Monoclonal antibody for resisting African swine fever virus D1133L protein and application thereof

Abstract

The invention discloses an anti-African swine fever virus D1133L protein monoclonal antibody and application thereof, and belongs to the technical field of monoclonal antibodies. The invention provides an anti-African swine fever virus D1133L monoclonal antibody 6E6. A heavy chain variable region of the monoclonal antibody 6E6 comprises a CDR1 of which the amino acid sequence is GFSLISYA, a CDR2 of which the amino acid sequence is IWTDGGT and a CDR3 of which the amino acid sequence is ARMDYYGCGDAMGY; a light chain variable region of the monoclonal antibody 6E6 comprises a CDR1 of which the amino acid sequence is QDIINY, a CDR2 of which the amino acid sequence is CTS and a CDR3 of which the amino acid sequence is KHYSKLPWA. The antibody disclosed by the invention has the characteristics of high purity, strong affinity, good specificity and the like, and can be widely applied to an immunodetection technology.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to anti-African swine fever virus D1133L protein monoclonal antibodies and applications thereof. Background Art

[0002] African swine fever virus (ASFV) is an acute, highly contagious animal disease that severely impacts the swine industry. Its pathogenic mechanism is complex, and currently no effective vaccine or drug is available for prevention and control. ASFV belongs to the genus Asfivirus in the family Asfarviridae and is the only known insect-borne double-stranded DNA virus. The ASFV virion has a complex structure, consisting of five layers: the viral nucleoid genome, the inner shell, the inner membrane, the capsid, and the outer membrane. The genome is approximately 170-190 kb in size and encodes over 150 proteins, including several structural and nonstructural proteins. ASFV is mainly transmitted through soft ticks and wild boar hosts. Once it enters the domestic pig population, it can cause acute or subacute infection with high mortality. Typical symptoms include high fever, subcutaneous hemorrhage, lymphadenopathy, pulmonary edema and disseminated intravascular coagulation (DIC), which seriously affects the development of the global pig industry.

[0003] In ASFV research, the D1133L protein is one of five helicases encoded by ASFV, expressed late in viral infection and highly conserved. The ASFV D1133L protein may be involved in viral transcriptional regulation, assembly, or virus-host interactions, and may play an important role in ASFV invasion of cells.

[0004] Currently, due to safety concerns, there is no widely available commercial ASFV vaccine. Therefore, current research focuses on clarifying the mechanisms of ASFV infection and replication, identifying key viral or host proteins involved in the infection process as antiviral drug targets, and developing anti-ASFV drugs. D1133L protein, one of the helicases encoded by ASFV, has been reported to have a monoclonal antibody that can significantly inhibit ASFV replication at the cellular level, suggesting that D1133L protein may be a key factor in the ASFV infection process and a potential target for the development of anti-ASFV drugs. The development of highly specific monoclonal antibodies against D1133L protein can not only be used to analyze the expression and distribution of D1133L using experimental methods such as Western blot and indirect immunofluorescence assay (IFA), but also provide new research tools for studying ASFV infection or replication mechanisms and antigen-antibody detection techniques.

[0005] The present invention uses prokaryotically expressed and purified ASFV D1133L protein to immunize mice, screen and obtain the hybridoma cell line 6E6 that secretes D1133L protein monoclonal antibodies. The antibodies can specifically recognize the D1133L protein in ASFV-infected cells and are suitable for Western blot and IFA detection, laying an important foundation for ASFV D1133L protein-related research and antigen-antibody detection methods. Summary of the Invention

[0006] The purpose of the present invention is to provide a monoclonal antibody against African swine fever virus D1133L protein and its application to solve the problems existing in the above-mentioned prior art. The monoclonal antibody against ASFV D1133L protein prepared by the present invention has the advantages of high antibody purity, strong affinity and good specificity, and provides a basis for the study of the structure and function of ASFV D1133L protein and the detection method based on D1133L protein.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an anti-African swine fever virus D1133L protein monoclonal antibody 6E6, wherein the heavy chain variable region of the monoclonal antibody 6E6 comprises a CDR1 with an amino acid sequence of GFSLISYA, a CDR2 with an amino acid sequence of IWTDGGT, and a CDR3 with an amino acid sequence of ARMDYYGCGDAMGY; the light chain variable region of the monoclonal antibody 6E6 comprises a CDR1 with an amino acid sequence of QDIINY, a CDR2 with an amino acid sequence of CTS, and a CDR3 with an amino acid sequence of KHYSKLPWA.

