A monoclonal antibody against L1R protein of bovine dermatophilus

Abstract

The present application belongs to the field of immunology and in vitro diagnosis technology, and relates to a monoclonal antibody against L1R protein of bovine nodular dermatopathy virus (LSDV). The present application expresses a recombinant L1R protein of LSDV with a deletion of a transmembrane region (182-204 aa); a monoclonal antibody 8D5 against the L1R protein of LSDV with good reactivity and specificity is screened after the protein is used to immunize mice; and the variable region sequences of the heavy chain and the light chain of the monoclonal antibody 8D5 are obtained; the monoclonal antibody 8D5 can specifically react with the L1R protein of LSDV, and the recombinant protein L1R can be specifically recognized by bovine LSDV positive serum; meanwhile, the monoclonal antibody 8D5 can be used to establish an LSDV ELISA antibody detection method, which lays a foundation for the diagnosis technology and vaccine research of LSDV.

Description

Technical Field

[0001] The invention belongs to the technical fields of immunology and in vitro diagnosis, and particularly relates to a monoclonal antibody against bovine lumpy skin disease virus L1R protein. Background Art

[0002] Lumpy skin disease (LSD) is a cattle infectious disease caused by the lumpy skin disease virus (LSDV). Its primary clinical feature is the appearance of widespread skin nodules that can penetrate the subcutaneous layer, reach the muscles, and even affect internal organs. In the later stages of infection, cows may experience decreased milk production and miscarriage, while bulls may become temporarily or permanently infertile. The incidence of LSD varies depending on the age, breed, and immune status of cattle, ranging from 5% to 45%, with an average mortality rate of approximately 5%. Due to its high contagiousness and severe harm to cattle, LSD poses a significant threat to the health and sustainability of the cattle industry, resulting in significant economic losses. LSD is classified as a Category II zoonotic disease in China and a notifiable animal disease by the World Organization for Animal Health (WOAH).

[0003] LSDV is a member of Capripoxvirus (CaPV) of Poxviridae, which also includes Goatpox virus (GTPV) and Sheeppox virus (SPPV). LSDV has high sequence similarity (about 97%) and antigen relationship with SPPV and GTPV nucleotide sequences, and cross-immune response. LSDV is an enveloped virus, and the virion is brick-shaped or short rod-shaped, with a size of about 300 nm x 260 nm; the genome of LSDV is 151 kbp, containing 156 open reading frames, of which 146 are conserved genes, encoding proteins involved in DNA replication, transcription, mRNA synthesis, nucleotide metabolism, structure formation and stability, virulence and host range. LSD currently lacks specific and effective treatment drugs, and rapid and accurate monitoring measures and vaccination are the key strategies for the prevention and control of LSD. LSDV L1R protein is encoded by L1R gene, which is a homolog of Vaccinia virus (VACV) L1R protein, and it has been reported that Vaccinia virus L1R protein is a protective antigen with good immunogenicity. L1R is a myristoylated protein responsible for virion assembly, present on the surface of late infection expressed mature virion (IMV), plays a role in IMV attachment or penetration, and is also an antigen target for inducing neutralizing antibodies. Poxvirus L1R contains an N-terminal disulfide-linked extracellular domain and a C-terminal hydrophobic transmembrane domain, the N-terminal of the extracellular domain is exposed on the surface of IMV and myristoylated, and the C-terminal transmembrane region anchors the protein to the envelope. Myristoylation can enhance the membrane binding ability of the protein, change the lipophilicity, promote the morphological change or assembly. L1R gene is highly conserved in all poxviruses, with a myristoylation motif and six cysteine residues, forming three disulfide bonds, which play a key role in virus morphogenesis and virion infection of cells.

