Monoclonal antibody for specifically recognizing epidemic hemorrhagic disease virus VP7 protein and application thereof

Abstract

The invention belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a monoclonal antibody for specifically recognizing epidemic hemorrhagic disease virus VP7 protein and application of the monoclonal antibody. According to the invention, a monoclonal antibody 5E11 for identifying EHDV (epidemic hemorrhagic disease virus) VP7 protein is prepared by a hybridoma technology; the monoclonal antibody 5E11 can recognize EHDV VP7 protein expressed by escherichia coli and can also be specifically combined with recombinant EHDV VP7 protein expressed in BHK-21 cells, and meanwhile, the monoclonal antibody does not react with recombinant bluetongue virus VP7 protein and recombinant African horse pestivirus VP7 protein; the monoclonal antibody 5E11 is used as a competitive antibody, a competitive ELISA antibody detection method for the epidemic hemorrhagic disease virus is established, and the monoclonal antibody 5E11 has important significance in diagnosis, prevention and control of EHDV.

Description

Technical Field

[0001] The present invention belongs to the technical fields of immunology and in vitro diagnosis, and particularly relates to a monoclonal antibody that recognizes the VP7 protein of epizootic hemorrhagic disease virus and its uses. Background Art

[0002] Epizootic haemorrhagic disease (EHD) is an arthropod-borne disease caused by epizootic haemorrhagic disease virus (EHDV), which is transmitted through the blood-sucking bites of Culicoides. It infects various ruminants such as cattle, sheep, and deer, seriously affecting the healthy development of the world's livestock industry. The clinical symptoms of this disease include fever, oral and nasal erosions, lameness, abortion, subcutaneous edema, and congestion in the mucosal area and hairless areas of the skin. Pathological changes include muscle degeneration in the larynx, pharynx, and esophagus, bleeding in the heart, lungs, and major blood vessels, as well as congestion and erosion in the abomasum. At least 9 serotypes of EHDV have been discovered, including serotypes 1, 2, 4, 5, 6, 7, 8, 9, and 10, and there is no cross-immune protection among different serotypes. Currently, EHDV is distributed between 35° south latitude and 49° north latitude, including parts of the Americas, Africa, Asia, the Middle East, Europe, and Oceania. In 2008, EHD was included in the list of notifiable animal diseases by the World Organization of Animal Health (WOAH).

[0003] EHDV belongs to the genus Orbivirus of the family Reoviridae and is similar to bluetongue virus and African horse sickness virus in the same genus. It has an icosahedral capsid structure with a diameter of about 80 nm. Its structure is divided into 3 consecutive protein layers: the outer capsid, the inner capsid, and the virus core. The genome is 10 segmented linear double-stranded RNAs, encoding 7 structural proteins (VP1, VP2, VP3, VP4, VP5, VP6, and VP7) and 5 non-structural proteins (NS1, NS2, NS3 / NS3A, NS4, and NS5). The virus core contains an RNA polymerase complex composed of VP1 (RNA polymerase), VP4 (capping enzyme), and VP6 (helicase) proteins. The inner capsid is composed of the structural proteins VP7 and VP3, and the outer capsid is composed of the structural proteins VP2 and VP5. The 5 non-structural proteins are mainly responsible for virus infection and replication and play important roles in intracellular transport, virus packaging and release, and anti-host immune responses. The VP7 protein is the most highly expressed structural protein in EHDV and is highly conserved among different serotypes. It is the main serogroup-specific antigen of EHDV and has important application value in the establishment of diagnostic methods and the development of vaccines for this disease.