[0009] Optionally, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6E6 is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody 6E6 is shown in SEQ ID NO: 2.

[0010] Optionally, the monoclonal antibody 6E6 produces a specific immune response with African swine fever virus or its D1133L protein antigen.

[0011] Optionally, the heavy chain subtype of the monoclonal antibody 6E6 is IgG2a, and the light chain subtype is Kappa.

[0012] The present invention also provides a nucleic acid molecule, which encodes the monoclonal antibody 6E6.

[0013] The present invention also provides a vector comprising the nucleic acid molecule.

[0014] The present invention also provides a host cell, which comprises the nucleic acid molecule or the vector.

[0015] The present invention also provides the use of the monoclonal antibody 6E6, the nucleic acid molecule, the vector, or the host cell in preparing a product for detecting African swine fever virus or its D1133L protein antigen.

[0016] Optionally, the product comprises reagents or kits for detection using Western-Blot or indirect immunofluorescence methods.

[0017] The present invention also provides a product for detecting African swine fever virus or its D1133L protein antigen, wherein the product comprises the monoclonal antibody 6E6 or the nucleic acid molecule or the vector or the host cell.

[0018] The present invention discloses the following technical effects:

[0019] The immunogen used in the present invention is the ASFV D1133L protein expressed and purified in prokaryotes.

[0020] The present invention provides monoclonal antibodies that can specifically react with ASFV and ASFV D1133L protein, have good immunogenicity, good specificity, and high sensitivity. They can be widely used in immunoassay techniques and can be used as antibodies in Western-blot and indirect immunofluorescence assays for identifying ASFV and D1133L protein and measuring ASFV viral content. The present invention lays an important foundation for the clinical detection of ASFV and its D1133L protein antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a Western Blot result diagram showing the expression of the ASFV D1133L prokaryotic protein expressed in Example 1 in the supernatant and precipitate after bacterial disruption;

[0023] Figure 2 This is the ELISA result of the reaction between the purified ASFV D1133L protein in Example 1 and clinical African swine fever virus-positive pig serum; wherein, the ASFV CP204L protein is used as a positive control;

[0024] Figure 3The ELISA test results of serum antibody titers of immunized mice in Example 1;

[0025] Figure 4 This is a diagram showing the results of IFA analysis of the D1133L monoclonal antibody in Example 2 (D1133L protein-expressing cells);

[0026] Figure 5 Figure 2 shows the Western-Blot analysis results of the D1133L monoclonal antibody in Example 2. pcaggs-D1133L: D1133L eukaryotic expression protein, pcaggs: negative control;

[0027] Figure 6 This is the result of IFA analysis of D1133L monoclonal antibody in Example 2 (virus-infected cells);

[0028] Figure 7 The Western-Blot analysis results of the D1133L monoclonal antibody in Example 2 are shown. Virus: cells infected with African swine fever virus; Mock: blank cell control.

[0029] Figure 8 This is a diagram showing the SDS-PAGE test results of the purification effect of the D1133L monoclonal antibody in Example 3;

[0030] Figure 9 The figure shows the potency of D1133L monoclonal antibody; NC is the negative control;

[0031] Figure 10 This is the Western-Blot result of D1133L protein fishing with D1133L monoclonal antibody in Example 3, anti-D1133L: D1133L antibody fishing, mIgG: mouse IgG fishing;

[0032] Figure 11 The results of PCR amplification of the variable regions in Example 4, M: Marker, VH: heavy chain of monoclonal antibody 6E6, VL: light chain of monoclonal antibody 6E6. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1 Hybridoma Preparation

