Monkeys pox virus a29l antibodies and uses thereof

Abstract

The application discloses a monkeypox virus A29L antibody and application thereof, and the A29L antibody or an antigen binding fragment thereof can be combined with the monkeypox virus A29L protein with high activity and specificity. The application also relates to a polynucleotide molecule, a vector and a host cell coding the A29L antibody or the antigen binding fragment thereof, and an antibody derivative or other products derived from the A29L antibody or the antigen binding fragment thereof according to the application.

Description

Technical Field

[0001] This invention belongs to the fields of cell biotechnology and immunology, and relates to monkeypox virus A29L antibody and its application. Background Technology

[0002] Monkeypox is a zoonotic disease caused by monkeypoxvirus (MPXV). Monkeypoxvirus is a double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. Its proteins are highly similar to those of other orthopoxviruses such as vacciniavirus (VACV) and variolavirus (VARV). Monkeypoxvirus has two forms of infectious viral particles: extracelular enveloped virus (EEV) particles and intracellular mature virus (IMV) particles. For MPXV, important neutralizing antibody targets identified include A35R and B6R for EEV, and M1R, A29L, E8L, and H3L for IMV.

[0003] Clinically, monkeypox presents with symptoms similar to smallpox, but its symptoms are milder. The incubation period is 5-12 days, mostly 6-13 days. Early symptoms include fever, headache, swollen lymph nodes, muscle aches, and severe fatigue, followed by a rash on the face and body. Monkeypox is a self-limiting disease, and patients usually recover spontaneously within two to three weeks. However, in children, pregnant women, or immunocompromised individuals due to other health conditions, monkeypox can lead to secondary infections such as pneumonia, sepsis, and encephalitis, which can be serious illnesses.

[0004] Because the clinical symptoms caused by monkeypox virus are difficult to distinguish from other poxviruses, diagnosing monkeypox based on clinical manifestations is very challenging and relies heavily on laboratory testing. The WHO recommends PCR detection of viral DNA as the preferred laboratory method for MPXV. However, existing PCR methods still have some limitations. Immunoassays, compared to traditional methods, offer significant advantages in accuracy and timeliness. The performance of immunoassays depends on the effectiveness of the antibodies against monkeypox virus.

[0005] Studies have shown that although the smallpox vaccine provides some protection against monkeypox, with the eradication of smallpox and the cessation of vaccination, most people (born after 1980) lack immunity to monkeypox virus, smallpox virus, and other orthopox viruses. Currently, although chemical drugs such as Tecovirimat and Brincidofovir have been approved for the treatment of smallpox, and vaccinia immunoglobulin (VIG) has been approved for complications caused by smallpox vaccination and can also be used for orthopox infections such as monkeypox, research on their efficacy against monkeypox remains limited. Currently, there are no effective drugs specifically targeting monkeypox virus for the treatment of the disease.

[0006] Therefore, there is a strong need in the field for antibodies that can effectively bind to and detect monkeypox virus, or antibodies that can treat diseases or symptoms caused by monkeypox virus infection or inhibit monkeypox virus activity. Summary of the Invention

[0007] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0008] The first aspect of the present invention provides an A29L antibody or an antigen-binding fragment thereof, wherein the A29L antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3.

[0009] Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as shown in SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are as shown in SEQ ID NO.4, 5 and 6 respectively.

[0010] In some embodiments, the heavy chain variable region of the A29L antibody or its antigen-binding fragment contains an amino acid sequence that is at least 90% identical to SEQ ID NO. 7, and the light chain variable region contains an amino acid sequence that is at least 90% identical to SEQ ID NO. 8.

[0011] In one specific embodiment, the heavy chain variable region of the A29L antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8.

[0012] In this invention, the A29L antibody or its antigen-binding fragment may be a monoclonal antibody, a domain antibody, a single-chain (scFv), a Fab fragment, an F(ab')2 fragment, a multispecific antibody, a single-domain heavy chain antibody, or a single-domain light chain antibody. In some embodiments, such antibodies or their antigen-binding fragments that bind to the monkeypox virus A29L protein are mouse, other rodent, chimeric, humanized, or fully human monoclonal antibodies.

