Diagnostic antigen and monoclonal antibody of bovine nodular dermatosis virus, and preparation method and application of diagnostic antigen and monoclonal antibody

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a diagnostic antigen and a monoclonal antibody for bovine nodular skin disease virus as well as a preparation method and application of the diagnostic antigen and the monoclonal antibody. The monoclonal antibody is secreted by hybridoma cells 14A2, the hybridoma cells 14A2 are preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the hybridoma cells 14A2 is CGMCC No.46313. According to the invention, a monoclonal antibody is prepared by aiming at ORF132 protein of bovine nodular skin disease virus. Meanwhile, by constructing a Pcold-TF-ORF132 prokaryotic expression vector, the ORF132-TF protein is expressed and used as an immunogen to prepare the monoclonal antibody, and the Pcold-TF-ORF132 prokaryotic expression vector has important significance for prevention and control of bovine nodular dermatosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a diagnostic antigen and monoclonal antibody of bovine lumpy dermatosis virus as well as a preparation method and application thereof. Background Art

[0002] Lumpy skin disease (LSD) is a highly host-specific and highly contagious viral disease of cattle caused by the bovine lumpy skin disease virus (LSDV). LSDV is a double-stranded DNA virus belonging to the Poxviridae family and the genus Capripox (Capx). The viral genome is large, comprising approximately 151,000 base pairs, encoding 156 putative viral genes, numerous proteins, and a complex pathogenic mechanism.

[0003] Currently, there is no specific treatment for bovine lumpy skin disease (BLD). Laboratory testing methods for diagnosing BLDV in China and abroad primarily include virus isolation and culture, electron microscopy, immunodiffusion, indirect immunofluorescence, and PCR. Each of these laboratory testing methods, depending on their specific characteristics, is suitable for different scenarios. For example, while virus isolation and culture is accurate, it is time-consuming and labor-intensive, requiring high laboratory standards. It is typically used for monitoring emerging outbreaks and is not suitable for rapid on-site testing. Electron microscopy is the most intuitive method, allowing direct observation of the virus, but it places the highest demands on laboratory personnel and equipment. Immunological tests such as immunodiffusion and indirect immunofluorescence are suitable for large-scale screening and are simple to perform, but antibody production is delayed after infection (with a window period of approximately 7 to 14 days), making early diagnosis impossible and difficult to distinguish from the same genus, the sheeppox virus. PCR testing is low-cost, simple, and highly specific, but it only detects viral nucleic acid and cannot confirm viral viability or infection status.

[0004] While the aforementioned detection methods are capable of laboratory testing for bovine nodular dermatitis, they all have varying degrees of limitations. Using monoclonal antibodies, unlike these methods, overcomes their respective limitations and offers advantages such as ease of use and low cost, making it of great significance for the prevention and treatment of bovine nodular dermatitis.

[0005] Currently, monoclonal antibodies developed against bovine lumpy skin disease virus have the problem of low titer. Summary of the Invention

[0006] In order to solve the problem of low titer of monoclonal antibodies developed for bovine lumpy skin disease virus in the prior art, the present invention provides a diagnostic antigen, monoclonal antibody, preparation method and application of bovine lumpy skin disease virus.

[0007] Currently, most antibodies developed against bovine lumpy skin disease virus are polyclonal antibodies, with only one monoclonal antibody being developed. This was disclosed in Wang Fangping's 2024 dissertation, "Preparation of Monoclonal Antibodies to Bovine Lumpy Skin Disease Virus ORF123 Protein and Epitope Analysis," published at Gansu Agricultural University. This method involved constructing a pet28a-ORF123 recombinant plasmid, expressing the ORF123 protein in prokaryotes, and using the ORF123 protein of the bovine lumpy skin disease virus as an immunogen to prepare a monoclonal antibody. These polyclonal antibodies suffer from low titers.

[0008] In order to solve the problem of low titer of the monoclonal antibodies developed against bovine nodular dermatitis virus as described in the monoclonal antibodies prepared publicly in the above-mentioned thesis, the present invention provides a monoclonal antibody against bovine nodular dermatitis virus, which is secreted by hybridoma cell 14A2. The hybridoma cell 14A2 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2024, with the deposit number CGMCC No. 46313.

[0009] The present invention provides a monoclonal antibody against bovine lumpy skin disease virus (BLVV). This monoclonal antibody has the advantage of high titer, which can solve the problem of low titer of monoclonal antibodies developed against BLVV in the prior art. Furthermore, the preparation process of the BLVV monoclonal antibody provided by the present invention is simple and low-cost.

