Recombinant antigenic protein p22, monoclonal antibody and application of african swine fever virus

Abstract

The application provides a recombinant antigen protein p22 of an African swine fever virus, a monoclonal antibody and application. The application provides a recombinant antigen protein of an African swine fever virus. The application provides an antibody or antigen binding fragment thereof of the recombinant antigen protein of the African swine fever virus. The application also provides a hybridoma cell. The application also provides a rapid detection test paper card of the African swine fever antibody. The recombinant antigen protein of the African swine fever virus provided by the application has higher affinity to the African swine fever antibody, higher specificity and better stability, and can be used for rapid and accurate detection of the African swine fever virus.

Description

[0001] (This application is a divisional application of the invention entitled “Recombinant antigen protein, monoclonal antibody and application of African swine fever virus”, with application number 202211168511.5 and application date of September 19, 2022. September 23, 2021 is reserved as the priority date.) Technical Field

[0002] This invention belongs to the field of animal disease detection technology, specifically relating to recombinant antigen proteins of African swine fever virus, as well as corresponding monoclonal antibodies and hybridoma cells. This invention also relates to the use of the recombinant antigen protein for detecting African swine fever virus. Background Technology

[0003] African swine fever (ASF) is a highly contagious and deadly disease caused by the African swine fever virus (ASFV), primarily infecting domestic pigs and various wild boars. It mainly infects pigs through contact with the ASF virus and is transmitted through contaminated materials (food waste, feed, water, pens, bedding, clothing, utensils, vehicles, etc.), with the digestive and respiratory tracts being the primary routes of infection. It can also be transmitted through the bites of vector insects such as soft ticks. The World Organisation for Animal Health (OIE) lists it as a notifiable animal disease, and it is also a Class A animal epidemic that my country is focusing on preventing and controlling. Its characteristics include a short onset time, with a mortality rate as high as 100% in the most acute and acute infections. Clinical manifestations include fever (reaching 40-42℃), rapid heartbeat, difficulty breathing, cough in some cases, serous or mucopurulent discharge from the eyes and nose, cyanosis of the skin, and significant hemorrhage in the lymph nodes, kidneys, and gastrointestinal mucosa, leading to immune escape or immunosuppression. The clinical symptoms of African swine fever are similar to those of classical swine fever, and diagnosis can only be made through laboratory monitoring.

[0004] First discovered in Kenya in 1921, ASFV has a high mortality rate and causes huge economic losses to the pig farming industry. Currently, ASF prevention and control has become a routine requirement in pig farming. "Preventing external importation, preventing internal spread, real-time monitoring, and culling infected pigs" has become the consensus for ASF prevention and control. Real-time monitoring and rapid, accurate detection are particularly important in pig farms. Therefore, rapid and reliable detection of ASFV is crucial. Rapid ASFV detection is not only necessary for ASF prevention and control but also essential for the differential diagnosis of pig diseases with similar clinical symptoms.

[0005] P30 is the main inducible protein of the ASFV viral particle, encoded by the CP204L gene, with a molecular weight of approximately 30 kDa, also known as the P32 protein. The P30 protein is a highly antigenic membrane protein that is expressed and secreted early in viral infection. It participates in the binding of the virus to cell surface receptors and inhibits the translation of host cell mRNA. Due to its early and high intracellular expression, using it as an antigen to detect ASFV antibodies results in detection approximately one week earlier than other antigens (such as P72).

[0006] P22 is a binding protein located on the inner envelope surrounding the viral nucleocapsid. The formation of the inner envelope is related to the endoplasmic reticulum. Currently, there is limited research on p22.

[0007] P72, sometimes referred to as p73, is a major structural protein of the viral particle and an important component of the icosahedral capsid, accounting for approximately 32% of the total viral particle protein. Encoded by the B646L gene, it has a molecular weight of approximately 73 kDa. Produced in the late stages of viral infection, this protein has a highly conserved sequence and excellent antigenicity. After viral infection, the body produces high titers of anti-p72 antibodies, which are often used for the serological diagnosis of ASF.

[0008] Currently, African swine fever virus (ASFV) detection methods are mainly divided into two categories: the first category is pathogen detection, including virus isolation detection, viral antigen detection, and genomic DNA detection, mainly using methods such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA); the second category is antibody detection. For subacute and chronic ASFV, the virulence decreases as the virus circulates and spreads. Pigs infected with ASFV can develop antibodies 7-10 days after infection, and these antibodies can last for a long time, making them suitable for large-scale antibody screening. Antibody detection can be used as a diagnostic basis for ASFV infection, mainly using methods such as indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blot assay.

[0009] While existing detection technologies offer high specificity and sufficient sensitivity, they require specialized laboratories and equipment. Furthermore, they demand highly skilled operators, involve lengthy testing times, and are costly. ELISA testing is prone to false positives due to cross-contamination, and PCR testing is susceptible to false negatives due to sample representativeness, inhibitors, and nucleic acid degradation. Additionally, the virulence of resistant pigs decreases as the virus circulates and spreads, rendering PCR ineffective in detecting it, thus posing a risk to pig farms. Moreover, the high cost of testing equipment makes widespread adoption difficult.

[0010] Therefore, none of the above methods are suitable for rapid detection or diagnosis at the grassroots level or on-site. Developing a simple and rapid real-time online detection technology that can detect African swine fever virus antibodies is an urgent technical problem to be solved.

[0011] Colloidal gold immunochromatography is a diagnostic technique that has developed rapidly in recent years. Its advantages include: (1) simple operation, diagnostic results can be obtained in minutes, convenient and fast, and no professional personnel are required; (2) less affected by external factors, one card per pig, avoiding cross-contamination, and high detection accuracy; (3) high cost-effectiveness, no need for other expensive instruments and professional laboratories, and low detection cost; (4) wide range of applications, easy to carry and store, suitable for farms of all sizes, and especially suitable for field and on-site diagnosis. At present, in the detection of African swine fever virus antibody colloidal gold test strips, there are colloidal gold immunochromatographic test strips based on one or two recombinant p30, p54 or p72 proteins. However, when detecting African swine fever virus antibody-negative or weakly positive pig serum or whole blood, the accuracy and sensitivity need to be improved, thus restricting the promotion and application of this method in the market.

[0012] Therefore, there is a current need for detection methods that can rapidly and accurately detect African swine fever virus. Summary of the Invention

[0013] Addressing the shortcomings of existing technologies, the main objective of this invention is to provide a recombinant antigen protein of African swine fever virus, along with corresponding monoclonal antibodies and hybridoma cells. This invention also relates to the use of the recombinant antigen protein, monoclonal antibodies, and hybridoma cells for detecting African swine fever virus. Furthermore, this invention provides a rapid test strip for African swine fever virus based on colloidal gold immunochromatography. The strip includes the following raw materials: a recombinant antigen protein of African swine fever virus and corresponding monoclonal antibodies.

