African swine fever p72 monoclonal antibody and application thereof

Abstract

The application provides a preparation of an African swine fever virus p72 monoclonal antibody and application of the African swine fever virus p72 monoclonal antibody in detection of p72 protein and establishment of an African swine fever virus latex chromatography test strip detection method. The application prepares an ASFV p72 protein monoclonal antibody hybridoma cell strain R3E9, and determines the variable region sequence of the monoclonal antibody. The monoclonal antibody can be used in IFA, recognizes the p72 protein expressed by a baculovirus and the p72 protein in ASFV infected cells, and can also be used in western blotting, and recognizes the denatured p72 protein. The African swine fever virus latex chromatography test strip prepared by using the monoclonal antibody has good specificity and sensitivity, and can realize the on-site rapid detection of ASFV without auxiliary equipment, provides important technical support for the rapid diagnosis of ASFV in pig farms in China, and will enhance the prevention and control ability of ASFV in China.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the preparation of a monoclonal antibody against African swine fever virus p72 and its application in detecting p72 protein and establishing a method for detecting African swine fever virus using chromatographic test strips. Background Technology

[0002] African swine fever (ASF) is a highly contagious and virulent disease caused by the African swine fever virus (ASFV) in pigs. Both domestic and wild pigs are susceptible to ASFV. Clinical symptoms vary depending on the virulence of the infecting strain: highly virulent strains cause acute cases, mainly characterized by high fever and widespread internal bleeding, with a rapid progression and death within 4-10 days, resulting in a 100% mortality rate; moderately virulent strains cause subacute cases with a prolonged incubation period and milder clinical symptoms, but significant internal bleeding and edema, with a mortality rate of 30-70%; attenuated strains cause chronic cases that can last 5 to 12 months, without causing internal bleeding, mainly characterized by skin necrosis and ulceration, joint inflammation and swelling, lymph node hyperplasia, and lung necrosis and mineralization, as well as growth retardation or even emaciation. Infected breeding pigs experience reproductive disorders. ASFV poses a significant threat to the pig industry, and the World Organisation for Animal Health (OIE) has listed it as a notifiable animal disease, severely impacting the pig farming sector.

[0003] ASFV is a linear double-stranded DNA virus, the sole member of the genus *African swine fevervirus* within the family African swine feverviridae. The viral genome is 170-194 kbp in length, encoding 151-167 open reading frames (ORFs). ASFV has an icosahedral morphology, with a particle diameter of 175-215 nm. Its structure is relatively complex, consisting of five layers from the inside out: a nucleoid containing the viral genome, an inner core shell, an inner envelope, a capsid, and an outer envelope. ASFV replicates within the cytoplasm of infected cells and releases mature viral particles with an outer envelope through budding. However, in addition to the mature viral particles, viral particles with a capsid layer formed within the cell are also infectious and can be released late in infection through cell lysis. The viral outer membrane contains the viral proteins p12 and CD2v, with CD2v exhibiting erythrocyte agglutination activity and playing a crucial role in viral pathogenesis. The icosahedral capsid of the virus contains five proteins: one major capsid protein, p72, and four minor capsid proteins, pH240R, p17, p49, and pM1249L. p72 is the most important structural component of the viral particle, accounting for nearly one-third of the total viral particle mass (Adv. VirusRes. 100, 41-74 (2018)). Simultaneously, p72 is also an important antigenic protein of the virus, serving as a primary basis for viral genotyping and a crucial target for ASFV diagnosis and subunit vaccine development.

