Monoclonal antibody targeting monkey pox virus A27L protein and application thereof

Abstract

The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a monoclonal antibody targeting monkey pox virus A27L protein and application of the monoclonal antibody. Specifically, the hybridoma cell strains are constructed by taking the monkey pox virus A27L protein as an immunogen, and monoclonal antibodies secreted by the cell strains have strong binding specificity with the monkey pox virus A27L protein, do not have cross reaction with other proteins, can be used for distinguishing monkey pox virus from other viruses, and can be used for detecting the monkey pox virus. The kit is used for early rapid and accurate detection of the monkey pox virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of monoclonal antibody preparation. Specifically relates to anti-monkeypox virus monoclonal antibody and its application. BACKGROUND

[0002] Monkeypox is a rare, sporadic, smallpox-like viral zoonosis caused by the poxvirus monkeypox virus (MPXV). There are two known different branches of monkeypox virus, respectively, the Congo Basin branch and the West African branch. The virus currently circulating belongs to the West African branch. It was mainly prevalent in Africa in the past, but in recent years, monkeypox cases have been found in many countries and regions around the world such as Europe, the Americas, and Australia, indicating that MPXV has occurred in human-to-human transmission. The global prevalence of monkeypox virus may pose a serious threat to public health.

[0003] At present, there is no specific treatment method for monkeypox, no specific MPXV vaccine, and it is a self-limiting disease. Therefore, it is urgent to develop effective prevention and control measures to prevent the spread of monkeypox. However, it is difficult to distinguish the clinical symptoms caused by monkeypox virus and other poxviruses, and differential diagnosis is difficult, so it is essential to develop a rapid and specific diagnostic method for early and accurate detection of MPXV. SUMMARY

[0004] On the basis of the prior art, the present application found that A27L is a candidate target gene for MPXV nucleic acid detection, and the conserved region of A27L gene is used as a target (Genebank: NP_536566.1). It is well amplified in different MPXV virus branches, and has no cross reaction with other pathogens, which can clearly distinguish monkeypox virus from other viruses. Therefore, the present application prepares anti-MPXV monoclonal antibody by constructing effective MPXV-A27L protein, and prepares MPXV antigen diagnostic kit based on the monoclonal antibody for early and accurate detection of MPXV.

[0005] In this regard, the technical solution of the present application includes but is not limited to the following: In one aspect, the present application provides a monoclonal antibody or antigen-binding fragment thereof that binds to monkeypox virus A27L protein, said monoclonal antibody or antigen-binding fragment thereof comprising: (1) Heavy chain CDR1-3: the amino acid sequences are respectively shown as GFTFSNYT, FSRDGYNT and AGGDFEV, and light chain CDR1-3: the amino acid sequences are respectively shown as QSLLYSSNQKNY, WAS and QQYYTYPLT; or (2) Heavy chain CDR1-3: amino acid sequences are shown as GFNIKDYF, IDPEDGES and TTLYGRGLDC, respectively, and light chain CDR1-3: amino acid sequences are shown as QNLLHSNGNTY, KVS and FQGSLVPWT, respectively.

[0006] In one aspect, the monoclonal antibody or its antigen-binding fragment according to the present invention comprises: (1) A heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 12; and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 13; or (2) Heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 14; and / or, light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 15.

[0007] In one aspect, the monoclonal antibody or its antigen-binding fragment according to the present invention comprises: (1) A heavy chain containing the amino acid sequence shown in SEQ ID NO: 5, and / or a light chain containing the amino acid sequence shown in SEQ ID NO: 7; or (2) Heavy chain containing the amino acid sequence shown in SEQ ID NO: 9, and / or light chain containing the amino acid sequence shown in SEQ ID NO: 11.

[0008] In one aspect, the amino acid sequence of the monkeypox virus A27L protein of the present invention is shown in Genebank accession number NP_536566.1.

[0009] In one aspect, the CDR described in this invention is defined according to the IMGT numbering scheme.

[0010] In one aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof described in the present invention.