[0014] The monoclonal antibody provided by this invention has a higher affinity for African swine fever virus, stronger specificity, and better stability, and can be used for rapid and accurate detection of African swine fever virus.

[0015] The objective of this invention is achieved through the following technical solution:

[0016] On one hand, the present invention provides a recombinant antigen protein p22 of African swine fever virus, said recombinant antigen protein comprising:

[0017] (i) The sequence shown in SEQ ID NO: 4;

[0018] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 4; or

[0019] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 4.

[0020] The present invention also provides an isolated nucleic acid molecule encoding the recombinant antigen protein p22 of the African swine fever virus; preferably, the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 3.

[0021] On the other hand, the present invention provides an antibody or antigen-binding fragment thereof of the recombinant antigen protein p22 of African swine fever virus, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein...

[0022] The heavy chain variable region includes the three CDRs contained in the heavy chain variable region shown in SEQ ID NO: 17; and the light chain variable region includes the three CDRs contained in the light chain variable region shown in SEQ ID NO: 19.

[0023] Preferably, the three CDRs contained in the heavy chain variable region and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system.

[0024] The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0025] (a) The chain variable region (VH) contains an amino acid sequence selected from the following:

[0026] (i) The sequence shown in SEQ ID NO: 17;

[0027] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 17; or

[0028] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 17;

[0029] And / or,

[0030] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0031] (iv) The sequence shown in SEQ ID NO: 19;

[0032] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 19; or

[0033] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 19;

[0034] Preferably, the substitution described in (ii) or (v) is a conservative substitution;

[0035] Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO: 17 and VL having the sequence shown in SEQ ID NO: 19.

[0036] On the other hand, the present invention also provides a hybridoma cell, the hybridoma cell having the accession number CCTCC NO: C202153.

[0037] The present invention also provides a monoclonal antibody against the recombinant antigen protein p22 of African swine fever virus, wherein the monoclonal antibody is secreted by hybridoma cells with accession number CCTCC NO: C202153.

[0038] The present invention also provides an isolated nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof as described in the present invention, or a heavy chain variable region and / or a light chain variable region thereof;

[0039] Preferably, the nucleic acid molecule is as shown in any one of SEQ ID NO: 18 or 20.

[0040] The present invention also provides a carrier comprising the isolated nucleic acid molecules described herein.

[0041] In another aspect, the present invention provides a kit for detecting African swine fever virus, which contains the recombinant antigen protein described in the present invention; or the antibody or its antigen-binding fragment described in the present invention;

[0042] Preferably, the kit further includes a second antibody and / or a third antibody;

[0043] Preferably, the antibody further comprises a detectable marker, such as colloidal gold particles, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin.

[0044] The present invention also provides the use of the recombinant antigen protein, antibody or antigen-binding fragment thereof in the preparation of a kit for detecting African swine fever virus.

[0045] The recombinant antigen protein provided by this invention can be used for research on African swine fever, such as pathogenic mechanisms and immune responses to antibodies; it can also be used for the development of diagnostic applications, such as ELISA detection kits and test strips for African swine fever; and for the development of African swine fever vaccines; and it can be applied to the detection of African swine fever using mixed antigens, making the detection more accurate and sensitive.

[0046] In one specific implementation, a test strip containing the recombinant antigen protein or antibody of the present invention can specifically detect antibodies against p22 in blood samples from pigs infected with African swine fever, thereby serving as a diagnostic tool for African swine fever. The p22 monoclonal antibody test strip exhibits high sensitivity and, as a relatively little-studied ASFV target, has significant potential research value.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0048] By designing different primers, expression vectors, and endonucleases, recombinant proteins can be constructed and immunized animals (including the steps and consumables used in the experiment, which can be adjusted to a certain extent) to obtain hybridoma cells that secrete the same monoclonal antibody.

[0049] ASFV antibody rapid test strips were prepared using different test strip materials, such as different types of card shells, PVC base plates, absorbent pads, sample pads, NC membranes, and fiber membranes, and obtained through different methods using recombinant p2 prokaryotic expression antigens. Attached Figure Description

[0050] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application, and do not constitute an undue limitation of this application.

[0051] Figure 1 is a map of the recombinant prokaryotic expression vector according to the present invention; wherein Figure 1AThe spectrum of p30-PET21-PEP Figure 1B The spectrum of p22-PET21-PEP Figure 1C The spectrum of p72-PET21-PEP;

[0052] Figure 2 This is a diagram showing the expression of ASFV p30, p22, and p72 proteins in E. coli after IPTG induction using recombinant plasmids. From right to left, p30 and p22 are represented by bacterial culture without recombinant plasmid, bacterial culture with recombinant plasmid protein, and Mark, respectively. For p72, from right to left, it is represented by Mark, bacterial culture without recombinant plasmid, and bacterial culture with recombinant plasmid protein, respectively.

[0053] Figure 3 These are SDS-PAGE images of purified ASFV p30, p22, and p72 recombinant proteins;

[0054] Figure 4 These are the results of ASFV p30, p22, and p72 antibody affinity assays; among them Figure 4 A represents the result of p30(e1)-PET21-PEP8F9G8; Figure 4 B represents the result of p22(e1)-PET21-PEP 7B12A1; Figure 4 C represents the result of p72(e1)-PET21-PEP6D8F2;

[0055] Figure 5 This is a schematic diagram of the structure of the ASFV antibody rapid detection test strip according to the present invention;

[0056] Figure 6 This is a schematic diagram illustrating the principle by which test strips determine the negative or positive results of blood samples.

[0057] Figure 7 This is a diagram showing the results of the specific detection test of the African swine fever antibody test strip of the present invention; from left to right, they are ASFV positive serum (sample #2), negative serum 1, CSFV positive serum 2, PRRSV positive serum 3, PCV2 positive serum 4, PRV positive serum 5, and PEDV positive serum 6.

[0058] Figure 8 This is a graph showing the results of the sensitivity test of the African swine fever antibody test strip of this invention; from left to right, the results are 1:50, 1:100, 1:200, and 1:400.

[0059] Figure 9 This is a diagram showing the repeatability test results of the African swine fever antibody test strip of this invention;

[0060] Figure 10This is a graph showing the results of the stability test of the African swine fever antibody test strip card of this invention;

[0061] Figure 11 This is a comparison test result diagram of similar products of the African swine fever antibody test strip, in which the test strip of this invention is on the far left.