[0039] 1. Preparation of immunogen

[0040] 1.1 Prokaryotic expression and purification of ASFV D1133L protein

[0041] ASFV D1133L, GenBank: ON380540., African swine fever virus isolateHB31A. First, specific primers were designed (see Table 1), and EcoRI and BamHI restriction sites were added to the 5' end of the primers. Using ASFV genomic DNA as a template, PrimeSTAR Max DNA polymerase was used to PCR amplify the D1133L coding region. The amplified product was purified by 1% agarose gel electrophoresis and ligated with the pET-28a(+) vector treated with EcoRI and BamHI double enzymes. The ligation product was transformed into Escherichia coli DH5α competent cells, and positive clones were screened on kanamycin plates. After plasmid extraction, the sequence was confirmed to be correct by Sanger sequencing to construct the prokaryotic expression plasmid PET-28a-D1133L-HIS.

[0042] The constructed prokaryotic expression plasmid PET-28a-D1133L-HIS vector was transformed into BL21 DE3 bacteria, evenly spread on the solid LB medium containing kanamycin resistance, and inverted in a 37°C incubator for overnight culture. When a single colony appears, pick a single colony and transfer it to the liquid LB medium with kanamycin resistance. Shake the bacteria for about 8 to 12 hours until the bacterial solution becomes turbid. At a ratio of 1:50, take the bacterial solution and inoculate it into the liquid LB medium with kanamycin resistance. Incubate at 37°C and 220×g for about 2.5 hours. At this time, test the OD value of the bacterial solution to be expressed. 600nm When the concentration is between 0.5-0.6, add sterile 100mM IPTG to make the final IPTG concentration of the bacterial solution to be expressed 0.5mM. Place the bacterial solution to be expressed on a shaker and induce at room temperature at 37°C and 220g for about 10 hours. After the induction expression is completed, collect the bacterial solution and place it in a centrifuge tube and centrifuge at 8000g for 10 minutes. After centrifugation, discard the supernatant, resuspend the precipitate with PBS and wash the bacteria. Add the bacterial solution to the centrifuge tube and centrifuge at 8000g for 10 minutes. Repeat the washing twice and add PBS to resuspend the bacterial precipitate. Perform Western Blot on the precipitate and supernatant to detect the expression of D1133L protein. The results are shown in Figure 1 After washing, the bacterial pellet was resuspended in PBS and then crushed under high pressure. After crushing, the product was added to a centrifuge tube and centrifuged at 12000g for 10 minutes. The precipitate was collected, dissolved in urea and renatured, and purified using a nickel column. The protein eluted from the nickel column was added to a boiled dialysis bag for dialysis. After dialysis, the protein was concentrated using an ultrafiltration tube of the corresponding size. After concentration, it was sterilized by filtration using a 0.22μm filter membrane and finally packaged and stored at -80℃.

[0043] Table 1 PET-28a-D1133L amplification primers

[0044]

[0045] 1.2D1133L protein reactogenicity verification - indirect ELISA test

[0046] The purified D1133L protein 1 μg / mL, 100 μL / well and the laboratory-stored P30 (CP204L) protein 1 μg / mL, 100 μL / well were coated on the ELISA plate at 4°C overnight. The ELISA plate was blocked with 5% skim milk powder at room temperature for 4 hours and washed with PBST buffer. African swine fever positive serum (reference article doi:10.3390 / vetsci11090403, laboratory-stored) was used as the primary antibody at 1:1000 and incubated at room temperature for 2 hours. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody. HRP-labeled goat anti-mouse was used as the secondary antibody at 1:6000 dilution and incubated at room temperature for 45 minutes. The ELISA plate was washed with PBST buffer, 100 μL / well of the color development solution was added and incubated at 37°C for 10 minutes, and then 50 μL / well of the stop solution was added. The results were observed by UV spectrophotometer at 450 nm.

[0047] ELISA test results showed (see Figure 2 ), the purified protein has good reactogenicity and can be used as an immunogen to immunize mice.

[0048] Preparation of monoclonal antibodies against ASFV D1133L protein

[0049] 2.1 Mouse immunization

[0050] Five female BALB / c mice were immunized with 50 μg of the purified D1133L protein per mouse. For the first immunization, the D1133L protein suspension was emulsified with Freund's complete adjuvant at a 1:1 ratio and injected subcutaneously at multiple points on the back of the mice. Two weeks later, the mice were immunized once with Freund's incomplete adjuvant emulsified with D1133L protein at a 1:1 ratio, followed by another immunization two weeks apart, for a total of three immunizations. Blood was collected from the tail vein 15 days after the third immunization.

