Sheep-derived anti-bluetongue virus recombinant neutralizing antibody and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a sheep-derived anti-bluetongue virus recombinant neutralizing antibody and a preparation method thereof. An anti-bluetongue virus specific B cell population is screened from sheep peripheral blood lymphocytes, a sheep anti-bluetongue virus antibody database is obtained through a single cell V (D) J sequencing technology, and the sheep recombinant antibody is successfully expressed after screening. Experiments such as Western-blot, cellular immunofluorescence, indirect ELISA and microneutralization prove that the antibody can be combined with bluetongue virus coat protein VP2 and has a good neutralization effect, a key tool is provided for rapid and typing detection of bluetongue, a foundation is laid for research and development of a novel efficient bluetongue vaccine, and the antibody has a good application prospect. The method is of great significance to further development of bluetongue prevention and control technology research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a sheep-derived anti-bluetongue virus recombinant neutralizing antibody and a preparation method thereof. Background Art

[0002] Bluetongue is an animal disease caused by bluetongue virus. Sheep are susceptible animals. After infection, they show clinical symptoms such as high fever, depression, facial edema, oral and nasal mucosal ulcers, drooling, lameness, etc., which can lead to death in severe cases. Cattle and deer often show latent infection. The disease is widely distributed in Africa, Asia, Europe, Oceania and the Americas. There are no related reports in Antarctica due to the lack of hosts and vectors. In my country, BTV-1 and BTV-16 are the main prevalent serotypes.

[0003] Bluetongue virus belongs to the genus Circovirus of the family Reoviridae. It is a non-enveloped double-stranded RNA virus. Its genome consists of 10 double-stranded RNA fragments, encoding a total of 7 structural proteins and 4 non-structural proteins. Among them, VP2 protein is the outermost structural protein of the virus, which can neutralize the virus and induce a protective immune response in animals.

[0004] With the development and application of high-throughput single-cell sequencing technology, antibody preparation technology has made new progress. In a large number of disease studies, single-cell V(D)J sequencing technology has been used to analyze and obtain human and mouse immune repertoires in a short period of time, and neutralizing antibodies and their epitope characteristics have been successfully screened. It is worth noting that although this technology has been reported in model animals such as cattle, pigs and zebrafish, the development of immune repertoires for sheep diseases is still blank. In addition, in the study of bluetongue virus antibodies, the neutralizing antibodies currently obtained mainly rely on mouse hybridoma technology. Although the relevant results have clarified the characteristics of some antigenic epitopes of VP2 protein, the neutralizing antibody repertoire produced in the natural host, the epitope recognition characteristics and the molecular mechanism of neutralization mediated by them have not been systematically studied so far.

[0005] In summary, the development of an anti-bluetongue virus antibody library derived from sheep and the preparation of neutralizing antibodies not only provide key tools for the rapid and accurate typing and detection of bluetongue disease, but also lay the foundation for the development of an efficient new bluetongue disease vaccine, which is of great significance for further research on bluetongue disease prevention and control technologies. Summary of the invention

[0006] In view of the above technical problems, the present invention aims to provide a sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 and its preparation method and use. Specifically, it includes the following contents:

[0007] In the first aspect, the present invention provides a sheep-derived recombinant anti-bluetongue virus neutralizing antibody, the amino acid sequence of the heavy chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown as SEQ ID NO.3.

[0008] In a second aspect, the present invention provides a nucleic acid encoding the light chain variable region sequence and the heavy chain variable region sequence of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody described in the first aspect.

[0009] Preferably, the nucleic acid sequence encoding the heavy chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown as SEQ ID NO.2, and the nucleic acid sequence encoding the light chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown as SEQ ID NO.4.

[0010] In a third aspect, the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid described in the second aspect.

[0011] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains the recombinant expression vector described in the third aspect.

[0012] In a fifth aspect, the present invention provides a method for screening sheep-derived anti-bluetongue virus recombinant neutralizing antibodies according to the first aspect, characterized in that the screening method comprises:

[0013] (1) Inactivated BTV-1 virus emulsified with ISA201 adjuvant was used to immunize sheep and isolate peripheral blood lymphocytes, which were then labeled and screened using antigens and fluorescent antibodies to obtain BTV-1 specific B cell populations;

[0014] (2) using sheep single-cell V(D)J nested primers to perform single-cell V(D)J sequencing on the BTV-1 specific B cell population described in step (1) to obtain sheep anti-bluetongue virus antibody library data information;

[0015] (3) The obtained sheep antibody library data was sorted according to the length of the heavy chain CDR3 region amino acid sequence, and antibody sequences with a length > 2 and a clonal type > 1 (i.e., antibodies with the same CDR3 region sequence are of the same clonal type) were screened out, thereby obtaining the heavy chain variable region and light chain variable region sequences of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody.