[0010] Preferably, the monoclonal antibody is prepared using ORF132-TF recombinant protein as an immunogen. The monoclonal antibody is obtained by immunization and screening using antigen protein.

[0011] The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is shown in SEQ ID NO.1.

[0012] Preferably, the amino acid sequence of the ORF132-TF recombinant protein is shown in SEQ ID NO. 2. A plasmid vector is constructed by homologous recombination for subsequent expression of the recombinant protein.

[0013] The second purpose of the present invention is to provide a diagnostic antigen of bovine lumpy skin disease virus, wherein the diagnostic antigen is the ORF132-TF recombinant protein.

[0014] The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is shown in SEQ ID NO.1.

[0015] The third object of the present invention is to provide a method for preparing the diagnostic antigen, wherein the diagnostic antigen is prepared using a prokaryotic expression system. Preferably, the nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into competent cells to induce expression to obtain the ORF132-TF recombinant protein, which is the diagnostic antigen.

[0016] Preferably, the expression vector comprises at least one of pCold-TF, pet-28a, pet-32a and pCold-sumo.

[0017] Preferably, during the preparation of the diagnostic antigen, a protein induction reagent (IPTG) is added to allow the plasmid to express the desired protein in large quantities.

[0018] A fourth object of the present invention is to provide the use of the monoclonal antibody or the diagnostic antigen in the preparation of a diagnostic kit for bovine nodular dermatitis, wherein the kit comprises the monoclonal antibody or the diagnostic antigen.

[0019] A fifth object of the present invention is to provide the use of the monoclonal antibody in the preparation of a drug for treating bovine lumpy skin disease virus.

[0020] Preferably, the drug contains the monoclonal antibody as an active ingredient.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a monoclonal antibody against bovine lumpy skin disease virus. The monoclonal antibody has the advantage of high titer and can solve the problem of low titer of monoclonal antibodies developed for bovine lumpy skin disease virus in the prior art.

[0022] 2. The monoclonal antibody to bovine lumpy skin disease virus provided by the present invention has the advantages of low cost and convenient construction of the detection method, which is conducive to the large-scale application of the technology for detecting bovine lumpy skin disease virus.

[0023] 3. The present invention uses a prokaryotic expression system to prepare ORF132-TF protein as an immunogen and screens out low-cost monoclonal antibodies against bovine nodular dermatitis ORF132 protein.

[0024] 4. The present invention utilizes a prokaryotic expression system to prepare immunogens. This method has the advantages of high protein yield, stable and convenient antigen preparation.

[0025] 5. The present invention utilizes hybridoma technology, cell subcloning and screening to efficiently prepare monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the PCR amplification of the ORF132-TF gene in the present invention; wherein, M: DL2000 DNA Marker; 1: LSDV-ORF132 gene.

[0027] Figure 2 This is the enzyme digestion of the PCold-TF empty plasmid in the present invention; wherein, M: DL15000 DNA Marker; 1: PCold-TF enzyme digested plasmid.

[0028] Figure 3 This is the identification of the PCold-TF-ORF132 recombinant plasmid in the present invention; wherein, M: DL2000 DNA Marker; 1, 2, 3: recombinant plasmid.

[0029] Figure 4 The figure is an SDS-PAGE diagram of the induced expression of PCold-TF-ORF132 in the present invention; wherein, M: protein marker; 1: bacterial supernatant; 2: BL21 (DE3) empty bacteria; 3: PCold-TF empty supernatant; 4: PCold-TF empty; 5: ORF132-TF whole bacteria; 6: ORF132-TF uninduced precipitate; 7: ORF132-TF induced precipitate 8; ORF132-TF uninduced supernatant 9: ORF132-TF induced supernatant.

[0030] Figure 5 The SDS-PAGE diagram of the induced expression of 5PCold-TF-ORF132 in the present invention at different times; wherein, M: protein marker; 1: induction time 10 h; 2: induction time 12 h; 3: induction time 14 h; 4: induction time 16 h; 5: induction time 18 h; 6: induction time 20 h; 7: induction time 22 h; 8: induction time 24 h; 9: induction time 26 h.

