Monoclonal antibody aiming at bovine nodular skin disease virus L1R protein

Abstract

The invention belongs to the technical field of immunology and in vitro diagnosis, and relates to a monoclonal antibody aiming at bovine nodular skin disease virus (LSDV) L1R protein. According to the invention, the LSDV recombinant L1R protein with deletion of a transmembrane region (182-204aa) is obtained through expression; after a mouse is immunized by the protein, a monoclonal antibody 8D5 which is good in reactivity and specificity and aims at the LSDV L1R protein is screened out; heavy chain and light chain variable region sequences for coding the monoclonal antibody 8D5 are obtained; the monoclonal antibody 8D5 can be subjected to specific reaction with LSDV L1R protein, the recombinant protein L1R can be specifically recognized by bovine LSDV positive serum, meanwhile, the monoclonal antibody 8D5 can be used for establishing an LSDV ELISA antibody detection method, and a foundation is laid for LSDV diagnostic technology and vaccine research and development.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology and in vitro diagnosis, and specifically relates to a monoclonal antibody against L1R protein of bovine nodular dermatosis virus. Background Art

[0002] Lumpy skin disease (LSD) is a cattle infectious disease caused by the lumpy skin disease virus (LSDV). Its main clinical feature is the appearance of extensive nodules on the skin. The nodules can penetrate the subcutaneous layer, reach the muscles, and even affect the internal organs. In the late stage of infection, cows may experience decreased milk production and miscarriage, and bulls may be temporarily or permanently infertile. The incidence of LSD varies depending on the age, breed, and immune status of cattle, ranging from 5% to 45%, and the average mortality rate is about 5%. Due to its high infectivity and serious harm to cattle, LSD poses a major threat to the health and sustainable development of the cattle industry, causing serious economic losses. LSD is listed as a Class II animal infectious disease in China, and the World Organization for Animal Health (WOAH) lists it as a notifiable animal disease.

[0003] LSDV is a member of the genus Capripoxvirus (CaPV) of the Poxviridae family, which also includes goatpox virus (GTPV) and sheeppox virus (SPPV). LSDV has a high sequence similarity (about 97%) and antigenic relationship with SPPV and GTPV nucleotide sequences, and has cross-immune reactions. LSDV is an enveloped virus, and the virus particles are brick-shaped or short rod-shaped, with a size of about 300nm×260nm; the LSDV genome is 151kbp, containing 156 open reading frames, of which there are 146 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 effective treatment drugs, and rapid and accurate monitoring measures and vaccination are key strategies for the prevention and control of LSD. LSDV L1R protein is encoded by the L1R gene and is a homolog of the L1R protein of vaccinia virus (VACV). It has been reported that the L1R protein of vaccinia virus is a protective antigen with good immunogenicity. L1R is a myristoylated protein responsible for the assembly of viral particles. It is present on the surface of mature viral particles (IMV) expressed in the late infection period and plays a role in the attachment or penetration of IMV. It is also an antigenic target for inducing the production of neutralizing antibodies. Poxvirus L1R contains an extracellular domain bound by an N-terminal disulfide bond and a C-terminal hydrophobic transmembrane domain. The N-terminus of the extracellular domain is exposed on the surface of the 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 lipophilicity, and promote morphological changes or assembly. The L1R gene is highly conserved in all poxviruses. It contains a myristoylation motif and six cysteine ​​residues, forming three disulfide bonds, and plays a key role in viral morphogenesis and the process of viral particles infecting cells.

[0004] Studies have shown that recombinant vaccines based on GTPV and VACV L1R proteins can induce strong and lasting immune responses in the host. The proteins also have good diagnostic antigenic properties and can be used to establish specific diagnostic methods. This not only lays a solid foundation for the development of LSD diagnostic reagents, but also provides application prospects for the development of new vaccines. Summary of the invention

[0005] In response to the above technical problems, the present invention targets the L1R protein of LSDV, cuts off 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, which is then used to immunize BALB / c mice, and hybridoma technology is used to successfully prepare specific monoclonal antibodies against the L1RΔ182-204aa protein, which 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] The detectable markers, drugs, gold nanoparticles / nanorods, magnetic nanoparticles, viral coat proteins or VLPs, or a combination thereof.