[0051] 2.2 Indirect ELISA detection of serum titer of immunized mice

[0052] The ELISA plate was coated with 1 μg / mL D1133L protein at 100 μL / well at 4°C overnight, and the ELISA plate was blocked with 5% skim milk powder at room temperature for 4 h. The ELISA plate was washed with PBST buffer, and the immunized mouse serum was diluted starting from 1:100 to 1:12800 as the primary antibody and incubated at room temperature for 2 h. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody. HRP-labeled goat anti-mouse was diluted 1:6000 as the secondary antibody and incubated at room temperature for 45 min. The ELISA plate was washed with PBST buffer, and 100 μL / well color development solution was added and incubated in a 37°C incubator for 10 min. Then 50 μL / well stop solution was added and the results were observed at 450 nm on an ELISA plate reader.

[0053] The results are as follows Figure 3 As shown, after mice were immunized with D1133L protein, antibodies against D1133L protein were detected in the mouse serum 15 days after the third immunization, and the subsequent cell fusion test could be performed.

[0054] 2.3 Cell fusion

[0055] The spleen cells of the boosted immunized mice were taken and fused with sp2 / 0 cells at a ratio of (10-15):1 in a 37°C water bath. PEG4000 was added for 1 minute, and the cells were allowed to stand for 1 minute. Then, 1 mL of 1640 medium was added for 1 minute. Next, 1 mL of 1640 medium was added for 20 seconds. Next, 30 mL of medium was added for 3 minutes. The cells were centrifuged at 800 rpm for 5 minutes and plated with 1×HAT 20% FBS 1640 medium. When the confluence of the successfully fused cells reached more than 50%, positive cell lines were screened.

[0056] 2.4 Hybridoma cell screening and cloning

[0057] Indirect immunofluorescence assays were performed on LLC-PK1 cells inoculated with ASFV virus solution. Cell culture supernatants collected 10 days after fusion were then used to screen for positive cell clones. Finally, hybridoma cells that reacted positively with ASFV-infected cells but negatively with LLC-PK1 blank cells were selected and subcloned using limiting dilution. After two rounds of subcloning and indirect immunofluorescence screening, a hybridoma cell line, 6E6, was obtained that stably secreted a monoclonal antibody against the D1133L protein.

[0058] Example 2 Verification of the specificity of hybridoma cells

[0059] 1. Identification of the reactivity of hybridoma supernatant with ASFV-D1133L protein

[0060] 1.1 Construction of eukaryotic expression plasmid for African swine fever D1133L protein

[0061] Using the African swine fever genome as a template, specific primers (see Table 2) were used to amplify the D1133L gene, resulting in a 3132-bp fragment. After double digestion with BamHI and NheI, the fragment was ligated into the pcaggs eukaryotic expression vector. Plasmid sequencing and amplification with specific primers confirmed the construct's correctness, resulting in the construction of the eukaryotic expression plasmid pcaggs-D1133L-Flag.

[0062] Table 2 Pcaggs-D1133L amplification primers

[0063] Primer name sequence D1133L-F CTAGCTAGCATGGCGTATCCCGAATTGGAT(SEQ ID NO:7) D1133L-R CGCGGATCCAAACCGGATAGGTGGGCGGAT(SEQ ID NO:8)

[0064] 1.2 Indirect immunofluorescence

[0065] HEK-293T cells were grown to a confluence of 80-90%, and then transfected with the D1133L protein eukaryotic expression plasmid. Indirect immunofluorescence assay was performed 28-30 hours later. After discarding the cell culture medium, wash once with PBS, add 4% paraformaldehyde and fix at room temperature for 15 minutes, discard the fixative, wash three times with PBS, use PBS containing 0.1% Triton X-100 as permeabilization solution, permeabilize at room temperature for 15 minutes, discard the permeabilization solution, wash three times with PBS, switch to PBS containing 5% BSA as blocking solution, block at room temperature for 2 hours, discard the blocking solution, add hybridoma cell culture supernatant and HA antibody as primary antibody, incubate at room temperature for 2 hours, wash three times with PBST, each time for 5 minutes, add Alexa Flour 488 donkey anti-mouse IgG (H+L) at a dilution of 1:500, incubate at room temperature for 45 minutes, wash three times with PBST, each time for 5 minutes, add ready-to-use DAPI to stain cell nuclei for 5 minutes, discard DAPI, wash three times with PBS, and observe the results under a fluorescence microscope.