[0004] Studies have shown that recombinant vaccines prepared based on GTPV and VACV L1R proteins can induce strong and persistent immune responses in hosts, and the protein also has good diagnostic antigen properties and can be used to establish specific diagnostic methods. This not only lays a solid foundation for the research and development of LSD diagnostic reagents, but also provides application prospects for the research and development of new vaccines. SUMMARY

[0005] In response to the above technical problems, the present invention targets the L1R protein of LSDV, truncates the transmembrane region (182-204aa) in the L1R protein, and successfully constructs a prokaryotic expression plasmid on this basis to express and purify the recombinant protein. Subsequently, it is used to immunize BALB / c mice, and specific monoclonal antibodies against the L1RΔ182-204aa protein are successfully prepared using hybridoma technology. This provides support for in-depth exploration of the biological function of the L1R protein and lays a solid foundation for the development of LSD diagnostic methods and new vaccines.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a monoclonal antibody that recognizes the L1R protein of bovine lumpy skin disease virus, wherein the monoclonal antibody comprises 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 as SEQ ID NO.7, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO.8.

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

[0012] Preferably, the nucleic acid comprises the sequences shown in SEQ ID NO.9 and SEQ ID NO.10.

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

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

[0015] In a fifth aspect, the present invention provides an immunoconjugate comprising:

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

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

[0018] Detectable markers, drugs, gold nanoparticles / nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or combinations thereof.

[0019] In a sixth aspect, the present invention provides use of the monoclonal antibody described in the first aspect above in the preparation of a reagent for detecting bovine lumpy skin disease virus.

[0020] In a seventh aspect, the present invention provides use of the monoclonal antibody described in the first aspect in preparing a test strip or a kit for detecting bovine lumpy skin disease virus.

[0021] In an eighth aspect, the present invention provides use of the monoclonal antibody described in the first aspect in in vitro detection of bovine lumpy skin disease virus for non-disease diagnosis purposes.

[0022] In a ninth aspect, the present invention provides an ELISA detection kit for bovine lumpy skin disease virus, the kit comprising the monoclonal antibody described in the first aspect above.

[0023] Preferably, the kit further comprises an enzyme-labeled plate, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

[0024] The beneficial effects of the present invention are as follows: the transmembrane region of the LSDVL1R gene is truncated (the corresponding gene fragment is 544-612 nt) and then cloned into the pET-28a prokaryotic expression vector, and the recombinant LSDVL1R protein (L1RΔ182-204aa) is obtained by inducing expression and purification; mice are then immunized with the recombinant LSDVL1R protein, and a hybridoma cell line 8D5 secreting anti-monoclonal antibodies is obtained through cell fusion and subcloning, and a monoclonal antibody against the recombinant LSDVL1R protein (L1RΔ182-204aa) is prepared; the monoclonal antibody can specifically react with the recombinant LSDVL1R protein (L1RΔ182-204aa), and the recombinant protein L1RΔ182-204aa can be specifically recognized by bovine LSDV positive serum, and can be used as a diagnostic antigen for antibody detection of bovine nodular dermatitis; at the same time, the monoclonal antibody 8D5 can be used as a competing antibody to establish a competitive ELISA detection method, which lays a foundation for the diagnostic technology and vaccine development of LSDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Expression results of the recombinant L1RΔ182-204aa protein; A shows the expression of the recombinant L1RΔ182-204aa protein induced at 37°C (M: protein molecular weight standard; 1: bacterial solution before induction; 2-5: bacterial solution samples at 2, 4, 6, and 8 hours of induction, respectively); B shows the solubility analysis of the recombinant L1RΔ182-204aa protein (M: protein molecular weight standard; 1: ultrasonic supernatant; 2: ultrasonic precipitate).

[0026] Figure 2 Identification results of the recombinant L1RΔ182-204aa protein, where M is the protein molecular weight standard and 1 is the sample induced with the recombinant L1RΔ182-204aa protein for 8 h;

[0027] Figure 3 Western-blot validation results of recombinant L1RΔ182-204aa protein and LSDV-positive bovine serum; A is LSDV-positive bovine serum, B is negative serum, M is the protein molecular weight standard, and 1 is the sample induced with recombinant L1RΔ182-204aa protein for 8 hours;

[0028] Figure 4 Purification results of recombinant L1RΔ182-204aa protein; where M is the protein molecular weight standard, 1-8 is the elution buffer, and 9 is the flow-through.