[0011] In one aspect, the polynucleotide of the present invention comprises: (1) A heavy chain coding sequence comprising the sequence shown in SEQ ID NO: 4, and / or a light chain coding sequence comprising the sequence shown in SEQ ID NO: 6; or (2) A heavy chain coding sequence comprising the sequence shown in SEQ ID NO: 8, and / or a light chain coding sequence comprising the sequence shown in SEQ ID NO: 10.

[0012] In one aspect, the present invention provides an expression vector comprising the polynucleotides described herein.

[0013] In one aspect, the present invention provides an engineered host cell comprising the polynucleotide or expression vector described in the present invention.

[0014] In one aspect, the present invention provides the use of the monoclonal antibody described herein or its antigen-binding fragment, polynucleotide, expression vector or engineered host cell in the preparation of a kit for detecting monkeypox virus A27L protein and / or monkeypox virus.

[0015] In one aspect, the present invention provides a kit for detecting monkeypox virus A27L protein or monkeypox virus, characterized in that the kit comprises the monoclonal antibody or its antigen-binding fragment described in the present invention, a polynucleotide, an expression vector, or an engineered host cell.

[0016] The beneficial effects of the present invention include, but are not limited to, the following: This invention uses monkeypox virus A27L protein as an immunogen to construct a hybridoma cell line. The monoclonal antibodies secreted by this cell line (especially 3-5H and 2-1G antibodies) have strong binding specificity to monkeypox virus A27L protein and no cross-reaction with other proteins. This can be used to distinguish monkeypox virus from other viruses and to enable early, rapid and accurate detection of monkeypox virus. Attached Figure Description

[0017] Figure 1 This shows a plasmid map that induces MPXV-A27L protein expression.

[0018] Figure 2 The image shows the SDS-PAGE electrophoresis results of MPXV-A27L protein induced expression and purified MPXV-A27L protein. Wherein, M: protein molecular weight standard; 1: pCold III-MPXV-A27L recombinant protein supernatant for column flow-through; 2: pCold III-MPXV-A27L recombinant protein 20mM / L imidazole washing buffer; 3: pCold III-MPXV-A27L recombinant protein 50mM / L imidazole washing buffer; 4-9: pCold III-MPXV-A27L recombinant protein 200mM / L imidazole elution buffer.

[0019] Figure 3 This shows the serum titer of monkeypox virus A27L monoclonal antibody.

[0020] Figure 4 This shows the titer of A27L ascites. Item 1: Ascites prepared from A27L monoclonal antibody strain A1; Item 2: Ascites prepared from A27L monoclonal antibody strain C2; Item 3: Ascites prepared from A27L monoclonal antibody strain E1; Positive: Positive serum from immunized mice; Negative: PBS.

[0021] Figure 5 Western blot identification of MPXV-A27L monoclonal antibody. Wherein, M: protein molecular weight standard; 1: porcine epidemic diarrhea virus nucleocapsid protein (PEDV N)-His recombinant protein; 2: MPXV-F3L-His recombinant protein; 3: other proteins of MPXV-A27L-His recombinant protein.

[0022] Figure 6 This diagram shows the heavy and light chain CDRs and variable region sequence structure of antibody 3-5H secreted by hybridoma A1 cell line.

[0023] Figure 7 This diagram shows the heavy and light chain CDRs and variable region sequence structure of antibody 2-1G secreted by hybridoma C2 cell line. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional channels, such as ordinary biochemical reagent stores.

[0025] Example 1: Construction of prokaryotic expression plasmid for MPXV-A27L protein Based on the West African branch monkeypox genome sequence published in GenBank, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the MPXV-A27L-His target gene (SEQ ID NO: 3) after codon optimization according to the E. coli host. Specific primers (SEQ ID NO: 1 and 2) were designed, and the designed upstream and downstream primers each carried approximately 15-20 bp homologous arms of the pCold III vector. The MPXV-A27L-His gene was cloned into the prokaryotic expression vector pCold III using the homologous recombinase 2×Basic Assembly Mix of Total Gold. The pCold III-A27L recombinant plasmid was obtained and transformed into E. coli competent cells DH5α. After plasmid sequencing verification, the recombinant expression strain carrying the MPXV-A27L gene was obtained by transforming it into prokaryotic expression strain BL21.

