Monoclonal antibody specifically recognizing VP5 protein of blue tongue virus

Abstract

The application belongs to the technical field of immunology and in vitro diagnosis, and relates to a monoclonal antibody capable of specifically recognizing bluetongue virus VP5 protein. A BTV-1 recombinant VP5 protein with N-terminal 1-79 amino acids deleted is expressed; after the protein is used to immunize mice, a monoclonal antibody 5H11 against the BTV-1 VP5 protein is screened, and the heavy chain and light chain variable region sequences coding the antibody are obtained; the monoclonal antibody 5H11 can specifically react with the BTV-1 VP5 protein, and can specifically recognize the natural VP5 protein in the BTV-1 infected cells; through twice truncation expression of the BTV-1 VP5 protein, a B cell linear epitope recognized by the 5H11 on the VP5 protein is identified, which lays a foundation for in-depth study on the structure and function of the BTV VP5 protein and development of a novel vaccine.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and in vitro diagnostic technology, and specifically relates to a monoclonal antibody that specifically recognizes the VP5 protein of bluetongue virus. Background Technology

[0002] Bluetongue disease (BT) is a non-contact infectious disease caused by bluetongue virus (BTV) that affects ruminants such as sheep, goats, cattle, and deer. The vector for transmission is the Culicoides midge. Culicoides Symptoms of BTV include high fever, rapid breathing, oral ulceration and congestion, runny nose, and frothing at the mouth. Some cases may present with lameness, and in severe cases, it can lead to death. At least 29 serotypes of BTV have been identified so far, and there is no cross-immunity between different serotypes.

[0003] BTV belongs to the Reovirus family ( Reoviridae ) Circovirus genus ( Orbivirus BTV (Bacterial Virus) is a non-enveloped virus. Its genome consists of 10 segmented (S1-S10) double-stranded RNAs (dsRNAs) of varying sizes, encoding 7 structural proteins (VP1, VP2, VP3, VP4, VP5, VP6, and VP7) and 5 non-structural proteins (NS1, NS2, NS3 / NS3A, NS4, and NS5). BTV is a double-capsidd virus. The outer capsid is composed of VP2 and VP5 proteins, which encapsulate the viral core particle. The inner capsid is composed of VP3 and VP7 proteins, which encapsulate the ten dsRNAs and the transcription complex. The polymerase VP1, the capping enzyme VP4, and the helicase VP6 constitute the BTV transcription complex.

[0004] VP5 protein is composed of S6 The gene encodes a protein approximately 59 kDa in size, which exists as a trimer. Each monomer consists of a dagger domain (M1~S68), an unfolded domain (K69~F354), and an anchoring domain (I355~A526). The VP5 protein is a major component of the outer capsid of BTV and is a key membrane-penetrating protein. It mediates viral entry into host cells by sensing low pH conditions in the endosomal environment and triggering conformational changes.

[0005] This invention first successfully expressed a truncated BTV-1 VP5 protein lacking 79 amino acids at the N-terminus, prepared a monoclonal antibody against the VP5 protein using hybridoma technology, and identified a B-cell linear epitope of 5H11 on the BTV-1 VP5 protein, laying the foundation for further research on the structure and function of the VP5 protein. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention targets the VP5 protein of BTV-1, truncating amino acids 1-79 from the N-terminus. After codon optimization based on E. coli preference, a truncated gene encoding the BTV-1 VP5 protein, VP5Δ79aa, is artificially synthesized and cloned into an expression vector. The recombinant VP5Δ79aa protein is then expressed and purified, and subsequently immunized BALB / c mice. Using hybridoma technology, a specific monoclonal antibody against the recombinant VP5Δ79aa protein is successfully prepared, providing support for in-depth exploration of the biological function of the VP5 protein and laying a solid foundation for the development of BT diagnostic methods and novel vaccines. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides a monoclonal antibody that specifically recognizes the VP5 protein of bluetongue virus, said monoclonal antibody comprising an antibody heavy chain and an antibody light chain;

[0008] The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3;

[0009] The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0010] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.8.

[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] Preferably, the nucleic acid comprises the sequences shown in SEQ ID NO.9 and SEQ ID NO.10.

[0013] Thirdly, the present invention provides a recombinant vector containing the nucleic acid described in the second aspect above.

[0014] Fourthly, the present invention provides a recombinant cell containing the recombinant vector described in the third aspect above.