[0062] Biological Preservation Information Description

[0063] Hybridoma cell line XjZ91 was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202152.

[0064] Hybridoma cell line XjH74 was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202153.

[0065] Hybridoma cell line XjL60 was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202154. Detailed Implementation

[0066] The following description of this application is only for illustrating various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. Those skilled in the art can readily derive various equivalents, variations, and modifications without departing from the scope of this application, and it should be understood that such equivalent embodiments are included within the scope of this invention. All documents cited in this application, including published materials, patents, and patent applications, are incorporated herein by reference in their entirety.

[0067] Example 1: Preparation of three hybridoma cells secreting monoclonal antibodies against African swine fever virus p30, p22, and p72

[0068] 1. Construction of recombinant protein expression vectors for African swine fever virus p30, p22, and p72

[0069] 1.1 Based on the data entered in NCBI, the gene sequence of the recombinant ASFV antigen to be expressed was selected, and the target gene was synthesized according to the publicly available gene fragment (Sangon Biotech (Shanghai) Co., Ltd.) as the PCR template strand:

[0070] The full-length (1-204) sequence of the ASFV CP204L gene (SEQ ID NO: 1) was selected as the target gene sequence encoding the p30 recombinant protein (SEQ ID NO: 2); the 29-177 interval sequence of the ASFV EP402R gene (SEQ ID NO: 3) was selected as the target gene sequence encoding the p22 recombinant protein (SEQ ID NO: 4); and the 420-646 interval sequence of the ASFV B646L gene (SEQ ID NO: 5) was selected as the target gene sequence encoding the p72 recombinant protein (SEQ ID NO: 6). The sequence structures are as follows.

[0071] SEQ ID NO: 1

[0072] ;

[0073] SEQ ID NO: 2

[0074] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSAHIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSESIHEKNDNETNECTSSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKNKLLSHLHLMF;

[0075] SEQ ID NO:3

[0076] TATAAAAAACAACAACCACCAAAAAAGGTCTGTAAAGTAGATAAAGATTGTGGTAGTGGAGAGCATTGTGTTCGTGGAACATGCAGCACATTGAGCTGCTTAGACGCTGTAAAAATGGACAAACGAAATATTAAGATAGATTCTAAGATTTCCTCATGTGAATTCACTCCCAATTTTTACCGTTTTACGGATACTGCTGCCGATGAGCAGCAAGAATTTGGAAAAACACGGCATCCTATAAAAATAACTCCATCTCCAAGTGAATCCCATAGCCCCCAAGAGGTGTGTGAAAAATATTGTTCATGGGGAACCGATGACTGTACAGGTTGGGAATATGTTGGTGATGAAAAGGAGGGAACATGTTATGTATATAATAATCCACATCACCCGGTTCTTAAATATGGTAAGGATCACATCATAGCCTTACCTAGAAATCATAAACATGCA;

[0077] SEQ ID NO:4

[0078] YKKQQPPKKVCKVDKDCGSGEHCVRGTCSTLSCLDAVKMDKRNIKIDSKISSCEFTPNFYRFTDTAADEQQEFGKTRHPIKITPSPSESHSPQEVCEKYCSWGTDDCTGWEYVGDEKEGTCYVYNNPHHPVLKYGKDHIIALPRNHKHA;

[0079] SEQ ID NO:5

[0080] ACCCCTGAAATACACAACCTTTTTGTAAAACGCGTTCGATTTTCCCTGATACGTGTCCATAAAACGCAGGTGACCCACACCAACAATAACCACCACGATGAAAAACTAATGTCTGCTCTTAAATGGCCCATTGAATATATGTTTATAGGATTAAAACCTACCTGGAACATCTCCGATCAAAATCCTCATCAACACCGAGATTGGCACAAGTTCGGACATGTTGTTAACGCCATTATGCAGCCTACTCACCACGCAGAGATAAGCTTTCAGGATAGAGATACAGCTCTTCCAGACGCATGTTCATCTATATCGGATATTAGCCCCGTTACGTATCCGATCACATTACCTATTATTAAAAACATTTCCGTAACTGCTCATGGTATCAATCTTATCGATAAGTTTCCATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCAATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGGTCATATTAACGTATCCAGAGCAAGAGAATTTTATATTAGTTGGGACACGGATTACGTGGGGTCTATCACTACGGCTGATCTTGTGGTATCGGCATCTGCTATTAACTTTCTTCTTCTTCAGAACGGTTCAGCTGTGCTGCGTTACAGTACC;

[0081] SEQ ID NO:6

[0082] TPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST。

[0083] 1.2 Designing amplification primers using Primers

[0084] (1) Primers for amplifying the p30 target band:

[0085] Upstream primer: SEQ ID NO: 7

[0086] GCCGTCTCGGATCCATGGATTTTATTTTAAATATATC;

[0087] Downstream primer: SEQ ID NO: 8

[0088] GCCGTCTCAAGCTTAAACATTAAATGTAGGTGAG;

[0089] (2) Primers for amplifying the p22 target band:

[0090] Upstream primer: SEQ ID NO: 9

[0091] GCCGTCTCGGATCCTATAAAAAACAACAACCAC;

[0092] Downstream primer: SEQ ID NO: 10

[0093] GCCGTCTCAAGCTTTGCATGTTTATGATTTCTA;

[0094] (3) Primers for amplifying the p72 target band:

[0095] Upstream primer: SEQ ID NO: 11

[0096] GCCGTCTCGGATCCACCCCTGAAATACACAACCTTTT;

[0097] Downstream primer: SEQ ID NO: 12

[0098] GCCGTCTCAAGCTTGGTACTGTAACGCAGCACAGC.

[0099] 1.3 In vitro PCR amplification of p30, p22, and p72 gene bands

[0100] PCR reaction system: 5 μL DNA template, 5 μL amplification primer mixture (2 μmol / L), 5 μL 10×PCS buffer, 5 μL 10×dNTPs (2.0 mmol / L) (Takara), 1.5 U Taq DNA polymerase (Takara), 28 μL H2O. All components are combined to make a total reaction volume of 50 μL. The mixture is incubated at 42℃ for 30-60 minutes, cooled on ice, centrifuged for a few seconds, and then PCR is performed.

[0101] PCR reaction conditions: 94℃ pre-denaturation for 2 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 40 seconds, for a total of 30 cycles; 72℃ extension for 5 minutes. After cycling, remove the reaction tubes and store them at 4℃ for later use.

[0102] 1.4 p30, p22, p72 genes and expression vector PET21-PEP double digestion and ligation

[0103] Double enzyme digestion reaction system for the target gene (20 μL): 16 μL target gene, Buffer O + 2 μL, BamHI 1 μL, HindIII 1 μL (NEB).