[0029] Figure 5 Western-blot identification results of monoclonal antibody 8D5;

[0030] Figure 6 Cell immunofluorescence identification results of monoclonal antibody 8D5. DETAILED DESCRIPTION

[0031] The following examples of the present invention are described in detail. It should be noted that the following examples are illustrative and are intended only to explain the present invention and are not to be construed as limiting the present invention. In addition, unless otherwise expressly stated, all reagents used in the following examples are commercially available or can be synthesized according to the literature or known methods. Reaction conditions not listed are readily available to those skilled in the art.

[0032] The cells, main reagents and experimental animals involved in the following implementation:

[0033] BL21 (DE3) competent cells and IPTG were purchased from Takara Biotechnology Co., Ltd.; High Affinity Ni-Charged Resin FF was purchased from GenScript; Freund's complete adjuvant and incomplete adjuvant were purchased from Sigma-Aldrich; Trition X-100, kanamycin, and bovine serum albumin (BSA) were purchased from Solebo; His-tag antibody was purchased from Beijing Zhongshan Jinqiao Company; goat anti-mouse IgG (HRP), goat anti-mouse IgG (Alexa Flour 488), and rabbit anti-bovine IgG (HRP) were purchased from Abcam; nitrocellulose (PVDF) membrane and carbonate-bicarbonate buffer were purchased from MERCK; SuperSignalWest Pico PLUS kit, Lipofectamine 2000, fetal bovine serum, Pierce BCA Protein Assay Kit, and DMEM medium were purchased from ThermoFisher. The experimental animals were 6-8 week old female BALB / c mice, and BHK-21 cells, mouse myeloma cells (SP2 / 0), and LSDV-infected cattle positive and negative sera were stored in our laboratory.

[0034] Example 1 Preparation of monoclonal antibody 8D5

[0035] 1. Construction of recombinant expression plasmid

[0036] With reference to the amino acid sequence of the LSDV (KSGP 0240 strain) L1R protein published in GenBank (GenBank accession number: AOE47636.1), the transmembrane region amino acids 182-204aa were truncated through protein hydrophobicity analysis, encoding a total of 222 amino acids with a molecular weight of 26 kDa (the gene sequence is shown in SEQ ID NO. 11). After optimizing the synonymous codons of the gene according to the codon preference of Escherichia coli (shown in SEQ ID NO. 12) and the codon preference of eukaryotic cells (shown in SEQ ID NO. 13), Wuhan Jinkairui Bioengineering Co., Ltd. was commissioned to synthesize and cloned into the pET-28a and pcDNA3.1 / myc-His vectors, respectively, to obtain the recombinant expression plasmids pET-28a-L1RΔ182-204aa and pcDNA3.1 / myc-His-L1RΔ182-204aa.

[0037] 2. Expression and solubility analysis of recombinant L1RΔ182-204aa protein

[0038] The pET-28a-L1RΔ182-204aa plasmid was transformed into BL21 (DE3) competent cells by ice bath and heat shock, and cultured overnight at 37°C. The cultured bacterial solution was added to LB medium containing kanamycin at a ratio of 1:100 and cultured at 37°C until the bacterial solution OD 600 After reaching a p-value of 0.6-0.8, IPTG was added to the culture medium to a final concentration of 0.5 mmol / L. The cells were cultured at 37°C for 8 hours. Every 2 hours, 1 mL of the culture was sampled for SDS-PAGE analysis to analyze protein expression at each hour. The cells were harvested by centrifugation after 8 hours of induction, resuspended in PBS, and then sonicated on ice for 30 minutes at 140 W, with a 3-second cycle and a 5-second pause. After sonication, the cells were centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant and precipitate were then sampled and analyzed for protein solubility by SDS-PAGE analysis.

