Anti-monkeypox virus antibody or its functional fragment, reagent and kit for detecting monkeypox virus

Abstract

The present invention discloses an anti-monkeypox virus antibody or a functional fragment thereof, a reagent and a kit for detecting monkeypox virus, and relates to the field of antibodies. The anti-monkeypox virus antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region, and the antibody provides an important source of raw materials for the detection of monkeypox virus and has improved affinity or activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to anti-monkeypox virus antibodies or functional fragments thereof, and reagents and kits for detecting monkeypox virus. Background Art

[0002] Monkeypox is caused by Monkeypox virus (MPXV). Monkeypox virus is a double-stranded DNA virus belonging to the genus Orthopoxvirus of the Poxviridae family. Monkeypox is a viral zoonosis that occurs mainly in the tropical rainforests of Central and West Africa and is occasionally exported to other regions. Current studies have found that the virus invades the human body through mucous membranes and broken skin. People are mainly infected through contact with lesions, blood, and other body fluids of infected animals, or through bites and scratches from infected animals. It is mainly transmitted from person to person through close contact, but can also be transmitted through droplets, contact with objects contaminated with the virus, and vertical transmission through the placenta. Sexual transmission cannot be ruled out.

[0003] In terms of clinical symptoms, the symptoms of monkeypox infection are similar to those of smallpox, but the clinical symptoms of monkeypox are milder, with an incubation period of 5-12 days, mostly 6-13 days. The initial symptoms of the patient are fever, headache, swollen lymph nodes, muscle aches, severe fatigue, and then a rash on the face and body. Monkeypox is a self-limiting disease, and patients usually recover on their own within two to three weeks; but for children, pregnant women, or people who are immunosuppressed due to other health conditions, monkeypox may cause other secondary infections, such as pneumonia, sepsis, and encephalitis, which are serious diseases.

[0004] At present, the detection methods of monkeypox virus are mainly PCR amplification and immunoassay. PCR amplification is the main detection method for monkeypox virus at home and abroad, but it has high requirements for instruments, equipment, detection sites and environmental conditions, and has disadvantages such as long detection time and low throughput, which is not convenient for large-scale detection of the population. The immunoassay based on colloidal gold has the advantages of being fast, convenient and easy to carry, so it has become a hot spot for research and development, and the detection performance of colloidal gold depends on the performance of antibodies against monkeypox virus.

[0005] Therefore, there is a strong need in the art for antibodies that effectively bind to and detect monkeypox virus.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide anti-monkeypox virus antibodies or functional fragments thereof, reagents and kits for detecting monkeypox virus or monkeypox virus antigens.

[0008] The present invention is achieved in that:

[0009] In the first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprises or is an amino acid sequence consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO:7; and the LCDR1, LCDR2, LCDR3 comprises or is an amino acid sequence consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO:8. .

[0010] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR1 to HCDR3 described in any of the above embodiments and the light chain variable region comprises the LCDR1 to LCDR3 described in any of the above embodiments; the heavy chain variable region is as shown in an amino acid sequence having at least 80% identity with SEQ ID NO:7 and / or the light chain variable region is as shown in an amino acid sequence having at least 80% identity with SEQ ID NO:8.

[0011] In a third aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is shown in SEQ ID NO:7, and the light chain variable region is shown in SEQ ID NO:8.

[0012] In a fourth aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:11; and the amino acid sequence of the light chain is shown in SEQ ID NO:12.

[0013] In a fifth aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or its functional fragment binds to the same epitope in the amino acid sequence of the monkeypox virus antigen protein as the antibody or its functional fragment described in any of the above embodiments; or, the antibody or its functional fragment competes with the antibody or its functional fragment described in any of the above embodiments for binding to the monkeypox virus antigen.

[0014] In a sixth aspect, an embodiment of the present invention provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiments.

[0015] In a seventh aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or its functional fragment as described in the preceding embodiments or the antibody conjugate as described in the preceding embodiments.

