Monoclonal antibodies against monkeypox virus a29l protein and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a monoclonal antibody against A29L protein of monkeypox virus and application thereof. The present application prepares the monoclonal antibody against A29L protein of monkeypox virus, and through optimization of antibody screening and expression technology, the antibody with high affinity and high specificity is screened, and then a new tool is provided for rapid detection, effective neutralization and treatment of monkeypox virus, and an important immunological basis is provided for vaccine research and development and treatment scheme design of monkeypox virus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody against monkeypox virus A29L protein and its application. Background Technology

[0002] Monkeypox is a viral disease caused by monkeypox virus (MPXV, genus Orthopoxvirus). The transmission routes and clinical symptoms of monkeypox virus are similar to those of smallpox, but are usually milder. MPXV is mainly transmitted through direct contact with the bodily fluids, broken skin, and respiratory secretions of infected animals or humans.

[0003] The monkeypox virus genome encodes several proteins associated with viral infection and immune evasion. Among them, the A29L protein, a surface glycoprotein of monkeypox virus, plays a crucial role in immune evasion. A29L interacts with immune molecules on the surface of host cells (such as MHC-I molecules), inhibiting host immune surveillance, reducing antigen presentation, and suppressing T-cell-mediated immune responses, thereby facilitating more efficient viral replication and spread within the host. Therefore, developing specific antibodies against the A29L protein has significant immunological and clinical implications.

[0004] Currently, no preventative vaccines or specific therapeutic drugs have been developed for monkeypox, and effective diagnostic and treatment methods for monkeypox virus are still under continuous exploration and research. In this process, monoclonal antibodies, due to their high specificity and effectiveness, have gradually become an important tool in research and clinical applications. The technology for preparing monoclonal antibodies originated in the 1970s, and the main process includes mouse immunization, hybridoma technology, and monoclonal antibody screening. Although this technology has made significant progress in the past few decades and has been widely used in many fields, it still faces many challenges in dealing with emerging viruses (such as monkeypox virus). Especially for viruses with complex structures and rapid mutation, existing technologies still have certain limitations in terms of antibody screening efficiency, antibody specificity, and broad spectrum. Therefore, developing more efficient and specific antibody screening platforms, as well as antibodies targeting specific targets of monkeypox virus, will be a key direction for future research.

[0005] Existing research on monoclonal antibodies against monkeypox virus mainly focuses on two aspects: first, developing monoclonal antibodies targeting the immunogenicity of monkeypox virus surface proteins; and second, research on the preparation and application of antibodies against the monkeypox virus A29L protein.

[0006] (1) Antibody development targeting monkeypox virus surface protein

[0007] The main goal of current research is to develop monoclonal antibodies that can recognize monkeypox virus surface proteins (such as A29L, B5R, and A35R). The B5R protein is a surface glycoprotein involved in the binding of the virus to host cells, thereby promoting viral invasion. Previous studies have successfully obtained monoclonal antibodies against the B5R protein by immunizing mice with monkeypox virus proteins; these antibodies have shown some neutralizing activity. However, these studies still have several limitations:

[0008] a. Reliance on traditional immunized animals: Current antibody development mainly relies on immunized animals such as mice and rabbits. However, these antibodies may not meet clinical needs in terms of affinity and specificity, especially when facing variants of monkeypox virus, where the broad spectrum and stability of the antibodies may be insufficient.

[0009] b. Low screening efficiency: Existing antibody screening processes usually take a long time to screen for antibodies with high affinity, and the screening process is inefficient.

[0010] c. Immunogenicity issues: Some monoclonal antibodies obtained by immunizing animals may trigger immune rejection reactions in clinical applications, thereby affecting treatment efficacy and patient safety.

[0011] (2) Antibody development targeting monkeypox virus A29L protein:

[0012] The A29L protein plays a crucial role in immune evasion in monkeypox virus, effectively inhibiting the host immune system's recognition and clearance of the virus. Therefore, developing specific antibodies against the A29L protein is key to improving the neutralizing efficacy of monkeypox virus and enhancing immune protection. Current research largely employs traditional hybridoma techniques, which involve inoculating immunized animals (such as mice) with monkeypox virus or the A29L protein and then screening for monoclonal antibodies that bind to the A29L protein. The main mechanism of action of these antibodies is to inhibit viral infection by preventing the virus from binding to receptors on the surface of host cells. However, existing approaches have the following limitations:

[0013] a. Low screening efficiency: Although some monoclonal antibodies against the A29L protein have been successfully screened, it is still difficult to screen for antibodies with high affinity and strong specificity due to the complex antigenic epitopes of the A29L protein, and some antibodies have insufficient specificity and affinity.

[0014] b. Incomplete immune response: The immune response of traditionally immunized animals is usually relatively simple, and the antibodies obtained may only target some epitopes of the A29L protein, making it difficult to cover all variants of the virus, which limits its wide application in different monkeypox virus strains.

[0015] c. Unstable neutralizing efficacy: Although some antibodies show strong binding ability in in vitro experiments, their neutralizing efficacy may be insufficient in vivo and they may not be able to effectively inhibit viral infection.

[0016] (3) Antibody screening scheme based on phage display technology:

[0017] In recent years, the application of phage display and single-cell antibody screening technologies has increased, providing new ideas for developing monoclonal antibodies against monkeypox virus and its important proteins (such as the A29L protein). Phage display technology can efficiently screen for antibodies that specifically bind to the A29L protein by displaying a large-scale antibody library (such as single-chain antibodies or Fab fragments). Compared with traditional hybridoma technology, this method has the following advantages: 1) By synthesizing a diverse antibody library, antibodies with high affinity and high specificity can be screened in a shorter time; 2) Overcoming the problem of incomplete immune response: Using human antibody libraries or humanized antibodies can effectively avoid immunogenicity issues, thereby improving the clinical safety of antibodies; 3) Flexible applicability: The screening conditions of phage display technology can be optimized according to needs, enabling the rapid discovery of specific antibodies against different monkeypox virus variants, thus broadening the scope of application. However, despite the significant advantages of phage display technology in antibody screening, it still faces some challenges: 1) Difficulty in library construction: The construction and screening of phage display libraries requires high-level technical and equipment support and is costly; 2) Complex screening process: Although phage display technology has high screening efficiency, for targets with complex antigenic epitopes such as A29L protein, continuous optimization is still required during the screening process to ensure the acquisition of antibodies with good neutralizing effects.