[0066] Observation under an inverted fluorescence microscope showed that D1133L protein reacted with the positive hybridoma supernatant in a specific antigen-antibody reaction. After adding the fluorescent secondary antibody, obvious specific fluorescence appeared under the field of view. The blank control well had no fluorescence. The results are as follows: Figure 4 As shown, this indicates that the antibody can specifically react with the D1133L protein of ASFV.

[0067] 1.3 Western Blot

[0068] Collect HEK 293T cell samples 28 hours after pcaggs-D1133L transfection, pre-treat and separate them by SDS-PAGE. Take a piece of film containing the target band and place it on the transfer apparatus. Transfer the membrane at 200mA for 1.5 hours. Block the PVDF membrane with 10% skim milk at room temperature for 2 hours. Wash with PBST 3 times, 5 minutes each time, then add hybridoma supernatant as the primary antibody and react slowly at 4°C overnight. Wash with PBST 3 times, 5 minutes each time, then incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45 minutes, and finally develop with ECL to observe the results. Figure 4 It can be seen that the ASFV D1133L monoclonal antibody can effectively bind to the D1133L eukaryotic expression protein, indicating that the antibody can react with the linearized D1133L protein.

[0069] 2. Identification of fusion hybridoma cells and ASFV reactivity

[0070] 2.1 Indirect immunofluorescence

[0071] LLC-PK1 cells were trypsinized and plated in 24-well plates. When the cells reached approximately 90% confluency, they were inoculated with ASFV virus solution and incubated at 37°C for 2 hours. The cells were then replaced with fresh 2% DMEM and cultured at 37°C for 36 hours before indirect immunofluorescence analysis. After discarding the cell culture medium, wash twice with PBS, add 4% paraformaldehyde and fix at room temperature for 15 minutes, discard the fixative, wash 3 times with PBS, use PBS containing 0.1% Triton 100 as permeabilization solution, permeabilize at room temperature for 15 minutes, discard the permeabilization solution, wash 3 times with PBS, switch to PBS containing 5% BSA as blocking solution, block at room temperature for 2 hours, discard the blocking solution, add hybridoma supernatant as primary antibody, incubate at room temperature for 2 hours, wash 3 times with PBST, each time for 5 minutes, add Alexa Flour488 donkey anti-mouse IgG (H+L) (1:500 dilution), incubate at room temperature for 45 minutes, wash 3 times with PBST, each time for 5 minutes, add ready-to-use DAPI to stain the cell nucleus for 5 minutes, discard DAPI, add PBS and wash 3 times, and observe the results under a fluorescence microscope. Observation under an inverted fluorescence microscope showed that the virus ASFV reacted specifically with the monoclonal antibody. After adding the fluorescent secondary antibody, obvious specific fluorescence appeared under the field of view. The results are as follows Figure 6 shown.

[0072] 2.2 Western Blot

[0073] Collect LLC-PK1 cell samples 36 hours after inoculation with ASFV virus liquid, pre-treat and separate them with SDS-PAGE, take a piece of film containing the target band and place it on the transfer apparatus, and transfer the membrane at 200mA for 1.5 hours. Block the PVDF membrane with 10% skim milk at room temperature for 2 hours. Wash 3 times with PBST for 5 minutes each time, add hybridoma supernatant as the primary antibody, and react slowly with shaking at 4°C overnight. Wash 3 times with PBST for 5 minutes each time, incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45 minutes, and finally develop with ECL to observe the results. Western-Blot identification results are as follows Figure 7 The results showed that the target band appeared at about 118.8 kDa, indicating that the prepared monoclonal antibody was able to produce a specific immune response with the ASFV virus.