[0104] Double enzyme digestion reaction system (20 μL) of plasmid PET21-PEP (Hunan Yuantai Biotechnology Co., Ltd.): 16 μL PET21-PEP, Buffer O + 2 μL, BamHI 1 μL, HindIII 1 μL (NEB).

[0105] Double enzyme digestion reaction conditions: digestion overnight at 37℃, then terminate the reaction by heating.

[0106] The ligation system for the target gene and expression vector consisted of: 2.5 μL of PET21-PEP DNA digestion reagent, 9.5 μL of the target gene fragment DNA digestion reagent, 1.5 μL of 10×T4 DNA ligase buffer, and 0.5 μL (3 U) of T4 DNA ligase (Takara).

[0107] Ligation reaction conditions: The Eppendorf tube was centrifuged for 2 seconds to concentrate the sample, and the ligation sample was placed in a constant temperature water bath at 16 ℃ overnight. The spectra of recombinant plasmids p30-PET21-PEP, p22-PET21-PEP, and p72-PET21-PEP are shown in Figure 1.

[0108] 2. Expression of recombinant p30, p22, and p72 proteins

[0109] Competent *E. coli* cells were prepared using CaCl2 solution. Recombinant expression vectors p30-PET21-PEP, p22-PET21-PEP, and p72-PET21-PEP were transformed into *E. coli* RST competent cells, respectively. Multiple colonies were picked from each cell and cultured in 50 mL centrifuge tubes in LB broth at 37 °C with shaking for 4 h. 5 mL of the culture was transferred to another centrifuge tube, and IPTG (AMRESCO) was added to a final concentration of 1 mM for induction expression. This was then combined with the remaining 15 mL of bacterial culture and cultured at 37 °C for another 4 h. After harvesting the bacteria, 1 mL of each of the induced and uninduced bacterial cultures were centrifuged to remove the culture medium. The precipitate was mixed with 1× Loading solution and subjected to SDS-PAGE gel electrophoresis to observe the expression results. The electrophoresis results are shown below. Figure 2 As shown.

[0110] Add 15 mL of uninduced bacterial culture to 1 L of LB liquid medium and shake for 4 h. After 4 h, induce expression with 1 mM IPTG. After 4 h, centrifuge at 3800 rpm for 10 min, discard the supernatant, and keep the precipitate.

[0111] 3. Purification of recombinant proteins p30, p22, and p72

[0112] The precipitated bacterial cells were ultrasonically disrupted using 50 mL of 8M urea (Beijing Chemical Industry Kaiyuan Company), then centrifuged at 18000 rpm for 30 min, and the supernatant was retained. The supernatant was then purified by affinity chromatography. After column chromatography, the affinity column was eluted with a gradient of 8M urea buffer containing 10 mM and 300 mM imidazole. Samples of the eluted liquids were analyzed by SDS-PAGE. The protein with the highest concentration and purity was dialyzed against PBS and concentrated with sucrose. The results are shown below. Figure 3 As shown, the electrophoretic purity of recombinant protein p30 was 95%; that of recombinant protein p22 was 80%; and that of recombinant protein p72 was 85%.

[0113] 4. Serological verification of p30, p22, and p72 recombinant proteins

[0114] Serological verification of recombinant p30, p22, and p72 proteins was performed using an indirect ELISA method. Three antigens were diluted to 1 μg / mL with coating buffer and added to each well of an ELISA plate at a rate of 100 μL / well. The plates were incubated overnight at 4°C. The liquid in the wells was discarded, and the plates were washed three times with washing buffer for 3 minutes each time, then patted dry. 300 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were patted dry. 100 μL of primary antibody (positive sera #1-5; negative sera #1-#5) at a specific dilution was added to each well, and the plates were incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were washed three times with washing buffer for 3 minutes each time, then patted dry. 50 μL of rabbit anti-pig IgG ELISA-labeled secondary antibody (SIGMA) at a 1:10000 dilution was added to each well, and the plates were incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were washed three times with washing buffer for 3 minutes each time, then patted dry. 100 μL of freshly prepared TMB (Beyotime Biotech) substrate chromogenic solution was added to each well, and the plates were incubated at 37°C for 15 minutes. 2 mol / L The reaction was terminated with H2SO4, and the OD value was read by an ELISA reader. The results are shown in Table 1.

[0115] Table 1. Results of ELISA serological validation assay for recombinant p30, p22, and p72 proteins.

[0116]

[0117] OD value analysis showed that all three recombinant expressed proteins could specifically bind to antibodies in positive serum of patients infected with African swine fever virus, but did not bind to other antibodies in negative serum of patients who had not been infected with African swine fever virus.

[0118] 5. Immunize mice by injecting recombinant p30, p22, and p72 proteins.

[0119] For the initial immunization, each BALB / c mouse (Hunan Slack Jingda Experimental Animal Co., Ltd.) was injected with 50 μg of recombinant protein and 1.5 mL of Freund's complete adjuvant (Sigma) via multiple subcutaneous injections. A second immunization was administered 3 weeks later, with the same dosage and route, but with Freund's incomplete adjuvant. A third immunization was administered 2 weeks later, with the same dosage but without adjuvant via intraperitoneal injection. Blood was collected 7 days later to measure the titer, and the immunization effect was detected using indirect ELISA. The results are shown in Table 2. A booster immunization was administered 3 days later, with a dose of 50 μg via intraperitoneal injection.

[0120] Reagents used in ELISA testing:

[0121] (1) p30 detection of coated antigen

[0122] Antigen 1 was a 2.0 μg / mL recombinant p30(e1)-PET21-PEP protein, and antigen 2 was a 2.0 μg / mL unrelated fusion protein with a 1861 tag.

[0123] (2) p22 detection of coated antigen

[0124] Antigen 1 was a 2.0 μg / mL recombinant p22(e1)-PET21-PEP protein, and antigen 2 was a 2.0 μg / mL unrelated fusion protein with a 1861 tag.

[0125] (3) p72 detection of coated antigen

[0126] Antigen 1 was a 2.0 μg / mL recombinant p72(e1)-PET21-PEP protein, and antigen 2 was a 2.0 μg / mL unrelated fusion protein with a 1861 tag.

[0127] (4) The primary antibody was obtained from mouse serum 10 days after the third immunization.

[0128] (5) The secondary antibody was HRP-labeled anti-mouse IgG (SIGMA): SIGMA, catalog number: A0168, production batch number: #068M4764V.