[0004] Currently, there are no effective vaccines or drugs available for ASFV, making detection technology crucial for its prevention and control. This invention prepares p72 protein using an E. coli expression system and uses p72 protein to immunize mice to generate monoclonal antibodies. Through screening and subclonal purification, a hybridoma cell line secreting p72 monoclonal antibodies was obtained. The monoclonal antibody secreted by this hybridoma cell line can be used to recognize p72 protein in Western blotting and can also specifically recognize p72 protein expressed in baculoviruses and p72 protein in ASFV-infected cells in indirect immunofluorescence experiments. An ASFV antigen test strip was prepared by labeling this monoclonal antibody with latex microspheres. This test strip exhibits good specificity and sensitivity, enabling rapid on-site detection of ASFV without auxiliary equipment, providing important technical support for rapid ASFV diagnosis in pig farms in my country and enhancing the country's ASFV prevention and control capabilities. Summary of the Invention

[0005] The purpose of this invention is to provide an ASFV p72 monoclonal antibody cell line and its applications. The antibody secreted by this monoclonal cell line can be used for Western blotting to detect p72 protein, and can also recognize p72 protein expressed in insect cells and p72 protein in ASFV-infected cells. Labeling this monoclonal antibody with latex microspheres can be used to develop a latex microsphere chromatography test strip for detecting ASFV.

[0006] In addition, the present invention also provides a method for preparing p72 protein and its application.

[0007] In addition, the present invention also provides an ASFV antigen latex chromatography test strip.

[0008] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: This invention involves cloning a p72 gene fragment into the prokaryotic expression vector pCold I, inducing expression at 16°C, and then denaturing, column-purifying, and renaturing the expressed p72 protein inclusion bodies to obtain purified p72 protein. Mice were immunized with p72 protein, and after a second immunization, mouse spleen cells were fused with SP2 / 0 cells. Positive hybridoma clones were screened using a p72 antibody ELISA, and a purified hybridoma cell clone was obtained by dilution. Indirect immunofluorescence experiments confirmed that this hybridoma antibody could bind to p72 protein expressed by baculovirus and p72 protein expressed on ASFV-infected porcine primary monocytes. An immunochromatographic method for ASFV antigen detection was established using latex microspheres conjugated with this monoclonal antibody as an immunoprobe.

[0009] The p72 protein used in this invention is prepared by cloning the p72 gene into the prokaryotic form vector pCold I, followed by low-temperature (16°C) induction, inclusion body denaturation, His column purification, and dialysis refolding.

[0010] The p72 latex microsphere chromatography test strip established in this invention is as follows: 300nm red latex microspheres labeled with p72 monoclonal antibody are used as the detection probe. A 4mm wide glass fiber strip is immersed in the probe solution and allowed to air dry to serve as the conjugate pad. 1.25mg / mL rabbit anti-p72 polyclonal antibody is sprayed onto the NC membrane as the detection line (T line), and 0.5mg / mL SPA protein is sprayed onto the same NC membrane as the quality control line (C line). The glass fiber strip is immersed in sample pad treatment solution and dried in a 37℃ oven to serve as the sample pad. The conjugate pad and absorbent pad are sequentially pasted onto both sides of the NC membrane, and the sample pad is pasted onto the other end of the conjugate pad. All pads overlap by 2mm and are assembled into a plastic slot to form the test strip. The sample is diluted 1:2 with sample diluent, and 3 drops are added to the sample application window of the test strip. After standing at room temperature for about 10 minutes, the color development results are observed. If both the T line and the C line are red, the result is positive; if the T line does not show color but the C line is red, the result is negative; if neither the T line nor the C line shows color, the test result is invalid. Attached Figure Description