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

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

[0021] In an eighth aspect, the present invention provides the use of the monoclonal antibody described in the first aspect in the 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 nodular dermatitis virus, the kit comprising the monoclonal antibody described in the first aspect above.

[0023] Preferably, the kit further comprises an ELISA 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 cut off (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; then the recombinant LSDVL1R protein is immunized with the recombinant mouse, and the hybridoma cell line 8D5 secreting the anti-monoclonal antibody is obtained by cell fusion and subcloning, and the 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 competitive antibody to establish a competitive ELISA detection method, which lays a foundation for the diagnosis technology and vaccine development of LSDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The expression results of the recombinant L1RΔ182-204aa protein; A is 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), and B is 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 mass 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 bovine positive serum; A is LSDV bovine positive serum, B is negative serum, M is 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 embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, if not explicitly stated, all reagents used in the following embodiments are commercially available, or can be synthesized according to text or known methods, and the reaction conditions not listed are easily available to those skilled in the art.

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

[0033] BL21 (DE3) competent cells and IPTG were purchased from Bao 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 Solebao; 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 culture medium were purchased from ThermoFisher Scientific Company; the experimental animals were 6-8 week old female BALB / c mice, and BHK-21 cells, mouse myeloma cells (SP2 / 0), positive serum and negative serum of LSDV infected cattle were preserved by 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 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 pET-28a and pcDNA3.1 / myc-His vectors, respectively, to obtain 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 0.6-0.8, add IPTG with a final concentration of 0.5mmol / L to the culture medium, culture at 37℃ for 8h, collect 1mL bacterial sample every 2h for SDS-PAGE, and analyze the expression of proteins at each hour. Collect the bacteria after 8h of induction by centrifugation, resuspend in PBS, add protease inhibitors, and ultrasonically break on ice for 30min, with a power of 140W, a working time of 3s, and a pause of 5s. After the ultrasonication is completed, centrifuge at 10000r / min for 10min at 4℃, take the ultrasonic supernatant and ultrasonic precipitate, prepare samples separately, and perform SDS-PAGE to analyze the solubility of the expressed protein.

[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 about 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 solution sample of Escherichia coli transformed with pET-28a-L1RΔ182-204aa for expression for 8 h was analyzed by SDS-PAGE and then transferred to a methanol-treated PVDF membrane. The membrane was blocked with 5% skim milk prepared in PBST for 1 h, incubated with mouse anti-His tag monoclonal antibody (1:5000) and LSDV bovine positive serum (1:200) as primary antibodies at 4°C overnight, washed three times with PBST, incubated with HRP-labeled goat anti-mouse IgG (1:10000) and rabbit anti-bovine IgG (1:2000) as secondary antibodies at 37°C for 1 h, washed three times with PBST, and after absorbing the water, SuperSignal West Pico PLUS chemiluminescent substrate was added for reaction in the dark for 2 min. The target bands were observed using the 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 the 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] Collect the bacterial sample induced for 8 hours at 37℃, add PBS to the precipitate for resuspending and ultrasonically disrupting, collect the disrupted precipitate by centrifugation, purify the target protein using HighAffinity Ni-Charged Resin FF, add 10mL of Binding buffer with a final concentration of 8M urea and 5mM imidazole to the ultrasonically disrupted precipitate to dissolve the inclusion body, bind the dissolved inclusion body to the His tag nickel column at 4℃ overnight, use Binding buffer as the eluent to remove impurity proteins; use Elution buffer with a final concentration of 500mM imidazole to elute, collect the target protein, and perform SDS-PAGE detection. According to the operating instructions of Pierce BCA ProteinAssay Kit, the concentration of the purified recombinant L1RΔ182-204aa protein sample was measured.