[0031] Figure 6 The figure shows the SDS-PAGE diagram of the induced expression of pCold-TF-ORF132 at different IPTG concentrations in the present invention; wherein, M: protein marker; 1: PCold-TF empty load; 2: induction concentration 0.1 mM; 3: induction concentration 0.2 mM; 4: induction concentration 0.3 mM; 5: induction concentration 0.4 mM; 6: induction concentration 0.5 mM; 7: induction concentration 0.6 mM; 8: induction concentration 0.8 mM; 9: induction concentration 0.9 mM.

[0032] Figure 7This is the purification of LSDV-ORF132 protein in the present invention; wherein, M: protein marker; 1: supernatant of bacterial liquid after ultrasonication in the induction group; 2: protein flow-through; 3: 20 mM imidazole elution; 4: 40 mM imidazole elution; 5: 60 mM imidazole elution; 6: 100 mM imidazole elution; 7: 200 mM imidazole elution; 8: 500 mM imidazole elution; 9: 600 mM imidazole elution.

[0033] Figure 8 Western blot analysis of the ORF132-TF protein in the present invention (His tag antibody; M: protein marker; I: purified ORF132-TF protein).

[0034] Figure 9 It is the growth status of hybridoma cells after cell fusion in the present invention. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0036] The recombinant plasmid PET-28a-ORF132-TF used in the examples of the present invention was synthesized by Sangon Biotechnology, and the plasmid pCold-TF was stored in the Laboratory of Animal Preventive Medicine and Public Health of Zhejiang Agriculture and Forestry University.

[0037] The materials and reagents used in the examples of the present invention are shown in Table 1.

[0038] Table 1 Main consumables The primers used in the prokaryotic expression of the LSDV ORF132-TF protein in the examples of the present invention are as follows: The nucleotide sequence of PCold-TF-ORF132-F is shown in SEQ ID NO.3: 5'-ATATGGAGCTCGGTACCCTCGAGATGACCAACACCTCCCTGC-3'.

[0039] The nucleotide sequence of PCold-TF-ORF132-R is shown in SEQ ID NO.4: 5'-TGCAGGTCGACAAGCTTGAATTCTCACTTGTAGAAGATGTGCTTCTTGAAC-3'.

[0040] Example 1 1. Construction and identification of PCold-TF recombinant plasmid 1.1. Amplification and identification results of target genes Using the target DNA as a template, the primers PCold-TF-ORF132-F and PCold-TF-ORF132-R designed by snapgene software were used to amplify the 132 gene of LSDV. DNA amplification was performed according to the PCR system in Table 2. The components shown in Table 2 were added to the PCR tube in sequence. As for the PCR instrument, the specific PCR instrument operation process was as follows: denaturation at 98°C for 30 seconds, annealing at 50°C for 15 seconds, extension at 68°C for 20 seconds, and 30 cycles. Agarose nucleic acid gel electrophoresis was then performed and the results were observed after running at 100V in the nucleic acid electrophoresis instrument for 15 minutes. The results are shown in Figure 2. Figure 1 shown.

[0041] The results showed that a very obvious band of about 550 bp was amplified between 500 bp and 700 bp, which was consistent with the size of the pre-extracted target band. The gel recovery and product purification and recombinant were performed.

[0042] The PCR system for amplification of the target gene is shown in Table 2.

[0043] Table 2 PCR system Note: The upstream primer refers to PCold-TF-ORF132-F; the downstream primer refers to PCold-TF-ORF132-R.

[0044] 1.2 Construction and identification of recombinant plasmids use ECOR Ⅰ and Xho I Two restriction endonucleases for the target gene ORF132 The product recovered from the gene gel and the prokaryotic expression vector PCold-TF were double-enzyme digested. In the clean bench, the enzyme digestion system components in Table 3 were added to the ep tube, 4 μL of 6× loading buffer was added, mixed and centrifuged, and nucleic acid electrophoresis was performed in a nucleic acid electrophoresis instrument at 100V for 20 minutes. The SDS-PAGE results are shown as follows. Figure 2 shown.

[0045] Afterwards, homologous recombination ligation was performed, and the obtained enzyme digestion product was connected with the DNA product. In the clean bench, it was added according to the content of the homologous recombination system in Table 4, reacted in a metal bath for 50 minutes and 15 minutes, and the DH5α plasmid was taken out from the -80℃ refrigerator, thawed on ice, and 10μL of the homologous recombination product was added. After reacting on ice for 30 minutes, it was added to a 42℃ water bath for heat shock for 90 seconds, and then 1ml of non-resistant LB liquid medium (composed of 1% tryptone, 0.5% yeast extract and 1% sodium chloride by mass ratio) was added and cultured in a shaker at 37℃ and 200rpm for 1 hour. Then 200μL was evenly spread on ampicillin-resistant LB solid medium (composed of 1% tryptone by mass ratio, 0.5% yeast extract by mass ratio, 1% sodium chloride by mass ratio and 1.5% agar by mass ratio) to obtain a culture plate with a single colony of the recombinant plasmid. Finally, the recombinant plasmid was identified by PCR as follows. Figure 3 shown.