[0013] The "complementarity-determining region" (CDR) or "hypervariant region" is a region in the antibody's variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0014] Based on the variable region amino acid sequence contained in the given A29L antibody or its antigen-binding fragment according to this invention, those skilled in the art can routinely determine the CDR contained therein. For example, the Kabat, AbM, Chothia, or Contact protocols can be used to define the CDR in the variable region amino acid sequence.

[0015] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0016] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0017] In some embodiments, functional variants of the A29L antibody or its antigen-binding fragment described herein are also included within the scope of protection of this invention. A “functional variant” refers to a protein having significant or marked sequence identity or similarity to the parent antibody, and retaining the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the A29L antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the parent antibody in terms of amino acid sequence.

[0018] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0019] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions can be: an acidic / negatively charged polar amino acid replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu); an amino acid with a nonpolar side chain replacing another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); an uncharged amino acid with a polar side chain replacing another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); an amino acid with a β-branched side chain replacing another amino acid with a β-branched side chain (e.g., He, Thr, and Val); and an amino acid with an aromatic side chain replacing another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0020] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 90% or more homology to the nucleotide sequence corresponding to the A29L antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned A29L antibody or its antigen-binding fragment, are all derived from and equivalent to the nucleotide sequence of this invention, and are also included within the scope of protection of this invention.

[0021] A second aspect of the present invention provides a polynucleotide molecule or a carrier comprising the polynucleotide molecule, said polynucleotide molecule encoding the A29L antibody or an antigen-binding fragment thereof described in the first aspect of the present invention.

[0022] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0023] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0024] In some implementations, the vector is introduced into the host cell by means including but not limited to physical methods, chemical methods, and biological methods.

[0025] In some implementations, the physical methods include, but are not limited to, calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation.

[0026] In some implementations, the chemical method includes, but is not limited to, colloidal dispersion systems and lipid-based systems.

[0027] In some implementations, the biological method includes, but is not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0028] In some embodiments, examples of vectors that can be used in this invention include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0029] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papillomavirus vectors.

[0030] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0031] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0032] A third aspect of the present invention provides a modified host cell or a population of host cells comprising the same, said modified host cell comprising the polynucleotide molecule or a carrier comprising the same as described in the second aspect of the present invention, the A29L antibody or an antigen-binding fragment thereof described in the first aspect of the present invention.

[0033] In some implementations, the host cell population may also include host cells other than the modified host cells.

[0034] In some implementations, the modified host cells include prokaryotic cells and eukaryotic cells.

[0035] In some implementations, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0036] In some implementations, the bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0037] In some implementations, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0038] The fourth aspect of the present invention provides an antibody derivative of an A29L antibody or an antigen-binding fragment thereof, the antibody derivative comprising a complex formed by direct or indirect coupling of the A29L antibody or an antigen-binding fragment thereof to a detectable marker as described in the first aspect of the present invention.

[0039] In this invention, the term "detectable marker" refers to a reagent that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, fluorescent dyes, enzymes, chemiluminescent markers, radioactive isotopes, electron-dense reagents, colored particles, biotin, or dioxigenin. Detectable markers tend to produce measurable signals, such as radioactivity, fluorescence, color, or enzyme activity. Examples of suitable fluorescent dyes include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride, and phycoerythrin; examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; chemiluminescent markers include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, acridine esters and their derivatives, adamantane, rare earth elements, and ruthenium bipyridine complexes; radioactive isotopes include, but are not limited to, those that ... 67 Ga、 68 Ga、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 13 N、 15 O、 186 Re、 110 In、 111 In.

[0040] Antibodies conjugated to detectable markers can be used for diagnostic or therapeutic purposes. Detectable markers can be directly attached to or conjugated to antibodies, or indirectly via intermediates such as linkers known in the art, using techniques known in the art.