[0011] Figure 1 PCR amplified ASFV p72 gene fragment, M: DNA Marker DL5000; 1: p72 gene fragment; Figure 2 Inducible expression of ASFV p72 truncated protein, where M: protein molecular weight standard; 1: pCold I empty vector; 2: pCold-p72 uninduced; 3: pCold-p72 induced bacterial cells; 4: sonicated supernatant of pCold-p72 induced bacteria; 5: sonicated precipitate of pCold-p72 induced bacteria. Figure 3 Purification of ASFV p72 truncated protein, M: protein molecular weight standard; 1: purified p72 truncated protein; Figure 4 Western blotting of p72 monoclonal antibody: M: protein molecular weight standard; 1: p54 protein; 2: p72 truncated protein; Figure 5 FA analysis of p72 protein expressed by baculovirus; Figure 6 IFA analysis of ASFV-infected cells: Left: Control cells; Right: ASFV-infected cells; Figure 7 SDS-PAGE image of R3E9 monoclonal antibody, M: protein marker; 2: R3E9 monoclonal antibody; Figure 8Colorimetric results of latex microspheres of different diameters: (a): p72 protein polyclonal antibody spray detection line; (b) Staphylococcus A protein spray detection line control line, 1: 200nm latex microspheres; 2: 300nm latex microspheres; 3: 400nm latex microspheres; Figure 9 C-line staining results for different SPA protein concentrations: 1: 0.125 mg / mL; 2: 0.25 mg / mL; 3: 0.5 mg / mL; Figure 10 T-line colorimetric results for different concentrations of p72 polyclonal antibody: 1: 0.5 mg / mL; 2: 0.75 mg / mL; 3: 1.0 mg / mL; 4: 1.25 mg / mL; Figure 11 Schematic diagram of latex microsphere chromatography test strip assembly; Figure 12 Specificity testing of test strips; Figure 13 Sensitivity detection of test strips, among which, 1: 512 ng / mL; 2: 256 ng / mL; 3: 128 ng / mL; 4: 64 ng / mL; 5: 32 ng / mL; 6: 16 ng / mL; 7: 8 ng / mL; 8: 4 ng / mL; 9: 2 ng / mL; 10: 1 ng / mL; 11: 0.5 ng / mL; 12: 0. 25 ng / mL; 0.125 ng / mL. Detailed Implementation

[0012] In the following examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in "A Concise Guide to Molecular Biology Experiments" (edited by FM Osber, RE Kingston, JG Seidman, et al., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).

[0013] ASFV is prevalent in my country. To provide a rapid on-site diagnostic method for ASFV, this invention prepares a truncated p72 protein using a prokaryotic expression system, and uses the p72 protein to immunize mice to prepare a monoclonal antibody. Through ELISA screening and subclonal purification, a hybridoma cell line secreting the p72 monoclonal antibody was obtained. The monoclonal antibody secreted by this hybridoma cell line can recognize the p72 protein in Western blotting, and can also bind to the p72 protein expressed by baculovirus and the p72 protein expressed by ASFV infection. A chromatographic test strip constructed using red latex microspheres labeled with this monoclonal antibody as a probe can specifically detect ASFV.

[0014] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0015] The present invention will be specifically illustrated below through examples.

[0016] The experimental materials used in the following embodiments of the present invention are as follows: BL21 competent cells were purchased from Takara; the p72 gene sequence was synthesized by General Biosystems (Anhui) Co., Ltd., and cloned into the pCold I vector; the His-tagged protein purification kit was purchased from Bio-Works.

[0017] Other reagents: Plasmid extraction kit, purchased from OMEGA; IPTG, BSA and TMB, purchased from Beyotime Biotechnology Co., Ltd.; skim milk, purchased from BD Biosciences, Inc.; Coomassie Brilliant Blue R-250, purchased from Biosharp; latex microspheres, Suzhou Weidu Biotechnology Co., Ltd.; Staphylococcus A protein (SPA), Beijing Wokai Biotechnology Co., Ltd.

[0018] Implementation Case 1. Expression and purification of truncated p72 protein 1.1 Construction of recombinant prokaryotic expression plasmid for p72 protein Hydrophilicity and antigenicity analysis of p72 were performed using Protean software in DNA STAR and TMHMM. Based on the structure of the p72 protein, amino acid positions 421-647 of the p72 protein were extracted for expression. Using the optimized p72 gene sequence synthesized in our laboratory as a template, primers P72O3F (5-ctccatatgCCCGAGATCCACAACCTGTTC-3) and P72O3R (5-gctctagattaGGTGCTGTACCTCAGCACGG-3) were designed and synthesized. A 681 bp p72 gene fragment was amplified by PCR. Figure 1 The p72 fragment and pColdI were digested with NdeI / XbaI, the digested p72 fragment and pColdI were recovered by gel ligation, and ligated with T4 DNA ligase. The mixture was then transformed into TOP10 competent cells, and the positive recombinant plasmid pCold-p72 was obtained by NdeI / XbaI double digestion screening and sequencing confirmation.