[0074] 3 Identification of monoclonal antibody types and subclasses

[0075] The subtype of D1133L monoclonal antibody was identified using the Mouse Mecrodinal Antibody Isotyping Elisa Kit (purchased from Proteintech), and the supernatant of positive subclones was collected.

[0076] The antibody type was detected using a kit, and the results showed that a monoclonal antibody cell line that stably secreted ASFV D1133L protein was successfully obtained, and the cell line secreted IgG2a type antibodies (Table 3), named 6E6.

[0077] Table 3 Identification of monoclonal antibody types and subclasses

[0078]

[0079] Note: + indicates a positive reaction, - indicates a negative reaction.

[0080] As shown in Table 3, the heavy chain subtype of monoclonal antibody 6E6 is IgG2a, and the light chain subtype is Kappa.

[0081] Example 3: Production of Monoclonal Antibodies by Hybridoma Cells

[0082] 1 Preparation and purification of ascites

[0083] Three BALB / c female mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Ten days later, approximately 2 × 10 6 Monoclonal cells in the logarithmic proliferation phase were injected into the peritoneal cavity of mice. After the abdomen of the mouse was significantly swollen, ascites was extracted and centrifuged at 10,000 rpm for 10 minutes to collect the supernatant. Monoclonal antibodies were purified using Protein A / G affinity chromatography.

[0084] The results are as follows Figure 8 As shown in the figure, the antibody protein secreted by the monoclonal antibody cell line was purified by Protein A / G affinity chromatography, and two bands appeared, one for the antibody heavy chain (55kDa) and the other for the antibody light chain (25kDa). There was no other impurity protein in the background, indicating a good purification effect.

[0085] 2 Ascites titer detection

[0086] The monoclonal antibody 6E6 purified from ascites and an irrelevant antibody (NC group) were serially diluted with PBST at 2 ng / μL, with a total of 8 dilution gradients. 50 μL / well of the diluted antibody was added to the D1133L protein-coated ELISA plate and reacted at RT for 2 hours. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:6000 dilution) was added and reacted at room temperature for 45 minutes. After washing four times with PBST, 100 μL / well of color development solution was added and incubated in a 37°C incubator for 10 minutes, and then 50 μL / well of stop solution was added, and the results were observed at 450 nm.

[0087] The results are as follows Figure 9 As shown, when the monoclonal antibody 6E6 was diluted to 0.015625 ng / μL, its OD 450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 0.015625 ng / μL.

[0088] 3 Antibody-based IP assays

[0089] PAMs were inoculated in a 6-well plate and cultured in an incubator for 12 hours before inoculation with African swine fever virus. 24 hours after virus infection, RIPA-lyzed cell samples were collected, and the supernatant of the cell lysate was taken. D1133L antibody and mouse IgG (negative control)-conjugated A+G agarose were added and incubated at 4°C for 12 hours. After that, the agarose-enriched sample was collected and silver-stained. The silver staining results are shown in Figure 2. Figure 10 As shown, the D1133L protein in virus-infected cells can be fished up by the 6E6 antibody.

[0090] The above results show that the antibody has a good affinity for the D1133L protein of virus infection in the natural state, and can serve as a good tool for subsequent basic research on ASFV D1133L protein and clinical diagnosis and prevention of African swine fever virus.

[0091] Example 4 PCR amplification and sequence determination of the variable region gene of the D1133L protein monoclonal antibody 6E6

[0092] RNA from the monoclonal antibody 6E6 hybridoma cells was extracted and converted to cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche). The variable region genes were amplified using nested PCR. First, the cDNA was used as a template to amplify the variable region genes using first-round mouse IgG and κ light chain primers. Second, the first-round product was used as a template to amplify the variable region genes using second-round mouse IgG and κ light chain primers. The PCR reaction system consisted of 25 μL of 2× PhantaFlash Master Mix (Vazyme), 1 μL each of Primer 1 and Primer 2, 1 μL of cDNA, and ddH2O to 50 μL. The reaction procedure was as follows: initial denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 50 s, followed by extension at 72°C for 10 min. The primers for antibody variable region gene amplification were referenced in the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.). After amplification, agarose gel electrophoresis was performed. The heavy chain and kappa light chain variable region genes of monoclonal antibody 6E6 were approximately 350 bp in size ( Figure 11 ), the target fragment was recovered by gel excision and inserted into the pMD-18T blunt-end vector for sequence determination.