[0046] The recombinant L1RΔ182-204aa protein in the precipitated sample was purified according to the High Affinity Ni-Charged Resin FF operating instructions, impurities were eluted with Binding Buffer, the target protein was eluted with Elution Buffer and SDS-PAGE was performed. The results are as follows: 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. The protein was emulsified with an equal amount of Freund's incomplete adjuvant and then boosted on the 14th, 28th, and 35th days after the initial immunization. The immunization dose was the same as the first immunization. After the immunization, the mouse tail vein blood was collected 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 determined by an enzyme marker. 450 Value, serum OD to be tested 450 / Negative control OD 450 When (S / N) ≥ 2.1, the maximum dilution factor was the antibody titer, and the mice with the highest antibody titer were selected for further preparation of monoclonal antibodies.

[0050] The results showed that after the fourth immunization, the recombinant L1RΔ182-204aa protein antibody titer in 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] The mouse with the highest titer of serum antibody against recombinant L1RΔ182-204aa protein was selected for cell fusion test. The spleen of the immunized mouse was removed on a sterile operating table, and the cells were gently ground with a syringe piston or forceps to release the cells. The suspension was filtered through a cell sieve to separate the spleen cells. Myeloma cells SP2 / 0 and spleen cells were fused at a ratio of 7:1 using PEG1000 as a fusion promoter. The fused cells were resuspended in HAT medium and inoculated into a 96-well cell culture plate. The 96-well plate was placed at 37°C and 5% CO 2 The cells were cultured in an 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 the growth wells is tested. The positive hybridoma cells are subcloned multiple times by limiting dilution method until all the hybridoma cell supernatants after subcloning are positive by indirect ELISA test. 450 The positive hybridoma single cell clones with high values ​​and good growth conditions were expanded and cultured to screen out the stable hybridoma cell line 8D5.

[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] 7-week-old BALB / c mice were selected and sensitized by intraperitoneal injection of sterile paraffin oil for 7-10 days. Then, 0.5 mL of 1×10 6 pcs / mL of hybridoma cells. The health status of the mice and the production of ascites were observed every day. 7-10 days after the injection of hybridoma cells, the abdomen of the mice swelled and the ascites could be collected. The collected ascites was centrifuged to remove the precipitate, and the supernatant was collected and purified 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 overnight at 4°C with monoclonal antibody 8D5 (1:1000) as the primary antibody. 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 drying, 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 primary antibody and goat anti-mouse IgG as secondary antibody. The results showed that 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 BHK-21 cells using lipofectamine2000. The negative control was the supernatant of SP2 / 0 cells. The 24-well plate was placed at 37°C and 5% CO. 2After culturing in a cell culture incubator for 24 hours, discard the old solution, wash the cells three times with PBS solution, then add 4% paraformaldehyde solution and fix them at room temperature for 10 minutes. Add 0.3% Triton X-100 solution and treat the cells for 10 minutes; block them with 3% BSA at room temperature for 1 hour, incubate them with monoclonal antibody 8D5 as primary antibody at 4℃ overnight, wash them with PBS three times, add goat anti-mouse IgG (Alexa Flour 488) (1﹕2000) as secondary antibody and incubate them at room temperature in the dark for 1 hour, then treat them with cell nuclear dye Hoechst (10μg / ml) reagent for 10 minutes, wash them with PBS three times, 5 minutes each time; observe and photograph the results 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 SP2 / 0 cell supernatant 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 light chain and heavy chain variable region sequences of monoclonal antibody 8D5

[0067] The total RNA of hybridoma cells secreting monoclonal antibody 8D5 was extracted by TRIzol lysis method, and then reverse transcribed and synthesized into cDNA using a reverse transcription kit. The obtained cDNA was used as a template for PCR amplification, and the amplified product was connected to a vector for sequencing to obtain the nucleotide sequence of the heavy chain and light chain variable region of the antibody.

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

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

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

[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 monoclonal antibody 8D5 were analyzed on the abysis.org website to obtain its CDR region.

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

[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 as follows:

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

Claims

1. A monoclonal antibody that recognizes the L1R protein of bovine nodular 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, characterized in that 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 antibody heavy chain of the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that 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, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, 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 nodular 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 nodular skin disease virus, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2.

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