[0019] 1.2 Preparation of p72 protein The recombinant plasmid pCold-p72 was transformed into BL21 competent cells. Single colonies were picked from the transformation plates and cultured in small batches until the OD600 reached 0.6. IPTG (final concentration 1 mM) was added, and the cells were incubated at 16°C for 16-20 hours to induce protein expression. The uninduced group was cultured under the same conditions without IPTG. The cells were collected by centrifugation at 6000 rpm for 5 min and resuspended in 1 mL PBS. The centrifuge tubes containing the bacterial culture were placed on ice and sonicated. The sonicated bacterial culture was centrifuged at 6000 rpm for 5 min at 4°C, and the supernatant and precipitate were collected separately. The precipitate was resuspended in 1 mL PBS. 40 μl of the supernatant and precipitate were each transferred to a 1.5 mL EP tube, 10 μl of 5× Loading Buffer was added, and the samples were boiled in water for 10 min. After cooling, the samples were centrifuged at 10000 rpm for 2 min. 10 µL of the sample was then subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining. SDS-PAGE results show ( Figure 2 After induction, the transformed bacteria expressed a truncated p72 protein of approximately 26 kDa, which was mainly present in the precipitate and expressed as inclusion bodies.

[0020] Transformed bacteria were cultured in large quantities and p72 protein expression was induced. After sonication to disrupt the bacterial cells, inclusion body precipitates were obtained by centrifugation. Inclusion body precipitates were collected from every 200 mL of bacterial culture and resuspended in 20 mL of inclusion body denaturation buffer (8 M urea, 100 mM Tris-HCl, 10 mM β-mercaptoethanol, 2 mM EDTA, 2 mM sodium deoxycholate), centrifuged at 1500 g for 30 min at 4 °C, and the supernatant was retained. The denatured p72 truncated protein was purified according to the His-tagged protein purification kit instructions. The purified protein was then placed in a dialysis bag and sequentially dialyzed through refolding buffers containing 6 M, 4 M, and 2 M urea, followed by PBS for refolding. The refolded protein solution was concentrated by centrifugation using a 10 kDa ultrafiltration tube and then dissolved in SDS. PAGE analysis of concentrated protein samples. SDS PAGE electrophoresis results ( Figure 3 The results showed that the purified p72 truncated protein appeared as a single band in the range of 25-30 kDa. The protein concentration was determined to be 1.8 mg / mL using a BCA protein assay kit. The p72 truncated protein was aliquoted and stored at -80°C for later use.

[0021] Implementation Case 2. Preparation and Identification of p72 Protein Monoclonal Antibody Hybridoma Cell Line 2.1 Mouse Immunization Five 6-8 week old Balb / c mice were initially immunized subcutaneously with p72 truncated protein emulsified with Freund's complete adjuvant. Twenty-one days after the initial immunization, they were subcutaneously immunized again with p72 truncated protein emulsified with Freund's incomplete adjuvant. Ten days after the second immunization, blood was collected via the tail vein to prepare serum, and antibody levels were detected using an indirect ELISA for p72 antibodies developed in our laboratory. ELISA results showed that the antibody levels in the serum of the five immunized mice reached a dilution of more than 1:64000. Twenty-one days after the second immunization, two mice with high p72 antibody levels were selected and boosted with purified p72 truncated protein via tail vein injection.

[0022] 2.2 Hybridoma cell fusion and screening Three days after the third booster immunization, mouse spleens were harvested to prepare mouse spleen cells. Following standard procedures, spleen cells were fused with myeloma cells SP2 / 0. The fused cells were seeded into 96-well plates containing feeder cells. Fifteen days post-fusion, the supernatant from wells containing hybridoma cells was collected for ELISA detection. Hybridoma cells from positive wells were purified using limiting dilution. After three rounds of monoclonal purification, the positive rate of the same hybridoma cell line in the 96-well plates reached 100%. The hybridoma cells were then expanded and preserved, resulting in 27 preserved hybridoma cell lines.