[0093] The sequencing results were compared and analyzed on the NCBI IGBLAST website. The sequencing results confirmed that the amplified sequences were DNA of the heavy and light chain variable regions of the monoclonal antibody. Specifically, the amino acid sequence of the heavy chain variable region of the mouse anti-ASFV D1133L protein monoclonal antibody 6E6 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the mouse anti-ASFV D1133L protein monoclonal antibody 6E6 is shown in SEQ ID NO: 2. The DNA sequence encoding the heavy chain variable region of the mouse anti-ASFV D1133L protein monoclonal antibody 6E6 is shown in SEQ ID NO: 3; and the DNA sequence encoding the light chain variable region of the mouse anti-ASFV D1133L protein monoclonal antibody 6E6 is shown in SEQ ID NO: 4.

[0094] SEQ ID NO: 1:

[0095] QVQLKESGPGLVAPSQSLSITCTVSGFSLISYAINWVRQPPGKGLEWLGVIWTDGGTNY NSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARMDYYGCGDAMGYWGQGTSVTV SS;

[0096] SEQ ID NO:2:

[0097] DIQMTQTTSSLSASLGDRVTISCRASQDIINYLNWYQQKPDGTIKLLIYCTSRLHSGVPS RFSGSGSGTDYSLTISNLEPEDIATYYCKHYSKLPWAFGGGTKVEIK;

[0098] SEQ ID NO:3:

[0099] CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAATCAGCTATGCTATAAACTGGGTTCGCCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGATGGAGGCACAAATTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAATGGATTACTACGGCTGCGGGGATGCTATGGGCTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0100] SEQ ID NO:4:

[0101] GATATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTATCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTATTAAACTCCTGATCTACTGCACATCAAGATT ACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTAAGCATTATAGTAAGCTTCCGTGGGCGTTCGGTGGAGGCACCAAGGTGGAAATCAAAC.

[0102] The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 6E6 are shown in Table 4 below.

[0103] Table 4 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy and light chain variable regions of monoclonal antibody 6E6

[0104] CDR1 CDR2 CDR3 Heavy chain GFSLISYA IWTDGGT ARMDYYGCGDAMGY light chain QDIINY CTS KHYSKLPWA

[0105] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An anti-African swine fever virus D1133L protein monoclonal antibody 6E6, characterized in that The heavy chain variable region of the monoclonal antibody 6E6 includes a CDR1 with an amino acid sequence of GFSLISYA, a CDR2 with an amino acid sequence of IWTDGGT, and a CDR3 with an amino acid sequence of ARMDYYGCGDAMGY; the light chain variable region of the monoclonal antibody 6E6 includes a CDR1 with an amino acid sequence of QDIINY, a CDR2 with an amino acid sequence of CTS, and a CDR3 with an amino acid sequence of KHYSKLPWA.

2. The monoclonal antibody 6E6 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6E6 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 6E6 is shown in SEQ ID NO:

2.

3. The monoclonal antibody 6E6 according to claim 1, characterized in that The monoclonal antibody 6E6 generates a specific immune response with African swine fever virus or its D1133L protein antigen.

4. The monoclonal antibody 6E6 according to claim 1, characterized in that The heavy chain subtype of the monoclonal antibody 6E6 is IgG2a, and the light chain subtype is Kappa.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the monoclonal antibody 6E6 according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 5 or the vector of claim 6.

8. Use of the monoclonal antibody 6E6 according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, or the vector according to claim 6, or the host cell according to claim 7 in the preparation of a product for detecting African swine fever virus or its D1133L protein antigen.

9. The use according to claim 8, characterized in that The product comprises reagents or kits for detection using Western-Blot or indirect immunofluorescence methods.

10. A product for detecting African swine fever virus or its D1133L protein antigen, characterized in that: The product comprises the monoclonal antibody 6E6 according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, or the vector according to claim 6, or the host cell according to claim 7.

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