[0046] The recombinant plasmids with positive identification results were sent to a sequencing company for sequencing. The sequencing results were compared using snapgene, and the comparison results showed that they were consistent with the target sequence.

[0047] The enzyme digestion system is shown in Table 3.

[0048] Table 3 Enzyme digestion system The homologous recombination system is shown in Table 4.

[0049] Table 4 Homologous recombination system 2. Expression and purification of ORF132-TF recombinant protein The recombinant plasmid pCold-TF-ORF132 was transformed into the BL21 (DE3) E. coli expression strain. 100 ml of expansion medium was incubated in a shaker at 37°C, 200 rpm for 4 hours. When the bacterial density reached an OD600 of 0.6, IPTG was added to a final concentration of 0.5 mM to induce expression. The induction conditions were: 16°C for 16 hours. After the 16-hour induction period, the bacterial culture was removed and collected by refrigerated centrifugation at 4°C. The specific steps are as follows:

[0050] Pre-cool the refrigerated centrifuge and centrifuge at 8000 rpm, 4°C, and 10 min. Resuspend the cells in 15 mL of 0.01 M PBS and sonicate for 3 seconds on, 5 seconds off, for a total of 10 min. Centrifuge again at 8000 rpm, 30 min, and 4°C. The centrifuged sample was separated from the precipitate and supernatant, 80 μL of the supernatant was added to 20 μL of 5× loading buffer, and boiled for 10 minutes. The sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific steps were as follows: first, the Novigene One-Step PAGE Gel Fast Preparation Kit was used for rapid preparation of polyacrylamide gel, 5 μL of 180 kDa Plus Prestained Protein Marker was loaded for protein marker calibration, and gel electrophoresis was performed at 120V for 90 minutes. After the electrophoresis, the gel was cut with a cutting plate and stained with Coomassie Brilliant Blue for 2 hours, and decolorized overnight with a destaining solution (70% water, 20% ethanol, and 10% acetic acid by volume).

[0051] Among them, the expansion culture medium is LB liquid culture medium, and its formula composition is 1% trypsin by mass ratio, 0.5% yeast extract by mass ratio and 1% sodium chloride by mass ratio.

[0052] The composition of 0.01mol PBS is: 8g sodium chloride, 0.24g potassium dihydrogen phosphate, 0.2g potassium chloride, and 1.4g disodium hydrogen phosphate added to 1L pure water.

[0053] (1) SDS-PAGE analysis of expression products The results of SDS-PAGE are as follows Figure 4 As shown, the ORF132-TF recombinant protein was primarily expressed as an albumin, with a target band appearing between 60 kDa and 85 kDa, consistent with the expected size of 79.4 kDa. The ORF132-TF recombinant protein was clearly expressed in the supernatant.

[0054] (2) Optimization of induction time of ORF132-TF recombinant protein In order to study the optimal expression conditions, the present invention uses different induction times to optimize the induction of the recombinant expression plasmid pcold-ORF132-TF. The induced expression recombinant bacterial solution is divided into 20 mL groups. At the same time, the ORF132-TF recombinant protein is induced to express for 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h and 26 h, and the recombinant bacterial solution samples at 9 different time points are taken and analyzed by SDS-PAGE electrophoresis. The results are as follows. Figure 5 shown.

[0055] Depend on Figure 5 It can be seen that the best expression effect is achieved when the induction time is 12h-16h.

[0056] (3) Optimization of IPTG concentration for induction of ORF132-TF recombinant protein In order to study the optimal expression conditions, the present invention uses different induction IPTG concentrations, and the induced expression recombinant bacterial solution is packaged into 20 mL groups. The IPTG concentrations of each group are different, namely: 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM and 0.8mM, and SDS-PAGE electrophoresis analysis is performed at the same time. The specific operation steps are as described above, and the results are as follows. Figure 6 .

[0057] Depend on Figure 6 It can be seen that the induction concentration of 0.3mM has the best expression effect.