[0041] The fifth aspect of the present invention provides a detection reagent comprising the A29L antibody or its antigen-binding fragment as described in the first aspect of the present invention, or an antibody derivative of the A29L antibody or its antigen-binding fragment as described in the fourth aspect of the present invention.

[0042] The sixth aspect of the present invention provides a conjugate of an A29L antibody or its antigen-binding fragment thereof, the conjugate comprising a conjugate formed by conjugating the A29L antibody or its antigen-binding fragment as described in the first aspect of the present invention with a therapeutic agent.

[0043] In some embodiments, the therapeutic agent includes, but is not limited to, cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0044] The seventh aspect of the present invention provides a pharmaceutical composition for treating diseases or symptoms caused by monkeypox virus infection or for inhibiting monkeypox virus activity, said pharmaceutical composition comprising the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or a conjugate of the A29L antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention.

[0045] In this invention, the term "treatment" refers to the improvement, prevention, relief, or reversal of a disease or condition or at least one identifiable symptom thereof, including treating patients at risk of or suspected of having the disease, as well as patients who have or have been diagnosed with the disease or condition, and includes suppressing clinical relapse. The term "prevention" as used herein refers to the complete prevention of symptoms of the disease, such as fever, swollen lymph nodes, rash, pneumonia, encephalitis, organ failure, etc. The term "relief" as used herein refers to a reduction in the severity or duration of disease symptoms. Relief includes, but does not require, complete recovery from or complete prevention of the disease or its symptoms.

[0046] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0047] In this invention, the term "excipient" refers to an additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API), also known as a pharmacological agent. General requirements for excipients include: stability, no incompatibility with the API, no side effects, no impact on efficacy, resistance to deformation, cracking, mold, and insect infestation at room temperature, harmlessness to the human body, no physiological effects, no chemical or physical reactions with the API, and no interference with the determination of the API's content.

[0048] In some embodiments, the excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants.

[0049] In some implementations, the diseases or symptoms caused by the monkeypox virus infection include fever, swollen lymph nodes, rash, pneumonia, encephalitis, and organ failure.

[0050] In some embodiments, the above-described pharmaceutical composition may be administered via any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration.

[0051] In some embodiments, the above-described compositions can be formulated into pharmaceutical compositions suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The pharmaceutical compositions may also contain solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as in the form of a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When administering the composition by infusion, it can be dispensed using infusion bottles containing sterile pharmaceutical-grade water or saline. When administering the composition by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

[0052] The eighth aspect of the present invention provides any one of the following methods, the method comprising:

[0053] (1) A method for preparing the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising the following steps: culturing the modified host cell or host cell population containing the third aspect of the present invention, and isolating the A29L antibody or antigen-binding fragment thereof according to the first aspect of the present invention from the culture.

[0054] (2) A method for detecting A29L or a fragment thereof in a test sample, the method comprising the following steps: contacting the test sample with the A29L antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of the A29L antibody or its antigen-binding fragment as described in the fourth aspect of the present invention, or contacting the test sample with a product for detecting A29L as described in the fifth aspect of the present invention, and detecting the formation of a complex of the A29L antibody or its antigen-binding fragment or an antibody derivative of the A29L antibody or its antigen-binding fragment with A29L or a fragment thereof;

[0055] (3) A method for preparing the modified host cell or host cell population containing the present invention according to the third aspect of the present invention, the method comprising the step of introducing the polynucleotide molecule or the vector containing the present invention according to the second aspect of the present invention into the host cell.

[0056] The ninth aspect of the present invention provides any of the following applications:

[0057] (1) The application of the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the antibody derivative of the A29L antibody or antigen-binding fragment thereof described in the fourth aspect of the present invention, and the detection reagent described in the fifth aspect of the present invention in the preparation of products for detecting A29L or fragment thereof;

[0058] (2) The use of the A29L antibody or its antigen-binding fragment described in the first aspect of the present invention, the antibody derivative of the A29L antibody or its antigen-binding fragment described in the fourth aspect of the present invention, and the detection reagent described in the fifth aspect of the present invention in the preparation of products for diagnosing whether a subject is infected with monkeypox virus or for diagnosing diseases or symptoms caused by monkeypox virus infection;

[0059] (3) The use of the A29L antibody or its antigen-binding fragment as described in the first aspect of the present invention, and the conjugate of the A29L antibody or its antigen-binding fragment as described in the sixth aspect of the present invention, in the preparation of a pharmaceutical composition for treating diseases or symptoms caused by monkeypox virus infection or for inhibiting monkeypox virus activity.