[0018] Existing methods for preparing monoclonal antibodies, especially traditional hybridoma techniques, while achieving significant progress in antibody development, still have several significant limitations when dealing with emerging viruses such as the novel coronavirus and monkeypox virus. The main problems include:

[0019] 1) Immunogenicity Differences: Traditional hybridoma technology typically relies on mouse immunization as the basis for antibody production. However, differences in immunogenicity among different viral strains or antigens can lead to insufficient or incomplete immune responses, which is particularly evident when facing some emerging viruses. For example, against highly variable pathogens such as the novel coronavirus or monkeypox virus, traditional immunization strategies may fail to generate sufficiently strong or efficient immune responses, making it difficult to screen for monoclonal antibodies with high affinity and high specificity, thus limiting their potential application in antiviral immunotherapy.

[0020] 2) Limited antibody diversity: Although hybridoma technology can effectively screen for monoclonal antibodies against specific antigens, the diversity and broad spectrum of the resulting antibodies are often insufficient due to the natural limitations of the mouse immune response. Especially when facing rapidly mutating viral strains, traditional hybridoma technology may not be able to recognize all viral subtypes or mutants, resulting in insufficient antibody recognition of different variants and inability to provide adequate immune protection.

[0021] 3) The screening process is cumbersome and time-consuming: The antibody screening process of traditional hybridoma technology involves multiple complex steps such as immunization, cell fusion, and clone screening. The entire process is not only time-consuming but also requires high-level experimental skills and is easily affected by environmental and operational conditions. This results in low antibody screening efficiency and makes it impossible to obtain sufficiently efficient antibodies in a short period of time, which is particularly lagging behind in responding to public health emergencies.

[0022] 4) Low antibody purity and production efficiency: Compared to modern recombinant antibody technology or humanized antibody technology, traditional hybridoma technology produces antibodies with lower purity and production efficiency. This directly affects the large-scale production capacity of antibodies and the feasibility of their clinical application, especially in the commercialization and widespread use of antibody drugs, where traditional methods face significant challenges.

[0023] In summary, although current research has made some progress in the development of monoclonal antibodies against monkeypox virus, challenges remain, including low antibody screening efficiency, incomplete immune responses, immunogenicity issues, and unstable neutralizing efficacy. Antibody screening protocols based on phage display technology offer a new approach to addressing these problems, but further optimization of screening strategies is still needed to ensure the acquisition of antibodies with high specificity, high affinity, and stable neutralizing effects. These advances will play a crucial role in the diagnosis, prevention, and treatment of monkeypox virus. Summary of the Invention

[0024] In view of this, the present invention provides a monoclonal antibody against the monkeypox virus A29L protein and its applications. The present invention prepares a monoclonal antibody against the monkeypox virus A29L protein, and through optimized antibody screening and expression techniques, selects antibodies with high affinity and high specificity. This provides a new tool for the rapid detection, effective neutralization, and treatment of monkeypox virus, and provides an important immunological basis for the development of monkeypox virus vaccines and the design of treatment regimens.

[0025] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0026] This invention provides a monoclonal antibody against monkeypox virus A29L protein, said monoclonal antibody comprising one or more of MPXVAntibody #5, MPXV Antibody #25, MPXV Antibody #28, MPXV Antibody #87, MPXVAntibody #94 or MPXV Antibody #111;

[0027] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of MPXV Antibody #5 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.4, LVS, and SEQ ID NO.5, respectively.

[0028] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of MPXV Antibody #25 are shown in SEQ ID NO.1, SEQ ID NO.6, and SEQ ID NO.3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.7, LVS, and SEQ ID NO.8, respectively.

[0029] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of MPXV Antibody #28 are shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.12, WAS, and SEQ ID NO.13, respectively.

[0030] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the MPXV Antibody #87 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.14, LVS, and SEQ ID NO.5, respectively.

[0031] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of MPXV Antibody #94 are shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.12, WAS, and SEQ ID NO.18, respectively.

[0032] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of MPXV Antibody #111 are shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown in SEQ ID NO.22, NAK, and SEQ ID NO.23, respectively.

[0033] In some specific embodiments of the present invention, the monoclonal antibody includes:

[0034] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO.27; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.29.

[0035] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #25 is shown in SEQ ID NO.31; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.33.

[0036] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #28 is shown in SEQ ID NO.35; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.37.

[0037] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #87 is shown in SEQ ID NO.39; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.41.

[0038] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #94 is shown in SEQ ID NO.43; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.45.

[0039] The amino acid sequence of the heavy chain variable region of the MPXV Antibody #111 is shown in SEQ ID NO.47; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.49.

[0040] The present invention also provides biomaterials, including any one of the following:

[0041] (I) The nucleic acid molecule encoding the monoclonal antibody;

[0042] (II) Expression vectors comprising the nucleic acid molecules described in (I);

[0043] (III) The host of the expression vector described in (II).

[0044] In some specific embodiments of the present invention, the nucleic acid molecule includes:

[0045] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO.26; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.28.

[0046] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #25 is shown in SEQ ID NO.30; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.32.

[0047] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #28 is shown in SEQ ID NO.34; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.36.

[0048] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #87 is shown in SEQ ID NO.38; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.40.

[0049] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #94 is shown in SEQ ID NO.42; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.44.

[0050] The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #111 is shown in SEQ ID NO.46; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.48.

[0051] In some specific embodiments of the present invention, the nucleotide sequence encoding the monkeypox virus core protein A29L is shown in SEQ ID NO.27.

[0052] The present invention also provides the use of any of the following in the preparation of products for detecting monkeypox virus:

[0053] (I) The monoclonal antibody;

[0054] (II) The biological material.

[0055] This invention also provides reagents or kits for detecting monkeypox virus, comprising any one of the following:

[0056] (I) The monoclonal antibody;

[0057] (II) The biological material.

[0058] The present invention also provides the use of any of the following in the preparation of products for the prevention or treatment of monkeypox virus:

[0059] (I) The monoclonal antibody;

[0060] (II) The biological material.

[0061] In some specific embodiments of the present invention, the product includes a vaccine or a drug.