[0058] (4) Purification of ORF132-TF recombinant protein After expressing the successfully expressed ORF132-TF protein in large quantities, it was purified using nickel column affinity chromatography. The specific steps are as follows: a. Place a white gasket at the bottom of the affinity chromatography column, add 3 mL of nickel column filler, add 6 mL of wash buffer to resuspend the filler, and cover the top with a white gasket.

[0059] b. Add the successfully expressed protein to the affinity chromatography column and incubate at 4°C overnight. Overnight refers to a time of ≥12 hours.

[0060] c. Collect the protein flow-through.

[0061] d. Elute with 20 mM, 40 mM, 60 mM, 100 mM, 200 mM, 500 mM and 600 mM imidazole, respectively, and collect the eluate.

[0062] e. Add the above eluates to SDS-PAGE 4× Loading Buffer respectively, mix well and boil for 10 minutes, then perform SDS-PAGE electrophoresis and observe the electrophoresis results.

[0063] f. Add the purified protein to a 50 kDa ultrafiltration tube and centrifuge horizontally at 4000 x g for 10 min at 4°C. Collect the liquid and store it at -80°C.

[0064] The recombinant protein ORF132-TF was purified by nickel column affinity chromatography to obtain a protein with higher purity. Figure 7 The purified ORF132-TF recombinant protein can be eluted at a concentration of 500 mM imidazole, with a clear band appearing at 76 kDa. There are no other protein bands at other positions in the purified protein sample.

[0065] The wash buffer balance solution consists of 8 g sodium chloride, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.4 g disodium hydrogen phosphate and 1.4 g imidazole added to 1 L of pure water.

[0066] 3. Western blot identification of ORF132-TF recombinant protein Western Blot analysis was performed using His tag antibody and purified ORF132-TF recombinant protein. Figure 8 As shown, a specific reaction occurs at , indicating that it can react specifically with the His tag antibody. The operation steps are as follows:

[0067] a. Add SDS-PAGE 5× Loading Buffer to the purified ORF132-TF recombinant protein, mix well, boil for 10 minutes, place on ice for 10 minutes, and then perform SDS-PAGE electrophoresis.

[0068] b. Electrophoresis: Perform electrophoresis at a voltage of 120V.

[0069] c. Transfer: Cut the gel of interest, then cut a PVDF membrane and four pieces of filter paper of the same size. Activate the PVDF membrane with methanol for 5 minutes. Then, soak the PVDF membrane, filter paper, and gel in transfer buffer for 20 minutes. Once the transfer time is up, place the two filter papers, PVDF membrane, gel, and two filter papers in this order in a semi-dry transfer apparatus.

[0070] d. Blocking: Wash the PVDF membrane three times with TBST, 10 min each time, and place it in blocking solution at 4°C overnight.

[0071] e. Incubation: After blocking, add the primary antibody Anti-His Mouse Monoclonal Antibody (1:5000 diluted in 10 ml of TBST) to the incubation solution and incubate overnight at 4°C. Wash three times with TBST, each wash for 10 minutes.

[0072] f. Incubate with HRP Goat Anti-Mouse IgG (HRP goat anti-mouse secondary antibody diluted 1:10,000 in 10 ml of TBST) for 1 hour, wash three times with TBST, each wash for 10 minutes, and finally perform chemiluminescence.

[0073] g. Exposure: Exposure in the dark, place the PVDF membrane in the luminescent solution, and perform the exposure procedure.

[0074] Western Blot was performed using His tag antibody and purified ORF132-TF protein. The results are as follows Figure 8 As shown, a specific reaction occurred at 76 kDa, indicating that it could react specifically with the His tag antibody.

[0075] The composition of TBST is as follows: 2.4 g tris (hydroxymethyl)aminomethane, 8.8 g sodium chloride are dissolved in 1 L pure water, and then 300 μL of Tween 20 is added.

[0076] The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is shown in SEQ ID NO.1: .

[0077] The amino acid sequence of the ORF132-TF recombinant protein is shown in SEQ ID NO.2: MTNTSLLLSYFFLFLYGNVFVKSDYNYLMYKINVFKNNESTIKCYHNNDIIFLSDDCVSFNSTYNTTVLNNDDVKTELVTLCDVSKEVQIFSLDNSYTGLFLTFLCNNNDSYWFVDILENGIGNNVPTCIDGEVNNKKVKSELQGSLDKEKVLLSTFCKVSHGDCRMWFKKHIFYK*.

