Monoclonal antibody specifically recognizing vp7 protein of epidemic hemorrhagic fever virus and use thereof

Abstract

The application belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a monoclonal antibody specifically recognizing VP7 protein of epidemic hemorrhagic disease virus and application thereof. The application prepares a monoclonal antibody 5E11 recognizing VP7 protein of epidemic hemorrhagic disease virus (EHDV) through hybridoma technology; the monoclonal antibody 5E11 can recognize EHDV VP7 protein expressed by E. coli, and can also specifically bind to recombinant EHDV VP7 protein expressed in BHK-21 cells, while the monoclonal antibody does not react with recombinant bluetongue virus VP7 protein and recombinant African horse sickness virus VP7 protein; the monoclonal antibody 5E11 is used as a competitive antibody to establish a competitive ELISA antibody detection method for epidemic hemorrhagic disease virus, which has important significance for diagnosis, prevention and control of EHDV.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, specifically relating to a monoclonal antibody that recognizes the VP7 protein of epidemic hemorrhagic disease virus and its uses. Background Technology

[0002] Epizootic hemorrhagic disease (EHD) is a vector-borne disease caused by the epizootic hemorrhagic disease virus (EHDV). It is transmitted through the blood-sucking bite of Culicoides midges and infects various ruminants, including cattle, sheep, and deer, seriously impacting the healthy development of global livestock farming. Clinical symptoms include fever, oral and nasal erosions, lameness, abortion, subcutaneous edema, and congestion in mucous membranes and hairless areas of the skin. Pathological changes include muscle degeneration in the larynx, pharynx, and esophagus; hemorrhage in the heart, lungs, and major blood vessels; and congestion and erosion of the abomasum. At least nine serotypes of EHDV have been identified, including serotypes 1, 2, 4, 5, 6, 7, 8, 9, and 10, with no cross-immunity between serotypes. Currently, EHDV is distributed between 35°S and 49°N latitude, including parts of the Americas, Africa, Asia, the Middle East, Europe, and Oceania. In 2008, EHD was included in the list of legally notifiable animal diseases by the World Organization of Animal Health (WOAH).

[0003] EHDV belongs to the genus *Circovirus* of the family Reoviridae. Similar to bluetongue virus and African horse sickness virus in the same genus, it has an icosahedral capsid structure with a diameter of approximately 80 nm. Its structure consists of three continuous protein layers: an outer capsid, an inner capsid, and a viral core. The genome is composed of 10 segmented linear double-stranded RNAs encoding seven structural proteins (VP1, VP2, VP3, VP4, VP5, VP6, and VP7) and five non-structural proteins (NS1, NS2, NS3 / NS3A, NS4, and NS5). The viral core contains an RNA polymerase complex composed of VP1 (RNA polymerase), VP4 (capping enzyme), and VP6 (helicase). The inner capsid is composed of structural proteins VP7 and VP3, while the outer capsid is composed of structural proteins VP2 and VP5. The five non-structural proteins are primarily responsible for viral infection and replication, playing crucial roles in intracellular transport, viral packaging and release, and anti-host immune responses. VP7 protein is the most expressed structural protein in EHDV and is highly conserved across different serotypes. It is the main serogroup-specific antigen of EHDV and has important application value in the establishment of diagnostic methods and vaccine development for this disease.

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

[0005] To address the aforementioned technical problems, this invention successfully expressed recombinant EHDV VP7 protein using an E. coli expression system. After purification, mice were immunized with the protein, and a highly specific monoclonal antibody against VP7 protein was successfully prepared using hybridoma technology. This provides a foundation for in-depth research into the biological function of EHDV VP7 protein and the development of EHD prevention and control technologies. Specifically, it includes the following:

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

[0007] 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;

[0008] 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.

[0009] Preferably, 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.

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

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

[0012] Thirdly, the present invention provides a recombinant vector containing the nucleic acid described in the second aspect above.

[0013] Fourthly, the present invention provides a recombinant cell containing the recombinant vector described in the third aspect above.