[0023] 2.3 Analysis of the biological characteristics of hybridoma antibodies The prepared p72 truncated protein was subjected to SDS-PAGE, then transferred to a membrane, and Western blotting analysis was performed using the 27 monoclonal antibodies from Example 2.2 as primary antibodies. Figure 4 As shown, all 27 monoclonal antibodies recognized the p72 truncated protein after denaturation electrophoresis.

[0024] BmN cells were infected with recombinant silkworm baculovirus rBm-p72 expressing ASFV p72 and B602L, constructed in our laboratory. After 48 hours, the cells were fixed, and indirect immunofluorescence (IFA) experiments were performed using monoclonal antibodies from the hybridoma cell culture supernatant as primary antibodies. The results are as follows: Figure 5 As shown, the monoclonal antibodies of six hybridoma cell lines (2E5, 5F3, H3E4, R1G4, R3E9, R3F7) can recognize the p72 protein expressed by baculovirus.

[0025] To further screen for monoclonal antibodies that can recognize p72 in ASFV-infected cells, porcine primary mononuclear cells were infected with ASFV in a P3 laboratory. After fixing the infected cells, indirect immunofluorescence analysis was performed using the supernatant from six cell hybridoma strains (2E5, 5F3, H3E4, R1G4, R3E9, and R3F7) as primary antibodies. Figure 6As shown, ASFV-infected cells exhibited specific fluorescence after incubation with R3E9 monoclonal antibody. This indicates that R3E9 binds to the p72 protein in ASFV-infected cells.

[0026] 2.4 Identification of R3E9 strain monoclonal antibody subtypes The isotype of the R3E9 strain monoclonal antibody was analyzed using a mouse monoclonal antibody isotype identification kit from ProteinTech. The culture supernatant of the R3E9 strain was identified according to the kit instructions. The values ​​measured by the kit (Table 1) show that the heavy chain of this monoclonal antibody is the IgG1 isotype, and the light chain is the Kappa isotype.