[0060] In some implementations, the product includes a reagent kit, a chip, or a test strip.

[0061] In some implementations, the kit includes, but is not limited to, ELISA detection kits, immunofluorescence detection kits, flow cytometry kits, and IHC detection kits.

[0062] In some embodiments, the kit may include a container, instructions for use, buffers, etc. In other embodiments, the kit may also include a lysis medium for dissolving the sample to be tested, universal reagents and buffers required for detection, such as various buffer solutions, detection labels, detection substrates, etc. This test kit can be an in vitro diagnostic device.

[0063] The tenth aspect of the present invention provides any of the following methods:

[0064] (1) A method for diagnosing whether a subject is infected with monkeypox virus, the method comprising contacting a test sample with the A29L antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of the A29L antibody or its antigen-binding fragment as described in the fourth aspect of the present invention, or contacting the test sample with the detection reagent as described in the fifth aspect of the present invention, detecting the formation of a complex of the A29L antibody or its antigen-binding fragment or the antibody derivative of the A29L antibody or its antigen-binding fragment with A29L or its fragment, and if the formation of the complex is detected, diagnosing the subject as infected with monkeypox virus;

[0065] (2) A method for treating monkeypox virus infection, the method comprising administering the A29L antibody or antigen-binding fragment thereof described in the first aspect of the present invention, a conjugate of the A29L antibody or antigen-binding fragment thereof described in the sixth aspect of the present invention, or a pharmaceutical composition described in the seventh aspect of the present invention to a subject.

[0066] The advantages and beneficial effects of this invention are as follows:

[0067] This invention discloses a monkeypox virus A29L antibody and its applications. The A29L antibody or its antigen-binding fragment of this invention can bind to the monkeypox virus A29L protein with high activity and specificity. This invention also relates to the polynucleotide molecule encoding the A29L antibody or its antigen-binding fragment, the vector, the host cell, and antibody derivatives or other products derived from the A29L antibody or its antigen-binding fragment according to this invention. Attached Figure Description

[0068] Figure 1 This is an image of the SDS-PAGE results after antibody expression and purification;

[0069] Figure 2 This is an electrophoresis image of the antibody after expression and purification;

[0070] Figure 3 This is a graph showing the results of antibody binding activity assay. Detailed Implementation

[0071] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0072] Example

[0073] 1. Immunogen Recombinant Expression

[0074] The A29L protein sequence of recombinant monkeypox virus was derived from strain ON563414.3. The A29L protein sequence of monkeypox virus membrane protein was synthesized and constructed into the pCDNA3.1 vector. Plasmid for transfection was extracted and transfected into HEK293 cells, which were cultured for 7 days. The supernatant was harvested, purified using a Ni column, and concentrated and replaced with a buffer to obtain the recombinant monkeypox virus A29L protein.

[0075] 2. Immunity

[0076] Immunization of mice: For the first immunization, Freund's complete adjuvant was used as the adjuvant, with each mouse receiving 100 μg of protein via intraperitoneal injection. The total dose of Freund's complete adjuvant and protein was 0.5 ml per mouse. The second immunization was administered 3 weeks after the first immunization. For the second immunization, Freund's incomplete adjuvant was used, with each mouse receiving 50 μg of protein. The total dose of Freund's incomplete adjuvant and protein was 0.5 ml per mouse. The third immunization was administered 2 weeks after the second immunization. Cell fusion was prepared 10 days after the third immunization.

[0077] SP2 / 0 fusion: Take feeder cells, and mix at 10...5 / Hole usage, lay 10 plates the day before fusion. 5 100 μl / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μl / well.