[0016] Preferably, in step (1), peripheral blood lymphocytes are enriched for specific B cell populations using a BD FACS AriaII flow sorter using biotin-labeled BTV-1 antigen, anti-IgM antibody, and anti-CD21 antibody according to the sorting principle of CD21+, IgM-, and BTV-1+;

[0017] Preferably, the cell viability of the specific B cell population reaches more than 85%, and the density reaches 1×10 6 cells / mL.

[0018] Preferably, the amplification primers for the single cell V(D)J nested PCR are shown in Table 1 below:

[0019] Table 1 Amplification primers for single cell V(D)J nested PCR

[0020]

[0021]

[0022] In a sixth aspect, the present invention provides a method for preparing the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to the first aspect, the method comprising:

[0023] (1) connecting the heavy chain variable region and light chain variable region sequences of the sheep anti-bluetongue virus recombinant neutralizing antibody to the heavy chain constant region and light chain constant region sequences of the sheep IgG1 antibody, respectively, and inserting them into the pcDNA3.4 expression vector to obtain pcDNA3.4-SH1-H and pcDNA3.4-SH1-L recombinant expression plasmids;

[0024] (2) The recombinant expression plasmid obtained in step (1) is co-transfected into HEK293 cells, and the cells and supernatant are collected after culturing for 8-10 days, and purified by Protein G affinity chromatography to obtain sheep-derived anti-bluetongue virus recombinant neutralizing antibodies.

[0025] In a seventh aspect, the present invention provides an immunoconjugate, comprising:

[0026] a. The sheep-derived anti-bluetongue virus recombinant neutralizing antibody described in the first aspect above;

[0027] b. and a coupling moiety selected from the group consisting of a detectable marker, a drug, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins, or a combination thereof.

[0028] In an eighth aspect, the present invention provides the use of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody described in the first aspect or the immunoconjugate described in the seventh aspect in the preparation of a reagent, test strip, or kit for detecting BTV-1.

[0029] In a ninth aspect, the present invention provides the use of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody described in the first aspect or the immunoconjugate described in the seventh aspect in the preparation of a drug for preventing or treating bluetongue disease.

[0030] In a tenth aspect, the present invention provides a reagent, comprising the sheep-derived anti-bluetongue virus recombinant neutralizing antibody described in the first aspect or the immunoconjugate described in the seventh aspect.

[0031] The beneficial effects of the present invention are:

[0032] 1. The present invention provides a sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1, which can bind to BTV-1VP2 protein and has good neutralizing activity, and can be used for the detection and diagnosis of bluetongue virus; it can also be used for the study of the neutralizing epitopes of bluetongue virus and the neutralization mechanism of sheep immune response, providing important information for the development of bluetongue virus vaccines, providing higher reliability for the accurate diagnosis of pathogens, and improving the applicability of antibodies in vaccine evaluation and prevention and control strategies, which is of great significance for the early diagnosis, prevention and control of BTV-1.

[0033] 2. The present invention provides a method for screening and preparing a sheep-derived recombinant neutralizing antibody SH1 against bluetongue virus. Specific B cell populations are enriched from peripheral blood lymphocytes of sheep immunized with inactivated virus BTV-1, and then a sheep-derived anti-bluetongue virus antibody database is obtained using single-cell V(D)J sequencing technology. The sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 with neutralizing activity is obtained by screening according to the length of the amino acid sequence in the heavy chain CDR3 region. Compared with traditional antibody preparation technology, this technology has the advantages of strong targeting and high efficiency, and provides a new technical means for studying the immune response mechanism of sheep and developing neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Flow cytometry results of enriched sheep anti-BTV-1 specific B cell population;

[0035] Figure 2 Map of recombinant expression plasmids of light and heavy chains of sheep recombinant neutralizing antibody SH1;

[0036] Figure 3 SDS-PAGE test results of sheep recombinant neutralizing antibody SH1;

[0037] Figure 4 Cellular immunofluorescence detection results of sheep recombinant neutralizing antibody SH1 binding to BTV-1;

[0038] Figure 5 Results of affinity test of sheep recombinant neutralizing antibody SH1 and BTV-1;

[0039] Figure 6 Indirect ELISA test results of sheep recombinant neutralizing antibody SH1 binding to BTV-1VP2;

[0040] Figure 7 Neutralization test results of sheep recombinant neutralizing antibody SH1. DETAILED DESCRIPTION

[0041] 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, if not explicitly stated, all reagents used in the following embodiments are commercially available, or can be synthesized according to text or known methods, and the reaction conditions not listed are easily available to those skilled in the art.