[0129] Table 2. Immunological effects detected by indirect ELISA

[0130]

[0131] Based on the OD value results, we can conclude that: the titer of mouse No. 3, immunized with recombinant p30(e1)-PET21-PEP protein, met the requirements, and the reaction with the tag protein was relatively weak, so mouse No. 3 was selected for fusion; the titer of mouse No. 4, immunized with recombinant p22(e1)-PET21-PEP protein, met the requirements, and the reaction with the tag protein was relatively weak, so mouse No. 4 was selected for fusion; the titers of all mice immunized with recombinant p72(e1)-PET21-PEP protein met the requirements, but the reaction results of the two antigens were almost the same, so the mice with higher OD values ​​were selected for fusion in sequence, starting with mouse No. 4.

[0132] 6. Cell fusion

[0133] (1) Preparation of feeder cells

[0134] BALB / c mice aged 6-10 weeks were euthanized by cervical dislocation, immersed in 75% alcohol for 3 min for disinfection, and the skin was cut open with sterile scissors to expose the peritoneum. 6 mL of culture medium was injected through a pipette, and the cells were repeatedly rinsed. The rinse solution was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium containing 20% ​​fetal bovine serum (GIBCO) to adjust the cell count to 1*10⁻⁶ cells / mL. 5 / mL. Take 100μL into a 96-well plate and incubate at 37℃.

[0135] (2) Preparation of myeloma cells

[0136] Expand the myeloma cell culture 36-48 hours before fusion. On the day of fusion, gently blow the cells off the flask wall using a bent-tip pipette and collect them in a 50 mL centrifuge tube or fusion tube. Centrifuge at 1000 rpm for 5-10 minutes, discard the supernatant, add 30 mL of incomplete culture medium, and wash once by centrifugation. Then resuspend the cells in 10 mL of incomplete culture medium and mix well. Take the myeloma cell suspension, add 0.4% tyrosine blue staining solution for viable cell counting, and use it for later use.

[0137] (3) Preparation of spleen cells

[0138] Immunized BALB / c mice were euthanized by cervical dislocation. The mice were then immersed in 75% alcohol for 5 minutes, fixed on a culture dish, and the skin on the left side of the abdomen was lifted to expose the spleen. Using ophthalmic forceps, the peritoneum was aseptically cut open in a laminar flow hood, and the spleen was removed. It was gently washed in a petri dish containing 10 mL of incomplete culture medium, and the surrounding connective tissue was carefully removed. The spleen cells were placed on a stainless steel sieve in the petri dish and ground into a cell suspension using a syringe needle core before counting. The spleen cells were then introduced into the incomplete culture medium in the petri dish. The cells were pipetted several times to prepare a single-cell suspension. Typically, 1 × 10⁶ cells were used per mouse. 8 -2.5×10 8 One spleen cell.

[0139] (4) Cell fusion

[0140] 1×10 8 Spleen cells and 1×10 7 Myeloma cells SP2 / 0 were mixed in a 50 mL fusion tube, and incomplete culture medium was added to a final volume of 30 mL. After thorough mixing, the tube was centrifuged at 1000 rpm for 5-10 minutes, and the supernatant was aspirated as completely as possible. The bottom of the fusion tube was gently tapped on the palm of the hand to loosen and evenly distribute the cell pellet. 1 mL of preheated 50% PEG (Sigma) was added over 30 seconds while gently stirring, and the mixture was aspirated and allowed to stand for 1 minute. Preheated incomplete culture medium was then added to stop the PEG reaction. This process was repeated every 2 minutes, adding 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 10 mL respectively. Finally, the tube was centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. 5 mL of complete culture medium was added, and the cell pellet was gently aspirated to suspend and mix the cells. The final volume of complete culture medium was then added to a final volume of 40-50 mL. 100 μL of the mixture was aliquoted into each well of a 96-well cell culture plate and incubated at 37°C in a 5% CO2 incubator. After 6 hours, 50 μL of selection medium was added to each well. Three days later, replace half of the medium with selective culture medium. When the medium has grown to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0141] 7. Selection of hybridoma cells

[0142] The effect of indirect ELISA on the secretion of antibody proteins by hybridoma cells was detected. Except for the primary antibody, which was obtained from the culture supernatant of hybridoma cells, all other reagents used were the same as those used in the ELISA serum detection after animal immunization. Dilute the antigen to 10 μg / mL with coating buffer and add 100 μL / well to each well of the ELISA plate. Incubate overnight at 4°C or for 2 hours at 37°C. Discard the liquid in the wells and wash three times with washing buffer for 3 minutes each time, then pat dry. Add 300 μL of blocking buffer to each well and block at 37°C for 1 hour. Discard the liquid in the wells and pat dry. Add 100 μL of hybridoma cell culture supernatant to each well, and set up positive, negative, and blank controls. Incubate at 37°C for 1 hour, then discard the liquid in the wells and wash three times with washing buffer for 3 minutes each time, then pat dry. Add 100 μL of enzyme-labeled secondary antibody to each well and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash three times with washing buffer for 3 minutes each time, then pat dry. Add 100 μL of freshly prepared TMB (Beyotime Biotech) substrate chromogenic solution to each well and incubate at 37°C for 15 minutes. Terminate the reaction with 2 mol / L H2SO4 and read the OD value using an ELISA reader.

[0143] Result interpretation: A P / N ratio ≥ 2.1 or P ≥ N + 3SD is considered positive. If the negative control wells are colorless or nearly colorless, and the positive control wells show clear color, the results can be observed directly with the naked eye. Hybridoma cells expressing the antibody protein positively and not cross-reacting with the tag protein are selected for cloning.

[0144] 8. Cloning of hybridoma cells (limiting dilution method)

[0145] Mouse spleen cells were prepared as feeder cells. Hybridoma cells were diluted with HT medium containing 20% ​​serum to prepare three different dilutions of hybridoma cells to be cloned, containing 5, 10, and 20 cells per milliliter. 5 × 10⁻⁶ cells were added per milliliter. 4 -1×10 5 The cell ratio was determined by adding peritoneal macrophages to the hybridoma cell suspensions described above. Each type of hybridoma cell was aliquoted into a 96-well plate (100 μL per well) and cultured at 37°C with 5% CO2 for 6 days. Antibody detection was initiated when visible clones appeared. Wells with only a single clone were marked under an inverted microscope, and the supernatant was used for antibody detection. Cells from antibody-positive wells were expanded and cryopreserved. The subclonal ELISA results are shown in Table 3.

[0146] Table 3 ELISA test results

[0147]

[0148] The results of subclonal supernatant detection showed that all 10 subclonal monoclonal antibodies prepared from the three antigens were positive and none of them cross-reacted with the tag protein. Based on the results, p30(e1)-PET21-PEP 8F9G8, p22(e1)-PET21-PEP 7B12A1, and p72(e1)-PET21-PEP 6D8F2 were selected for preservation and subsequent experiments.