[0039] The expression results are as follows Figure 1 As shown in Figure A, the recombinant L1RΔ182-204aa protein was successfully expressed in E. coli, with the highest expression level at 8h. Its size was approximately 26kDa, which was consistent with the expected size. The bacterial sample induced for 8h was ultrasonically disrupted, and the precipitate and supernatant after ultrasonication were collected for SDS-PAGE. The results are shown in Figure 2. Figure 1 As shown in B, the L1RΔ182-204aa protein was mainly expressed in the form of inclusion bodies.

[0040] 3. Identification of recombinant L1RΔ182-204aa protein

[0041] The bacterial culture samples transformed with pET-28a-L1RΔ182-204aa and expressed in Escherichia coli for 8 h were analyzed by SDS-PAGE and then transferred to a methanol-treated PVDF membrane. The membrane was blocked with 5% skim milk in PBST for 1 h and incubated with mouse anti-His tag monoclonal antibody (1:5000) and LSDV bovine positive serum (1:200) as primary antibodies at 4°C overnight. The membrane was washed three times with PBST and incubated with HRP-conjugated goat anti-mouse IgG (1:10000) and rabbit anti-bovine IgG (1:2000) as secondary antibodies at 37°C for 1 h. The membrane was washed three times with PBST, and the water was aspirated and SuperSignal West Pico PLUS chemiluminescent substrate was added for reaction in the dark for 2 min. The target bands were visualized using a ChemiDoc XRS+ imaging system (Bio-Rad).

[0042] The results of identifying the recombinant L1RΔ182-204aa protein with His tag antibody are as follows Figure 2 As shown, it was shown that the recombinant L1RΔ182-204aa protein with a His tag was successfully obtained.

[0043] The results of Western-blot verification of recombinant L1RΔ182-204aa protein and LSDV bovine positive serum are as follows Figure 3 As shown, the results showed that the recombinant L1RΔ182-204aa protein reacted with LSDV bovine positive serum and produced a target band consistent with the expected size, but did not react with negative serum, indicating that the recombinant L1RΔ182-204aa protein with good reactivity was successfully obtained.

[0044] 4. Purification of recombinant L1RΔ182-204aa protein

[0045] After inducing expression at 37°C for 8 hours, a sample of the bacterial suspension was collected. The pellet was resuspended in PBS and sonicated. The pellet was collected by centrifugation and the target protein was purified using High Affinity Ni-Charged Resin FF. Inclusion bodies were solubilized by adding 10 mL of Binding Buffer (8 M urea and 5 mM imidazole) to the sonicated pellet. The solubilized inclusion bodies were then bound to a His-tag nickel column overnight at 4°C. Impurities were removed using Binding Buffer. The target protein was eluted using Elution Buffer (500 mM imidazole) and analyzed by SDS-PAGE. The concentration of the purified recombinant L1RΔ182-204aa protein sample was determined according to the Pierce BCA Protein Assay Kit instructions.

[0046] The recombinant L1RΔ182-204aa protein in the precipitated sample was purified according to the High Affinity Ni-Charged Resin FF operating instructions. Contaminants were eluted with Binding Buffer, and the target protein was eluted with Elution Buffer and subjected to SDS-PAGE. The results are shown in Figure 2. Figure 4 As shown, a recombinant L1RΔ182-204aa protein of high purity and consistent with the expected size was successfully obtained.

[0047] 5. Monoclonal Antibody Preparation

[0048] (1) Mouse immunization

[0049] The purified recombinant L1RΔ182-204aa protein was used as an antigen, diluted with PBS to an appropriate concentration, and an equal amount of Freund's complete adjuvant was added for emulsification. The immunization dose was 50 μg per mouse. BALB / c mice were immunized by multiple subcutaneous injections at the back, and two negative controls were set up. On the 14th, 28th, and 35th days after the initial immunization, the protein was emulsified with an equal amount of Freund's incomplete adjuvant and then boosted with the same immunization dose as the first immunization. After the immunization, blood was collected from the tail vein of the mice to separate the serum. The purified recombinant L1RΔ182-204aa protein was used as the coating antigen. The serum titer was determined by indirect ELISA, and the OD was measured by microplate reader. 450 Value, serum OD to be tested 450 / Negative control OD 450 The maximum dilution factor when (S / N) ≥ 2.1 was the antibody titer, and the mouse with the highest antibody titer was selected for further preparation of monoclonal antibodies.