[0042] Example 1 Preparation of sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1

[0043] 1. Construction of a single-cell V(D)J antibody library against bluetongue virus in sheep

[0044] Sheep were immunized with inactivated virus BTV-1 emulsified with ISA201 adjuvant, and peripheral blood lymphocytes were isolated. The peripheral blood lymphocytes were sorted by BD FACSAriaII flow cytometry using biotin-labeled BTV-1 antigen, anti-IgM antibody, and anti-CD21 antibody according to the sorting principle of CD21+, IgM-, and BTV-1+ to enrich specific B cell populations ( Figure 1 The cells were collected and resuspended in FACS buffer, and then the cell viability and cell density were detected by cell counter. The final cell viability was 86% and the cell density was 1.03×10 6 / mL. The cells were sent to Jinweizhi Biotechnology Co., Ltd. for single-cell V(D)J sequencing, and the sheep anti-bluetongue virus antibody library data were obtained by analysis. The amplification primers for the single-cell V(D)J nested PCR are shown in Table 1 above.

[0045] 2. Analysis and screening of sheep antibody library

[0046] The obtained sheep antibody library data were sorted according to the amino acid length of the heavy chain CDR3 region, and antibody sequences with a length > 27 and a clonal type > 1 (i.e., antibodies with the same CDR3 region sequence are of the same clonal type) were screened out. Finally, the sheep antibody SH1 was obtained, whose heavy chain CDR3 region amino acid length was 28 and the number of clonal types was 2.

[0047] SH1-VH amino acid sequence: QVRLQESGPSLVKPSQTLSLTCTVSGFSLSSNTVVWVRQAPGKA PEWVGGIDNDGHEGYNSALKSRLSIGRDTSKSQVSLSLRSVSNKDTAVYYCGRINFDYYSGYGYAYGWWTMISYYGVDVWGRGLLVTVSS (shown in SEQ ID NO.1);

[0048] SH1-VH nucleic acid sequence: CAGGTGCGGCTGCAGGAGTCGGGACCCAGCCTGGTGAAGCCCT CACAGACCCTCTCCCTCACCTGCACGGTCTCTGGATTCTCATTAAGCAGCAATACTGTAGTCTGGGTCCGCCAGGCTCCAGGAAAGGCGCCGGAGTGGGTTGGTGGTATTGATAATGATGGACACGAGGGCTATAATTCAGCCCTTAAGTCCCGGCTCAGCATCGGCAGGGACACCTCCAAGAGTCAAGTCTCCCTGTCACTGAGAAGCGTGTCAAATAAGGACACGGCCGTGTACTACTGTGGAAGAATCAACTTCGATTATTATAGTGGTTATGGTTATGCTTATGGTTGGTGGACAATGATCTCGTACTACGGTGTAGATGTGTGGGGCCGAGGACTCCTGGTCACCGTCTCCTCA (shown as SEQ ID NO.2);

[0049] SH1-VL amino acid sequence: QTVLPQPSSVSRSLGQSVSITCSGNSSNVGYGNYVTWYQQVPGS APKLLIYGATSRASGVPDRFSGSRSGNTATLIITSLQAEDEADYYCASYDSDNYNGVFGSGT RLIVL (shown as SEQ ID NO.3);

[0050] SH1-VL nucleic acid sequence: CAAACTGTGCTGCCTCAGCCGTCCTCCGTGTCCAGGTCCCTGGG CCAGAGTGTCTCCATCACTTGTTCGGGAAACAGCAGTAACGTTGGATATGGTAATTATGTCACCTGGTACCAACAGGTCCCAGGATCAGCCCCCAAGCTCCTCATCTATGGTGCAACCAGTCGAGCCTCGGGGGTCCCCGACCGAT TCTCCGGCTCCAGGTCTGGCAACACAGCGACTCTAATTATCACCTCGCTCCAGGCTGAGGACGAGGCCGATTATTACTGTGCATCTTATGACAGTGATAATTATAATGGTGTTTTCGGCAGCGGGACCAGGCTGATCGTCCTG(SEQ ID NO.4 shown).