[0149] 9. Identification of Ig classes and subclasses of monoclonal antibodies

[0150] The ELISA plate was coated with 10 μg / mL antigen, with 50 μL added to each well and incubated overnight at 4°C. After washing, 100 μL of the monoclonal antibody sample to be tested was added to each well and incubated at 37°C for 1 hour; negative and positive control wells were included. After washing, 100 μL of HRP-labeled anti-mouse and subclass Ig antibody reagent was added to each well and incubated at 37°C in the dark for 20 minutes; the reaction was terminated with 2 mol / L H2SO4, and the antibody subtype was determined based on the color. p30(e1)-PET21-PEP 8F9G8 was identified as IgG1; p22(e1)-PET21-PEP 7B12A1 as IgG1; and p72(e1)-PET21-PEP 6D8F2 as IgG1.

[0151] Finally, hybridoma cells that stably expressed clonal antibodies against ASFV p30, p22, and p72 were selected, preserved, and sequenced. Among them, hybridoma cell XjZ91 secreted a monoclonal antibody against the recombinant antigen protein p72 and was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202152.

[0152] A monoclonal antibody secreted by hybridoma cells XjH74 containing the recombinant antigen protein p22 was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202153.

[0153] A monoclonal antibody secreted by hybridoma cells XjL60 containing the recombinant antigen protein p30 was deposited on March 10, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC No: C202154.

[0154] The cDNA sequences of the heavy chain variable region and the light chain variable region corresponding to the secreted antibody were obtained by sequencing, as shown in Table 4 below.

[0155] Table 4. Antibody variable region and expression gene sequence of the present invention

[0156]

[0157] 10. Non-competitive ELISA method for antibody affinity determination

[0158] 10.1 Determination of the optimal antigen coating concentration

[0159] Dissolve the antigen in coating buffer to prepare solutions with concentrations of 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, and 0.3125 mg / L. Add 100 μl to each well of the ELISA plate, and set up internal controls with 3 wells per concentration. Seal and incubate overnight at 4°C. Discard the coating buffer, add 200 μl of blocking buffer to each well, and block at 37°C for 2 h. Discard the liquid in the plate, wash the plate 3 times with washing buffer (300 μl per well, 5 min each time), add 100 μl of 10 mg / L antibody to each well, and incubate at 37°C for 60 min. Discard the liquid in the plate, wash the plate 3 times with washing buffer (300 μl per well, 5 min each time), pat dry, and add 50 μl of Anti-Pig to each well. IgG-HRP antibody, diluted 1:3000, was incubated at 37°C for 30 min. The liquid in the plate was discarded, and the plate was washed three times with washing buffer (300 μl per well, 5 min each time). 100 μl of TMB solution was added to each well and the plate was developed at room temperature in the dark for 15 min. 50 μl of stop solution was added to each well, and the results were read at 450 nm using a microplate reader. The results are expressed as OD values.

[0160] 10.2 Non-competitive ELISA assay procedure

[0161] Dissolve the antigen in coating buffer to prepare solutions with concentrations of 5 mg / L, 2.5 mg / L, 1.25 mg / L, and 0.625 mg / L. Add 100 μL to each well of the ELISA plate, with two wells for each concentration as an internal control. Seal and incubate overnight at 4°C. Discard the coating buffer, add 200 μL of blocking buffer to each well, and incubate at 37°C for 2 hours. Discard the liquid in the plate, wash the plate three times with 300 μL of washing buffer for 5 minutes each time, and add 100 μL of solutions with concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, and 1.5625 × 10⁻⁶ mg / L to each well. -1 7.8×10 -2 3.9×10 -2 1.95×10 -2 9.75×10 -3 4.88×10 -3 2.44×10 -3 1.22×10-3 6.1×10 -4 Add 100 μL of the mg / L antibody solution to each well, two wells per concentration, and incubate at 37°C for 60 min. Discard the liquid in the plate, wash the plate three times with washing buffer (300 μL per well, 5 min each time), pat dry, and add 50 μL of Anti-Pig IgG-HRP antibody to each well (antibody dilution ratio 1:3000). Incubate at 37°C for 30 min. Discard the liquid in the plate, wash the plate three times with washing buffer (300 μL per well, 5 min each time), and add 100 μL of TMB solution to each well. Develop the color at room temperature in the dark for 15 min. Add 50 μL of stop solution to each well and read the value using a microplate reader at 450 nm. The result is expressed as OD value.

[0162] The antibody concentration corresponding to half of the maximum OD value (i.e., OD50%) is obtained by plotting a graph. The affinity constant is calculated using the formula K = (n - 1) / 2(nAb′ - Ab), where Ab and Ab′ represent the antibody concentrations (mol / L) that produce the half-maximum absorbance when the antigen concentrations are Ag and Ag′, respectively, and n = Ag / Ag′. Then, pairwise comparisons are made: when n = 2, three K values ​​are obtained; when n = 4, two K values ​​are obtained; and when n = 8, one K value is obtained. The average of the six K values ​​is then used as the final result.

[0163] 10.3 Results

[0164] 10.3.1 Optimal Antigen Coating Concentration

[0165] The p30(e1)-PET21-PEP, p22(e1)-PET21-PEP, and p72(e1)-PET21-PEP antigens were administered at concentrations of 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, and 0.3125 mg / L, respectively. Six serial dilutions were used to coat ELISA plates, and the final OD450 values ​​were as follows: p30(e1)-PET21-PEP: 2.656, 2.396, 1.903, 1.779, 1.257, 0.724; p22(e1)-PET21-PEP: 2.474, 2.123, 1.602, 1.344, 0.619, 0.489; p72(e1)-PET21-PEP: 2.785, 2.644, 2.404, 1.899, 1.206, 0.680. () Select OD450 values. 450The coating concentration was between 0.15 and 2.5 mg / L. Under this series of dilution gradients, the antigen coating concentration and OD value showed a good linear relationship. For each of the three antigens, four concentration gradients of 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, and 0.3125 mg / L were used for coating ELISA plates, and the antigen-antibody reaction curves were measured.