[0050] The results showed that after the fourth immunization, the recombinant L1RΔ182-204aa protein antibody titer of the immune serum of mouse No. 3 was the highest, at 1:128000, indicating that the purified recombinant L1RΔ182-204aa protein exhibited good immunogenicity and could effectively induce the production of high-titer antibodies in mice.

[0051] (2) Cell fusion

[0052] Mice with the highest serum antibody titers against the recombinant L1RΔ182-204aa protein were selected for cell fusion assays. Spleens were removed from immunized mice under a sterile operating table and gently triturated with a syringe plunger or forceps to release the cells. The suspension was then filtered through a cell sieve to isolate splenocytes. Myeloma SP2 / 0 cells were fused with splenocytes at a ratio of 7:1 using PEG1000 as a fusion promoter. The fused cells were resuspended in HAT medium and seeded into 96-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator and replaced with HT medium after 7 days.

[0053] (3) Screening and subcloning of hybridoma cells

[0054] When the cell growth area reaches 1 / 3-1 / 2 of the area at the bottom of the culture well, the culture medium of all growth wells is tested. The positive hybridoma cells are subcloned multiple times by limiting dilution method until all hybridoma cell supernatants after subcloning are positive by indirect ELISA test. 450 The positive hybridoma single cell clone with high value and good growth condition was expanded and cultured, and the stable hybridoma cell line 8D5 was screened.

[0055] Table 1 Antibody screening results

[0056] Monoclonal antibody 8D5 Negative control 1.8330 / 1.8395 0.0703 / 0.0779

[0057] (4) Preparation of mouse monoclonal antibody ascites

[0058] Seven-week-old BALB / c mice were selected and sensitized by intraperitoneal injection of sterile paraffin oil for 7 to 10 days. Then, 0.5 mL of 1×10 6 Mix the hybridoma cells at 100 μg / mL. Observe the mice's health and ascites production daily. Seven to ten days after hybridoma cell injection, the mice's abdomens will swell and ascites can be collected. The collected ascites is centrifuged, the precipitate removed, and the supernatant collected for purification using a Protein G-agarose affinity chromatography column.

[0059] 6. Biological characteristics of monoclonal antibodies

[0060] (1) Western-blot identification

[0061] The purified recombinant L1RΔ182-204aa protein was analyzed by SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h and incubated with monoclonal antibody 8D5 (1:1000) as the primary antibody at 4°C overnight. The membrane was washed three times with PBST and HRP-labeled goat anti-mouse IgG (1:10000) as the secondary antibody. The membrane was washed three times with PBST. After absorbing the water, SuperSignal West Pico PLUS chemiluminescent substrate was added and reacted in the dark for 2 min. The target bands were observed using the ChemiDoc XRS+ imaging system (Bio-Rad).

[0062] The results are as follows Figure 5 As shown, the purified recombinant L1RΔ182-204aa protein was subjected to Western-blot analysis, with monoclonal antibody 8D5 as the primary antibody and goat anti-mouse IgG as the secondary antibody. The results showed that the monoclonal antibody 8D5 could specifically react with the purified recombinant L1RΔ182-204aa protein.