[0016] In an eighth aspect, an embodiment of the present invention provides a method for detecting monkeypox virus or monkeypox virus antigen, which comprises: contacting the antibody or its functional fragment, antibody conjugate or reagent or kit as described in the above embodiments with the monkeypox virus or monkeypox virus antigen in the sample to be tested to form an immune complex.

[0017] In a ninth aspect, an embodiment of the present invention provides a nucleic acid, a vector, and a cell.

[0018] In a tenth aspect, an embodiment of the present invention provides a method for preparing the antibody or a functional fragment thereof described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0019] In the eleventh aspect, the embodiments of the present invention provide the use of the antibodies or functional fragments thereof, antibody conjugates, reagents or kits as described in the preceding embodiments in detecting monkeypox virus or monkeypox virus antigens or in preparing products for detecting monkeypox virus or monkeypox virus antigens.

[0020] The present invention has the following beneficial effects:

[0021] The anti-monkeypox virus antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of monkeypox virus or monkeypox virus antigen and has improved affinity or activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The figure shows the reducing SDS-PAGE result of Anti-MPXV 2F19. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0026] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0027] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0028] In the present invention, the heavy chain complementary determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3.

[0029] In the first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprise or are amino acid sequences consistent with HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO:7; and the LCDR1, LCDR2, LCDR3 comprise or are amino acid sequences consistent with LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO:8.

[0030] The definition methods of CDRs are well known in the art. Common ones include Kabat, Chothia, AbM, Contact or IMGT. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. There are other CDRs definition methods that may not strictly follow one of the above methods, but will still overlap with at least a portion of the CDRs defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups, such as shortening the C-termini of HCDR3 and LCDR3 defined by Kabat by 2 amino acids each. As described herein, CDRs may refer to CDRs defined by any method known in the art, including a combination of methods.

[0031] In alternative embodiments, the CDRs are defined by the Kabat, Chothia, AbM, Contact or IMGT numbering systems.

[0032] In an alternative embodiment, the CDRs are based on the CDRs defined by the Kabat numbering system, with the C-termini of HCDR3 and LCDR3 each being shortened by 2 amino acids.

[0033] In an optional embodiment, the amino acid sequences of HCDR1, HCDR2 and HCDR3 include SEQ ID NOs: 1 to 3 in sequence or are shown in sequence as SEQ ID NOs: 1 to 3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 include SEQ ID NOs: 4 to 6 in sequence or are shown in sequence as SEQ ID NOs: 4 to 6.

[0034] In an alternative embodiment, the antibody or a functional fragment thereof further comprises a framework region.

[0035] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region (HFR) can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region (LFR) can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0036] The heavy chain variable region is obtained by connecting HCDR and HFR in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting LCDR and LFR in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In the present invention, "-" represents a peptide bond. The combined fragments are arranged in sequence from upstream (N-terminus) to downstream (C-terminus).

[0037] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR1 to HCDR3 described in any of the above embodiments and the light chain variable region comprises the LCDR1 to LCDR3 described in any of the above embodiments; the heavy chain variable region is as shown in an amino acid sequence having at least 80% identity with SEQ ID NO:7 and / or the light chain variable region is as shown in an amino acid sequence having at least 80% identity with SEQ ID NO:8.

[0038] In an alternative embodiment, the heavy chain variable region is as shown in an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7; and the light chain variable region is as shown in an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0039] On the other hand, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is shown in SEQ ID NO:7; and the light chain variable region is shown in SEQ ID NO:8.

[0040] In an alternative embodiment, the antibody or a functional fragment thereof further comprises a constant region.

[0041] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0042] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0043] In an optional embodiment, the species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

[0044] In an alternative embodiment, the heavy chain constant region is as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto.

[0045] In an alternative embodiment, the light chain constant region is as shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

[0046] In alternative embodiments, the "having at least 80% identity" can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0047] On the other hand, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain is shown in SEQ ID NO:11, and the light chain is shown in SEQ ID NO:12.