[0026] Example 2 Expression and purification of MPXV-A27L protein (1) The recombinant expression bacteria constructed in Example 1 were activated and transferred to LB liquid medium containing ampicillin and cultured overnight in a shaker (37 ℃, 220 rpm). The bacterial culture was then transferred to new LB medium containing ampicillin at a volume ratio of 1:100 and cultured in a shaker until the OD600 value was ≈0.8. After cooling at room temperature for 30 min, IPTG was added to a final concentration of 0.5 mM and expression was induced at 16 ℃ and 220 rpm for 24 h. The bacterial cells were collected by centrifugation at 4 ℃ and 8000 rpm for 10 min. The bacterial cells were washed twice with PBS and resuspended with 1 / 10 volume of the original bacterial culture lysis buffer. The bacterial culture was sonicated at 200 W for 3 s and rest for 6 s until the bacterial culture was clear. The supernatant and precipitate were separated by centrifugation at 4 ℃ and 12000 rpm for 20 min. 100 μL of the supernatant was taken for SDS-PAGE electrophoresis to verify the protein expression. The results showed that the MPXV-A27L recombinant protein (approximately 13.6 kDa) could be expressed in soluble form in E. coli BL21, as shown in the following figures. Figure 2 As shown.

[0027] (2) Purification of the supernatant obtained in (1) using an HIS-tagged nickel affinity chromatography column. The protein supernatant was added to an HIS-tagged nickel affinity chromatography column and incubated overnight at 4 °C; impurities were eluted with 20 and 50 mM imidazole washing buffer, and the target protein was eluted with 200 mM; the eluted protein was high-purity MPXV-A27L protein obtained by ultrafiltration and centrifugation to remove imidazole. The protein purification status was detected by SDS-PAGE, and the results showed that MPXV-A27L-HIS recombinant protein could be effectively purified. The results are as follows. Figure 2 As shown.

[0028] Example 3: Preparation of MPXV-A27L protein monoclonal antibody (1) Mouse immunization Purified MPXV-A27L protein (50 µg / mouse) was added to an equal volume of Freund's adjuvant (CFA for primary immunization, IFA for secondary immunization), emulsified, and injected subcutaneously at multiple sites into two 8-week-old female 0BALB / c mice. Mice injected with PBS emulsified with an equal volume of adjuvant served as controls. Immunization was repeated every two weeks for a total of three immunizations. Furthermore, to ensure immunization efficacy, a booster immunization of MPXV-A27L protein (100 µg / mouse) was administered intraperitoneally three days before fusion. Serum titers were measured from immunized mice.

[0029] (2) Detection of antibody titer using indirect ELISA method MPXV-A27L protein was diluted to 0.5 µg / mL using coating buffer, and 100 µL / well was added to a 96-well microplate. The plate was incubated overnight at 4°C, washed three times with PBST, and then blotted dry. 200 µL / well of freshly prepared 5% skim milk powder was added, and the plate was blocked at 37°C for 2 h. The plate was then blotted dry and stored at 4°C for later use. Mouse serum samples were processed and added to 96-well microplates, with a negative serum control included. The plates were incubated at 37°C for 1 h, washed three times with PBST, and then blotted dry. Goat anti-mouse IgG-HRP was diluted 1:5000 with PBS, and 100 µL / well was added to a 96-well microplate. The plates were incubated at 37°C for 1 h, washed five times with PBST, and then blotted dry.

[0030] Add 100 μL of TMB enzyme substrate reaction solution to each well and incubate at room temperature for 5–8 min. Add 100 μL of 2 M H₂SO₄ stop solution to each well. Immediately after adding the stop solution, use a microplate reader to detect the OD of each well. 450 Value, record the data. If the OD of the hole to be measured... 450 A P / N value greater than or equal to 2.1 times that of the negative control (i.e., P / N value > 2.1) is considered a positive result, confirming the presence of anti-MPXV-A27L protein antibody in mouse serum. Additionally, serum titers were measured in two mice, and the results are as follows: Figure 3 As shown.