[0166] 10.3.2 Antibody Affinity Test

[0167] The experimental results are shown in Tables 5-7 below. Figure 4 As shown. The experimental results show that:

[0168] The concentration of p30(e1)-PET21-PEP 8F9G8 at the OD50% of the 2.5μg coating curve was 13μg / L (0.87×10⁻⁶). - 10 The concentration at OD50% of the 1.25 μg coating curve was 23 μg / L (1.53 × 10⁻⁶ mol / L). -10 The concentration at 0.625 μg coating curve (mol / L) was 55 μg / L (3.67 × 10⁻⁶ mol / L). -10 mol / L), the concentration at OD50% of the 0.3125 μg coating curve is 60 μg / L (4 × 10⁻⁶ mol / L). -10 (mol / L). Substituting into the formula K = (n - 1) / 2(nAb′ - Ab), the affinity constant is calculated to be 1.256 × 10⁻⁶. 9 L / mol.

[0169] The concentration of p22(e1)-PET21-PEP 7B12A1 at the OD50% of the 2.5 μg coating curve is 21 μg / L (1.4 × 10⁻⁶). - 10 The concentration at OD50% of the 1.25 μg coating curve was 38 μg / L (2.53 × 10⁻⁶ mol / L). -10 The concentration at 0.625 μg coating curve (mol / L) was 46 μg / L (3.07 × 10⁻⁶ mol / L). -10 The concentration at 0.3125 μg coating curve (mol / L) was 52 μg / L (3.47 × 10⁻⁶ mol / L). -10 (mol / L). Substituting into the formula K = (n - 1) / 2(nAb′ - Ab), the affinity constant is calculated to be 1.347 × 10⁻⁶. 9 L / mol.

[0170] The concentration of p72(e1)-PET21-PEP 6D8F2 at the OD50% of the 2.5 μg coating curve is 55 μg / L (3.67 × 10⁻⁶). -10 mol / L), the concentration at OD50% of the 1.25 μg coating curve is 60 μg / L (4 × 10⁻⁶ mol / L). -10 The concentration at 0.625 μg coating curve (mol / L) was 69 μg / L (4.6 × 10⁻⁶ mol / L). -10 mol / L), the concentration at OD50% of the 0.3125 μg coating curve is 90 μg / L (6 × 10⁻⁶ mol / L). -10 (mol / L). Substituting into the formula K = (n - 1) / 2(nAb′ - Ab), the affinity constant is calculated to be 8.914 × 10⁻⁶. 8 L / mol.

[0171] Table 5. P30 ELISA test results

[0172]

[0173] Table 6P22 ELISA Detection Results

[0174]

[0175] Table 7P72 ELISA Detection Results

[0176]

[0177] Example 2: Preparation of a test strip for the specific detection of African swine fever antibodies

[0178] The preparation of the ASFV antibody rapid detection test strip, specifically the structure is as follows: Figure 5 As shown, it includes: a base plate A, a 25mm long solid nitrocellulose membrane (chromatographic membrane) 3 is attached to a 60mm wide rigid plastic base plate 1 in the middle of the base plate A, an absorbent filter paper (absorbent pad) 2 about 18mm long is attached and overlaps the solid nitrocellulose membrane 3 by 2mm, a 10mm long gold label pad 4 overlaps the solid nitrocellulose membrane 3 by 2mm, a glass cellulose membrane sample pad 5 about 15mm long overlaps the gold label pad 4 by 3mm, and a microcomputer automatic chopping machine cuts it into 3mm wide strips B, and the strips are covered with a protective plastic shell C.

[0179] The solid nitrocellulose membrane (chromatographic membrane) 3 is provided with a control line 7 and a detection line 8; the detection line 8 is located near the sample pad 5 and is formed by spraying mouse anti-pig IgG antibody; the control line 7 is located near the absorbent pad 4 and is formed by spraying goat anti-mouse IgG antibody.

[0180] The gold-labeled pad 4 is coated with the antigen protein P22 prepared in Example 1, which binds to colloidal gold, and can be used for ASFV monoclonal antibody detection.

[0181] The above-mentioned rapid test strip for detecting ASFV antibodies is prepared through the following steps:

[0182] 1. Preparation of colloidal gold-labeled antigens

[0183] Add 2 mL of 0.5% trisodium citrate solution to 50 mL of boiling 0.01% chloroauric acid aqueous solution. The resulting colloidal gold particles have a diameter of about 15 nm. Adjust the pH of the colloidal gold to 8.5 with 0.1 mol / L K2CO3 to obtain gold sol.

[0184] The prepared recombinant antigen protein P22 was added to a gold sol at a labeling ratio of 1:1000 and stirred slowly for 10 min. 20% PEG10000 was added to a final concentration of 0.05%, and the mixture was centrifuged at 4℃ and 1500 rpm for 20 min to remove unbound colloidal gold particles. The mixture was then centrifuged at 4℃ and 15000 rpm for 1 h to remove the supernatant, thus obtaining a preliminarily purified colloidal gold-labeled antigen mixture. The mixture was then separated and purified by propylene dextran S-400 column chromatography to obtain the colloidal gold-labeled antigen.

[0185] 2. Test strip preparation and assembly

[0186] Preparation of gold-labeled pad 4: The obtained colloidal gold-labeled antigen was diluted to 1 mg / mL at a ratio of 1:100 and then uniformly sprayed onto a glass fiber membrane at a rate of 1 μL / cm. After drying at 56 °C for 1 h, gold-labeled pad 4 was obtained. It was then vacuum sealed, dried, and stored for later use.

[0187] Preparation of sample pad 5: After soaking glass wool in buffer solution, dry it at 56 ℃ for 1 h to obtain sample pad 5; vacuum seal it and dry it at 4 ℃ for later use; the buffer solution is 0.01 mol / L PBS, 0.1% Triton×100 or 4% Tween-20, 0.01% sodium azide;

[0188] Preparation of the chromatographic membrane 3: 2 mg / mL of mouse anti-pig IgG antibody was pumped into sample cell A of the XYZ3000 three-dimensional spray membrane apparatus, and 2 mg / mL of goat anti-mouse IgG antibody was pumped into sample cell B; the parameters were set to 50 dots / mL / cm; mouse anti-pig IgG antibody detection lines and goat anti-mouse IgG antibody control lines were sprayed onto the nitrocellulose membrane, with the two lines parallel and approximately 0.5 cm apart; the nitrocellulose membrane was dried in a 42 ℃ drying oven to obtain the chromatographic membrane; it was then vacuum-sealed, dried, and stored for later use.

[0189] Assembly and Cutting: Attach the chromatography membrane 3 to the base plate 1; attach an absorbent pad 2, which is cut from absorbent filter paper, to one end of the chromatography membrane 3; the absorbent pad 2 overlaps the chromatography membrane 3 by 2 mm; then attach a gold label pad 4 to the other end of the chromatography membrane 3, with the gold label pad 4 overlapping the chromatography membrane 3 by 2 mm; finally attach a sample pad 5, with the sample pad 5 overlapping the gold label pad 4 by 3 mm; place the assembled test strip into a cutting machine and cut it into test strips B with a width of 3 mm, then seal the surface with a plastic mold 6; the test strip can also be placed in a card holder to make a test card.