[0015] Fifthly, the present invention provides an immunoconjugate comprising:

[0016] (i) The monoclonal antibody described in the first aspect above;

[0017] (ii) and the coupling portion selected from the following group:

[0018] It can detect markers, drugs, gold nanoparticles / nanorobars, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0019] Sixthly, the present invention provides that the monoclonal antibody described in the first aspect above has any of the following uses:

[0020] (1) Application in the preparation of reagents for detecting bluetongue virus;

[0021] (2) Application in in vitro detection of bluetongue virus for non-disease diagnosis purposes;

[0022] (3) Application in identifying B-cell linear epitopes of bluetongue virus VP5 protein.

[0023] Preferably, the reagent for detecting bluetongue virus includes test strips or kits.

[0024] In a seventh aspect, the present invention provides an ELISA detection kit for bluetongue virus, the kit comprising the monoclonal antibody described in the first aspect above.

[0025] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, enzyme-labeled secondary antibody, washing solution, chromogenic agent, and stop solution.

[0026] Eighthly, the present invention provides a B-cell linear epitope peptide of bluetongue virus VP5 protein, wherein the B-cell linear epitope peptide is located at 164-183aa of bluetongue virus VP5 protein, and its sequence is: TEEDLQMRRLATALQKEIGE.

[0027] In a ninth aspect, the present invention provides the application of the B-cell linear epitope peptide described in the eighth aspect above in the preparation of BTV epitope vaccines.

[0028] The beneficial effects of this invention are:

[0029] (1) In this invention, the VP5 sequence of the standard reference strain of bluetongue virus type 1 in GenBank (GenBank accession number: FJ969723.1) was deleted, and the N-terminal amino acids 1-79 were deleted. After optimizing the gene codon according to the Escherichia coli preference, the truncated gene VP5Δ79aa encoding the BTV-1 VP5 protein was artificially synthesized and cloned into the expression vector pET-28a-sumo. The recombinant VP5Δ79aa protein was obtained by inducing expression and purification.

[0030] (2) Recombinant VP5Δ79aa protein was used to immunize 6-8 week old female BALB / c mice. Their spleen cells were then fused with mouse myeloma cells (SP2 / 0) to obtain a hybridoma cell line that could stably secrete VP5 protein. A monoclonal antibody 5H11 against recombinant VP5 protein (VP5Δ79aa protein) was prepared.

[0031] (3) The monoclonal antibody 5H11 can specifically react with recombinant VP5Δ79aa protein and can specifically recognize the natural VP5 protein in BTV-1 infected cells, laying the foundation for in-depth research on the structure and function of BTV VP5 protein as well as the diagnostic technology and vaccine development of BTV-1.

[0032] (4) Studies have shown that a region at the amino terminus of VP5 can directly affect the neutralization of bluetongue virus. This invention provides experimental materials for in-depth analysis of the structure and function of VP5 protein, and will also lay a theoretical foundation for further discovery of VP5 protein antigenic epitopes and vaccine development.

[0033] (5) This invention identifies a B-cell linear epitope on the BTV-1 VP5 protein by performing two truncated expression of the BTV-1 VP5 protein: 164 TEEDLQMRRLATALQKEIGE 183 This lays the foundation for in-depth research into the structure and function of the BTV VP5 protein, as well as for the development of diagnostic technologies and vaccines for BTV-1. Attached Figure Description

[0034] Figure 1 Inducible expression of recombinant VP5Δ79aa protein at 16℃ (A and B), where M is the molecular weight standard of the protein; 1 is the uninduced sample; and 2~9 are samples induced at 16℃ for 2, 4, 6, 8, 10, 12, 14, and 16 h.

[0035] Figure 2 Solubility analysis of recombinant VP5Δ79aa protein induced at 16 ℃; where M is the molecular weight standard of protein; 1 is the uninduced sample; 2 is the expression culture before ultrasonic disruption; 3 is the supernatant after ultrasonic disruption; 4 is the precipitate after ultrasonic disruption.

[0036] Figure 3 SDS-PAGE and Western blot identification results of purified recombinant VP5Δ79aa protein; where M is the molecular weight standard of protein; 1 is the purified recombinant VP5Δ79aa protein.