[0014] Fifthly, the present invention provides an immunoconjugate comprising:

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

[0016] (ii) and the coupling portion selected from the following group:

[0017] It can detect markers, drugs, gold nanoparticles / nanorobars, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0018] In a sixth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect above in the preparation of reagents for detecting epidemic hemorrhagic disease virus.

[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 test strips or kits for detecting epidemic hemorrhagic disease virus.

[0020] Eighthly, the present invention provides the application 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, the kit comprising the monoclonal antibody described in the first aspect above.

[0022] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, enzyme-labeled secondary antibody, washing solution, chromogenic agent, and stop solution.

[0023] The beneficial effects of this invention are as follows: Based on the gene sequence published in GenBank, the EHDV VP7 gene was artificially synthesized after codon optimization. This gene was cloned into an expression plasmid to construct the recombinant plasmid pET-EHDV VP7. This plasmid was transformed into competent E. coli cells, and expression was induced by IPTG with optimized expression conditions. The expressed VP7 protein was purified by Ni-NTA affinity chromatography and renatured by dialysis with different concentrations of imidazole. The renatured VP7 protein was used to immunize BALB / c mice, and a monoclonal antibody against the VP7 protein was prepared using hybridoma technology. This monoclonal antibody not only reacts with the recombinant EHDV VP7 protein expressed in E. coli, but also binds to the recombinant EHDV VP7 protein expressed in eukaryotic cells. Simultaneously, it does not react with the recombinant bluetongue virus (BTV) VP7 protein or the recombinant African horse sickness virus (AHSV) VP7 protein, exhibiting good specificity. This lays the foundation for studying the structure and function of the EHDV VP7 protein and establishing diagnostic methods. Attached Figure Description

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

[0025] Figure 2 The purification and refolding results of EHDV VP7 protein; where M is the molecular weight standard of the protein, and 1 to 3 are the samples after the first to third imidazole elutions, respectively;

[0026] Figure 3 Western blot identification results of EHDV VP7 refolded protein, where M is the molecular weight standard of protein, and 1-4 are the samples after the first to fourth imidazole elutions, respectively.

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

[0028] Figure 5 Results of immunofluorescence identification of monoclonal antibodies; Detailed Implementation

[0029] The embodiments of the present invention are 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 limiting the present invention. In addition, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized according to methods described in the text or known methods. For reaction conditions not listed, they are readily available to those skilled in the art.

[0030] The following are the cells, main reagents, and experimental animals involved in the implementation:

[0031] BHK-21 cells, mouse myeloma cells (SP2 / 0), recombinant BTV VP7 protein and recombinant AHSV VP7 protein expressed and purified from E. coli, goat anti-EHDV positive serum, EHDV negative serum control, and EHDV positive serum control were all preserved in our laboratory; E. coli BL21(DE3) competent cells and isopropyl-β-D-thiogalactoside (IPTG) were 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) were purchased from Abcam; kanamycin, Triton X-100, and bovine serum albumin (BSA) were purchased from Solarbio; Lipofectamine 2000 Reagent, Dulbecco's Modified Eagle Medium (DMEM) culture medium, fetal bovine serum (FBS), and protein quantification kits were purchased from Thermo Fisher Scientific. NP-40 lysis buffer was purchased from Beyotime; SuperKine TM The hypersensitive ECL luminescent solution was purchased from Abbkine; the PVDF transfer film was purchased from MERCK; Ni-NTA agarose was purchased from QIAGEN; Freund's adjuvant and Freund's incomplete adjuvant were both purchased from Sigma; the experimental animals were approximately 8-week-old female BALB / c mice.

[0032] Example 1: Preparation of monoclonal antibody 5E11

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

[0034] Based on the full-length EHDV VP7 gene sequence published in GenBank (accession number: AM744983.1), the VP7 gene codons were optimized according to the gene expression bias of E. coli and eukaryotic cells. The VP7 gene was synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. and cloned into the plasmid vectors pET-28 and pcDNA3.1 / myc-His, and named pET-EHDV VP7 and pcDNA3.1 / myc-His-EHDV VP7, respectively.