[0062] This invention provides the following beneficial effects:

[0063] This invention optimizes traditional hybridoma technology, improving the efficiency and quality of antibody development, specifically including:

[0064] 1) Enhancing antibody immunoreactivity and affinity: By optimizing immunization strategies, employing novel adjuvants and different immunization routes, the immune response of mice to monkeypox virus was enhanced. This approach helps improve the specificity and affinity of monoclonal antibodies, thereby obtaining more efficient antibodies.

[0065] 2) Expanding the diversity and broad spectrum of antibodies: By improving traditional hybridoma technology and combining it with advanced methods such as single-cell antibody screening, the diversity and broad spectrum of antibody libraries will be further expanded. This measure will enable the antibody library to screen for antibodies that can recognize different subtypes and mutants when faced with viral variants, thereby enhancing the adaptability and breadth of antibodies and providing a more effective immune intervention for dealing with emerging viral strains.

[0066] 3) Shorten antibody screening cycle: By optimizing cell fusion, screening, and cloning processes, the efficiency of the antibody screening process can be improved, shortening the preparation cycle. This measure will enable monoclonal antibodies to be deployed to practical applications more quickly, especially in response to public health emergencies, providing rapid responses and solutions to ensure timely countermeasures against viral threats.

[0067] Through the aforementioned innovative measures, this invention overcomes the limitations of traditional hybridoma technology, improving the efficiency, precision, and broad-spectrum nature of antibody development, particularly in the face of emerging viruses such as monkeypox virus. These improvements will provide a more efficient, precise, and widely applicable solution for antibody drug development, promote the application of monoclonal antibodies in the public health field, and provide a more powerful immune intervention tool for the prevention and control of emerging viruses, thereby effectively addressing the threat of emerging viruses globally. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0069] Figure 1 This is a schematic diagram of the antibody-encoding gene structure (including the 5' and 3' non-coding regions).

[0070] Figure 2 These are the SDS-PAGE results of molecular sieve purification of monkeypox A29L protein;

[0071] Figure 3 This is the SDS-PAGE result of the anti-monkeypox A29L monoclonal antibody. Detailed Implementation

[0072] This invention discloses a monoclonal antibody against monkeypox virus A29L protein and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0073] The main technical problem this invention aims to solve is to develop a monoclonal antibody with high affinity and high specificity targeting the A29L protein of monkeypox virus (MPXV) to effectively detect monkeypox virus infection and address the shortcomings of existing diagnostic and treatment methods. Specifically, the main technical problems to be solved include:

[0074] 1) Limited Recognition Scope of Existing Antibodies: The development and research of monoclonal antibodies against monkeypox virus are still in their early stages. Existing antibodies have significant shortcomings in terms of specificity and affinity, making it difficult to meet the needs of clinical diagnosis and treatment. This limitation makes it difficult for existing antibodies to provide sufficient sensitivity and broad spectrum, especially in the recognition of diverse monkeypox virus variants.

[0075] 2) Lack of highly effective and specific neutralizing antibodies: The A29L protein, as an important structural component of monkeypox virus, plays a crucial role in viral pathogenicity and its evasion of the host immune response. Developing specific monoclonal antibodies against the A29L protein is of great significance. Antibodies capable of recognizing and neutralizing the A29L protein can not only effectively block the binding of the virus to host cells but also significantly enhance the immune defense against monkeypox virus infection. Therefore, the lack of such highly effective neutralizing antibodies is a key technical obstacle in current anti-monkeypox virus therapy.

[0076] 3) Insufficient sensitivity and specificity of existing diagnostic tools: Current methods for diagnosing monkeypox virus infection have not yet reached the standards of speed and accuracy, especially in the early diagnosis of viral infection. Existing detection methods are often limited by factors such as antibody specificity, sensitivity, and operational complexity, affecting their widespread application in clinical practice. Developing a monoclonal antibody with high affinity and high specificity can significantly improve the detection sensitivity and specificity of viral infection, which is crucial for rapid diagnosis and early intervention.

[0077] In summary, the main objective of this invention is to prepare a monoclonal antibody against the A29L protein of monkeypox virus, and to screen for antibodies with high affinity and high specificity through optimized antibody screening and expression techniques. This provides a new tool for the rapid detection, effective neutralization, and treatment of monkeypox virus, and offers an important immunological basis for vaccine development and treatment design. Therefore, the technical solution provided by this invention will significantly advance the diagnosis, prevention, and treatment of monkeypox virus infection.

[0078] The traditional hybridoma technology used in this invention has a long history and certain advantages in the preparation of monoclonal antibodies. Although many emerging technologies such as single-cell antibody screening and phage display technology have emerged and been widely used with the advancement of science and technology, the traditional hybridoma technology still has irreplaceable advantages in the following aspects.

[0079] 1) Mature technology and rich experience

[0080] High reliability: Because hybridoma technology has been widely used and validated, the prepared antibodies usually have high stability and consistency; Rich experience: Laboratories and companies have accumulated a wealth of experience, and technicians can reduce the risk of experimental failure through standardized operations.

[0081] 2) Generate antibodies with high affinity and high specificity.

[0082] High affinity: Hybridoma technology can screen for antibodies with very high affinity, which is crucial for many applications that require precise identification (such as diagnosis and treatment); Monoclonal antibody stability: The prepared antibodies can be stably produced during long-term culture without cross-reactivity and have good specificity.

[0083] 3) Applicable to multiple antigen types

[0084] Wide applicability: Hybridoma technology can handle complex membrane proteins, whole antigens, and simple small molecule antigens; Effective for both natural and artificial antigens: It does not require many optimization steps and has good adaptability to both naturally occurring antigens (such as pathogens and tumor cells) and synthetic antigens (such as artificial vaccines).

[0085] 4) High antibody diversity and selectivity

[0086] Natural immune diversity: By immunizing mice or other species, different types of antibodies can be screened from a variety of cells, covering a wider range of antigenic epitopes; High selectivity: Through a rigorous screening process, highly selective monoclonal antibodies can be obtained, suitable for a variety of complex applications.

[0087] 5) High output and large-scale production

[0088] High yield: By optimizing culture conditions and using suitable cell lines (such as SP2 / 0, NS0, etc.), large quantities of monoclonal antibodies can be obtained; Adaptable to industrial production: The production process of hybridoma technology has been industrialized, facilitating large-scale, long-term production.