[0037] Figure 4IFA was used to identify the reactivity of monoclonal antibodies with native VP5 protein; A1 and B1 are BHK-21 cells infected with BTV-1, with Hoechst 33342 staining of the cell nuclei; A2 is the supernatant of negative cells; B2 is the monoclonal antibody 5H11; A3 is a combined image of A1 and A2; B3 is a combined image of B1 and B2.

[0038] Figure 5 Western blot was used to identify the specificity of the monoclonal antibody 5H11; where M is the protein molecular weight standard; 1 is BHK-21 cells; 2 is BHK-21 cells infected with BTV-1; and 3 is recombinant AHSV VP5 protein.

[0039] Figure 6 Epitope identification of monoclonal antibody 5H11 on VP5; where A is a schematic diagram of VP5 epitope identification; B is the verification of primary truncated antigenic epitopes; and C is the verification of secondary truncated antigenic epitopes. Detailed Implementation

[0040] 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.

[0041] The following are the cells, main reagents, and experimental animals involved in the implementation:

[0042] BTV-1 strain, BHK-21 cells, mouse myeloma cells (SP2 / 0), recombinant AHSV VP5 protein, and BTV-1 positive serum were all preserved by the National Key Laboratory for Animal Disease Control. BL21(DE3) competent cells and IPTG were purchased from Takara Bio Inc. (Dalian). 6×His-tagged monoclonal antibody, goat anti-mouse IgG (HRP), and goat anti-mouse IgG (Alexa Flour 568) were purchased from Abcam. DMEM medium, fetal bovine serum, Super Signal West Pico PLUS kit, and Hoechst 33342 were purchased from Thermo Fisher Scientific. Kanamycin, Triton X-100, and bovine serum albumin (BSA) were purchased from Solarbio. Carbonate-bicarbonate buffer and PVDF membranes were purchased from Merck. Ni-NTA AAgarose was purchased from Qiagen. The mouse monoclonal antibody Ig subclass identification ELISA kit was purchased from Fondide Technology (Wuhan) Co., Ltd.

[0043] Example 1: Preparation of monoclonal antibody 5H11

[0044] 1. Construction of recombinant expression plasmids

[0045] Based on the amino acid sequence (GenBank accession number: ACR58462.1) and nucleotide sequence (GenBank accession number: FJ969723.1) of the VP5 protein of the bluetongue virus type 1 standard reference strain in GenBank, the sequence encoding the first 79 amino acids of the N-terminus was truncated (gene sequence shown in SEQ ID NO.11). Codon optimization was performed according to the codon bias of E. coli expression (shown in SEQ ID NO.12). The target sequence was amplified by Wuhan Jinkairui Biotechnology Co., Ltd. and ligated into the expression vector pET-28a-sumo to synthesize a truncated recombinant plasmid, which was named pET-sumo-VP5Δ79aa.

[0046] 2. Expression and identification of recombinant VP5Δ79aa protein

[0047] The recombinant plasmid pET-sumo-VP5Δ79aa was transformed into BL21 (DE3) competent cells and cultured overnight at 37 °C in LB medium containing kanamycin. The cells were then seeded at a volume ratio of 1:100 into LB (K+) medium and cultured at 37 °C until OD (open-cell growth) occurred. 600 When the concentration of the protein was 0.6–0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16 °C. Bacterial culture was collected every two hours. The expression of the target protein was analyzed using SDS-PAGE and Western blot. Simultaneously, to determine whether the truncation of the first 79 amino acids at the N-terminus affected the reactivity of the recombinant VP5Δ79aa protein, BTV-1 positive serum (1:500) and VP5 polyclonal antibody (1:500) were used as primary antibodies, and HRP-labeled rabbit anti-goat IgG (1:10000) and HRP-labeled goat anti-rabbit IgG (1:10000) were used as secondary antibodies. Western blot was used to identify the reactivity of the recombinant VP5Δ79aa protein.

[0048] Western blot results are as follows Figure 1 As shown in Figure A, the recombinant VP5Δ79aa protein can be expressed in large quantities at 16 ℃, with the optimal induction at 16 ℃ for 16 h using 0.5 mM IPTG. The size is approximately 70 kDa, which is consistent with the expected size.

[0049] Reactivity results as follows Figure 1 As shown in Figure B, the recombinant VP5Δ79aa protein can react with VP5 polyclonal antibody and BTV-1 positive serum.

