Monoclonal antibody for specifically recognizing bluetongue virus VP5 protein

Abstract

The invention belongs to the technical field of immunology and in vitro diagnosis, and relates to a monoclonal antibody for specifically recognizing bluetongue virus VP5 protein. According to the invention, BTV-1 recombinant VP5 protein with deletion of 1-79 amino acids at the N terminal is obtained through expression; immunizing a mouse with the protein, screening out a monoclonal antibody 5H11 aiming at the BTV-1VP5 protein, and obtaining heavy chain and light chain variable region sequences for coding the antibody; the monoclonal antibody 5H11 can be subjected to a specific reaction with BTV-1 VP5 protein, and can be used for specifically recognizing natural VP5 protein in BTV-1 infected cells; by carrying out truncation expression on the BTV-1VP5 protein twice, the B cell linear epitope identified by 5H11 on the VP5 protein is identified, and a foundation is laid for deeply researching the structural function of the BTV VP5 protein and researching and developing a novel vaccine.

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 specifically recognizes the VP5 protein of Bluetongue virus. Background Art

[0002] Bluetongue (BT) is a non-contact infectious disease that infects ruminants such as sheep, goats, cattle, and deer caused by Bluetongue virus (BTV). The transmission vector is Culicoides. The morbidity characteristics include high fever, rapid breathing, oral ulceration and congestion, runny nose, foaming at the mouth, etc. Some cases will show lameness, and in severe cases, it will lead to the death of animals. Currently, at least 29 serotypes of BTV have been discovered, and there is no cross-immune protection between different serotypes.

[0003] BTV belongs to the genus Orbivirus of the family Reoviridae, without an envelope. Its genome consists of 10 segments of double-strand RNA (dsRNA) of different sizes (S1 - S10), encoding 7 structural proteins (VP1, VP2, VP3, VP4, VP5, VP6, and VP7) and 5 non-structural proteins (NS1, NS2, NS3 / NS3A, NS4, NS5). BTV is a double-layer capsid virus. The outer capsid is composed of VP2 protein and VP5 protein, which encapsulates the viral core particles; the inner capsid is composed of VP3 protein and VP7 protein, which encapsulates ten dsRNAs and the transcription complex; the polymerase VP1, capping enzyme VP4, and helicase VP6 constitute the BTV transcription complex.

[0004] The VP5 protein is encoded by the S6 gene, with a size of approximately 59 kDa, existing in the form of a trimer. Each monomer consists of a dagger domain (M1 - S68), an extended domain (K69 - F354), and an anchoring domain (I355 - A526). The VP5 protein is the main component of the outer capsid of BTV and is a key membrane penetration protein. It triggers conformational changes by sensing the low pH environment of the endosome and mediates the entry of the virus into host cells.

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

[0006] To solve the above technical problems, the present invention targets the VP5 protein of BTV-1. After truncating the N-terminal 1-79 amino acids of the VP5 protein and optimizing the gene codons according to the codon bias of Escherichia coli, a truncated gene VP5Δ79aa encoding the BTV-1 VP5 protein is artificially synthesized and cloned into an expression vector. Then, the recombinant VP5Δ79aa protein is expressed and purified, and subsequently used to immunize BALB / c mice. Using the hybridoma technology, a specific monoclonal antibody against the recombinant VP5Δ79aa protein is successfully prepared, providing support for further exploring the biological function of the VP5 protein and laying a solid foundation for the development of BT diagnostic methods and new vaccines. The specific contents are as follows:

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

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

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

[0010] Preferably, 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.

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

[0013] In the third aspect, the present invention provides a recombinant vector that contains the nucleic acid described in the second aspect above.

[0014] In the fourth aspect, the present invention provides a recombinant cell that contains the recombinant vector described in the third aspect above.

[0015] In the fifth aspect, the present invention provides an immunoconjugate, and the immunoconjugate includes:

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

[0017] (ii) and a coupling moiety selected from the group consisting of:

[0018] a detectable label, a drug, gold nanoparticles / nanorods, magnetic nanoparticles, a virus capsid protein or VLP, or a combination thereof.

[0019] In a sixth aspect, the present invention provides the following uses of the monoclonal antibody described in the first aspect above:

[0020] (1) Application in the preparation of a reagent for detecting Bluetongue virus;

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

[0022] (3) Application in the identification of the B-cell linear epitope of Bluetongue virus VP5 protein.

[0023] Preferably, the reagent for detecting Bluetongue virus includes a test strip or a kit.

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

[0025] Preferably, the kit further includes an enzyme-linked immunosorbent assay (ELISA) plate, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a chromogenic agent, and a stop solution.