[0051] 3. Expression and purification of sheep-derived recombinant neutralizing antibody SH1 against bluetongue virus

[0052] The heavy chain variable region sequence and light chain variable region sequence obtained in 2 were added to the sheep IgG1 constant region sequence, and the secretion signal peptide sequence was introduced at the N-terminus, and the 6×His tag for easy detection and purification was introduced at the C-terminus. The synthesized antibody SH1 light chain and heavy chain sequences were inserted into the pcDNA3.4 expression vector, and the constructed expression plasmids were named pcDNA3.4-SH1-H and pcDNA3.4-SH1-L recombinant expression plasmids ( Figure 2 The two recombinant expression vectors were mixed with the transfection reagent PEI at a ratio of 1:3 according to a heavy chain to light chain ratio of 1:2, and incubated at room temperature for 15 minutes; then slowly added to HEK293 cells for expression, and 3% SMS293-SUPI feed solution, 0.2% enhancer 1, and 0.8% enhancer 2 were added after 24 hours, and 1% feed solution was added every 48 hours thereafter; cells and supernatant were collected after continued culture at 37°C for 8-10 days.

[0053] 4. Purification of sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1

[0054] The cells expressing the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 and the supernatant were ultrasonically disrupted and centrifuged at 12000 rpm for 10-15 minutes; after filtering with a 0.45 μm filter, the antibody was purified using 1 mL of Protein G affinity chromatography.

[0055] The eluted sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 was collected into a dialysis bag and dialyzed at 4°C for more than 8 hours, and the PBS buffer was replaced every 2-4 hours. After the dialysis, the antibody was concentrated in PEG 6000. Finally, the concentrated sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 was detected by SDS-PAGE and stored at -80°C. Figure 3 As shown, the present invention successfully obtained the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1.

[0056] Example 2 Identification of sheep-derived recombinant neutralizing antibodies against bluetongue virus

[0057] 1. Cellular immunofluorescence detection of sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1

[0058] BSR cells infected with BTV-1 were fixed with 4% paraformaldehyde for 15 min, washed 3 times with PBS, and then treated with 0.5% TritonX-100 for 10 min. After washing 3 times with PBS, they were blocked with 3% BSA for 1 h. After aspiration, purified sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 and PBS negative control were added, respectively, and incubated at 4°C overnight; mouse anti-His antibody (1:5000 dilution) was incubated at room temperature for 1 h, washed 3 times with PBS, and Alexa Fluor was added. 568 labeled goat anti-mouse IgG antibody (1:1000 dilution) was incubated at room temperature in the dark for 1 hour, washed 3 times with PBS, Hoechst 33342 was added to stain the cell nucleus for 7 minutes, washed 3 times with PBS, and the slides were sealed and observed under a fluorescence microscope to collect images. Figure 4 As shown, the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 can bind to the BTV-1 virus in BSR cells.

[0059] 2. Indirect ELISA detection of sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1

[0060] (1) Verify the affinity between BTV-1 and sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1, the specific method is as follows:

[0061] Coating: Add 100 μL of 5×10 cells / mL diluted in CBS buffer (0.05 M carbonate-bicarbonate buffer pH 9.6) to each well. 3 TCID 50 The virus solution was coated at 4°C overnight; the plate was washed 5 times with PBST buffer;

[0062] Blocking: Block the ELISA plate with PBST buffer containing 5% skim milk powder, 200 μL / well, incubate at 37°C for 1 h; wash the plate 5 times with PBST buffer;

[0063] Detection: The sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 and sheep negative serum were diluted 2-fold and added to each well at 100 μL. The plates were incubated at 37°C for 1 h and washed 5 times with PBST buffer.

[0064] Add enzyme-labeled secondary antibody: add 100 μL of mouse anti-His antibody diluted at a ratio of 1:10000, incubate at 37°C for 1 h; wash the plate 5 times with PBST buffer, then add 100 μL of rabbit anti-mouse IgG-HRP antibody diluted at a ratio of 1:40000, incubate at 37°C for 1 h; wash the plate 5 times with PBST buffer;

[0065] Color development: Use TMB color development solution in the dark, incubate at 37℃ for 10min; add stop solution and read OD 450 value.

[0066] The results are as follows Figure 5 As shown, Cut off value (positive judgment value) = 2.1 × N (N is the OD value of the negative control). According to the antibody concentration corresponding to the Cutoff value as the judgment value, compared with the negative control, the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 described in the present application can bind to BTV-1 and has a certain affinity.