[0048] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof binds to the same epitope in the amino acid sequence of the monkeypox virus antigen protein as the antibody or the functional fragment thereof described in any of the above embodiments; or, the antibody or the functional fragment thereof competitively binds to the monkeypox virus antigen with the antibody or the functional fragment thereof described in any of the above embodiments.

[0049] In an optional embodiment, the antibody or its functional fragment has a KD≤6.34×10 -8 Affinity binding of M to monkeypox virus antigens.

[0050] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -08 M, KD≤10 -09 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 Affinity binding of M to monkeypox virus antigens.

[0051] In an optional embodiment, the antibody or its functional fragment has a KD≤1.14×10 -11 Affinity binding of M to monkeypox virus antigens.

[0052] There are many methods for determining antibody affinity (KD), which can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).

[0053] In an alternative embodiment, KD is determined using a kinetic assay; preferably, a surface plasmon resonance assay, such as by using a surface plasmon resonance assay such as Biosensor system of the system.

[0054] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0055] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0056] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0057] On the other hand, an embodiment of the present invention further provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiment.

[0058] In an optional embodiment, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.

[0059] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0060] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0061] In an optional embodiment, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

[0062] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0063] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0064] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0065] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0066] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0067] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0068] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0069] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0070] In an optional embodiment, the colloidal metal is colloidal gold.

[0071] In an optional embodiment, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or its functional fragment. In the antibody conjugate, the antibody is coupled to the solid phase carrier.

[0072] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0073] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0074] In an optional embodiment, the solid phase carrier is a nitrocellulose membrane.

[0075] On the other hand, an embodiment of the present invention further provides a reagent or a kit, which includes the antibody or its functional fragment as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments.

[0076] In an optional embodiment, the reagent or kit further comprises a second antibody, which binds to the antibody or a functional fragment thereof;

[0077] In an optional embodiment, the reagent or kit further comprises a second antibody that binds to the monkeypox virus or a monkeypox virus antigen.

[0078] In an alternative embodiment, the reagent or kit has improved detection sensitivity or specificity.

[0079] On the other hand, an embodiment of the present invention further provides a method for detecting monkeypox virus or monkeypox virus antigen, comprising:

[0080] The antibody or functional fragment thereof as described in any of the foregoing embodiments, the antibody conjugate as described in any of the foregoing embodiments, or the reagent or kit as described in any of the foregoing embodiments is contacted with the monkeypox virus or monkeypox virus antigen in the sample to be detected to form an immune complex.

[0081] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0082] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the monkeypox virus or a monkeypox virus antigen.

[0083] On the other hand, an embodiment of the present invention also provides the use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in detecting monkeypox virus or monkeypox virus antigen or preparing a product for detecting monkeypox virus or monkeypox virus antigen.

[0084] On the other hand, an embodiment of the present invention also provides the use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in the preparation of a product having at least one of the following uses, wherein the uses include: diagnosing or assisting in diagnosing diseases related to monkeypox virus infection, and predicting or assisting in predicting at least one of the prognosis and efficacy of diseases related to monkeypox virus infection.

[0085] In an optional embodiment, the related diseases caused by the monkeypox virus infection include at least one of fever, headache, swollen lymph nodes, muscle aches, severe fatigue, pneumonia, sepsis and encephalitis.

[0086] In an alternative embodiment, the product comprises a reagent or a kit.

[0087] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in any of the aforementioned embodiments.

[0088] On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the above embodiments.

[0089] On the other hand, an embodiment of the present invention further provides a cell, which contains the isolated nucleic acid described in any of the foregoing embodiments or the vector described in any of the foregoing embodiments.

[0090] On the other hand, an embodiment of the present invention further provides a method for preparing the antibody or a functional fragment thereof as described in any of the foregoing embodiments, comprising: culturing the cell as described in any of the foregoing embodiments.