[0078] Screening: Positive wells were screened using ELISA. Recombinant monkeypox virus A29L protein was plated into 96-well plates and incubated overnight. The plates were washed, blocked with skim milk powder, and incubated at 37°C for 1 hour. After washing, 100 μL of 96-well culture supernatant was added and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse secondary antibody was added and incubated at 37°C for 30 minutes. After washing, chromogenic buffer was added and incubated for 10 minutes. Stop solution was added, and OD values ​​were read. 450 The numerical value; screening for high expression levels of cell lines for subcloning.

[0079] 3. Sequence Fishing

[0080] Subcloned cells were collected, RNA was extracted from the cells, reverse transcribed into the RNA, amplification primers were designed, PCR was performed using the amplification primers, the PCR product was ligated into a vector, the vector was transformed into recombinant host cells, cultured overnight, and clones were picked the next day for sequencing. The antibody sequence information for clone number 3E5 is shown in Table 1, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0081] Table 1. Amino acid sequences of monkeypox virus A29L protein and its antibody

[0082]

[0083] 4. Antibody expression

[0084] The 3E5 antibody was expressed by transiently transfecting the plasmid of the 3E5 antibody into HEK293 cells via PEI, and the expression level was detected to be 328 mg / L.

[0085] 5. Physicochemical property testing

[0086] The purity of the 3E5 antibody was detected by SDS-PAGE, and the experimental results are as follows: Figure 1 As shown, the antibody purity is greater than 95%.

[0087] The purity of the 3E5 antibody was determined by HPLC, and the experimental results are as follows: Figure 2 As shown, the antibody purity is greater than 95%.

[0088] 6. Combined with activity detection

[0089] Antibody activity was detected by ELISA: A29L protein was diluted in 0.05M carbonate buffer and coated at a concentration of 1 μg / ml, 100 μl / well, and incubated overnight at 4°C; after washing the plate and removing residual buffer, 300 μl / well of 5% skim milk powder was added for blocking and incubated at 37°C for 1 h; after washing the plate and removing residual buffer, the 3E5 antibody concentration was diluted to 10 μg / ml, and 11 4x dilutions were performed. 100 μl / well of 3E5 antibody was added and incubated at 37°C for 1 h; 100 μl / well of anti-mouse IgG-HRP-labeled secondary antibody was added and incubated at 37°C for 30 min; after washing the plate and removing residual buffer, 100 μl / well of chromogenic buffer was added and chromogenic reaction was performed for 10 min; 100 μl / well of stop solution was added and OD was read. 450 We will analyze the numerical values.

[0090] Experimental results are as follows Figure 3 As shown, the 3E5 antibody binds well to the A29L antibody, EC 50 It is 0.1410 ug / ml.

[0091] 7. Specific detection

[0092] The specificity of the antibodies was detected by ELISA: The plates were coated with monkeypox virus A29L, A35R, B6R, smallpox virus A29L, A35R, B6R, and vaccinia virus proteins A27, A35R, B6R. 3E5 antibody was added, and the plates were incubated at 37°C for 1 hour. After washing, HRP-labeled secondary antibody was added, and the plates were incubated at 37°C for 30 minutes. After washing again, chromogenic buffer was added, and the plates were incubated for 10 minutes. Stop solution was added, and the OD values ​​were read. 450 We will analyze the numerical values.

[0093] Experimental results: Only monkeypox virus A29L showed normal color development, while the other antigens showed no positive reaction, indicating that the antibody provided in this application has good specificity.

[0094] In summary, the antibody provided in this application can bind to the monkeypox virus A29L protein with high activity and high specificity.

[0095] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An A29L antibody or its antigen-binding fragment, characterized in that, The A29L antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3. Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as shown in SEQ ID NO.1, 2 and 3 respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are as shown in SEQ ID NO.4, 5 and 6 respectively.

2. The A29L antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the A29L antibody or its antigen-binding fragment contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO.7, and the light chain variable region contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO.

8.

3. The A29L antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain variable region of the A29L antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.

8.