[0004] The laboratory diagnostic techniques for EHDV mainly include virus isolation, neutralization test, agar gel immunodiffusion test (AGID), competitive enzyme-linked immunosorbent assay (C-ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR (qPCR), etc. Among them, C-ELISA is the EHDV antibody detection method preferentially recommended by the World Organization for Animal Health (OIE). Summary of the Invention

[0005] In view of the above technical problems, the present invention successfully expressed the recombinant EHDV VP7 protein using the Escherichia coli expression system. After purification, mice were immunized, and monoclonal antibodies against the VP7 protein with good specificity were successfully prepared by hybridoma technology, providing a basis for in-depth study of the biological function of EHDV VP7 protein and the development of EHD prevention and control technologies. The specific contents are as follows:

[0006] In the first aspect, the present invention provides a monoclonal antibody that specifically recognizes the VP7 protein of epizootic hemorrhagic disease virus, and the monoclonal antibody includes an antibody heavy chain and an antibody light chain;

[0007] The variable region CDRs of the antibody heavy chain include CDR1 with the amino acid sequence shown in SEQ ID NO.5, CDR2 with the amino acid sequence shown in SEQ ID NO.6, and CDR3 with the amino acid sequence shown in SEQ ID NO.7;

[0008] The variable region CDRs of the antibody light chain include CDR1 with the amino acid sequence shown in SEQ ID NO.8, CDR2 with the amino acid sequence shown in SEQ ID NO.9, and CDR3 with the amino acid sequence shown in SEQ ID NO.10.

[0009] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is as shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the antibody light chain is as shown in SEQ ID NO.4.

[0010] Preferably, the nucleic acid includes the sequences shown in SEQ ID NO.1 and SEQ ID NO.3.

[0011] In the second aspect, the present invention provides a nucleic acid that encodes the light chain and heavy chain of the monoclonal antibody described in the first aspect above.

[0012] In a third aspect, the present invention provides a recombinant vector, which contains the nucleic acid described in the second aspect above.

[0013] In a fourth aspect, the present invention provides a recombinant cell, which contains the recombinant vector described in the third aspect above.

[0014] In a fifth aspect, the present invention provides an immunoconjugate, which comprises:

[0015] (i) the monoclonal antibody described in the first aspect above;

[0016] (ii) and a coupling moiety selected from the group consisting of:

[0017] a detectable label, a drug, gold nanoparticles / nanorods, magnetic nanoparticles, a virus capsid protein or VLP, or a combination thereof.

[0018] In an eighth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect above in the in vitro detection of epidemic hemorrhagic disease virus for non-disease diagnosis purposes.

[0019] In a seventh aspect, the present invention provides the use of the monoclonal antibody described in the first aspect above in the preparation of a test strip or kit for detecting epidemic hemorrhagic disease virus.

[0020] In an eighth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect above in the in vitro detection of epidemic hemorrhagic disease virus for non-disease diagnosis purposes.

[0021] In a ninth aspect, the present invention provides an ELISA detection kit for epidemic hemorrhagic disease virus, which comprises the monoclonal antibody described in the first aspect above.

[0022] Preferably, the kit further comprises an enzyme-linked immunosorbent assay (ELISA) plate, a blocking solution, a dilution solution, an enzyme-labeled secondary antibody, a washing solution, a chromogenic agent, and a termination solution.

[0023] The beneficial effects of the present invention are as follows: The EHDV VP7 gene was artificially synthesized by codon optimization with reference to the published gene sequences in GenBank. This gene was cloned into an expression plasmid to construct the recombinant plasmid pET-EHDV VP7. The recombinant plasmid was transformed into competent Escherichia coli cells, induced for expression with IPTG, and the expression conditions were optimized. The expressed VP7 protein was purified by Ni-NTA affinity chromatography and dialyzed and refolded with different concentrations of imidazole. The refolded VP7 protein was used to immunize BALB / c mice, and monoclonal antibodies against the VP7 protein were obtained by hybridoma technology. The monoclonal antibodies can not only react with the recombinant EHDV VP7 protein expressed in Escherichia coli, but also bind to the recombinant EHDV VP7 protein expressed in eukaryotic cells. At the same time, they do not react with the recombinant bluetongue virus (BTV) VP7 protein and the recombinant African horse sickness virus (AHSV) VP7 protein, showing good specificity, which lays a foundation for studying the structural function of the EHDV VP7 protein and establishing diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Results of inducing the expression of EHDV VP7 protein at 37°C. Among them, M is the protein molecular weight standard, 1 is the uninduced bacterial liquid sample, and 2-5 are the bacterial liquid samples induced for 2 h, 4 h, 6 h, and 8 h, respectively.