[0089] Traditional hybridoma technology has advantages such as maturity, reliability, high yield, and wide applicability, especially in terms of yield and stability, making it suitable for long-term commercial production. However, the objectives of this invention can be achieved by using emerging technologies such as single-cell antibody screening, phage display, and nanobody technology.

[0090] The amino acid sequence information of the CDR region of the antibody described in this invention is shown in Table 1:

[0091] Table 1. Amino acid sequence of the CDR region of the antibody

[0092]

[0093] The main instruments, reagents and consumables used in this invention are shown in Tables 2 and 3.

[0094] Table 2 Main Instruments

[0095]

[0096]

[0097] Table 3 Main reagents and consumables

[0098]

[0099] Unless otherwise specified, the monoclonal antibody against monkeypox virus A29L protein provided by this invention and the raw materials and reagents used in its application are all commercially available.

[0100] The present invention will be further illustrated below with reference to the embodiments:

[0101] Example 1: Preparation of monkeypox virus hybridoma antibody

[0102] I. Preparation of Antigen Protein:

[0103] (1) Plasmid synthesis: After codon optimization, the A29L gene fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the expression vector pGEX-4T-1.

[0104] The nucleotide sequence of A29L (codon optimized) is as follows (SEQ ID NO.24):

[0105]

[0106] (2) Protein expression and purification.

[0107] 1. Transform the synthesized plasmid into competent cells, plate the cells, pick single clones to confirm plasmid resistance, and incubate overnight with gentle shaking. Preserve the culture using glycerol solution.

[0108] 2. Glyceryl bacteria were induced by adding 0.5, 1.0, and 1.5 mmol of IPTG and incubated overnight in a shaker at 37°C.

[0109] 3. The induced bacterial culture was lysed by ultrasonication. The supernatant was soluble protein and the precipitate was inclusion bodies.

[0110] 4. Purify the target protein using an affinity chromatography Ni-NTA column, and elute the target protein with reduced glutathione.

[0111] 5. Dialyze to buffer solution, check concentration and purity, verify by gel electrophoresis, and store at -80℃ after passing the test. The monkeypox A29L fusion protein (fused with GST tag) prepared in this invention was analyzed by SDS-PAGE electrophoresis (e.g., Figure 2 The protein showed a clear band at 40 kD, with the correct size and no impurities, indicating high purity of monkeypox A29L protein.

[0112] Preparation of soluble histidine-tagged recombinant proteins:

[0113] (1) Transform the synthesized plasmid into competent cells, plate the cells, pick single clones to confirm plasmid resistance, and then shake them overnight. Use glycerol solution to preserve the culture mixture.

[0114] (2) Glyceryl bacteria were induced by adding 0.5, 1.0, and 1.5 mmol of IPTG and incubated overnight in a shaker at 37°C.

[0115] (3) The induced bacterial culture was lysed by ultrasonication and used as the protein to be purified for the next step.

[0116] (4) The amount of purification packing material used is calculated based on a loading of 10 mg / ml;

[0117] (5) Add the purification packing material to the gravity column. After the 20% ethanol of the preservation solution has been added, add 10 column volumes of ultrapure water to rinse the ethanol off. Finally, add 10 column volumes of PBS pH 7.5 to balance the gravity column.

[0118] (6) Plug the lower end of the gravity column with a plug, and after suspending the packing material evenly with the sample to be purified, mix it with the sample, put it into the binding shaker, and place the binding shaker in a 4°C refrigerator. The binding time shall not be less than 30 min.

[0119] (7) Remove the combined sample from the binding shaker and let it stand in a 4℃ refrigerator for 5-10 min. Then use a pipette to add the mixture of sample and packing material into the empty column and collect the flow-through sample (FT). Take 40 μl of the sample (FT) for SDS-PAGE analysis.

[0120] (8) Wash the column with 10 column volumes of PBS pH 7.5 to elute non-specifically bound host proteins, collect the sample (W1), and take 40 μl of sample (W1) for SDS-PAGE detection.

[0121] (9) Gradient rinsing with imidazole solutions of different concentrations;

[0122] (10) Wash one tube with PBS pH 7.5 + high concentration imidazole for each column volume, for a total of 9 tubes. Take 40 μl of sample from each tube and record it as E1-E9 for SDS-PAGE detection.

[0123] (11) Collect the target protein based on the SDS-PAGE results.

[0124] The amino acid sequence of the purified A29L antigen protein is as follows (SEQ ID NO.25):

[0125] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKL VCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKHNDEVLFRLENHAETLRAAMISLAKKIDVQTGRRPYEEFGGSHHHHHH

[0126] II. Animal Immunization:

[0127] The immunogen (A29L fusion protein), adjuvants (first immunization used complete Freund's adjuvant (paraffin oil + lanolin + BCG), the next three immunizations used incomplete Freund's adjuvant (paraffin oil + lanolin)), immunized animals, and immunization strategies are shown in Table 4 below:

[0128] Table 4 Animal Immunity

[0129]

[0130] III. Fusion Experiment:

[0131] (1) Balb / c mice that had been boosted with immunization (purchased from the Wuhan BASF Model Biology Center) were fixed in place, and their eyeballs were removed to collect blood. The mice were then euthanized by cervical dislocation and disinfected in 75% alcohol for at least 30 seconds.

[0132] (2) Mouse whole blood was left to stand at 20℃±5℃ for 1 hour, and then stored at 4℃ overnight. The next day, mouse whole blood was centrifuged at 3000rpm for 15 min, and the upper serum was carefully aspirated as a positive control for hybridoma screening. It was then aliquoted and stored at -20℃.

[0133] (3) Fix the mouse’s limbs with pins so that it is facing upwards. Use the first set of tweezers and scissors to cut open the epidermis, use the second set of tweezers and scissors to cut open the abdominal wall muscle layer, and use the third set of tweezers and scissors to separate and remove the spleen.

[0134] (4) Take three culture dishes with a diameter of 10 cm and add 10 ml of 1640 basic culture medium. Rinse the spleen once in the first culture dish; in the second culture dish, use tweezers to remove the residual connective tissue on the surface of the spleen (be careful not to tear the spleen capsule); in the third culture dish, gently grind the spleen with the ground surfaces of two glass slides to break the spleen capsule, and then obtain spleen cells.