[0026] In an eighth aspect, the present invention provides a B-cell linear epitope peptide of Bluetongue virus VP5 protein, which B-cell linear epitope peptide is located at positions 164 - 183 aa 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 a BTV epitope vaccine.

[0028] The beneficial effects of the present invention are:

[0029] (1) Referring to the VP5 sequence of the Bluetongue virus type 1 standard reference strain in GenBank (GenBank accession number: FJ969723.1), the N-terminal 1 - 79 amino acids were deleted, and after optimizing the gene codons 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, and the recombinant VP5Δ79aa protein was obtained by induced expression and purification;

[0030] (2) Female BALB / c mice aged 6 - 8 weeks were immunized with the recombinant VP5Δ79aa protein. Spleen cells were taken from them and fused with mouse myeloma cells (SP2 / 0) to obtain a hybridoma cell line that could stably secrete monoclonal antibodies against the VP5 protein, and monoclonal antibody 5H11 against the recombinant VP5 protein (VP5Δ79aa protein) was prepared.

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

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

[0033] (5) In the present invention, the BTV-1 VP5 protein was truncated and expressed twice, and 1 B-cell linear epitope of the monoclonal antibody 5H11 described in this application on the BTV-1 VP5 protein was identified: 164 TEEDLQMRRLATALQKEIGE 183 aa, laying a foundation for the in-depth study of the structure and function of the BTV VP5 protein and the diagnosis technology and vaccine development of BTV-1. Brief Description of the Drawings

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

[0035] Figure 2 Solubility analysis of the recombinant VP5Δ79aa protein induced at 16 °C; among them, M is the protein molecular weight standard; 1 is the uninduced sample; 2 is the expression bacterial liquid before ultrasonic disruption; 3 is the supernatant after ultrasonic disruption; 4 is the precipitate after ultrasonic disruption.

[0036] Figure 3 Identification results of SDS-PAGE (A) and Western-blot (B) of the purified recombinant VP5Δ79aa protein; among them, M is the protein molecular weight standard; 1 is the purified recombinant VP5Δ79aa protein.

[0037] Figure 4IFA identification of the reactivity of monoclonal antibodies with native VP5 protein; among them, A1 and B1 are BHK-21 cells infected with BTV-1, and the nuclei are stained with Hoechst 33342; A2 is the negative cell supernatant; B2 is the monoclonal antibody 5H11; A3: merged image of A1 and A2; B3 is the merged image of B1 and B2.

[0038] Figure 5 Western-blot identification of the specificity of monoclonal antibody 5H11; among them, M is the protein molecular weight standard; 1 is BHK-21 cells; 2 is BHK-21 cells infected with BTV-1; 3 is the recombinant AHSV VP5 protein.

[0039] Figure 6 Epitope identification of monoclonal antibody 5H11 on VP5; among them, A is the schematic diagram of VP5 epitope identification; B is the verification of primary truncated antigen epitopes; C is the verification of secondary truncated antigen epitopes. Detailed implementation manners

[0040] The 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 to 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 literature or known methods, and for the reaction conditions not listed, they are easily obtained by those skilled in the art.

[0041] Cells, main reagents and experimental animals involved in the following experiments:

[0042] The BTV-1 strain, BHK-21 cells, mouse myeloma cells (SP2 / 0), recombinant AHSV VP5 protein, and BTV-1 positive serum are all stored in the National Key Laboratory for Animal Disease Prevention and Control. BL21(DE3) competent cells and IPTG are purchased from Takara Bio Inc. (Dalian). The 6×His-tag monoclonal antibody, goat anti-mouse IgG (HRP), and goat anti-mouse IgG (AlexaFlour 568) are purchased from Abcam. DMEM medium, fetal bovine serum, Super Signal West Pico PLUS kit, and Hoechst 33342 are purchased from Thermo Fisher Scientific. Kanamycin, Triton X-100, and bovine serum albumin (BSA) are purchased from Solarbio. The carbonate-bicarbonate buffer and PVDF membrane are purchased from MERCK. Ni-NTA Agarose is purchased from QIAGEN. The mouse monoclonal antibody Ig subclass identification ELISA kit is purchased from Fuyinde Technology (Wuhan) Co., Ltd.