[0027] Table 1. Identification of monoclonal antibody subtypes in R3E9 strain

[0028] 2.5R3E9 strain monoclonal antibody variable region gene sequence R3E9 hybridoma cells cultured in 6-well plates were lysed after the supernatant was removed, with 1 mL of Trizol added to each well. The lysed samples were collected in 1.5 mL centrifuge tubes and temporarily stored at -80°C. The samples were then submitted to a biotechnology company for monoclonal antibody variable region sequencing of mouse hybridoma cells. The sequencing results are as follows: The nucleotide sequence of the heavy chain variable region (VH) is as follows: 5- CAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCGGGATACACATTCACTGACTATGTTATCACCTGGGTGAAGCAGAGAAATGGACAGGCCTTGAATGGATTGGAGAGATTTATCCTGGAATTGGTA GTACTTATTACAATGACAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGTAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAACAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3 (SEQ ID NO.1) in, Backbone region 1 (FR1): 5-CAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCG-3 (SEQ ID NO.2) Complementarity Determining Region 1 (CDR1): 5- GGATACACATTCACTGACTATGTT-3 (SEQ ID NO. 3) FR2: 5- ATCACCTGGGTGAAGCAGAGAAATGGACAGGGCCTTGAATGGATTGGAGAG-3 (SEQ ID NO. 4) CDR2: 5- ATTTATCCTGGAATTGGTAGTACT-3 (SEQ ID NO. 5) FR3: 5- TATTACAATGACAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGTAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGT-3 (SEQ ID NO. 6) CDR3: 5- GCAACAGACTAC-3 (SEQ ID NO. 7) FR4: 5- TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3 (SEQ ID NO. 8) The corresponding amino acid sequence is as follows: VH: QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVITWVKQRNGQGLEWIGEIYPGIGSTYYNDKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFCATDYWGQGTTLTVSS (SEQ ID NO. 9) FR1: QVQLQQSGPELVKPGASVKMSCKAS (SEQ ID NO. 10) CDR1: GYTFTDYV (SEQ ID NO. 11) FR2: ITWVKQRNGQGLEWIGE (SEQ ID NO. 12) CDR2: IYPGIGST (SEQ ID NO. 13) FR3: YYNDKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFC (SEQ ID NO. 14) CDR3: ATDY (SEQ ID NO. 15) FR4: WGQGTTLTVSS (SEQ ID NO. 16) The nucleotide sequence of light chain variable region (VL) is as follows: 5-GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGCACATAGTAATGGAAACACCTATTTACAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTATCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3 (SEQ ID NO. 17) wherein, FR1: 5-GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT-3 (SEQ ID NO. 18) CDR1: 5- CAGAGCATTGCACATAGTAATGGAAACACCTAT-3 (SEQ ID NO. 19) FR2: 5- TTACAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC-3 (SEQ ID NO. 20) CDR2: 5- AAAGTTTCC-3 (SEQ ID NO. 21) FR3: 5-AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC-3 (SEQ ID NO. 22) CDR3: 5-TATCAAGGTTCACATGTTCCTCCGACG-3 (SEQ ID NO. 23) FR4: 5- TTCGGTGGAGGCACCAAGCTGGAAATCAAA-3 (SEQ ID NO. 24) The corresponding amino acid sequence is as follows: VL: DVLMTQTPLSLPVSLGDQASISCRSSQSIAHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCYQGSHVPPTFGGGTKLEIK (SEQ ID NO.25) FR1: DVLMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO.26) CDR1: QSIAHSNGNTY (SEQ ID NO.27) FR2: LQWYLQKPGQSPKLLIY (SEQ ID NO.28) CDR2: KVS (SEQ ID NO.29) FR3: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO.30) CDR3: YQGSHVPPT(SEQ ID NO.31) FR4: FGGGTKLEIK(SEQ ID NO.32) Implementation Example 3: Application of Monoclonal Antibody R3E9 in Immunochromatographic Test Strips 3.1 Preparation and purification of R3E9 ascites fluid Five 8-week-old female BALB / c mice were intracavitarily injected with 0.5 mL of phenazine. One week later, 0.5 mL (1 × 10⁻⁶) of R3E9 hybridoma cells were injected into the abdomen of each mouse. 6 (Number of cells). Approximately 7 days after hybridoma cell inoculation, mice showed significant abdominal distension, disheveled fur, and lethargy. Mice were euthanized, soaked in 70% ethanol for 10 minutes, and the abdominal skin was cut open. Ascites fluid was aspirated using a pipette tip, centrifuged at 2000 rpm for 10 minutes, and the supernatant was aliquoted into 1.5 mL EP tubes and stored at -20°C. The collected ascites fluid was serially diluted with 5% skim milk starting at a ratio of 1:100, and the antibody titer was determined to be 1:51200 using an indirect ELISA method.

[0029] Antibodies from R3E9 ascites fluid were purified using a Protein G antibody purification kit. A small amount of the purified antibody was then subjected to SDS-PAGE electrophoresis for identification. The results are as follows: Figure 7 As shown, the purified antibody exhibits specific bands at 55 kDa and 24 kDa, consistent with the characteristics of antibody heavy and light chains. The concentration of the purified antibody was determined using the BCA method, and the R3E9 antibody concentration was 0.83 mg / mL.