[0135] (5) Use a 10 ml pipette to draw up the thoroughly ground spleen cell suspension, filter it through a cell sieve, and transfer it to a 50 ml sterile centrifuge tube.

[0136] (6) Take another 10 ml of 1640 basic culture medium and rinse the culture dish 2-3 times. Then filter it through a cell sieve and transfer it to a 50 ml sterile centrifuge tube in step (5). Centrifuge at 1500 rpm for 6 min.

[0137] (7) Discard the supernatant, and repeatedly pipette 10-15 times with 10 ml of 1640 basic culture medium to fully suspend the spleen cell pellet. Then add 30 ml of 1640 basic culture medium and pipette 5 times to mix well. Centrifuge at 1500 rpm for 6 min.

[0138] (8) Repeat step (7) once.

[0139] (9) Discard the supernatant and resuspend the spleen cell pellet by repeatedly pipetting 10-15 times with 5 ml of 1640 basic culture medium. Take about 0.2 ml of cell suspension, dilute it 20-40 times, and count the spleen cells. Place the cells at 20℃±5℃ before fusion.

[0140] (10) During the intercentrifugation period, sp2 / 0 cells were collected in a 50 ml sterile centrifuge tube and centrifuged at 1000 rpm for 5 min.

[0141] (11) Discard the supernatant, and repeatedly pipette the myeloma cells (purchased from AtaGenix Laboratories Co., Ltd. (Wuhan), Wuhan, PR China) precipitate with 10 ml of 1640 basic culture medium 10-15 times. Add 30 ml of 1640 basic culture medium and pipette 5 times to mix well. Centrifuge at 1000 rpm for 5 min.

[0142] (12) Repeat step (11) once.

[0143] (13) Discard the supernatant and resuspend the myeloma cell pellet by repeatedly pipetting 10-15 times with 5 ml of 1640 basic culture medium. Take out about 0.2 ml of cell suspension, dilute it 10-20 times and count the cells. Place the cells at 20℃±5℃ before fusion.

[0144] (14) Before fusion begins, turn on the constant temperature water bath and set the temperature to 37°C. Place the PEG and 1640 basic culture medium in the water bath for preheating.

[0145] (15) Based on the cell count results, mix the required spleen cells and myeloma cells separately and then mix them in a 50 ml centrifuge tube at a ratio of 5:1.

[0146] (16) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently tap the centrifuge tube wall to loosen the cell pellet.

[0147] (17) Place the centrifuge tube in a 37°C water bath and add the preheated PEG (1 ml within 1 min) evenly into the cell pellet. While adding the PEG, gently stir with the tip of the pipette while rotating the centrifuge tube and let it stand for 90 seconds.

[0148] a. Add the preheated 1640 basic culture medium at a constant rate (first time): add 1 ml over 1 min while gently stirring.

[0149] b. Add the preheated 1640 basic culture medium (second time) at a uniform rate: add 2 ml over 1 min while gently stirring.

[0150] c. Add the preheated 1640 basic culture medium (third time) at a uniform rate: add 9 ml over 3 min while gently stirring.

[0151] d. Add the preheated 1640 basic culture medium at a constant rate, stirring gently while adding until 40 ml is reached. Place the centrifuge tube in a 37°C water bath and let it stand for 3 min.

[0152] (18) The fused cell suspension was centrifuged at 800 rpm for 5 min, the supernatant was removed, and the cell pellet was loosened.

[0153] (19) Add 5 ml of HAT medium, gently pipette the cell pellet 10 times, add an appropriate amount of HAT medium according to the number of spleen cells, pipette to mix well, and inoculate into a 96-well cell culture plate.

[0154] IV. Subcloning Screening:

[0155] (1) Preparation of cell suspension: under a microscope, select hybridoma cells in good growth condition to prepare cell suspension.

[0156] (2) The number of cells in the cell suspension is accurately counted according to the cell counting method, which is generally around 10 cells / ml.

[0157] (3) Place a new 24-well culture plate in a clean bench. Add 900 μl of 15% HT selective medium to wells A1, A2, and A3 respectively. Mix the hybridoma cells from the limited dilution 24-well culture plate and take 100 μl of the cell suspension. Add the suspension to well A1 of the new 24-well culture plate. Use a 1 ml pipette to repeatedly pipette about 10 times. Then, take 100 μl from well A1 using a single-channel pipette (20-100 μl) and transfer it to well A2. Repeat this process until well A3 is reached.

[0158] (4) Take 120 cells from well A3 and place them into a V-shaped groove. Use a 10ml pipette to aspirate 15% HT selective culture medium into the V-shaped groove twice, so that the total volume of the culture medium in the V-shaped groove is 16ml. Repeat the pipetting process about 8 times. When seeding 96-well culture plates, add 200 μl / well to columns 1-6, which is 1.5 cells per well. Add another 6.4ml of 15% HT selective culture medium to the remaining 6.4ml of cell suspension, repeat the pipetting process about 8 times, and add 200 μl / well to columns 7-12, which is 0.75 cells per well.

[0159] (5) Place in a 37℃ 8% CO2 incubator and incubate for 5 days. On the seventh day, small cell clones can be seen under an inverted microscope. Mark "1" on the plate cover if there is a single cell clone cluster, and mark "√" on the plate cover if there are two or more cell clone clusters. Record the results and make statistics.

[0160] (6) The culture supernatant can be harvested around day 8-9 for antibody detection.

[0161] (7) Select positive cells that grow well in single clonal growth wells, transfer them to 24-well plates and then clone them for culture or expand culture to obtain hybridoma cells.

[0162] V. Antibody Production:

[0163] Subtype identification and antibody production were performed on the selected cell lines, and the antibodies were purified using the protein G method.

[0164] 1. Centrifuge 5000g of the sample to be purified for 5 min and collect the supernatant.

[0165] 2. Add the supernatant to the protein G column and collect it by flow-through into a 15 ml centrifuge tube.

[0166] 3. After loading the sample, wash the column with 10 column volumes of PBS.

[0167] 4. While washing the column, prepare 1.5 ml sterile EP tubes and pre-add 50 μl of 1M pH 9.0 Tris-HCl to each collection tube.