[0063] (2) Cell immunofluorescence analysis

[0064] BHK-21 cells were plated in a 24-well plate. When the cells grew to 80% to 90% confluency, the recombinant expression plasmid pcDNA3.1 / myc-His-L1RΔ182-204aa was transfected into the BHK-21 cells using lipofectamine 2000. The negative control was the supernatant of SP2 / 0 cells. The 24-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours, after which the old fluid was discarded. The cells were washed three times with PBS solution, and then 4% paraformaldehyde solution was added and fixed at room temperature for 10 minutes. The cells were treated with 0.3% Triton X-100 solution for 10 minutes, blocked with 3% BSA at room temperature for 1 hour, incubated with monoclonal antibody 8D5 as the primary antibody at 4°C overnight, washed three times with PBS, and incubated with goat anti-mouse IgG (Alexa Flour 488) (1:2000) as the secondary antibody at room temperature in the dark for 1 hour. The cells were then treated with the nuclear dye Hoechst (10 μg / ml) reagent for 10 minutes and washed three times with PBS for 5 minutes each time. The results were observed and photographed using an inverted fluorescence microscope.

[0065] The results are as follows Figure 6 As shown, the monoclonal antibody 8D5 prepared by the present invention can react with the recombinant L1RΔ182-204aa protein expressed in BHK-21 cells, and green fluorescence can be observed, while BHK-21 cells incubated with the supernatant of SP2 / 0 cells have no green fluorescence, indicating that the LSDV L1R protein monoclonal antibody 8D5 prepared by the present invention has good specificity.

[0066] 7. Amplification of the light and heavy chain variable region sequences of monoclonal antibody 8D5

[0067] Total RNA was extracted from hybridoma cells secreting the monoclonal antibody 8D5 using the TRIzol lysis method, and then reverse transcribed using a reverse transcription kit to synthesize cDNA. The resulting cDNA was used as a template for PCR amplification. The amplified product was ligated into a vector and sequenced to obtain the nucleotide sequences of the antibody's heavy and light chain variable regions.

[0068] The cDNA of the heavy chain variable region of the monoclonal antibody 8D5 is: GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAGGTTCCAGGACAAGGCCACTATAACAACAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTATTAGAAGGGGATTGGGCCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (shown in SEQ ID NO. 9);

[0069] The cDNA of the light chain variable region of the monoclonal antibody 8D5 is: GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAGGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGCAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAACAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTCTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG (shown in SEQ ID NO. 10).

[0070] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8D5 is as follows, as sequenced:

[0071] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGNT KYDPRFQDKATITTDTSSNTAYLQLSSLTSEDTAVYYCIRRGLGPDYWGQGTTLTVSS (shown in SEQ ID NO.7);

[0072] The amino acid sequence of the light chain variable region of the monoclonal antibody is as follows:

[0073] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQRNYLTWYQQKPGQPPKLLIYGAS TRQSGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDHSSPFTFGSGTKLEIKR (shown in SEQ ID NO. 8).

[0074] The sequenced light chain and heavy chain variable region sequences of the monoclonal antibody 8D5 were analyzed on the abysis.org website to obtain its CDR regions.

[0075] The sequences of the three complementarity determining regions (CDRs) of the heavy chain variable region are shown below:

[0076] CDR1: DTYIH (shown in SEQ ID NO. 1);

[0077] CDR2: RIDPANGNTKYDPRFQD (shown in SEQ ID NO. 2);

[0078] CDR3: RGLGPDY (shown in SEQ ID NO. 3);

[0079] The sequences of the three complementarity determining regions (CDRs) of the light chain variable region are shown below:

[0080] CDR1: KSSQSLLNSGNQRNYLT (shown in SEQ ID NO. 4);

[0081] CDR2: GASTRQS (shown in SEQ ID NO. 5);

[0082] CDR3: QNDHSSPFT (shown in SEQ ID NO. 6).

[0083] 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 aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes the L1R protein of bovine lumpy skin disease virus, characterized in that: The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.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 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.

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 includes 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 conjugated moiety selected from the group consisting of a detectable label, a drug, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or VLPs, or a combination thereof.

8. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent, a test strip or a kit for detecting bovine lumpy skin disease virus.

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

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

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