing the vector described in (d4); (d9) Microorganisms containing the nucleic acid molecules described in (d1); (d10) Microorganisms containing the expression cassette described in (d2); (d11) Microorganisms containing the carrier described in (d3); (d13) Microorganisms containing the carrier described in (d4); (d14) A virus containing the nucleic acid molecule described in (d1); (d15) A virus containing the expression cassette described in (d2); (d16) A virus containing the vector described in (d3); (d17) is a virus containing the vector described in (d4).

5. A coupling agent, characterized in that, It consists of the monoclonal antibody as described in claim 1 or 2 or the recombinant protein as described in claim 3, and the conjugated portion; The coupling portion is at least one of the following: a detectable marker, biotin / antibiotin, a protein radionuclide, and gold nanoparticles / nanorobars.

6. The use of the monoclonal antibody as described in claim 1 or 2, or the biomaterial as described in claim 4, in the preparation of drugs for the treatment and / or prevention of diseases caused by monkeypox virus infection, or in reagents, detection plates, kits, or detection chips for the detection and diagnosis of corresponding antigen proteins, viruses, or diseases.

7. The application as described in claim 6, characterized in that, The drug has the function of e1: e1 for the prevention and / or treatment of monkeypox virus infection; The reagent, detection plate, kit, or detection chip has the function of f1: f1 detects monkeypox virus.

8. A pharmaceutical composition, characterized in that, It comprises the monoclonal antibody as described in claim 1 or 2, the biological material as described in claim 4, and a pharmacologically acceptable carrier; The pharmaceutical composition has the function of e1: e1 is for the prevention and / or treatment of monkeypox virus infection.

9. A testing product, characterized in that, It includes the monoclonal antibody as described in claim 1 or 2 and the biological material as described in claim 4; The testing product has function f1: f1 detects monkeypox virus.

10. The testing product as described in claim 9, characterized in that, The testing products are reagents, testing plates, testing chips, or reagent kits.

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