[0168] 5. Elute with 1 ml of 0.1 M glycine solution (pH 2.5) and collect the eluent in a 1.5 ml EP tube. Immediately after collection, invert the EP tube to mix thoroughly and test the pH of the eluent with pH paper to ensure it is neutral.

[0169] 6. After elution, add 3 column volumes of 0.1M glycine solution (pH 2.5) to the protein G column to completely elute the column. Then wash the column with 5 column volumes of dihydrochloric acid, followed by 2 column volumes of 20% ethanol. After the elution is complete, tighten the cap at the bottom of the column and add 2 column volumes of 20% ethanol to store the column at 4°C.

[0170] 7. Centrifuge the eluted antibody at 12000 rpm for 2-3 min at room temperature, discard the precipitate, and measure the OD value of the supernatant to calculate the antibody concentration. Formula: Antibody concentration (mg / ml) = OD280 / 1.4 × dilution factor. If the concentration cannot be displayed, it indicates that the concentration is too high. Use a multi-channel pipette to aspirate 200 μl back into the original tube, then aspirate 20 μl of the original antibody solution, add 180 μl of 1*PBS to dilute 10 times, mix well, and perform the detection.

[0171] 8. Calculate the antibody concentration based on the measured OD280, and take 2 μg of antibody for SDS-PAGE detection.

[0172] SDS-PAGE detection experimental steps:

[0173] (1) Mixing adhesives (top layer adhesive, bottom layer adhesive);

[0174] (2) Antibody denaturation (after adding sample buffer, heat in a metal bath at 95-100℃ for 5 minutes).

[0175] (3) SDS-electrophoresis (start with 120V for electrophoresis, then adjust to 200V after the proteins run to the stacking gel until electrophoresis is complete).

[0176] (4) Coomassie brilliant blue staining and destaining;

[0177] (5) Imaging analysis results.

[0178] 9. Submit antibody samples for indirect ELISA testing to determine antibody titer:

[0179] (1) Coating: A29L antigen, concentration 2μg / ml, 100μl / well, 37℃, 2h.

[0180] (2) Blocking: 3% BSA-PBS, 300 μl / well, 37℃, 1.5h.

[0181] (3) Washing: PBST, 300 μl / well, twice.

[0182] (4) Add primary antibody: Dilute with primary antibody dilution buffer (MPXV Antibody #5, MPXV Antibody #25, MPXV Antibody #28, MPXV Antibody #87, MPXV Antibody #94, MPXV Antibody #111) to 1 μg / ml to 0.015 μg / ml, 100 μl / well, 37℃, 1h.

[0183] The primary antibody diluent used was the universal antibody diluent for ELISA, purchased from Wuhan BOSTER Biotechnology Co., Ltd. (Catalog No.: AR1106-2).

[0184] (5) Washing: PBST, 300 μl / well, 3 times.

[0185] (6) Add secondary antibody: Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG Fc (purchased from Jackson Immuno Research, Code: 115-035-071, https: / / www.jacksonimmuno.com / catalog / products / 115-035-071), 1:10000, 100 μl / well, 37℃, 30 minutes.

[0186] (7) Washing: PBST, 300 μl / well, 3 times.

[0187] (8) Add TMB substrate: 100 μl / well, 37℃, 10 min.

[0188] (9) Termination: 2 mol HCl, 50 μl / well.

[0189] (10) Microplate reader reading: OD450-OD620.

[0190] 10. The concentration of the tested samples is as follows:

[0191] Ab#5 antibody concentration: 5.35 mg / ml;

[0192] Ab#25 antibody concentration: 2.56 mg / ml;

[0193] Ab#28 antibody concentration: 3.89 mg / ml;

[0194] Ab#87 antibody concentration: 5.5 mg / ml;

[0195] Ab#94 antibody concentration: 3.5 mg / ml;

[0196] The Ab#111 antibody concentration was 0.62 mg / ml.

[0197] 11. Perform SDS-PAGE detection on the antibodies.

[0198] Example 2: Sequencing of hybridoma cell lines

[0199] 1. Total RNA was extracted from the hybridoma cell line obtained in Example 1, and cDNA was amplified by reverse transcription PCR. The variable region sequence of the antibody was amplified by PCR using subtype-specific primers, and the products were subcloned into T vectors and sequenced for analysis. A schematic diagram of the primary structure of the cDNA encoding the antibody is shown below. Figure 1 As shown.

[0200] 2. Sequencing methods:

[0201] Total RNA was extracted from hybridoma cell lines and sequenced by AtaGenix Laboratories Co., Ltd. (Wuhan, PR China).

[0202] Total cDNA was amplified by RT-PCR using oligo-dT primers. Using cDNA as a template, nested-PCR amplification was performed on VH and VL cells using forward degenerate primers (designed according to the V or L gene) and reverse constant region-specific primers, respectively. The PCR products were ligated into a T vector, transformed, and subjected to colony PCR. Positive clones were selected for sequencing analysis. The sequences obtained from sequencing were analyzed using the online software Vbase2 and IMGT / V Quest.

[0203] 3. The sequencing results are analyzed as follows:

[0204] MPXV Antibody #5-VH:

[0205] DNA sequence (SEQ ID NO.26):

[0206] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGTCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGAGCATTAATCCTACCTATGGT GATACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0207] amino acid sequence:

[0208] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.27):

[0209] QVQLQQPGAELVKPGSSVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGS INPTYGDT NFTEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYC TRWDY WGQGTTLTVSS

[0210] MPXV Antibody #5-VL:

[0211] DNA sequence (SEQ ID NO.28):

[0212] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGTTGGCAAGGTACACATTTTCCCCAAACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0213] amino acid sequence:

[0214] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.29):

[0215] DVVMTQTPLTLSVTIGQPASISCKSSQ SLLESDGKTY LNWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQT FGGGTKLEIK

[0216] MPXV Antibody #25-VH:

[0217] DNA sequence (SEQ ID NO.30):

[0218] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGGGGATTAATCCTACCAGTGGTGCTACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGTTCAGCCTACCTGCAACTCAGCAGCCTGGCATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0219] amino acid sequence:

[0220] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.31):

[0221] QVQLQQPGAELVKPGASVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGG INPTSGAT NFTEKFKTKATLTVDKSSSSAYLQLSSLASEDSAVYYC TRWDY WGQGTTLTVSS

[0222] MPXV Antibody #25-VL:

[0223] DNA sequence (SEQ ID NO.32):

[0224] GGTGTTGTGATGACCCAGACTCCACTCACTTTGTCGATTACCCTTGGACAACCAGCCTCCATTTCTTGCAAGTCAGGTCAGAGCCTCTTAGATAGTAATGGAAAGGCATACTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0225] amino acid sequence:

[0226] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.33):

[0227] GVVMTQTPLTLSITLGQPASISCKSG QSLLDSNGKAY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGTHFPHT FGGGTKLEIK

[0228] MPXV Antibody #28-VH:

[0229] DNA sequence (SEQ ID NO.34):

[0230] CAGGTTATTCTGAAAGAGTCTGGCCCTGGAATATTGCAGCCCTCTCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTTAATACTTATAATACAGCTGTGAACTGGATTCGTCAGCCTTCTGGAAAGGGTCTGGAGTGGTTGGCACTAATCGGGTCAGATGATAATGAACTCTATAACCCATTTCTGAAAAGTCGAATCACAATCTCCAAGGATGCCTCCAACAGCCAGGTATTCCTCAAGATCACTAGTGTGGACACTCAAGATTCTGCCACATACTACTGTGTTAACGGGGGATTACGACGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0231] amino acid sequence:

[0232] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.35):

[0233] QVILKESGPGILQPSQTLSLTCSFS GFSLNTYNTA VNWIRQPSGKGLEWLAL IGSDDNE LYNPFLKSRITISKDASNSQVFLKITSVDTQDSATYYC VNGGLRRAWFAY WGQGTLVTVSA

[0234] MPXV Antibody #28-VL:

[0235] DNA sequence (SEQ ID NO.36):

[0236] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATTAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTTACAGGGAGTGGATATGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0237] amino acid sequence:

[0238] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.37):

[0239] DIVMTQSHKFMSTLVGDRVSITCKAS QDVGTA IVWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGYGTDFTLTISNVQSEDLADYFC QQYSSYPLT FGSGTKLEIK

[0240] MPXV Antibody #87-VH:

[0241] DNA sequence (SEQ ID NO.38):

[0242] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGTCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGAGCATTAATCCTACCTATGGTGATACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0243] amino acid sequence:

[0244] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.39):

[0245] QVQLQQPGAELVKPGSSVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGS INPTYGDT NFTEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYC TRWDY WGQGTTLTVSS

[0246] MPXV Antibody #87-VL:

[0247] DNA sequence (SEQ ID NO.40):

[0248] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGTTGGCAAGGTACACATTTTCCCCAAACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0249] amino acid sequence:

[0250] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.41):

[0251] DVVMTQTPLTLSVTIGQPASISCKSS QSLLESDGKTY LNWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQT FGGGTKLEIK

[0252] MPXV Antibody #94-VH:

[0253] DNA sequence (SEQ ID NO.42):

[0254] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGAGCTATGGTTACGACGGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0255] amino acid sequence:

[0256] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.43):

[0257] EVQLVESGGGLVKPGGSLKLSCAAS GFTFSDYY MYWVRQTPEKRLEWVAT ISDGGSYT YYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYC ARELWLRRGWFAY WGQGTLVTVSA

[0258] MPXV Antibody #94-VL:

[0259] DNA sequence (SEQ ID NO.44):

[0260] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATTTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0261] amino acid sequence:

[0262] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.45):

[0263] DIVMTQSHKFMSTSVGDRVSITCKAS QDVGTA VAWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGFGTDFTLTISNVQSEDLADYFCQ QYSSYLT FGAGTKLELK

[0264] MPXV Antibody #111-VH:

[0265] DNA sequence (SEQ ID NO.46):

[0266] CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAACAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACTTATACTGGTAATACAAACTACAACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGGGGAATCTACAATGACTACGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0267] amino acid sequence:

[0268] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.47):

[0269] QVQLQQSGPELVRPGVSVKISCKGS GYTFTDYA MHWVKQSHAKSLEWIGV ISTYTGNT NYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYC ARGESTMTTTY WGQGTLVTVSA

[0270] MPXV Antibody #111-VL:

[0271] DNA sequence (SEQ ID NO.48):

[0272] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAATTATTACTGTCAACATTTTTGGAGAACTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0273] amino acid sequence:

[0274] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.49):

[0275] DIQMTQSPASLSASVGETVTITCRAS GNIHNY LAWYQQKQGKSPQLLVY NAK TLADGVPSRFSGSGSGTQYSLKINSLQPEDFGNYYC QHFWRTPPT FGGGTKLEIK

[0276] Example 1: Indirect ELISA detection of antibody titer

[0277] The antibody titer was detected by indirect ELISA using the method described in Example 1, and the results are shown in Table 5:

[0278] Table 5. Indirect ELISA detection of antibody titer

[0279]

[0280] Note: OD values ​​are read; 450nm is specific absorption, and 620nm is non-specific absorption. Dilution concentrations from 1μg / ml to 0.015μg / ml are converted to the corresponding dilution ratios. Non-specific nm values ​​are removed by subtracting the absorbance at 620nm (OD620) from the absorbance at 450nm. Wells with OD values ​​higher than twice that of the blank control are considered positive. The dilution factor of the lightest-colored positive well represents the titer of the target antibody in the serum sample. Results show a titer greater than 1:128000.

[0281] Example 2: Antibody subtype detection results

[0282] Antibody subtypes were detected using a mouse monoclonal antibody subtype identification kit (Cat no: PK20002, https: / / www.ptgcn.com / products / Mouse-Monoclonal-Antibody-Isotyping-Kit-PK20002.htm). The results are shown in Table 6.

[0283] Table 6 Antibody Subtypes

[0284]

[0285] Example 3: SDS-PAGE electrophoresis analysis

[0286] The monkeypox-specific monoclonal antibody prepared in this invention was analyzed by SDS-PAGE electrophoresis using the method described in Example 1 (e.g., ...). Figure 3 The antibody showed distinct bands at 25kb and 55kb, with correct size and no extraneous bands. The full-length antibody was 250kb, with correct size and no extraneous bands. This indicates that the monkeypox-specific monoclonal antibody has high purity.