4. A polynucleotide molecule or a carrier comprising the thereof, characterized in that, The polynucleotide molecule encodes the A29L antibody or its antigen-binding fragment as described in any one of claims 1-2.

5. A modified host cell or a population of host cells containing the same, characterized in that, The modified host cell comprises the A29L antibody or its antigen-binding fragment as described in any one of claims 1-3, the polynucleotide molecule as described in claim 4, or a vector containing the same.

6. The host cell or host cell population comprising the host cell according to claim 5, characterized in that, The host cell population also includes host cells other than the modified host cells.

7. The host cell or host cell population comprising the host cell according to claim 5, characterized in that, The modified host cells include prokaryotic cells and eukaryotic cells.

8. The host cell or a population of host cells containing the host cell according to claim 7, characterized in that, The prokaryotic cells include bacteria.

9. The host cell or a population of host cells containing the host cell according to claim 7, characterized in that, The prokaryotic cells include actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae.

10. The host cell or a population of host cells comprising the host cell according to claim 8, characterized in that, The bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

11. The host cell or a population of host cells containing the host cell according to claim 7, characterized in that, The eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

12. An A29L antibody or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The antibody derivative includes the A29L antibody or its antigen-binding fragment as described in any one of claims 1-3, which is directly or indirectly coupled to a detectable marker to form a complex.

13. The A29L antibody or an antibody derivative thereof with an antigen-binding fragment according to claim 12, characterized in that, Detectable markers include fluorescent dyes.

14. The A29L antibody according to claim 12, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, Detectable markers include enzymes.

15. The A29L antibody according to claim 12, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, Detectable markers include chemiluminescent markers, radioactive isotopes, electron-dense reagents, biotin, or digoxin.

16. The A29L antibody according to claim 13, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The fluorescent dyes include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, and phycoerythrin.

17. The A29L antibody according to claim 14, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

18. The A29L antibody according to claim 15, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The chemiluminescent markers include luminol and its derivatives, isoluminol and its derivatives, acridine esters and their derivatives, adamantane, rare earth elements, and bipyridine ruthenium complexes.

19. The A29L antibody according to claim 15, or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The radioactive isotope includes 67 Ga、 68 Ga、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 13 N、 15 O、 186 Re、 110 In、 111 In.

20. A detection reagent, characterized in that, The detection reagent includes the A29L antibody or its antigen-binding fragment as described in any one of claims 1-3, or an antibody derivative of the A29L antibody or its antigen-binding fragment as described in any one of claims 12-19.

21. A method for preparing the A29L antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The method comprises the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in any one of claims 5-11, and isolating the A29L antibody or its antigen-binding fragment as described in any one of claims 1-3 from the culture.

22. A method for in vitro detection of A29L or fragments thereof in a sample for non-diagnostic purposes, characterized in that, The method includes the following steps: contacting the sample to be tested with the A29L antibody or its antigen-binding fragment as described in any one of claims 1-3, or contacting the sample to be tested with an antibody derivative of the A29L antibody or its antigen-binding fragment as described in any one of claims 12-19, or contacting the sample to be tested with the detection reagent as described in claim 20, and detecting the formation of a complex of the A29L antibody or its antigen-binding fragment or an antibody derivative of the A29L antibody or its antigen-binding fragment with A29L or its fragment.

23. A method for preparing the modified host cell or host cell population containing the present invention as described in any one of claims 5-11, characterized in that, The method includes the following steps: introducing the polynucleotide molecule of claim 4 or a vector containing it into a host cell.

24. The use of the A29L antibody or antigen-binding fragment thereof according to any one of claims 1-3, the antibody derivative thereof of the A29L antibody or antigen-binding fragment thereof according to any one of claims 12-19, and the detection reagent according to claim 20 in the preparation of products for detecting A29L.

25. The application according to claim 24, characterized in that, The products include reagent kits, chips, or test strips.

26. The application according to claim 25, characterized in that, The kits include ELISA detection kits, immunofluorescence detection kits, flow cytometry kits, and IHC detection kits.

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