[0035] 2. Inducible expression of VP7 protein

[0036] The recombinant plasmid pET-EHDV VP7 was transformed into E. coli BL21(DE3) to expand the bacterial culture. When the bacterial culture D 600 When the concentration reached 0.6, the bacterial culture before induction was used as a control. IPTG inducer was added to LB medium to a final concentration of 0.5 mmol / L. VP7 protein expression was induced at 37℃. Samples of uninduced bacterial culture and bacterial culture at 2, 4, 6 and 8 hours after induction were taken, centrifuged at 10,000 r / min, and the supernatant was discarded. The precipitated samples were resuspended with PBS to observe and analyze the expression of VP7 protein.

[0037] The results are as follows Figure 1 As shown, the recombinant EHDV VP7 protein was successfully expressed, with a molecular weight of 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 large-scale induction of bacterial expression, the bacterial pellet was collected, sonicated, and resuspended in PBS. The target protein was purified according to the Ni-NTA agarose instructions. Impurities were removed with 25 column volumes of binding buffer, and the target protein was eluted with elution buffer. The purified protein was then concentrated by dialyzing at 4°C, and the dialysis solution was filtered through a 0.22 μm filter. After filtration, the protein was bound again to a Ni-NTA affinity column, and impurities were eluted sequentially with low concentrations of imidazole (20 mmol / L, 60 mmol / L, 200 mmol / L). The target protein was eluted with 500 mmol / L imidazole, and the concentration was determined. The protein purification and renaturation results were analyzed by SDS-PAGE. Finally, Western blotting analysis was performed using goat anti-EHDV positive serum and HRP-labeled rabbit anti-goat IgG as primary and secondary antibodies, respectively.

[0040] Following the Ni-NTA agarose affinity chromatography procedure, VP7 protein from the precipitated sample after ultrasonic lysis following 8 hours of induction at 37°C was purified and refolded. The protein solution remained clear and transparent throughout, with no obvious precipitation or protein denaturation or aggregation. SDS-PAGE results are shown below. Figure 2 As shown, high-purity 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 (As shown).

[0041] 4. Mouse immunization with VP7 protein

[0042] Six 8-week-old BALB / c mice were selected. Purified and refolded VP7 protein was used as the antigen, with each mouse receiving 50 μg. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until the water-emulsion phase did not separate. The mice were immunized via subcutaneous injection at multiple sites. Subsequent immunizations were performed using Freund's incomplete adjuvant emulsified with the same dose of VP7 protein, with three booster immunizations administered two weeks apart. Seven days after the final immunization, blood was collected from the tail vein, and serum was separated to determine antibody titers. Purified and refolded VP7 protein was used as the coating antigen, and the mouse serum served as the primary antibody. HRP-labeled goat anti-mouse IgG was used as the secondary antibody. Indirect ELISA was performed, and antibody titers were measured using a microplate reader. 450 Value, test sample D 450 / Negative Control D 450 The dilution with a value ≥2.1 is the antibody titer. The mice with the highest antibody titer are selected to prepare monoclonal antibodies.

[0043] 5. Monoclonal antibody preparation

[0044] Spleen cells from immunized mice were isolated, ground into a single-cell suspension, and fused with SP2 / 0 cells in the logarithmic growth phase. The cell supernatant was measured using an indirect enzyme-linked immunosorbent assay (ELISA) to screen for hybridoma-positive cells. These cells were then clonalized using a limiting dilution method to identify positive cell lines that stably secreted monoclonal antibodies against the VP7 protein, which were subsequently amplified. Liquid paraffin oil was injected intraperitoneally into BALB / c mice. Hybridoma-positive cells were inoculated on day 8, and ascites was collected on day 20 when the mice's abdomens expanded. The antibody titer of the obtained monoclonal antibody 5E11 is shown in Table 1, demonstrating good reactivity and specificity to the EHDVVP7 protein.

[0045] Table 1. Antibody titer test results

[0046] Monoclonal antibody 5E11 negative control 3.5083 0.3732

[0047] 6. Western blot identification

[0048] The results are as follows Figure 4As shown, the purified monoclonal antibody was validated using Western blot. Samples were prepared using recombinant EHDV VP7 protein, BTV VP7 protein, and AHSV VP7 protein expressed in *E. coli* for Western blot identification. The results indicate that the monoclonal antibody 5E11 prepared in this invention can specifically react with EHDV VP7 protein, exhibiting good specificity.