[0050] 3. Solubility analysis and purification of recombinant VP5Δ79aa protein

[0051] The bacterial culture after induction was collected by centrifugation, resuspended in PBS, and then disrupted using an ultrasonic homogenizer. The ultrasonic power was 140 W, the ultrasonic time was 3 s, the interval was 3 s, and the working time was 40 min. After centrifugation, the precipitate and supernatant were collected separately, and their solubility was analyzed by SDS-PAGE. The precipitate after ultrasonic disruption was purified by Ni-NTA affinity chromatography. The purified VP5 protein was refolded at 4 ℃ using urea solutions of different concentrations (pH 8.0) prepared with PBS and 1 mM EDTA. The urea concentrations were 6M, 4M, 3M, 2M, 1M, 0.5M, and 0M, respectively. The solution was changed every 12 h. The VP5 protein after refolding was analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown.

[0052] The induced bacterial culture was sonicated, and the supernatant and precipitate were analyzed by SDS-PAGE. The results are as follows: Figure 3 As shown, recombinant VP5Δ79aa protein was mainly expressed in the form of inclusion bodies at 16 ℃. The precipitate after ultrasonic disruption was purified according to the Ni-NTA Agarose instructions. The purified recombinant VP5Δ79aa protein was then refolded in urea solutions of different concentration gradients. Identification was performed by SDS-PAGE and Western blot. The results showed that highly pure VP5Δ79aa protein was obtained after purification and refolding, and this protein reacted with BTV-1 positive serum.

[0053] 4. Animal immunization

[0054] Using purified VP5 protein as the antigen, six 6-8 week old BALB / c mice were immunized, with one mouse serving as a negative control. Each mouse in the experimental group was initially immunized with 80 µg of recombinant VP5Δ79aa protein emulsified with Freund's complete adjuvant at a 1:1 ratio via multiple subcutaneous injections. At 2, 4, and 5 weeks, mice were re-immunized with the same dose of recombinant VP5Δ79aa protein emulsified with Freund's incomplete adjuvant at a 1:1 ratio. Seven days after the fourth immunization, tail vein blood was collected from the mice. Purified recombinant VP5Δ79aa protein (500 ng / mL) was coated onto an ELISA plate. Serum from immunized mice was used as the primary antibody, HRP-labeled goat anti-mouse IgG as the secondary antibody, and serum from unimmunized mice as the negative control. Serum titers were measured. OD was determined using an ELISA reader. 450 nm Value, serum OD of the test 450 nm / Negative control OD450 nm When (S / N) ≥ 2.1, the maximum dilution factor is the antibody titer. Mice with high antibody titers are selected to prepare monoclonal antibodies.

[0055] The antibody titer in the serum of mice immunized with recombinant VP5Δ79aa protein was determined by indirect ELISA. Spleen cells with the highest VP5 antibody titer (1:128000) in the immune serum of mice after the fourth immunization were selected for the preparation of monoclonal antibody 5H11.

[0056] 5. Preparation of monoclonal antibodies

[0057] The day before cell fusion, feeder cells were prepared. Healthy, unimmunized female BALB / c mice had their spleens removed under aseptic conditions. A single-spleen cell suspension was prepared using HAT medium containing 20% ​​fetal bovine serum and seeded into 96-well plates (100 µL per well). The plates were then incubated at 37°C in a 5% CO2 incubator. Seven days after the fourth immunization, the spleens were removed under aseptic conditions, and a single-cell suspension was prepared. This suspension was mixed with SP2 / 0 cells at a ratio of 5:1 to 10:1. The mixture was centrifuged at low speed to obtain a cell pellet. The pellet was washed once with DMEM, centrifuged again, and the culture medium was discarded. The cell pellet was then gently loosened by stirring the bottom of the centrifuge tube. 1 mL of preheated (37°C) 50% PEG1450 was slowly and evenly added to the centrifuge tube over 1 minute to initiate cell fusion. After 1 minute, preheated (37°C) DMEM was slowly added to terminate the fusion. During this process, the mixture could be gently stirred with a pipette tip to ensure even mixing. After low-speed centrifugation, the cells were gently resuspended and mixed in HAT medium containing 20% ​​fetal bovine serum. 100 µL of the mixture was then seeded into pre-prepared feeder cell plates and cultured at 37 ℃ in a 5% CO2 incubator. After successful cell fusion, the supernatant of unfused SP2 / 0 cells was used as a negative control, and positive hybridoma cells were screened using an indirect ELISA method. The selected positive hybridoma cells were subjected to three consecutive subclonal purifications using limiting dilution to obtain a stable hybridoma cell line secreting VP5 protein, which was then expanded for culture. The hybridoma cell supernatant was collected, and the cell subtype was identified using a mouse monoclonal antibody subtype identification kit. 6–8 week old BALB / c mice were intraperitoneally injected with sterile paraffin oil, followed by intraperitoneal injection of hybridoma cells one week later. Ascites fluid was collected 1–2 weeks later after the mice's peritoneum expanded.