[0091] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0092] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0094] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0095] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0096] Example 1 Preparation of Anti-MPXV 2F19 Monoclonal Antibody

[0097] 1. Expression plasmid construction

[0098] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by a gene sequencing company.

[0099] 1.1 Anti-MPXV 2F19 antibody gene preparation

[0100] mRNA was extracted from the hybridoma cell line secreting Anti-MPXV 2F19 monoclonal antibody, and the DNA product was obtained by RT-PCR. It was inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, 4 positive clones of the Heavy Chain and Light Chain genes were taken and sent to a gene sequencing company for sequencing.

[0101] 1.2 Sequence analysis of the variable region gene of Anti-MPXV 2F19 antibody

[0102] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. In the gene fragment amplified by the Light Chain, the VL gene sequence was 339 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 360 bp, belonging to the VH1 gene family, preceded by a 57 bp leader peptide sequence.

[0103] 1.3 Construction of recombinant antibody expression plasmid

[0104] pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction sites and is subsequently referred to as 3.4A expression vector; based on the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the Anti-MPXV 2F19 antibody were designed, with restriction endonuclease cleavage sites and protective bases at both ends, respectively, and the 0.74KB Light Chain gene fragment and the 1.42kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0105] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

[0106] 2. Recombinant Antibody Sample Preparation

[0107] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of cells after transfection, and place them in a 35℃ constant temperature incubator for culture, speed 120rmp, CO2 content 8%, and centrifuge after 13 days. The supernatant of centrifugation was affinity purified using protein A affinity chromatography column. Take 6μg of purified antibody for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0108] 3. Affinity and activity optimization

[0109] Although the Anti-MPXV 2F19 monoclonal antibody prepared above has the ability to bind to monkeypox virus antigens, its affinity and antibody activity are not ideal, so the applicant performs directed mutation on the variable region of the antibody. That is, the computer is used to simulate the structure of the antibody variable region, the structure of the antigen-antibody variable region complex, the key amino acid analysis of the antibody and the mutation design, and the bidirectional primers covering the mutation site are designed and synthesized according to the mutation scheme, the primers at both ends of the target DNA are synthesized, and a high-fidelity PCR reaction is performed. The PCR product is cloned into a vector, and then the mutant antibody is prepared according to the method of step 2 above. After screening, a monoclonal antibody with significantly improved affinity and antibody activity was obtained, and it was named Anti-MPXV2F19RMb1, and its heavy chain and light chain amino acid sequences are as follows.

[0110] Table 1 Antibody sequences

[0111] Sample name Heavy chain number Light chain number Anti-MPXV 2F19RMb1 SEQ ID NO:11 SEQ ID NO:12

[0112] Example 2 Affinity Analysis

[0113] The purified antibody was diluted in advance, and the monkeypox recombinant antigen (purchased from Feipeng Bio) was diluted in gradients at the same time; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using a CM5 chip that had been pre-coupled with goat anti-mouse IgG, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents the binding rate; and kd represents the dissociation rate).

[0114] Table 2 Affinity analysis data

[0115] Sample name KD(M) ka kd Control Antibodies 6.34E-08 1.26E+06 7.99E-02 Anti-MPXV 2F19RMb1 1.14E-11 2.11E+06 2.40E-05

[0116] Example 3 Activity Identification

[0117] The coating solution (main component NaHCO3) was diluted with recombinant monkeypox recombinant antigen (purchased from Feipeng Bio) to 1ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+Nacl), and patted dry; blocking solution (20% BSA+80% PBS) was added, 120uL per well, incubated at 37°C for 1h, and patted dry; the diluted purified antibody and control antibody were added, 100uL / well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution, and patted dry; goat anti-mouse IgG-HRP was added, 100uL per well, incubated at 37°C for 30min; the cells were washed 5 times with washing solution, and patted dry; color developing solution A (50uL / well) and color developing solution B (50uL / well) were added, and the cells were incubated for 10min; the stop solution was added, 50uL / well; the OD value was read at 450nm (reference 630nm) on the microplate reader.