[0078] 4. Detection of serum titer of immunized BLAB / c female mice Immunization of BLAB / c female mice was performed according to the following protocol: a. Take ORF132-TF protein at a concentration of 1.0 μg / mL and dilute it with physiological saline to obtain a mixed solution. Add an equal amount of Freund's complete adjuvant to the mixed solution and shake thoroughly to emulsify it (it is best if the drop is placed on the liquid surface and does not dissipate) to obtain the emulsified sample.

[0079] b. The emulsified sample was injected three times from the neck to the back of BLAB / c female mice, with 100 μg protein per BLAB / c female mouse.

[0080] c. Record the immunization time and collect blood one week after immunization.

[0081] d. Take 1.0 μg / mL of ORF132-TF protein and dilute it with PBS to obtain a mixed solution; add an equal amount of Freund's incomplete adjuvant to the mixed solution, shake it thoroughly, and emulsify it to obtain an emulsified sample.

[0082] e. The emulsified sample was injected multiple times into the neck and back of BLAB / c female mice, with 100 μg protein per mouse.

[0083] f. Record the immunization intervals one month apart and collect blood samples one week after immunization.

[0084] Three days before cell fusion, BLAB / c female mice were boosted intraperitoneally with 300 μg of ORF132-TF protein injected intraperitoneally into each BLAB / c female mouse.

[0085] 7±1-week-old BLAB / c female mice were immunized with purified and ultrafiltered ORF132-TF protein. A second immunization was administered one month after the first, and a third immunization was administered one month after the second. Seven days after the second or third immunization, orbital blood was collected from the BLAB / c female mice, and serum was isolated and assayed for antibody levels.

[0086] 5. Indirect ELISA method to detect the titer level of immune serum The coated ORF132-TF protein at a concentration of 1.0 μg / mL was added to the CB solution for gradient dilution. After dilution at 1:1000, 1:3000, 1:9000 and 1:27000, 100 μL was added to each well and placed in a 4°C refrigerator for 12 h. The cells were washed 3 times with PBST and 150 μL of 5% skim milk (2 g skim milk was added to 40 mL of PBST) was added to each well and incubated at 37°C for 1 h. Wash three times with PBST; perform serial dilutions of serum collected from orbital blood (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 128000) and add 100 μL to each well, incubate at 37°C for 1 hour, wash three times with PBST, then add 1:5000 goat anti-mouse secondary antibody, then add 100 μL to each well of 1:5000 goat anti-mouse secondary antibody, incubate at 37°C for 1 hour, wash three times with PBST; add TMB colorimetric solution, 100 μL per well, develop for 15 minutes; finally, stop the color development with 100 μL stop solution per well; measure OD 450 , record the OD value.

[0087] The composition of CB solution is: 1.6g sodium carbonate and 2.9g sodium bicarbonate dissolved in 1L pure water.

[0088] The composition of PBST is: 8 g sodium chloride, 0.24 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.4 g disodium hydrogen phosphate, followed by the addition of 300 μL of Tween 20.

[0089] Stop solution composition: 108 mL of 98% concentrated sulfuric acid is added to 892 mL of pure water.

[0090] The test results are shown in Tables 5 to 7.

[0091] Table 5 Serum titer test results of mice immunized four times with ORF132-TF protein-1 Table 6 Serum titer test results of mice immunized four times with ORF132-TF protein-2 Table 7 Serum titer test results of mice immunized four times with ORF132-TF protein-3 ELISA results showed that the serum antibody level of experimental mice after the second immunization was significantly lower than that after the third immunization.

[0092] 6. Results of cell fusion and fused cell screening Three days before cell fusion, serum titers were tested on pre-immunized BLAB / c female mice, and high-titer BLAB / c female mice were selected as experimental mice.

[0093] Cell fusion was performed as follows: a. Preheat 40 mL of HAT culture medium, 15 mL of basal culture medium, and 0.8 mL of polyethylene glycol to a beaker of water at 37°C. The HAT culture medium contains 5 mM hypoxanthine, 20 mM aminopterin, and 0.8 mM thymidine. The basal culture medium is prepared from the following ingredients:

[0094] b. Aspirate approximately 3×10 myeloma cells 7 Splenic lymphocytes 10 7 Place both cells in a 50mL centrifuge tube, mix thoroughly, and then add basal culture medium to 40mL to obtain a cell suspension. Myeloma cells were obtained from laboratory storage. Splenic lymphocytes were obtained from the spleens of immunized mice.

[0095] c. Centrifuge the myeloma cell and spleen lymphocyte suspensions at 1000 rpm for 10 minutes, discard the supernatant, and gently tap the bottom of the tube to mix the two cell pellets into a paste.