[0058] 6. Identification of monoclonal antibodies

[0059] (1) Indirect ELISA

[0060] The purified recombinant VP5Δ79aa protein was diluted to 500 ng / mL with carbonate-bicarbonate buffer and coated onto an ELISA plate at 100 µL per well, incubated overnight at 4 °C. After washing three times with PBST, 200 µL of 5% skim milk powder was added to each well, and the plate was blocked at 37 °C for 1 h. After washing three times with PBST, 100 µL of supernatant from hybridoma cells was added to each well as a negative control (SP2 / 0 cell supernatant), and the plate was incubated at 37 °C for 1 h. After washing three times with PBST, HRP-labeled goat anti-mouse IgG (1:100000) was added, and the plate was incubated at 37 °C for 1 h. After washing three times with PBST, 100 µL of TMB chromogenic solution was added to each well, and the plate was incubated at 37 °C in the dark for 15 min. The reaction was then stopped by adding 2M H2SO4, and the OD was measured using an ELISA reader. 450nm value.

[0061] The results showed that monoclonal antibody 5H11 reacted well with recombinant VP5Δ79aa protein (Table 1). The subtypes of the supernatants from the two hybridoma cell lines were identified using a mouse monoclonal antibody subtype identification kit, and the results indicated that the heavy chain of monoclonal antibody 5H11 was IgG2b.

[0062] Table 1. ELISA results of the reactivity of monoclonal antibody 5H11 with recombinant VP5 protein.

[0063]

[0064] (2) Immunofluorescence (IFA)

[0065] BHK-21 cells were seeded into 24-well cell culture plates with prepared slides. After 24 h, BTV-1 cells were seeded, with blank BHK-21 cells used as a negative control. After 24 h of culture, the culture medium was discarded; cells were fixed for 30 min with pre-chilled 4% paraformaldehyde; permeabilized with 0.2% Triton X-100 for 10 min; washed 3 times with PBS, and blocked with 3% BSA at room temperature for 1 h; washed 3 times with PBS, and incubated overnight at 4 ℃ with the supernatant of the hybridoma cells to be tested; washed 3 times with PBS, and incubated for 1 h at room temperature with Alexa Fluor 568-labeled goat anti-mouse IgG (1:1000); washed 3 times with PBS, and incubated for 10 min at room temperature in the dark with Hoechst 33342 (1:2000); washed 3 times with PBS, and mounted. The results were observed under an inverted fluorescence microscope.

[0066] The results are as follows Figure 4As shown, BHK-21 cells infected with BTV-1 showed red fluorescence after incubation with monoclonal antibody 5H11, while cells incubated with negative cell supernatant did not show red fluorescence, indicating that the monoclonal antibody 5H11 described in this application can specifically recognize the natural VP5 protein in BTV-1 infected cells.

[0067] (3) Western blot (WB)

[0068] BHK-21 cells, BTV-1, and recombinant AHSV / VP5 protein were sequentially added to sample wells for SDS-PAGE electrophoresis. The antigens were then transferred to PVDF membranes via electroblotting. The membranes were blocked with 5% skim milk at room temperature for 1 h. The supernatant of hybridoma cells 5H11 was used as the primary antibody (1:500), and the membranes were incubated overnight at 4 °C. The membranes were washed three times with PBST for 10 min each time, and HRP-labeled goat anti-mouse IgG (1:10000) was added and incubated at room temperature for 1 h. The membranes were washed three times with PBST for 10 min each time. The reaction was performed using a Super Signal WestPico PLUS chemiluminescent substrate in the dark, and the target bands were observed using a ChemiDoc XRS+ imaging system (Bio-Rad).

[0069] The results are as follows Figure 5 As shown, the monoclonal antibody 5H11 specifically recognizes the VP5 protein in BTV-1 and does not react with blank BHK-21 cells, recombinant BTV-16 VP5Δ41aa protein, VP5 protein in EHDV, or recombinant AHSV VP5 protein. A single target band is visible at 59 kDa.