[0190] Example 3: Specificity detection of African swine fever antibody test strips

[0191] Using recombinant antigen protein p22, ASFV antibody detection was performed on the test strips prepared above. The serum samples to be tested were: ASFV antibody positive porcine serum, porcine negative serum, classical swine fever virus (CSFV) positive serum, porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, porcine circovirus (PCV2) positive serum, pseudorabies virus (PRV) positive serum, and porcine epidemic diarrhea virus (PEDV) positive serum. The detection methods are as follows:

[0192] 1. Place the test strip card flat on a dry surface.

[0193] 2. Using a disposable capillary tube, draw 10 μL of the liquid to be tested, then move the capillary tube directly above the sample application hole (S end) of the test strip card and squeeze to apply the sample.

[0194] 3. Add 2 drops of buffer solution vertically into the sample well.

[0195] 4. Time the test for 15 minutes and judge the results according to the result judgment method.

[0196] The determination result can be one of the following situations, and the principle diagram is shown below. Figure 6 As shown:

[0197] (1) Positive result: Two red lines, one red reaction line at the test line (T) and one red reaction line at the control line (C), indicate that the antibody concentration in the sample is equal to or higher than the detection limit;

[0198] (2) Negative result: Only one red reaction line appears at the control line (C), indicating that the antibody concentration in the sample is below the detection limit;

[0199] (3) Invalid result: No red reaction line appears at the control line (C), the test is invalid. When an invalid result occurs, please check whether the test method is correct and retest with a new test strip, paying particular attention to whether the sample volume is sufficient;

[0200] Specific test results are as follows: Figure 7As shown, ASFV positive serum exhibits two dark reaction bands simultaneously at both the test line (T line) and the control line (C line), indicating a positive result. Porcine negative serum, classical swine fever virus (CSFV) positive serum, porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, porcine circovirus (PCV2) positive serum, pseudorabies virus (PRV) positive serum, and porcine epidemic diarrhea virus (PEDV) positive serum only show color at the control line (C line), indicating a negative reaction. The test results demonstrate that the test strips prepared as described above can accurately detect ASFV positive serum without interference from other virus-positive serums, exhibiting high specificity and no cross-reaction.

[0201] Example 4: Sensitivity Detection of African Swine Fever Antibody Test Strip

[0202] Using porcine negative serum as a control, standard ASFV antibody positive serum was diluted 1:50, 1:100, 1:200, and 1:400 times, respectively, and tested using the recombinant antigen protein p22 test strip prepared above.

[0203] Specific test results are as follows: Figure 8 As shown, when detecting ASFV antibody-positive serum at dilutions of 1:50, 1:100, and 1:200, the prepared test strips showed two reaction bands simultaneously at the test line and control line, indicating a positive result. When detecting a sample diluted 400 times, only one reaction band appeared at the control line, indicating a negative result. This demonstrates that the test strips prepared as described above exhibit excellent sensitivity for ASFV-positive serum at a dilution of 400.

[0204] Example 5: Repeatability Test of African Swine Fever Antibody Test Strip

[0205] Standard positive serum was diluted to three concentrations (high, medium, and low). Each serum gradient was tested three times in parallel using test strips prepared from the same batch. The color development of the C and T lines was observed, and the uniformity of color development was visually assessed. The test results are as follows: Figure 9 As shown, the colorimetric results of the tests were consistent and the repeatability was good.

[0206] Example 6: Stability Test of African Swine Fever Antibody Test Strip

[0207] The prepared ASFV antibody test strips were placed at a constant temperature of 37℃ for 0, 1, 5, 11, 14, 15, and 16 days. The high and low concentrations of the negative control serum and the ASFV positive standard were then measured, and the tests were repeated three times. The results were then observed. Figure 10 As shown in the figure, the test results indicate that the test strip card has good stability.

[0208] Example 7: African Swine Fever Antibody Test Strip Compliance Rate Detection

[0209] Commercially available ELISA kits were purchased and used to test 52 clinically positive and 100 clinically negative samples. A comparative experiment was conducted using the ASFV antibody test strip prepared according to this invention, and the concordance rate between the two methods was calculated to determine the accuracy of the test strip. The concordance rate calculation method is shown in Table 8 below:

[0210] Table 8 Compliance Rate Calculation Method

[0211]

[0212] Positive compliance rate calculation formula = 100% [a / (a+c)]

[0213] The formula for calculating the negative compliance rate is: 100% [d / (b + d)]

[0214] Overall success rate calculation formula = 100% [(a+d) / n]

[0215] The results of the compliance test are shown in Table 9.

[0216] Table 9. Test strip compatibility rate results

[0217]

[0218] The positive concordance rate was 96.15%, the negative concordance rate was 100%, and the overall concordance rate was 98.68%.

[0219] Example 8: Comparison Test of African Swine Fever Antibody Test Strips with Similar Products

[0220] The ASFV antibody test strips prepared above were compared with test strips developed by two other companies, simultaneously testing four ELISA-positive swine serum samples (samples #2, #3, #4, and #5 shown in the image). The results are as follows: Figure 11 As shown in the figure, the test strip product developed in this patent is on the left. It has a 100% accuracy rate in detecting four clinical positive samples, and the T line is more obvious than the other two products, indicating that the test strip of this invention has higher sensitivity.

[0221] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof of the recombinant antigen protein p22 of African swine fever virus, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

19.

2. A hybridoma cell, characterized in that, The hybridoma cells are deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C202153.

3. A monoclonal antibody against the recombinant antigen protein p22 of African swine fever virus, characterized in that, The monoclonal antibody is produced by the hybridoma cells as described in claim 2.

4. An isolated nucleic acid molecule, characterized in that, For a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment as described in claim 1; the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO: 18, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:

20.

5. A carrier, characterized in that, It contains the isolated nucleic acid molecules as described in claim 4.

6. A kit for detecting African swine fever virus, characterized in that, It contains the antibody or its antigen-binding fragment as described in claim 1.

7. The reagent kit according to claim 6, characterized in that, The kit also includes a second antibody and / or a third antibody.

8. The reagent kit according to claim 6, characterized in that, The antibody contains a detectable marker.

9. The reagent kit according to claim 6, characterized in that, The kit is a colloidal gold kit, wherein the antibody or its antigen-binding fragment as described in claim 1 is loaded onto the test strip of the colloidal gold kit.

10. Use of the antibody or antigen-binding fragment thereof as described in claim 1 in the preparation of a kit for detecting African swine fever virus.

Images