[0096] d. Place the centrifuge tube in a preheated beaker, draw 0.8 mL of PEG-4000, and add it to the precipitate. Stir while adding for 1 minute.

[0097] e. Incubate in a water bath for 90 seconds. Dilute PEG-4000 with 15 mL of pre-warmed basal medium. Add 1 mL during the first 30 seconds, 3 mL during the last 30 seconds, and the remaining 11 mL within 2 minutes.

[0098] f. Add basal culture medium until 40 mL is reached, centrifuge at 1000 rpm for 10 min, discard the supernatant and keep the paste-like cell pellet for later use.

[0099] g. Gently resuspend the cells in 40 mL of HAT selective culture medium containing 20% ​​(m / v) newborn calf serum.

[0100] h. Mix the feeder cells and fused cells, add 200 μL to each well of a 96-well plate using a dispenser, and culture in a 37°C, 5% CO2 incubator. Observe daily.

[0101] i. Observe the cell colonies under a microscope when they have grown to a certain size.

[0102] Microscopic observation results Figure 9 shown.

[0103] Depend on Figure 9It was found that cell fusion between mouse-derived spleen cells and immortalized myeloma cells was successfully achieved.

[0104] 7. Indirect enzyme-linked immunosorbent assay (ELISA) screening of positive cell wells After coating 1.0 μg of ORF132-TF protein with CB solution, 50 μL of supernatant from each well of the 96-well plate was aspirated and added to the ELISA plate. The plate was incubated at 37°C for 1 hour and washed three times with PBST. Then, 100 μL of goat anti-mouse secondary antibody (1:5000 (1 μL of secondary antibody diluted in 10 mL of PBST)) was added to each well. The plate was incubated at 37°C for 1 hour and washed three times with PBST. TMB color development solution was added to each well at 100 μL, and color development was performed for 15 minutes. Finally, 100 μL of stop solution was added to each well to stop color development. The OD value was measured. 450 , record the OD value, and retain the wells with higher OD value and good cell growth for subsequent screening of positive clones.

[0105] The results are shown in Table 8. Four positive clones were identified in the first round of screening.

[0106] 8. Cloning of positive cells by limiting dilution method The cloning method of positive cells is as follows: a. According to the test results, select OD 450 The wells with higher values ​​and good cell growth were selected as positive wells.

[0107] b. Use a micropipette to disperse the cells in the well to be cloned, take 100 μL and transfer it into an EP tube, aspirate an appropriate amount and count.

[0108] c. Dilute to 10% in EP tube with HT medium. 3 / mL. The formula components of HT culture medium are: xanthine (Hypoxantin, H) and thymidine (Thymidin, T).

[0109] d. Fill three 4 mL centrifuge tubes with 3.8 mL, 2.4 mL, and 2.0 mL of culture medium, respectively, for dilution.

[0110] e. Take 200 μL and dilute to 10 3 / mL of cells were placed in the first centrifuge tube, mixed thoroughly, 1.6mL of the mixture was pipetted and added to the second centrifuge tube, mixed again, and 2mL of the mixture was pipetted and added to the third centrifuge tube.

[0111] f. Divide the cells in the centrifuge tube into 96-well feeder cell plates at 100 μL / well. Place one column in the first centrifuge tube, two columns in the second centrifuge tube, and five columns in the third centrifuge tube. Transfer the excess cells in the EP tube to a 12-well plate.

[0112] g. Thereafter, observe the status of cells in the clone wells daily.

[0113] The secondary and tertiary cloning screening methods are the same as the above methods, until 80%-90% of the wells on the entire plate are positive, which is considered to be the selection of single clone cells.

[0114] The results showed that three monoclonal cell lines (2E6, 7B3, and 10C7) turned into negative cell lines, and 14A2 turned into a positive cell line.

[0115] From the above results, it can be seen that one positive cell line (14A2) was successfully screened.

[0116] Table 8 Changes in hybridoma antibody secretion levels 9. Large-scale preparation of monoclonal antibodies 0.5 mL of liquid paraffin was injected into the peritoneal cavity of BLAB / c mice, and hybridoma cells (5 × 10 5 After 10 days, the BLAB / c mice were injected intraperitoneally with 500 cells (100 cells / mL) of BLAB / c cells. After 7 days, the abdomen of the BLAB / c mice would be swollen to varying degrees. The condition of the BLAB / c mice should be monitored closely and the ascites should be extracted promptly. The extracted ascites should be centrifuged at 1000 rpm for 10 minutes. After the ascites is extracted, it should be mixed and aliquoted for subsequent use.