[0049] 7. Immunofluorescence analysis

[0050] BHK-21 cells (80%–90% confluence) were transfected with the recombinant plasmid pcDNA3.1 / myc-His-EHDV VP7. After 24 hours of transfection, the cells were washed three times with PBS, 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, washed three times with PBS, and incubated overnight at 4°C with the prepared anti-VP7 protein monoclonal antibody 5E11. After washing three times with PBS, the cells were incubated for 1 hour at room temperature in the dark with goat anti-mouse IgG (Alexa Flour 488) antibody (1:1000 dilution), and then incubated with Hoechst 33342 nuclear staining solution at room temperature for 15 minutes. After washing with PBS, the cells were observed using an inverted fluorescence microscope.

[0051] The results are as follows Figure 5 As shown, the monoclonal antibody 5E11 prepared in this invention can react with the recombinant VP7 protein expressed in BHK-21 cells and show 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 hybridoma cells secreting the monoclonal antibody 5E11 using the TRIzol lysis method, and then cDNA was synthesized by reverse transcription using a reverse transcription kit. The obtained cDNA was used as a template for PCR amplification, and the amplification product was ligated into a vector for sequencing to obtain the nucleotide sequences of the antibody's heavy and light chain variable regions.

[0054] The DNA sequence encoding the heavy chain variable region of the monoclonal antibody, as determined by sequencing, is as follows:

[0055] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAG

[0056] TCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTTTGGTATAGGAGTAGGCTGGCT

[0057] TCGTCAGTCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGAATGATAAAA

[0058] AGTACTATAACACAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAACAAC

[0059] CAGGTATTCCTCAAGATCGCCACTGTGGACACTACAGATACTGCCACATACTACTGTGGT

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

[0061] The DNA sequence encoding the light chain variable region of the monoclonal antibody is as follows:

[0062] GATATTGTACTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAACTATTAGCAACAACCTACACTGGTTTCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCAT CTCTGGGATCCCCTCCAGGTTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCACCAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGACTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG(SEQ ID NO.3 shown);

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

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

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

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

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

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

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

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

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

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

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

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

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

[0076] 1. Establishment of detection methods

[0077] Coating: The expressed EHDV VP7 recombinant protein was diluted to 0.5 μg / mL with CBS buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6), and 100 μL / well was coated into the microplate and incubated overnight at 4°C; the plate was washed 3 times with PBST buffer.

[0078] Blocking: Block the microplate with microplate 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 per well). Then add diluent (0.01M PBS pH 7.2) to the microplate at 50 μL / well. Add the EHDV negative serum to be tested to wells C1 and C2, and the EHDV positive serum to wells D1 and D2 at 50 μL / well. Incubate at 37°C for 30 min.

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

[0081] Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:100000 (0.01M PBS pH7.2) to the microplate, incubate at 37℃ for 30 min, and wash the plate 3 times with PBST buffer.

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

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

[0084] The results are shown in Table 2. The competitive ELISA detection method for epidemic 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 Results of Competitive ELISA Detection

[0086]

[0087] The above description is merely a preferred embodiment of the present invention and is 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody recognizing VP7 protein of epidemic hemorrhagic fever 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 comprises 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 comprises 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 of claim 1, wherein 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.

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

4. The nucleic acid of claim 3, wherein, the nucleic acid comprises sequences as 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 of claim 3 or 4.

6. A recombinant cell, characterized in that, the recombinant cell contains the recombinant vector of claim 5.

7. Use of the monoclonal antibody of claim 1 or 2 in the preparation of a reagent for detecting epidemic hemorrhagic fever virus.

8. Use of the monoclonal antibody of claim 1 or 2 in the in vitro detection of epidemic hemorrhagic fever virus for purposes other than disease diagnosis.

9. An ELISA test kit for the detection of epizootic hemorrhagic disease virus, characterized in that, the kit comprises the monoclonal antibody of claim 1 or 2.

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