[0031] (3) Hybridoma cell preparation The day before fusion, a healthy, unvaccinated Kunming mouse was euthanized by cervical indwelling, and feeder cells were added to a 96-well plate. Buffed mice were euthanized by enucleation to remove blood, and serum was collected as a positive control. The mouse spleen was removed and placed on a sterile dish. An appropriate amount of DMEM medium was drawn up using a syringe, and the spleen was repeatedly defrosted to prepare a single-cell suspension. The spleen cell suspension and SP2 / 0 cells were then fused at a 5:1 ratio. The cells were gently resuspended in HAT selective medium and added at 100 μL / well to the feeder cells prepared the day before. The wells were then incubated at 37 ℃ in a 5% CO2 incubator. Cell status was observed approximately 4 days later.

[0032] (4) Subcloning screening When the hybridoma cells reached 30%, positive wells with strong antibody secretion ability were screened out by indirect ELISA. Subcloning was performed by limiting dilution. After 6 subclonings, 3 stable antibody-secreting cell lines were obtained, namely A1, C2 and E1.

[0033] (5) Large-scale preparation of monoclonal antibodies MPXV-A27L protein mAb was prepared in large quantities using an in vivo ascites induction method. Two weeks prior to preparation, 1 mL of ascites adjuvant was intraperitoneally injected into BALB / c multiparous female mice. Hybridoma cell lines A1, C2, and E1, which were in a logarithmic growth phase, were resuspended and counted. Each female mouse was then intraperitoneally injected with 1 x 102 cells. 6 Cells. Ascites fluid was collected and purified approximately 10 days later. Indirect ELISA showed that the titer of purified ascites fluid induced by A1 and C2 hybridoma cell lines was >1 / 256000 (see [link to ELISA]). Figure 4 The monoclonal antibody subclass was identified as IgG1 using a monoclonal antibody detection kit (Biodragon).

[0034] Example 4: Monoclonal Antibody Identification and Sequencing MPXV-A27L protein was subjected to SDS-PAGE electrophoresis. High-titer antibodies from the ascites fluid corresponding to hybridomas of strains A1 and C2 were selected as primary antibodies, diluted 1:4000, and incubated overnight at 4 °C. Then, incubation and color development were performed using HRP-labeled mouse secondary antibody. The color development results are shown below. Figure 5 The results show that the A1 hybridoma only binds to the MPXV-A27L protein, and does not bind to the MPXV-F3L protein, porcine epidemic diarrhea virus nucleocapsid protein, or HIS tag, demonstrating good specificity.