[0043] Preparation of Monoclonal Antibody 5H11 in Example 1

[0044] 1. Construction of Recombinant Expression Plasmid

[0045] Referring to 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, after truncating the base sequence encoding the first 79 amino acids at the N-terminus (the gene sequence is shown in SEQ ID NO.11), codon optimization was performed according to the codon preference of Escherichia coli expression (shown in SEQ ID NO.12). Wuhan Jinkairui Bioengineering Co., Ltd. was commissioned to amplify the target sequence and ligate it to the expression vector pET-28a-sumo to synthesize a recombinant plasmid for truncated expression, 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. Inoculated into LB(K+) medium at a volume ratio of 1:100 and cultured at 37°C until OD 600 = 0.6 - 0.8, IPTG with a final concentration of 0.5 mM was added, and induction expression was carried out at 16°C respectively. Bacterial liquid was collected every two hours. SDS-PAGE and Western-blot were used to analyze the expression of the target protein. At the same time, to determine whether the truncation of the first 79 amino acids at the N-terminus would affect the reactivity of the recombinant VP5Δ79aa protein, BTV-1 positive serum (1:500) and VP5 polyclonal antibody (1:500) were used as the primary antibodies, and HRP-labeled rabbit anti-goat IgG (1:10000) and HRP-labeled goat anti-rabbit IgG (1:10000) were used as the secondary antibodies to identify the reactivity of the recombinant VP5Δ79aa protein by WB.

[0048] The results of Western-blot are as shown in Figure 1 A below. The recombinant VP5Δ79aa protein can be highly expressed at 16°C. The best induction condition is 16 h at 16°C with 0.5 mM IPTG, and its size is about 70 ku, which is consistent with the expected size.

[0049] The results of reactivity are as shown in Figure 1 B below. 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 solution after induced expression was collected by centrifugation, resuspended in PBS, and then disrupted using an ultrasonic crusher with an ultrasonic power of 140 W, an ultrasonic time of 3 s, an interval time of 3 s, and a working time of 40 min. After centrifugation, the precipitate and supernatant after ultrasonication were collected separately, and their solubility was analyzed by SDS-PAGE. The precipitate after ultrasonic disruption was purified according to Ni-NTA affinity chromatography. The purified VP5 protein was renatured at 4°C, and the renaturation solution was urea solutions with different concentrations (pH 8.0) prepared with PBS and 1 mM EDTA. The urea concentrations were 6 M, 4 M, 3 M, 2 M, 1 M, 0.5 M, and 0 M in sequence. The liquid was changed every 12 h, and the renatured VP5 protein was analyzed by SDS-PAGE. The results are as Figure 2 shown.

[0052] The bacterial solution after induced expression was ultrasonically disrupted, and the supernatant and precipitate after ultrasonication were separately taken for SDS-PAGE analysis. The results are as Figure 3 shown. Under the condition of 16°C, the recombinant VP5Δ79aa protein was mainly expressed in the form of inclusion bodies ( Figure 3 as shown in A). The precipitate after ultrasonic disruption was purified according to the operation instructions of Ni-NTA Agarose. The purified recombinant VP5Δ79aa protein was renatured in urea solutions with different concentration gradients. Through SDS-PAGE and Western-blot identification, the results showed that a VP5Δ79aa protein with relatively high purity was obtained after purification and renaturation, and this protein could react with BTV-1 positive serum ( Figure 3 as shown in B).

[0053] 4. Animal immunization

[0054] Using the purified VP5 protein as an antigen, 6 six- to eight-week-old BALB / c mice were selected for immunization, and one mouse was not immunized as a negative control. In the experimental group, each mouse was emulsified with 80 μg of recombinant VP5Δ79aa protein and Freund's complete adjuvant in equal volume (1:1) and immunized by subcutaneous multi-point injection for the first time. After 2, 4, and 5 weeks respectively, the mice were immunized again with the same dose of recombinant VP5Δ79aa protein emulsified with Freund's incomplete adjuvant in equal volume (1:1). Seven days after the fourth immunization, the tail vein blood of the mice was collected. The purified recombinant VP5Δ79aa protein (500 ng / mL) was coated on an enzyme-linked immunosorbent assay (ELISA) plate. The serum of the immunized mice was used as the primary antibody, HRP-labeled goat anti-mouse IgG was used as the secondary antibody, and the serum of the non-immunized mice was used as the negative control to determine the serum titer of the immunized mice. The OD 450nm value was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The OD 450nm of the test serum / OD of the negative control450nm When (S / N) ≥ 2.1, the maximum dilution multiple is the antibody titer. Select mice with high antibody titers to prepare monoclonal antibodies.