[0118] Table 3 Activity data

[0119] Concentration (ng / ml) 15.63 7.81 3.91 1.95 0.98 0.00 Control Antibodies 1.123 0.701 0.412 0.256 0.151 0.022 Anti-MPXV 2F19RMb1 1.921 1.254 0.763 0.447 0.270 0.021

[0120] Example 4 Antibody Stability Assessment

[0121] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the expressed antibodies were stable. The following table shows the OD results of the enzyme immunoassay activity test after 21 days of testing.

[0122] Table 4 Stability data

[0123] Sample concentration (ng / ml) 7.81 3.91 0.00 4℃, 21-day samples 1.267 0.766 0.019 -80℃, 21 days sample 1.229 0.738 0.018 37℃, 21 days sample 1.234 0.741 0.017

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0125] The amino acid sequence involved in this application is as follows:

[0126]

Claims

1. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 7; The LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:8; The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by the Kabat, Chothia, AbM, Contact or IMGT numbering systems.

2. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1 to 3, respectively; the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 4 to 6, respectively.

3. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region comprises the HCDR1, HCDR2, HCDR3 described in any one of claims 1 to 2, and the light chain variable region comprises the LCDR1, LCDR2, LCDR3 described in any one of claims 1 to 2; the heavy chain variable region is shown as an amino acid sequence that is at least 80% identical to SEQ ID NO:7, and the light chain variable region is shown as an amino acid sequence that is at least 80% identical to SEQ ID NO:

8.

4. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region is shown in SEQ ID NO:7; the light chain variable region is shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

7. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The constant region includes a heavy chain constant region and / or a light chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region is shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto.

10. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The light chain constant region is shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

11. An anti-monkeypox virus antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is shown in SEQ ID NO:

12.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 11, characterized in that: The antibody or antigen-binding fragment thereof has a KD of ≤6.34×10 -8 Affinity binding of M to monkeypox virus antigens.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 11, wherein the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

14. An antibody conjugate, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. The antibody conjugate according to claim 14, characterized in that: The antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

16. The antibody conjugate according to claim 14, characterized in that: The antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

17. The antibody conjugate according to claim 14, characterized in that: The antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

18. The antibody conjugate according to claim 17, characterized in that The marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

19. The antibody conjugate according to claim 17, characterized in that: The label is colloidal gold.

20. A reagent or a kit, characterized in that: It includes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the antibody conjugate according to any one of claims 14 to 19.

21. The reagent or kit according to claim 20, characterized in that The reagent or kit also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

22. The reagent or kit according to claim 20, characterized in that The reagent or kit also includes a second antibody that binds to the monkeypox virus or a monkeypox virus antigen.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the antibody conjugate according to any one of claims 14 to 19, or the reagent or kit according to any one of claims 20 to 22 in the preparation of a product for detecting monkeypox virus or monkeypox virus antigen, characterized in that: It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the antibody conjugate according to any one of claims 14 to 19, or the reagent or kit according to any one of claims 20 to 22 is contacted with the monkeypox virus or monkeypox virus antigen in the sample to be detected to form an immune complex.

24. The use according to claim 23, characterized in that The immune complex also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

25. The use according to claim 23, characterized in that The immune complex also includes a second antibody that binds to the monkeypox virus or a monkeypox virus antigen.

26. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

27. A carrier, characterized in that It contains the nucleic acid according to claim 26.

28. A cell, characterized in that It contains the nucleic acid according to claim 26 or the vector according to claim 27.

29. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, characterized in that: It includes: Cultivate the cell of claim 28.

30. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the antibody conjugate according to any one of claims 14 to 19, or the reagent or kit according to any one of claims 20 to 22 in the preparation of a product for detecting monkeypox virus or monkeypox virus antigen, or in detecting monkeypox virus or monkeypox virus antigen for non-disease diagnosis purposes.

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