[0035] The A1 monoclonal cell line was sent to a sequencing company for sequencing, and the gene sequences of the heavy and light chains of the A27L monoclonal antibody (3-5H) were obtained as shown in SEQ ID NO: 4 and SEQ ID NO: 6, and the corresponding amino acid sequences of the heavy and light chains are shown in SEQ ID NO: 5 and SEQ ID NO: 7. Figure 6 As shown, further analysis revealed that the amino acid sequences of CDR1-3 in the heavy chain variable region of the 3-5H antibody are GFTFSNYT, FSRDGYNT, and AGGDFEV, respectively; and the amino acid sequences of CDR1-3 in the light chain variable region are QSLLYSSNQKNY, WAS, and QQYYTYPLT, respectively (according to the IMGT numbering scheme). The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0036] The C2 monoclonal cell line was sent to a sequencing company for sequencing, and the gene sequences of the heavy and light chains of the anti-A27L monoclonal antibody (2-1G) were obtained as shown in SEQ ID NO: 8 and SEQ ID NO: 10, and the corresponding amino acid sequences of the heavy and light chains are shown in SEQ ID NO: 9 and SEQ ID NO: 11. Figure 7As shown, further analysis revealed that the amino acid sequences of CDR1-3 in the heavy chain variable region of the 2-1G antibody are: GFNIKDYF, IDPEDGES, and TTLYGRGLDC, respectively; and the amino acid sequences of CDR1-3 in the light chain variable region are: QNLLHSNGNTY, KVS, and FQGSLVPWT, respectively (according to the IMGT numbering scheme). The amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0037] sequence list The primer pairs (including homologous arms) for amplifying the MPXV-A27L gene in SEQ ID NO.1 and 2 are as follows: Forward primer: AATCACAAAGTGCATATGATGGATGGCACCCTGTTTC (SEQ ID NO.1) Reverse primer: AGACTGCAGGTCGACAAGCTTTTATTCATACGGGCGGCGG (SEQ ID NO.2) SEQ ID NO.3 MPXV-A27L-His target gene: ATGGATGGCACCCTGTTTCCGGGCGATGACGATCTGGCGATTCCGGCGACCGAATTTTTAGCACCAAAGCGGCGAAAAACCCGGAAACCAAACGCGAAGCGATTGTGAAAGCGTATGGCGATGATAACGAAGAAACCCTGAAACAGCGCCTGACCAACCTGGAAAAAAAAAAATTACCA ACATTACCACCAAATTTGAACAGATTGAAAAATGCTGCAAACATAACGATGAAGTGCTGTTTCGCCTGGAAAACCATGCGGAAACCCTGCGCGCGGCGATGATTAGCTGGCGAAAAAAATTGATGTGCAGACCGGCCGCCGCCCGTATGAActcgagcaccaccaccaccaccactga The coding sequence of the antibody heavy chain SEQ ID NO: 4 3-5H: SEQ ID NO: 5 The amino acid sequence of the 3-5H antibody heavy chain, with the underlined portion representing the variable region sequence of the heavy chain: EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYTLSWVRQTPAKRLEWVATFSRDGYNTYYSDNMKGRFT ISRDTARNTLYLQMSSLRSEDTAMYYCA GGDFEVWGTGTTVTVSSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK The coding sequence of the antibody light chain SEQ ID NO: 6 3-5H: GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATACCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGATGCTCAGCCATCTGTCTTTCTCTTCCAACCATCTCTGGACGAGTTACATACAGGAAGTGCCTCTATCGTGTGCATATTGAATGACTTCTACCCCAAAGAGGTCAATGTCAAGTGGAAAGTGGATGGCGTAGTCCAAAACAAAGGCATCCAGGAGAGCACCACAGAGCAGAACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACGGAGTACCAAAGTCATGAAAAGTTCTCCTGCGAGGTCACTCACAAGAGCCTGGCCTCCACCCTCGTCAAGAGCTTCAACAGGAGCGAGTGTCAGAGAGAGTA Amino acid sequence of the light chain of the 3-5H antibody. The underlined part is the light chain variable region sequence: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFT GSGSGTDFTLTISSVKAEDLAVYYCQQYYTY PLTFGAGTKLELKDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE Coding sequence of the heavy chain of the 2-1G antibody: The amino acid sequence of the antibody heavy chain of SEQ ID NO: 9 2-1G, with the underlined portion representing the variable region sequence of the heavy chain: EVQLRQSGADLVRPGAAVKLSCTASGFNIKDYFIHWVKQRPEQGLEWIGKIDPEDGESEYVPKFQNKAT VTADTSSNTAYLHLSSLTSEDTAVYYCTTLYGRGLDCWGQGTSVTVSS MVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLMITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQ FNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK The coding sequence of the antibody light chain SEQ ID NO: 10 2-1G: GATCTTTTGCTGACCCAAACTCCGCTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTACATCTCTTGTACCTCTAATCAGAATCTTTTACATAGTAATGGAAACACCTATTTAGACTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCTTCCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACACATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTATTGCTTTCAAGGTTCACTTGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGATGCTCAGCCATCTGTCTTTCTCTTCCAACCATCTCTGGACGAGTTACATACAGGAAGTGCCTCTATCGTGTGCATATTGAATGACTTCTACCCCAAAGAGGTCAATGTCAAGTGGAAAGTGGATGGCGTAGTCCAAAACAAAGGCATCCAGGAGAGCACCACAGAGCAGAACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACGGAGTACCAAAGTCATGAAAAGTTCTCCTGCGAGGTCACTCACAAGAGCCTGGCCTCCACCCTCGTCAAGAGCTTCAACAGGAGCGAGTGTCAGAGAGAGTA Amino acid sequence of the light chain of the 2-1G antibody, with the underlined part being the light chain variable region sequence: DLLLTQTPLSLPVSLGDQAYISCTSNQNLLHSNGNTYLDWYLQKPGQSPKLLIYKVSFRFSGVPDRFSG SGSGTHFTLKISRVEAEDLGVYYCFQGSLVPWTFGGGTKLEIK ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 12 Heavy chain variable region of the 3-5H antibody EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYTLSWVRQTPAKRLEWVATFSRDGYNTYYSDNMKGRFTISRDTARNTLYLQMSSLRSEDTAMYYCA SEQ ID NO: 13 Variable region of the light chain of 3-5H antibody DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTY SEQ ID NO: 14 Variable region of the heavy chain of 2-1G antibody EVQLRQSGADLVRPGAAVKLSCTASGFNIKDYFIHWVKQRPEQGLEWIGKIDPEDGESEYVPKFQNKATVTADTSSNTAYLHLSSLTSEDTAVYYCTTLYGRGLDCWGQGTSVTVSS SEQ ID NO: 15 Variable region of the light chain of 2-1G antibody DLLLTQTPLSLPVSLGDQAYISCTSNQNLLHSNGNTYLDWYLQKPGQSPKLLIYKVSFRFSGVPDRFSGSGSGTHFTLKISRVEAEDLGVYYCFQGSLVPWTFGGGTKLEIK