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

[0056] 5. Preparation of Monoclonal Antibodies

[0057] One day before cell fusion, feeder cells were prepared. Take healthy BALB / c female mice that have not been immunized, remove their spleens under sterile conditions, make a single-cell spleen cell suspension with HAT medium containing 20% fetal bovine serum, spread it in a 96-well plate, 100 μL per well, and culture it in an incubator at 37°C and 5% CO2. Seven days after the fourth immunization of the mice, the spleens were removed under sterile conditions, prepared into single-cell suspensions, mixed evenly with SP2 / 0 cells at a ratio of 5:1 - 10:1, centrifuged at low speed to obtain cell pellets, washed once with DMEM, centrifuged again, discard the medium completely, and gently stir the bottom of the centrifuge tube to loosen the cell pellets. Slowly and evenly add 1 mL of 50% PEG1450 preheated at 37°C to the centrifuge tube within 1 minute for cell fusion. After 1 minute, slowly add preheated DMEM at 37°C to terminate the fusion. During this period, gently stir with a pipette tip to make it act evenly. After low-speed centrifugation, gently suspend and mix with HAT medium containing 20% fetal bovine serum, and spread it into the pre-prepared feeder cell plate at a volume of 100 μL per well, and culture it in an incubator at 37°C and 5% CO2. After successful cell fusion, the supernatant of unfused SP2 / 0 cells was used as a negative control, and positive hybridoma cells were screened by the indirect ELISA method. The selected positive hybridoma cells were subcloned and purified continuously 3 times by the limited dilution method to obtain a hybridoma cell line that stably secretes antibodies against the VP5 protein, and it was expanded and cultured. Collect the supernatant of hybridoma cells and identify its subtype with a mouse monoclonal antibody subtype identification kit. Select 6 - 8-week-old BALB / c mice, inject them intraperitoneally with sterilized paraffin oil, inject hybridoma cells intraperitoneally 1 week later, and collect ascites 1 - 2 weeks later when the mice's abdomens are distended.

[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 on an ELISA plate at 100 μL per well overnight at 4°C. After washing 3 times with PBST, 5% skim milk powder was added at 200 μL per well and blocked at 37°C for 1 h. After washing 3 times with PBST, the supernatant of the hybridoma cells to be tested was added into the ELISA plate, and at the same time, the supernatant of SP2 / 0 cells was added as a negative control, 100 μL per well, and incubated at 37°C for 1 h. After washing 3 times with PBST, HRP-labeled goat anti-mouse IgG (1:100000) was added and incubated at 37°C for 1 h. After washing 3 times with PBST, TMB chromogenic solution was added, 100 μL per well, and incubated at 37°C in the dark for 15 min, then 2 M H2SO4 was added to terminate the reaction, and the OD 450nm value was measured using an ELISA reader.

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

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

[0063] Monoclonal antibody 5H11 Negative control 1.04415 0.069

[0064] (2) Immunofluorescence (IFA)

[0065] BHK-21 cells were seeded in a 24-well cell culture plate with coverslips. After 24 h, BTV-1 was inoculated, and blank BHK-21 cells were used as a negative control. After culturing for 24 h, the medium was discarded; pre-cooled 4% paraformaldehyde was added to fix the cells for 30 min; 0.2% Triton X-100 was used for membrane penetration for 10 min; washed 3 times with PBS, 3% BSA was added and blocked at room temperature for 1 h; washed 3 times with PBS, the supernatant of the hybridoma cells to be tested was added and incubated overnight at 4°C; washed 3 times with PBS, AlexaFluor 568-labeled goat anti-mouse IgG (1:1000) was added and incubated at room temperature for 1 h; washed 3 times with PBS, Hoechst 33342 (1:2000) was added and incubated at room temperature in the dark for 10 min; after washing 3 times with PBS, the coverslip was sealed, and the results were observed under an inverted fluorescence microscope.

[0066] The results were as Figure 4As shown, red fluorescence could be observed in BHK-21 cells infected with BTV-1 after incubation with monoclonal antibody 5H11, while no red fluorescence appeared in cells incubated with the supernatant of negative cells, indicating that the monoclonal antibody 5H11 described in this application could specifically recognize the native VP5 protein in BTV-1-infected cells.

[0067] (3) Western-blot (WB)

[0068] BHK-21 cells, BTV-1, and recombinant AHSV / VP5 protein were added to the sample wells in sequence for SDS-PAGE electrophoresis, and the antigen was transferred to a PVDF membrane by electroblotting. Block with 5% skim milk at room temperature for 1 h; use the supernatant of hybridoma cell 5H11 as the primary antibody (1:500) and incubate overnight at 4°C; wash the membrane 3 times with PBST, 10 min each time, add HRP-labeled goat anti-mouse IgG (1:10000) and incubate at room temperature for 1 h; wash the membrane 3 times with PBST, 10 min each time, react with Super Signal West Pico PLUS chemiluminescent substrate in the dark, and observe the target band using a ChemiDoc XRS+ imaging system (Bio-Rad).