[0025] Figure 2 Results of purification and refolding of EHDV VP7 protein; among them, M is the protein molecular weight standard, and 1-3 are the samples after the 1st-3rd imidazole elution, respectively.

[0026] Figure 3 Western-blot identification results of the refolded EHDV VP7 protein. Among them, M is the protein molecular weight standard, and 1-4 are the samples after the 1st-4th imidazole elution, respectively.

[0027] Figure 4 Western blot identification results of monoclonal antibody 5E11; M is the protein molecular weight standard, 1 is the recombinant EHDV VP7 protein, 2 is the recombinant BTV VP7 protein, and 3 is the recombinant AHSV VP7 protein.

[0028] Figure 5 Results of cellular immunofluorescence identification of monoclonal antibodies. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] Embodiments of the present invention will be described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. Additionally, if not explicitly stated, all reagents used in the following embodiments are commercially available or can be synthesized according to the text or known methods. For reaction conditions not listed, they are easily obtained by those skilled in the art.

[0030] Cells, main reagents and experimental animals involved in the following experiments:

[0031] BHK-21 cells, mouse myeloma cells (SP2 / 0), recombinant BTV VP7 protein and recombinant AHSV VP7 protein expressed and purified by Escherichia coli, goat anti-EHDV positive serum, EHDV negative serum control, and EHDV positive serum control are all stored in our laboratory; E. coli BL21(DE3) competent cells and isopropyl-β-D-thiogalactoside (IPTG) are both purchased from Takara Bio Inc. (Dalian); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, HRP-labeled rabbit anti-goat IgG, and goat anti-mouse IgG (Alexa Flour 488) are all purchased from Abcam; kanamycin, Triton X-100, and bovine serum albumin (BSA) are all purchased from Solarbio; Lipofectamine 2000 Reagent, Dulbecco's Modified Eagle Medium (DMEM) medium, fetal bovine serum (FBS), and protein quantification kit are all purchased from Thermo Fisher Scientific; NP-40 lysis buffer is purchased from Beyotime; SuperKine TM Ultra-sensitive ECL chemiluminescence solution is purchased from Abbkine; transfer membrane (PVDF) is purchased from MERCK; Ni-NTA Agarose is purchased from QIAGEN; Freund's adjuvant and Freund's incomplete adjuvant are both purchased from Sigma; the experimental animals are female BALB / c mice at about 8 weeks old.

[0032] Example 1 Preparation of monoclonal antibody 5E11

[0033] 1. Design and codon optimization of EHDV VP7 gene

[0034] Referring to the published full-length gene sequence of EHDV VP7 in GenBank (accession number: AM744983.1), the codons of the VP7 gene were optimized according to the codon bias of Escherichia coli and eukaryotic cell gene expression. It was synthesized by Wuhan Kingcare Bioengineering Co., Ltd. and cloned into plasmid vectors pET-28 and pcDNA3.1 / myc-His, named pET-EHDV VP7 and pcDNA3.1 / myc-His-EHDV VP7 respectively.

[0035] 2. Induced expression of VP7 protein

[0036] The recombinant plasmid pET-EHDV VP7 was transformed into E. coli BL21(DE3), and the bacterial liquid was expanded. When the OD of the bacterial liquid 600 reached 0.6, the bacterial liquid before induction was used as a control. IPTG inducer was added to the LB medium with a final concentration of 0.5 mmol / L, and VP7 protein was induced to express at 37 °C. Bacterial liquid samples of the non-induced bacterial liquid and the bacterial liquid at 2, 4, 6, and 8 hours after induction were taken respectively. After centrifugation at 10,000 r / min, the supernatant was discarded, and the precipitate samples were resuspended with PBS to observe and analyze the expression of VP7 protein.