[0287] 1) The MPXV Antibody #5 showed distinct bands for its light and heavy chains at 25 KD and 55 KD respectively in lane 2 of the first gel (left), with correct size and no extraneous bands. The full-length antibody showed a distinct band at 250 KD in lane 6 of the first gel (left), with correct size and no extraneous bands, as expected.

[0288] 2) The light and heavy chains of the MPXV antibody #25 showed distinct bands at 25 KD and 55 KD respectively in lane 3 of the first gel (left). The full-length antibody showed a distinct band at 250 KD in lane 7 of the first gel (left), with the correct size and no extraneous bands, as expected.

[0289] 3) The light and heavy chains of antibody MPXV #28 showed distinct bands at 25 KD and 55 KD respectively in lane 4 of gel 1 (left). The full-length antibody showed a distinct band at 250 KD in lane 8 of gel 1 (left), which was of the correct size and free of extraneous bands, as expected.

[0290] 4) The light and heavy chains of antibody MPXV #87 showed distinct bands at 25 kDa and 55 kDa respectively in lane 5 of gel 1 (left). The full-length antibody showed a distinct band at 250 kB in lane 9 of gel 1 (left), with the correct size and no extraneous bands, as expected.

[0291] 5) The light and heavy chains of antibody MPXV #94 showed distinct bands at 25 KD and 55 KD respectively in lane 9 of the second gel (right). The full-length antibody showed a distinct band at 250 KD in lane 11 of the second gel (right), with the correct size and no extraneous bands, as expected.

[0292] 6) The light and heavy chains of the MPXV antibody #111 showed distinct bands at 25 KD and 55 KD respectively in lane 10 of the second gel (right), with correct size and no extraneous bands. The full-length antibody showed a distinct band at 250 KD in lane 12 of the second gel (right), with correct size and no extraneous bands, as expected.

[0293] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the A29L protein of monkeypox virus, characterized in that, the monoclonal antibody comprises one or more of MPXV Antibody #5, MPXV Antibody #25, MPXV Antibody #28, MPXV Antibody #87, MPXV Antibody #94, or MPXV Antibody #111; the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the MPXV Antibody #5 are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the MPXV Antibody #5 are shown in SEQ ID NO. 4, LVS, and SEQ ID NO. 5, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the MPXV Antibody #25 are shown in SEQ ID NO. 1, SEQ ID NO. 6, and SEQ ID NO. 3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the MPXV Antibody #25 are shown in SEQ ID NO. 7, LVS, and SEQ ID NO. 8, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the MPXV Antibody #28 are shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the MPXV Antibody #28 are shown in SEQ ID NO. 12, WAS, and SEQ ID NO. 13, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the MPXV Antibody #87 are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the MPXV Antibody #87 are shown in SEQ ID NO. 14, LVS, and SEQ ID NO. 5, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the MPXV Antibody #94 are shown in SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID NO. 17, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region of the MPXV Antibody #94 are shown in SEQ ID NO. 12, WAS, and SEQ ID NO. 18, respectively; The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the MPXV Antibody #111 are shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the MPXV Antibody #111 are shown in SEQ ID NO. 22, NAK and SEQ ID NO. 23, respectively.

2. The monoclonal antibody of claim 1, wherein, comprising: the amino acid sequence of the heavy chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO. 27; the amino acid sequence of the light chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO. 29; the amino acid sequence of the heavy chain variable region of the MPXV Antibody #25 is shown in SEQ ID NO. 31; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 33; the amino acid sequence of the heavy chain variable region of the MPXV Antibody #28 is shown in SEQ ID NO. 35; the amino acid sequence of the light chain variable region of the MPXV Antibody #28 is shown in SEQ ID NO. 37; the amino acid sequence of the heavy chain variable region of the MPXV Antibody #87 is shown in SEQ ID NO. 39; the amino acid sequence of the light chain variable region of the MPXV Antibody #87 is shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain variable region of the MPXV Antibody #94 is shown in SEQ ID NO. 43; the amino acid sequence of the light chain variable region of the MPXV Antibody #94 is shown in SEQ ID NO. 45; the amino acid sequence of the heavy chain variable region of the MPXV Antibody #111 is shown in SEQ ID NO. 47; the amino acid sequence of the light chain variable region of the MPXV Antibody #111 is shown in SEQ ID NO.

49.

3. Biomaterials characterized in that, comprising any one of: (I) a nucleic acid molecule encoding the monoclonal antibody according to claim 1 or 2; (II) an expression vector comprising the nucleic acid molecule according to (I); (III) a host comprising the expression vector according to (II).

4. The biomaterial of claim 3, wherein The nucleic acid molecule comprises: the nucleotide sequence of the heavy chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO. 26; the nucleotide sequence of the light chain variable region of the MPXV Antibody #5 is shown in SEQ ID NO. 28; the nucleotide sequence of the heavy chain variable region of the MPXV Antibody #25 is shown in SEQ ID NO. 30; the nucleotide sequence of the light chain variable region of the MPXV Antibody #25 is shown in SEQ ID NO. 32; The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #28 is shown as SEQ ID NO. 34; the nucleotide sequence of the light chain variable region of the MPXV Antibody #28 is shown as SEQ ID NO. 36; The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #87 is shown as SEQ ID NO. 38; the nucleotide sequence of the light chain variable region of the MPXV Antibody #87 is shown as SEQ ID NO. 40; The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #94 is shown as SEQ ID NO. 42; the nucleotide sequence of the light chain variable region of the MPXV Antibody #94 is shown as SEQ ID NO. 44; The nucleotide sequence of the heavy chain variable region of the MPXV Antibody #111 is shown as SEQ ID NO. 46; the nucleotide sequence of the light chain variable region of the MPXV Antibody #111 is shown as SEQ ID NO.

48.

5. Use of any one of: (I) the monoclonal antibody according to claim 1 or 2; (II) the biological material according to claim 3 or 4 in the manufacture of a reagent or kit for detecting monkeypox virus.

6. A reagent or kit for detecting monkeypox virus, characterized in that, comprising any one of: (I) the monoclonal antibody according to claim 1 or 2; (II) the biological material according to claim 3 or 4.

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