[0030] 3.2 Latex microsphere labeling of R3E9 monoclonal antibody p72 monoclonal antibody was labeled with red latex microspheres. The labeling steps were as follows: (1) Add 1 mL of coupling buffer (50 mM MES, pH=6.0) to a 2.0 mL EP tube, and then add 0.125 mL of microsphere (4% solid content) suspension. (1) Sonicate to mix, centrifuge at 20000g for 10 min at 15℃, and discard the supernatant; (2) Add 1 mL of coupling buffer, sonicate to mix, add 3.5 μL of LEDC solution (10 mg / mL EDC) to the EP tube, vortex to mix, then add 33 μL of NHS solution (10 mg / mL NHS), sonicate to mix; (3) Place the EP tube on a spin mixer, activate at 37℃, 40 r / min for 20 min, centrifuge at 15℃, 20000g for 10 min, and remove the supernatant; (4) After activating the latex microspheres, wash them once with coupling buffer, resuspend them with coupling buffer, add R3E9 monoclonal antibody (50 μg), mix, place on a spin mixer, and couple at 37℃, 40 r / min for 2 h; (5) Add 0.5 mL of microsphere blocking solution to the EP tube, vortex to mix, place the EP tube on a spin mixer, and couple at 37℃, 40 r / min for 1 h. h; (6) After sealing, the latex microspheres are washed twice with microsphere washing solution, and finally 0.5 mL of microsphere preservation solution (final concentration of microspheres 1 mg / mL) is added to the EP tube, mixed well, and stored at 4℃ for later use.

[0031] 3.3 Optimization and Assembly of Chromatographic Test Strips Glass fibers were cut into strips 2.2 cm wide, then soaked in sample pad treatment solution and dried in an oven at 37°C for 5 hours to obtain the sample pad for the antigen test strip. Untreated glass fibers were cut into strips 4 mm wide, thoroughly soaked in the latex microsphere labeling solution prepared in Example 3.2, and then allowed to dry naturally at room temperature to obtain the binding pad for the antigen test strip.

[0032] Latex microspheres with particle sizes of 200 nm, 300 nm, and 400 nm were selected to label p72 monoclonal antibodies. After separately spraying the detection line (p72 protein polyclonal antibody) and control line (staphylococcal A protein) onto an NC membrane, test strips were initially assembled. Baculovirus-expressed p72 protein was then added to the test strips. The results are shown in the figure. Line C (… Figure 8 a) and T-line ( Figure 8 (b) Bands appeared in all samples, but the latex marker with a diameter of 300 nm showed the best color development and clearest bands. Therefore, the optimal diameter for latex microspheres is 300 nm.

[0033] Staphylococcal protein A (SPA) was used as the control line (C line) for the test strips. SPA protein was diluted to concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively. Each concentration of SPA protein was sprayed onto an NC membrane and allowed to air dry at room temperature for preliminary assembly of the test strips. Diluted p72 antigen was then added to the sample pad, as shown in the image. Figure 9 As shown, the C-line for different concentrations of SPA all exhibited clear bands, but the band at 0.5 mg / mL was the darkest. Therefore, a SPA concentration of 0.5 mg / mL was selected for the C-line spray film.

[0034] Using laboratory-prepared p72 protein polyclonal antibody as the detection line (T line), the polyclonal antibody was diluted to 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, and 1.25 mg / mL, respectively. Each concentration of polyclonal antibody was sprayed onto an NC membrane and allowed to air dry at room temperature to preliminarily assemble the test strips. p72 antigen prepared using baculovirus expression was then added to the sample pad. The results are as follows: Figure 10 As shown, bands appeared in the T line for different concentrations of p72 polyclonal antibody, but the test strip showed the best color development when the polyclonal antibody concentration was 1.25 mg / mL. Therefore, the spray film concentration of p72 polyclonal antibody was determined to be 1.25 mg / mL.

[0035] The NC membrane with the sprayed detection and control lines is pasted onto the PVC base plate. The absorbent pad is cut into strips with a width of 1.7 cm. Then, the bonding pad and the absorbent pad are pasted onto both sides of the NC membrane in sequence. The sample pad is then pasted on top of the bonding pad, with each pad overlapping the others by 2 mm. Figure 11 Cut the assembled test strips into 4 mm wide strips using a cutter, pack them into a plastic casing, and seal them in a vacuum-sealed bag for later use.