[0037] The results are as Figure 1 shown. The recombinant EHDV VP7 protein was successfully expressed, and its molecular weight was approximately 40 kDa; the size of the target protein was consistent with the expected size.

[0038] 3. Purification and refolding of VP7 protein

[0039] After a large amount of induced expression of the bacteria, the bacterial cell precipitate was collected and ultrasonically broken. The precipitate was resuspended with PBS, and the target protein was purified according to the Ni-NTA Agarose instruction manual. 25 column volumes of binding buffer were used to remove the miscellaneous proteins, and the target protein was eluted with elution buffer. Then, the purified target protein was dialyzed and concentrated at 4 °C, and the target protein solution in the dialysis bag was filtered through a 0.22 μm filter membrane. After filtration, it was bound to the Ni-NTA affinity column again, and miscellaneous proteins were eluted successively with low-concentration imidazole (20 mmol / L, 60 mmol / L, 200 mmol / L), and the target protein was eluted with 500 mmol / L imidazole, and the concentration was measured. The results of protein purification and refolding were analyzed by SDS-PAGE. Finally, goat anti-EHDV positive serum and HRP-labeled rabbit anti-goat IgG were used as the primary antibody and the secondary antibody respectively for protein immunoblot analysis.

[0040] Purify and refold the VP7 protein in the precipitated sample after ultrasonic lysis of the induced expression at 37°C for 8 h according to the operation instructions of Ni-NTA Agarose affinity chromatography. The protein solution always remained clear and transparent, without obvious precipitation, and no protein denaturation and aggregation occurred. The SDS-PAGE results are as Figure 2 shown. A highly pure recombinant EHDV VP7 protein was obtained through purification and refolding, and the refolded VP7 protein could be recognized by goat anti-EHDV positive serum ( Figure 3 shown).

[0041] 4. Immunization of mice with VP7 protein

[0042] Select 6 BALB / c mice at about 8 weeks old. Use the purified and refolded VP7 protein as the antigen, and immunize each mouse with 50 μg. Mix the antigen and Freund's complete adjuvant in the same volume and emulsify until the water-in-oil phase does not separate. Immunize the mice by subcutaneous multi-point injection. For subsequent immunizations, emulsify Freund's incomplete adjuvant with the same dose of VP7 protein and perform 3 booster immunizations at intervals of 2 weeks. Collect blood from the tail vein 7 days after the last immunization, separate the serum and measure the antibody titer. Use the purified and refolded VP7 protein as the coating antigen, the serum of the immunized mice as the primary antibody, and HRP-labeled goat anti-mouse IgG as the secondary antibody to perform indirect ELISA. Measure the D 450 value with an enzyme-linked immunosorbent assay reader, and detect the dilution factor when the sample D 450 / negative control D 450 ≥ 2.1 as the antibody titer, and select the mouse with the highest antibody titer to prepare monoclonal antibodies.

[0043] 5. Preparation of monoclonal antibodies

[0044] Isolate the spleen cells of the immunized mice, grind them into a single-cell suspension, and fuse them with SP2 / 0 cells in the logarithmic growth phase. Measure the cell supernatant after fusion by indirect enzyme-linked immunosorbent assay, screen out the hybridoma positive cells, perform clonal culture by the limiting dilution method, screen out the positive cell line that stably secretes monoclonal antibodies against the VP7 protein, and perform clonal amplification. Finally, inject liquid paraffin oil intraperitoneally into BALB / c mice, inoculate the hybridoma positive cells on the 8th day, and the mouse's abdomen will swell on the 20th day, then the ascites can be collected. The antibody titer detection results of the obtained monoclonal antibody 5E11 are shown in Table 1, and it has good reactivity and specificity with the EHDV VP7 protein.