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that binds to a monkeypox virus A27L protein, characterized in that, The monoclonal antibody or antigen-binding fragment thereof comprises: (1) heavy chain CDR1-3: amino acid sequences are shown as GFTFSNYT, FSRDGYNT and AGGDFEV, respectively, and light chain CDR1-3: amino acid sequences are shown as QSLLYSSNQKNY, WAS and QQYYTYPLT, respectively; or (2) heavy chain CDR1-3: amino acid sequences are shown as GFNIKDYF, IDPEDGES and TTLYGRGLDC, respectively, and light chain CDR1-3: amino acid sequences are shown as QNLLHSNGNTY, KVS and FQGSLVPWT, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 12; and / or, a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13; or (2) a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 14; and / or, a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:

15.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 5, and / or, a light chain comprising an amino acid sequence as shown in SEQ ID NO: 7; or (2) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 9, and / or, a light chain comprising an amino acid sequence as shown in SEQ ID NO:

11.

4. A polynucleotide, comprising: The polynucleotide encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3.

5. An expression vector, characterized by, The expression vector comprises the polynucleotide according to claim 4.

6. An engineered host cell, characterized in that, The engineered host cell comprises the polynucleotide according to claim 4 or the expression vector according to claim 5.

7. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, the polynucleotide according to claim 4, the expression vector according to claim 5 or the engineered host cell according to claim 6 in the preparation of a kit for detecting or titrating monkeypox virus A27L protein.

8. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, the polynucleotide according to claim 4, the expression vector according to claim 5 or the engineered host cell according to claim 6 in the preparation of a kit for detecting monkeypox virus.

9. A kit for detecting or titrating monkeypox virus A27L protein, characterized in that, The kit comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, the polynucleotide according to claim 4, the expression vector according to claim 5 or the engineered host cell according to claim 6.

10. A kit for detecting monkeypox virus, characterized by, The kit comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, the polynucleotide according to claim 4, the expression vector according to claim 5 or the engineered host cell according to claim 6.

Images