[0070] (4) Monoclonal antibody titer determination

[0071] The titer of anti-BTV VP5 monoclonal antibody 5H11 was detected by ELISA, and the results are shown in Table 2. The results show that the titer of this monoclonal antibody can reach 1:25600.

[0072] Table 2. Results of valence determination

[0073]

[0074] 7. Amplification of the variable region sequences of the 5H11 light and heavy chains of monoclonal antibody

[0075] Total RNA was extracted from hybridoma cells secreting the monoclonal antibody 5H11 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.

[0076] The cDNA of the heavy chain variable region of the monoclonal antibody 5H11 is: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATAACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGG TAAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTGGACACCTCTGCCAGCTCTGCCTATTTGCGGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGGTTGGACTGGGAGGGAAACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.9 shown);

[0077] The cDNA of the light chain variable region of the monoclonal antibody 5H11 is: GATGTTGTGATGACCCAAAGTCCATTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGGCTGTTCACAGTAATGGAAACACCTATTCACATTGGTACCTGCAGAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTA CAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGTTCACGTTCGACTCGGGGACAAAGTTGGAAATAAAACGG(SEQ ID NO.10 shown).

[0078] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5H11, as determined by sequencing, is shown below:

[0079] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGKPTYADDFKGRFAFSLDTSASSAYLRINNLKNEDTATYFCARLDWEGNFDYWGQGTTLTVSS (shown in SEQ ID NO.7);

[0080] The amino acid sequence of the light chain variable region of the monoclonal antibody is shown below:

[0081] DVVMTQSPFSLPVSLGDQASISCRSSQRLVHSNGNTYSHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPFTFDSGTKLEIKR (shown in SEQ ID NO. 8).

[0082] The light and heavy chain variable region sequences of the monoclonal antibody 5H11 obtained from sequencing were analyzed on the abysis.org website to determine its CDR region.

[0083] The sequences of the three complementarity-determining regions (CDRs) of the heavy chain variable region are shown below:

[0084] CDR1: DYSMH (shown as SEQ ID NO.1);

[0085] CDR2: WINTETGKPTYADDFKG (shown as SEQ ID NO.2);

[0086] CDR3: LDWEGNFDY (shown as SEQ ID NO.3);

[0087] The sequences of the three complementarity-determining regions (CDRs) of the light chain variable region are shown below:

[0088] CDR1: RSSQRLVHSNGNTYSH (shown as SEQ ID NO.4);

[0089] CDR2: KVSNRFS (shown in SEQ ID NO.5);

[0090] CDR3: SQSTHVPPFT (shown in SEQ ID NO.6).

[0091] 8. Recognition of the 5H11 antigenic epitope of monoclonal antibodies

[0092] The VP5Δ79aa sequence was initially truncated into three fragments (F1: 80-233aa, F2: 213-368aa, F3: 348-526aa), and GST fusion expression was performed using the expression vector pGEX-6p-1. Then, the F1 fragment was further truncated into four fragments (F11: 80-132aa, F12: 112-164aa, F13: 144-202aa, F14: 182-233aa) and GST fusion expression was performed using the pGEX-6p-1 vector. The reactivity with the monoclonal antibody 5H11 was then tested. The empty vector pGEX-6p-1 served as a control.

[0093] like Figure 6 As shown, this invention uses the epitope walking method to identify the antigenic epitopes corresponding to 5H11 on the VP5 protein, and the amino acid sequences of the epitopes are as follows: 164 TEEDLQMRRLATALQKEIGE 183 This provides a theoretical basis for the design of BTV epitope vaccines and the establishment of novel diagnostic methods.

[0094] 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 specifically recognizing VP5 protein of blue tongue 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.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2 and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; the variable region CDR of the antibody light chain comprises CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5 and CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

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.7, and the amino acid sequence of the variable region of the antibody light chain is as shown in SEQ ID NO.

8.

3. A nucleic acid, characterized in that, the nucleic acid encodes the heavy chain variable region and the light chain variable region 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.9 and SEQ ID NO.

10.

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.The monoclonal antibody of claim 1 or 2 has any one of the following uses: (1) use in preparing a reagent for detecting blue tongue virus; (2) use in in vitro detection of blue tongue virus for non-disease diagnosis purposes; (3) use in identifying B cell linear epitope of VP5 protein of blue tongue virus.

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