[0045] Table 1 Antibody titer detection results

[0046] Monoclonal antibody 5E11 Negative control 3.5083 0.3732

[0047] 6. Western-blot identification

[0048] The results are as Figure 4The results showed that the purified monoclonal antibody was verified by Western blot. Samples prepared with recombinant EHDV VP7 protein, BTV VP7 protein, and AHSV VP7 protein expressed by Escherichia coli were used for Western-blot identification. The results indicated that the monoclonal antibody 5E11 prepared in the present invention could specifically react with EHDV VP7 protein, demonstrating good specificity.

[0049] 7. Immunofluorescence analysis

[0050] The recombinant plasmid pcDNA3.1 / myc-His-EHDV VP7 was transfected into BHK-21 cells (80% - 90% confluence). The transfected cells were rinsed 3 times with PBS 24 hours after transfection, fixed with 4% PFA for 15 minutes, incubated with 1% Triton X-100 at room temperature for 10 minutes, blocked with 3% BSA at room temperature for 30 minutes, rinsed 3 times with PBS, and then incubated with the prepared anti-VP7 protein monoclonal antibody 5E11 overnight at 4°C. After rinsing 3 times with PBS, goat anti-mouse IgG (Alexa Flour 488) antibody (diluted 1:1000) was added and incubated at room temperature in the dark for 1 hour. The cells were then incubated with Hoechst 33342 nuclear staining solution at room temperature for 15 minutes, washed with PBS, and observed under an inverted fluorescence microscope.

[0051] The results are as Figure 5 shown. The monoclonal antibody 5E11 prepared in the present invention could react with the recombinant VP7 protein expressed in BHK-21 cells, showing green fluorescence, while no fluorescence was observed in BHK-21 cells transfected with pcDNA3.1 / myc-His.

[0052] 8. Amplification of the variable region sequences of the light and heavy chains of monoclonal antibody 5E11

[0053] Total RNA was extracted from the hybridoma cells secreting monoclonal antibody 5E11 using the TRIzol lysis method, and then reverse transcribed into cDNA using a reverse transcription kit. PCR amplification was performed using the obtained cDNA as a template, and the amplified product was ligated to a vector for sequencing to obtain the nucleotide sequences of the heavy and light chain variable regions of the antibody.

[0054] After sequencing, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody was as follows:

[0055] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAG

[0056] TCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTTTGGTATAGGAGTAGGCTGGCT

[0057] TCGTCAGTCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGAATGATAAAA

[0058] AGTACTATAACACAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAACAAC

[0059] CAGGTATTCCTCAAGATCGCCACTGTGGACACTACAGATACTGCCACATACTACTGTGGT

[0060] CGGAACTTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG (shown as SEQ ID NO.1);

[0061] The coding DNA sequence of the light chain variable region of the monoclonal antibody is

[0062] GATATTGTACTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAACTATTAGCAACAACCTACACTGGTTTCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCACCAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGACTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (shown as SEQ ID NO.3);

[0063] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows:

[0064] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTFGIGVGWLRQSSGKGLEWLAHIWWNDK KYYNTALKSRLTISKDTSNNQVFLKIATVDTTDTATYYCGRNFFDYWGQGTTLTVSS (as shown in SEQ ID NO.2);

[0065] The amino acid sequence of the light chain variable region of the monoclonal antibody is as follows: DIVLTQSPATLSVTPGDSVSLSCRASQTISNNLHWFQQKSHESPRLLIKYASQSISGIPSRFSGS GSGTDFTLSITSVETEDFGMYFCQQTNSWPLTFGAGTKLELKR (as shown in SEQ ID NO.4).

[0066] The sequences of the light chain and heavy chain variable regions of the monoclonal antibody 5E11 obtained by sequencing were analyzed on the website abysis.org to obtain its CDR regions.

[0067] The sequences of the three complementarity-determining regions (CDRs) of the heavy chain variable region are as follows:

[0068] CDR1: TFGIGVG (as shown in SEQ ID NO.5);

[0069] CDR2: HIWWNDKKYYNTALKS (as shown in SEQ ID NO.6);

[0070] CDR3: NFFDY (as shown in SEQ ID NO.7);

[0071] The sequences of the three complementarity-determining regions (CDRs) of the light chain variable region are as follows:

[0072] CDR1: RASQTISNNLH (as shown in SEQ ID NO.8);

[0073] CDR2: YASQSIS (as shown in SEQ ID NO.9);

[0074] CDR3: QQTNSWPLT (as shown in SEQ ID NO.10).