[0067] (2) Verify the reactivity of BTV-1 VP2 protein with sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1, the specific method is as follows:

[0068] Coating: Add BTV-1VP2 protein expressed by the baculovirus expression system to the ELISA plate at a concentration of 0.5 μg / mL and coat overnight at 4°C; wash the plate 5 times with PBST buffer;

[0069] Blocking: Block the ELISA plate with PBST buffer containing 5% skim milk powder, 100 μL / well, incubate at 37°C for 1 h; wash the plate 5 times with PBST buffer;

[0070] Detection: The sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 and sheep negative serum were diluted with PBST and added to the ELISA plate, 100 μL / well, incubated at 37°C for 1 h, and the plate was washed 5 times with PBST buffer;

[0071] Add enzyme-labeled secondary antibody: dilute rabbit anti-sheep IgG1-HRP antibody at a ratio of 1:40000 and add to the ELISA plate, 100 μL / well, incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer;

[0072] Color development: Use TMB color development solution in the dark, incubate at 37℃ for 10min; after adding stop solution, read OD 450 value.

[0073] The results are as follows Figure 6As shown, S / N>2.1, indicating that the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 described in the present application can bind to the BTV-1VP2 protein.

[0074] 3. Micro-neutralization test of sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1

[0075] Vero cells were seeded in 96-well culture plates and cultured at 37°C and 5% CO 2 The sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 prepared in Example 1 was diluted 2-fold in a serum-free medium (starting from 1:2, actually 1:4), and each dilution was repeated at least 4 times. 50 μL of 100 TCID 50 BTV-1 virus solution was incubated at 37°C for 1 h to promote neutralization reaction. 50 The virus-antibody mixture and the control group were added to the cell culture plate and incubated at 37°C with 5% CO 2 The culture was continued under the same conditions for 3-4 days.

[0076] The virus-induced cytopathic effect (CPE) was observed using an inverted microscope and the situation of each well was recorded. 50 BTV-1 control wells were completely lesioned, 0.1 TCID 50 The final judgment standard was that the BTV-1 control well had no lesions. The culture supernatant was discarded, and 10% formaldehyde fixative was added for 1 hour. Then 100 μL of crystal violet solution was added to each well for staining for 30 minutes. The endpoint titer was calculated as the ability to neutralize 50% of 100 TCID 50 The antibody dilution required for BTV-1 infection of cells was finally expressed as IC 50 Indicates antibody titer. Figure 7 As shown, the sheep-derived anti-bluetongue virus recombinant neutralizing antibody SH1 of the present invention has good neutralizing activity against BTV-1, and its IC 50 It is 274ng / mL.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sheep-derived recombinant neutralizing antibody against bluetongue virus, characterized in that: The amino acid sequence of the heavy chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown in SEQ ID NO.

3.

2. A nucleic acid, characterized in that The nucleic acid encodes the light chain variable region sequence and the heavy chain variable region sequence of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid sequence encoding the heavy chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown in SEQ ID NO.2, and the nucleic acid sequence encoding the light chain variable region of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody is shown in SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to claim 2 or 3.

5. A host cell, characterized in that The host cell contains the recombinant expression vector according to claim 4.

6. The method for preparing the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1, characterized in that: The method comprises: (1) connecting the heavy chain variable region and light chain variable region sequences of the sheep anti-bluetongue virus recombinant neutralizing antibody to the heavy chain constant region and light chain constant region sequences of the sheep IgG1 antibody, respectively, and inserting them into the pcDNA3.4 expression vector to obtain pcDNA3.4-SH1-H and pcDNA3.4-SH1-L recombinant expression plasmids; (2) The recombinant expression plasmid obtained in step (1) is co-transfected into HEK293 cells, and the cells and supernatant are collected after culturing for 8-10 days, and purified by Protein G affinity chromatography to obtain sheep-derived anti-bluetongue virus recombinant neutralizing antibodies.

7. An immunoconjugate, characterized in that: The immunoconjugate comprises: a. The sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1; b. and a coupling moiety selected from the group consisting of a detectable marker, a drug, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins, or a combination thereof.

8. Use of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1 or the immunoconjugate according to claim 7 in the preparation of a reagent, test strip, or kit for detecting BTV-1.

9. Use of the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1 or the immunoconjugate according to claim 7 in the preparation of a drug for preventing or treating bluetongue disease.

10. A reagent, characterized in that The reagent includes the sheep-derived anti-bluetongue virus recombinant neutralizing antibody according to claim 1 or the immunoconjugate according to claim 7.

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