[0117] 10. Indirect ELISA test for ascites After coating ORF132-TF protein in the same manner as above, 100 μL of mouse ascites was aspirated and added to the ELISA plate. The plate was incubated at 37°C for 1 hour and washed 3 times with PBST. Then, 100 μL of 1:5000 goat anti-mouse secondary antibody was added to each well. The plate was incubated at 37°C for 1 hour and washed 3 times with PBST. TMB colorimetric solution was added to each well at 100 μL and color was developed for 15 minutes. Finally, 100 μL of stop solution was added to each well to stop color development. OD was measured. 450 , record the OD value.

[0118] The results are shown in Table 9. The ascites titer can reach 16,000.

[0119] From the above results, it can be seen that the antibodies secreted by the 14A2 cell line have a high titer.

[0120] Table 9 14A2 ascites indirect ELISA test results In summary, there is currently no specific drug for bovine lumpy skin disease virus. This invention uses the ORF132 protein of bovine lumpy skin disease virus (BLV) to construct a prokaryotic expression vector expressing the ORF132-TF protein as an immunogen to prepare monoclonal antibodies. The monoclonal antibodies prepared by this invention have high potency, low cost, and high yield.

[0121] Comparative Example 1 Zhou Xuemei once published a paper on the cloning and prokaryotic expression of the ORF132 protein of bovine lumpy dermatitis virus in the Chinese Journal of Preventive Veterinary Medicine. The results showed that the ORF132 protein has good immunogenicity, but the recombinant plasmid ORF13 constructed in this study expressed the protein in the form of inclusion bodies, and no antibodies were prepared.

[0122] Comparative Example 2 Feng Chunyan once published the recombinant expression and monoclonal antibody preparation of bovine nodular dermatitis A27L protein in the Chinese Journal of Zoonotic Infectious Diseases. The study used prokaryotic expression plasmids to prepare recombinant protein antigens, but the titer of the antigen after immunization was low.

[0123] From the above experimental results, it can be seen that the preparation of monoclonal antibodies against bovine lumpy skin disease virus in the present invention has the characteristics of high yield, low cost and convenient operation, providing technical support for the establishment of a detection method for bovine lumpy skin disease virus.

[0124] The present invention provides a monoclonal antibody against bovine lumpy skin disease virus. The monoclonal antibody has the advantage of high titer and can solve the problem of low titer of monoclonal antibodies developed against bovine lumpy skin disease virus in the prior art.

[0125] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0126] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A monoclonal antibody to bovine lumpy skin disease virus, characterized in that The monoclonal antibody is secreted by hybridoma cell 14A2, and the hybridoma cell 14A2 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2024, with the deposit number CGMCC No.46313.

2. The monoclonal antibody against bovine lumpy skin disease virus according to claim 1, characterized in that The monoclonal antibody is prepared using ORF132-TF recombinant protein as an immunogen; The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is shown in SEQ ID NO.

1.

3. The monoclonal antibody against bovine lumpy skin disease virus according to claim 2, characterized in that The amino acid sequence of the ORF132-TF recombinant protein is shown in SEQ ID NO.

2.

4. A diagnostic antigen for bovine lumpy skin disease virus, characterized in that The diagnostic antigen is the ORF132-TF recombinant protein according to claim 2; The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is shown in SEQ ID NO.

1.

5. A method for preparing the diagnostic antigen according to claim 4, characterized in that: The diagnostic antigen is prepared by using a prokaryotic expression system.

6. The method according to claim 5, characterized in that The nucleotide sequence of the gene encoding the ORF132-TF recombinant protein is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into competent cells to induce expression to obtain the ORF132-TF recombinant protein, which is the diagnostic antigen.

7. The method according to claim 6, characterized in that The expression vector includes at least one of pCold-TF, pet-28a, pet-32a and pCold-sumo.

8. Use of the monoclonal antibody according to claim 1 or the diagnostic antigen according to claim 4 in preparing a diagnostic kit for bovine nodular dermatitis, characterized in that: The kit includes the monoclonal antibody or the diagnostic antigen.

9. Use of the monoclonal antibody according to claim 1 in the preparation of a medicament for treating bovine lumpy skin disease virus.

10. The use according to claim 9, characterized in that The drug uses the monoclonal antibody as an active ingredient.

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