[0075] Example 2 Establishment of a competitive ELISA detection method using monoclonal antibody 5E11

[0076] 1. Establishment of the detection method

[0077] Coating: Dilute the expressed recombinant EHDV VP7 protein with CBS buffer (0.05M carbonate-bicarbonate buffer, pH 9.6) to 0.5 μg / mL, and coat 100 μL / well onto the ELISA plate. Incubate overnight at 4°C; wash the plate 3 times with PBST buffer.

[0078] Blocking: Block the ELISA plate with an ELISA plate stabilizer, 200 μL / well, incubate at 37°C for 1 h, empty and store at -4°C.

[0079] Detection: Add EHDV negative serum control to wells A1 and A2, and EHDV positive serum control to wells B1 and B2 (100 μL / well). Subsequently, add the dilution solution (0.01M PBS, pH 7.2) to the ELISA plate, 50 μL / well. Add the EHDV negative serum to be tested to wells C1 and C2, and EHDV positive serum to wells D1 and D2, 50 μL / well. Incubate at 37°C for 30 min.

[0080] Adding competitive antibody: Dilute the monoclonal antibody 5E11 prepared in the example of this application at 0.5 mg / mL in a ratio of 1:4000 (the dilution solution is 0.01M PBS, pH 7.2), then add it to the ELISA plate, 100 μL / well. Incubate at 37°C for 30 min, and wash the plate 3 times with PBST buffer.

[0081] Adding enzyme-labeled secondary antibody: Add the HRP-labeled goat anti-mouse secondary antibody diluted at 1:100000 (0.01M PBS, pH 7.2) to the ELISA plate, 100 μL / well. Incubate at 37°C for 30 min, and wash the plate 3 times with PBST buffer.

[0082] Color development: Develop color with TMB color developer in the dark, 100 μL / well, incubate at 37°C for 10 min; add 100 μL / well of the stop solution and read the OD 450 value.

[0083] Judgment method: PI% = (1 - OD value of sample / OD value of negative control) × 100%. When PI ≥ 60%, it is judged as positive; when PI < 60%, it is judged as negative. The experiment is valid when the OD value of the negative control is greater than 0.7 and the ratio of the OD value of the positive control to the OD value of the negative control < 0.3.

[0084] The results are shown in Table 2. The competitive ELISA detection method for epizootic hemorrhagic disease virus (EHDV) established using the monoclonal antibody 5E11 described in this application can be used for the detection of EHDV serum antibodies.

[0085] Table 2 Competitive ELISA detection results

[0086]

[0087] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody that recognizes the VP7 protein of epidemic hemorrhagic disease virus, characterized in that: The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.5, CDR2 with an amino acid sequence as shown in SEQ ID NO.6, and CDR3 with an amino acid sequence as shown in SEQ ID NO.7; The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.8, CDR2 with an amino acid sequence as shown in SEQ ID NO.9, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

10.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

4.

3. A nucleic acid, characterized in that The nucleic acid encodes the light chain and antibody heavy chain of the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid includes the sequences shown in SEQ ID NO.1 and SEQ ID NO.

3.

5. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid according to claim 3 or 4.

6. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 5.

7. An immunoconjugate, characterized in that: The immunoconjugate comprises: (i) the monoclonal antibody according to claim 1 or 2; (ii) and a conjugated moiety selected from the group consisting of a detectable label, a drug, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

8. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent, paper strip or kit for detecting epidemic hemorrhagic disease virus.

9. Use of the monoclonal antibody according to claim 1 or 2 in in vitro detection of epidemic hemorrhagic disease virus for non-disease diagnosis purposes.

10. An ELISA detection kit for epidemic hemorrhagic disease virus, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2.

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