[0069] The results are as Figure 5 shown. The monoclonal antibody 5H11 specifically recognized the VP5 protein in BTV-1, did not react with blank BHK-21 cells, recombinant BTV-16VP5Δ41aa protein, the VP5 protein in EHDV, or recombinant AHSV VP5 protein, and a single target band was visible at 59 ku.

[0070] (4) Determination of the titer of monoclonal antibody

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

[0072] Table 2 Results of titer determination

[0073] Primary antibody concentration <![CDATA[OD 450 value]]> 1：1600 0.9634 1：3200 0.5572 1：6400 0.3515 1：12800 0.2163 1：25600 0.1508 1：51200 0.1118 1：102400 0.0980 1：204800 0.0961 Negative control 0.0798

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

[0075] Total RNA was extracted from hybridoma cells secreting monoclonal antibody 5H11 using the TRIzol lysis method, and then reverse transcription was performed using a reverse transcription kit to synthesize cDNA. PCR amplification was carried out using the obtained cDNA as a template, and the amplification product was ligated to a vector for sequencing to obtain the nucleotide sequences of the heavy and light chain variable regions of the antibody.

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

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

[0078] After sequencing, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5H11 is as follows:

[0079] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETG KPTYADDFKGRFAFSLDTSASSAYLRINNLKNEDTATYFCARLDWEGNFDYWGQGTTLTVS S (as shown in SEQ ID NO.7);

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

[0081] DVVMTQSPFSLPVSLGDQASISCRSSQRLVHSNGNTYSHWYLQRPGQSPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPFTFDSGTKLEIKR (as shown in SEQ ID NO.8).

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

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

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

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

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

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

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

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

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

[0091] 8. Identification of the epitope of monoclonal antibody 5H11

[0092] The VP5Δ79aa sequence was initially truncated into three fragments (F1: 80 - 233aa, F2: 213 - 368aa, F3: 348 - 526aa), and GST fusion expression was carried out using the expression vector pGEX-6p-1. Then, the F1 fragment was truncated again 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 to detect its reactivity with the monoclonal antibody 5H11. The empty vector pGEX-6p-1 was used as a control.

[0093] As Figure 6 shown, the present invention identified the antigenic epitopes corresponding to 5H11 on the VP5 protein by epitope walking method, and the amino acid sequences of the epitopes are: 164 TEEDLQMRRLATALQKEIGE 183 aa, which provides a theoretical basis for the design of BTV epitope vaccines and the establishment of new diagnostic methods.

[0094] 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 in the protection scope of the present invention.

Claims

1. A monoclonal antibody that specifically recognizes the VP5 protein of bluetongue virus, characterized in that the monoclonal antibody comprises an antibody heavy chain and an antibody light chain; the variable region CDRs of the antibody heavy chain include CDR1 with the amino acid sequence shown in SEQ ID NO.1, CDR2 with the amino acid sequence shown in SEQ ID NO.2, and CDR3 with the amino acid sequence shown in SEQ ID NO.3; the variable region CDRs of the antibody light chain include CDR1 with the amino acid sequence shown in SEQ ID NO.4, CDR2 with the amino acid sequence shown in SEQ ID NO.5, and CDR3 with the amino acid sequence shown in SEQ ID NO.

6.

2. The monoclonal antibody according to claim 1, wherein, 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.

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

4. The nucleic acid according to claim 3, wherein The nucleic acid comprises the sequences 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 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 conjugate moiety selected from the group consisting of: a detectable label, a drug, gold nanoparticles / nanorods, magnetic nanoparticles, a viral capsid protein or VLP, or a combination thereof.

8. The monoclonal antibody according to claim 1 or 2 has any of the following uses: (1) Application in the preparation of a reagent for detecting bluetongue virus; (2) Application in the in vitro detection of bluetongue virus for non-disease diagnosis purposes; (3) Application in the identification of B cell linear epitopes of the bluetongue virus VP5 protein.

9. A B cell linear epitope peptide of Bluetongue virus VP5 protein, characterized in that, The B cell linear epitope peptide is located at 164-183aa of the bluetongue virus VP5 protein, and its sequence is: TEEDLQMRRLATALQKEIGE.

10. Application of the B cell linear epitope peptide according to claim 9 in the preparation of a BTV epitope vaccine.

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