[0036] 3.4 Criteria for Interpreting ASFV Antigen Test Strip Results Add the inactivated ASFV virus to the sample application port and let it stand at room temperature for 5-10 minutes, observing the color development of the test strip. A positive result is indicated when both the T and C lines on the test strip are red; a negative result is indicated when the T line is not colored but the C line is red; and an invalid result is indicated when neither the T nor C lines are colored.

[0037] 3.5 Specificity and Sensitivity of ASFV Antigen Test Strips ASFV inactivated virus, as well as cell culture supernatants of PRV, PRRSV, PEDV, PCV, and PDCoV, were diluted with sample diluent and added dropwise to the sample application port of the test strip. The strips were incubated at room temperature for 5-10 minutes. Results were as follows: Figure 12As shown, only the test strips containing ASFV inactivated virus showed positive results, while the test strips containing cell culture supernatant containing PRV, PRRSV, PCV, and PEDV showed negative results, indicating that the antigen detection test strips have good specificity.

[0038] The p72 protein expressed by baculovirus was diluted to concentrations of 512 ng / mL, 256 ng / mL, 128 ng / mL, 64 ng / mL, 32 ng / mL, 16 ng / mL, 8 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL, respectively, and added to the sample outlet of the antigen test strip. The strips were incubated at room temperature for 5-10 minutes. The results are as follows: Figure 13 As shown, when the concentration of p72 antigen in the sample is ≥1 ng / mL, the T line of the test strip develops color; when the concentration of p72 antigen in the sample is 0.5 ng / mL, the T line of the test strip does not develop color, indicating that the detection limit of the antigen test strip is 1 ng / mL.

[0039] Through the above embodiments, this invention prepared a hybridoma cell line R3E9 containing a monoclonal antibody against ASFV p72 protein and determined the variable region sequence of this monoclonal antibody. This monoclonal antibody can be used for in vitro anabolism (IFA) to recognize p72 protein expressed by baculovirus and p72 protein in ASFV-infected cells; it can also be used for western blotting to recognize denatured p72 protein. In particular, labeling this monoclonal antibody with latex microspheres can be used to prepare latex chromatography test strips for detecting ASFV antigen.

Claims

1. A monoclonal antibody against the p72 protein of African swine fever virus, characterized in that, The antibody or its functional fragment has a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the amino acid sequences of the complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are as follows: HCDR1 is shown in SEQ ID NO.11, HCDR2 is shown in SEQ ID NO.13, and HCDR3 is shown in SEQ ID NO.15; LCDR1 is shown in SEQ ID NO.27, LCDR2 is shown in SEQ ID NO.29, and LCDR3 is shown in SEQ ID NO.31, wherein the sequence of SEQ ID NO.29 is KVS.

2. The antibody or its functional fragment according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region are as follows: the heavy chain variable region is shown in SEQ ID NO.9, and the light chain variable region is shown in SEQ ID NO.

25.

3. The antibody or its functional fragment according to claim 2, characterized in that, The light chain constant region of the antibody is a κ-type light chain constant region.

4. The antibody or its functional fragment according to claim 2, characterized in that, The heavy chain constant region of the antibody is the heavy chain constant region of the IgG antibody.

5. The antibody or its functional fragment according to any one of claims 1-4, characterized in that, The functional fragments are Fab, Fab', F(ab')2, Fv, or ScFv fragments.

6. An isolated nucleic acid molecule, characterized in that, It encodes an antibody or a functional fragment thereof as described in any one of claims 1-5.

7. A recombinant vector containing the nucleic acid molecule of claim 6.

8. Recombinant cells containing the recombinant vector of claim 7.

9. The use of the antibody or its functional fragment as described in any one of claims 1-5, the nucleic acid molecule as described in claim 6, the recombinant vector as described in claim 7, or the recombinant cell as described in claim 8 in the preparation of a competitive ELISA detection kit for detecting African swine fever virus.

10. An ASFV antigen latex chromatography test strip, characterized in that... The latex chromatography test strip contains a monoclonal antibody against the African swine fever virus p72 protein as described in claim 1.

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