Recombinant antigen protein p22 of African swine fever virus, monoclonal antibody and application

Abstract

The invention provides a recombinant antigen protein p22 of an African swine fever virus, a monoclonal antibody and application. The invention provides a recombinant antigen protein of African swine fever virus. The invention provides an antibody or an antigen binding fragment of a recombinant antigen protein of African swine fever virus. The invention also provides a hybridoma cell. The invention also provides a rapid test paper card for the African swine fever antibody. The recombinant antigen protein of the African swine fever virus, provided by the invention, has higher affinity, stronger specificity and better stability on an African swine fever antibody, and can be used for rapidly and accurately detecting 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”. The application number of the original application is 202211168511.5, the application date is September 19, 2022, and September 23, 2021 is reserved as the priority date.) Technical Field

[0002] The present invention belongs to the technical field of animal disease detection, and specifically relates to a recombinant antigen protein of African swine fever virus and corresponding monoclonal antibodies and hybridoma cells. The present invention also relates to the use of the recombinant antigen protein for detecting African swine fever virus. Background Art

[0003] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV) that mainly infects domestic pigs and various wild boars. It is mainly transmitted through contact with African swine fever virus and through African swine fever virus contaminants (kitchen waste, feed, drinking water, pens, bedding, clothing, utensils, vehicles, etc.). The digestive tract and respiratory tract are the main routes of infection; it can also be transmitted through the bites of vector insects such as blunt-edged soft ticks. The World Organization for Animal Health (OIE) has listed it as a notifiable animal disease, and it is also a type of animal epidemic that my country focuses on preventing. It is characterized by a short course of disease, with the most acute and acute infection mortality rate as high as 100%. Clinical manifestations include fever (up to 40-42°C), rapid heartbeat, difficulty breathing, some coughs, serous or mucopurulent secretions from the eyes and nose, cyanosis of the skin, and obvious bleeding 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 swine fever and can only be confirmed by laboratory monitoring.

[0004] ASFV was first discovered in Kenya in 1921. It has a high mortality rate and has caused huge economic losses to the pig farming industry. At present, ASF prevention and control has become a normal requirement for pig farming. "Preventing external imports, preventing internal spread, real-time monitoring, and tooth extraction for infection" has become a consensus for ASF prevention and control. Real-time monitoring and rapid and accurate detection are particularly important in pig farms. Therefore, rapid and reliable detection of ASFV is crucial. Rapid detection of ASFV is not only necessary for ASF prevention and control, but also necessary for differential diagnosis of pig diseases with similar clinical symptoms.

[0005] P30 is the main inducing protein of ASFV virus particles, encoded by the CP204L gene, with a molecular weight of about 30ku, also known as P32 protein. P30 protein is a highly antigenic membrane protein that is expressed and secreted in the early stages of viral infection. P30 protein is involved in the binding of the virus to cell surface receptors and inhibits the translation of host cell mRNA. Due to its early expression and high expression level in cells, using it as an antigen to detect ASFV antibodies is about a week earlier than other antigens (such as P72).

[0006] P22 is a binding protein located on the inner envelope membrane wrapped around the outer side of the viral nucleocapsid. The formation of the inner envelope membrane is related to the endoplasmic reticulum of the cell. Currently, there is little research on p22.

[0007] P72, sometimes also called p73 protein, is the main structural protein of the virus particle and an important component of the icosahedral capsid of the virus particle, accounting for about 32% of the entire virus particle protein. It is encoded by the B646L gene and has a molecular weight of about 73ku. It is produced in the late stage of virus infection. The protein sequence is highly conserved and has good antigenicity. After the virus infects the body, it can produce high titers of anti-p72 antibodies and is often used in ASF serological diagnosis.

[0008] At present, African swine fever virus detection methods are mainly divided into two categories: the first category is pathogen detection, including virus isolation detection, virus antigen detection and genomic DNA detection, which are mainly detected by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and other methods; the second category is antibody detection. For subacute and chronic African swine fever, as the virus circulates and spreads, its virulence will decrease. Antibodies can appear 7-10 days after pigs are infected with ASFV. The antibodies can last for a long time and are suitable for large-scale antibody screening. Antibody detection can be used as a basis for diagnosis of ASFV infection. It is mainly detected by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA) and immunoblotting methods.

[0009] Although the existing detection technology has high detection specificity and can meet the needs of sensitivity, it requires professional laboratories and experimental equipment. At the same time, it has high technical requirements for operators, long detection time and high cost. ELISA testing is prone to false positive results due to cross-contamination, and false negative results are prone to occur during PCR testing due to the representativeness of sampling, inhibitors and nucleic acid degradation. At the same time, as the virus circulates and spreads in pigs, its virulence will decrease, and PCR testing cannot effectively detect it, resulting in certain risks in pig farms. In addition, the detection instruments and equipment are expensive, making it difficult to use them comprehensively.

[0010] Therefore, the above methods are not suitable for rapid detection or diagnosis at the grassroots or on-site. Developing a simple and rapid real-time online detection technology that can detect African swine fever virus antibodies is a technical problem that urgently needs to be solved.

[0011] Colloidal gold immunochromatography is a diagnostic technology that has developed rapidly in recent years. Its main advantages are: (1) simple operation, can obtain diagnostic results within a few minutes, convenient and fast, and does not require professional personnel; (2) less affected by external factors, one pig one card, avoid cross contamination, and high detection accuracy; (3) cost-effective, no need for other expensive instruments and professional laboratories, low detection cost; (4) wide range of use, easy to carry and store, suitable for farms of all sizes, 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 immunochromatography test strips based on recombinant p30, p54 or p72 single or two proteins, but when detecting African swine fever virus antibody negative or weakly positive pig serum or whole blood, the accuracy and sensitivity need to be improved, which restricts the promotion and application of this method in the market.

[0012] Based on this, there is currently a need for a detection method that can quickly and accurately detect African swine fever virus. Summary of the invention

[0013] Based on the deficiencies of the prior art, the main purpose of the present invention is to provide a recombinant antigen protein of African swine fever virus and corresponding monoclonal antibodies and hybridoma cells. The present invention also relates to the use of the recombinant antigen protein, monoclonal antibodies and hybridoma cells for detecting African swine fever virus. The present invention also provides a rapid detection test paper card for African swine fever virus based on colloidal gold immunochromatography technology. It includes its raw materials: recombinant antigen protein of African swine fever virus and corresponding monoclonal antibodies.

[0014] The monoclonal antibody provided by the present invention has 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 the present invention is achieved through the following technical solutions:

[0016] In one aspect, the present invention provides a recombinant antigen protein p22 of African swine fever virus, wherein the recombinant antigen protein comprises:

[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., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) 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 to the sequence shown in SEQ ID NO:4.

[0020] The present invention also provides an isolated nucleic acid molecule, which encodes 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 an antigen-binding fragment thereof to the recombinant antigen protein p22 of African swine fever virus, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

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

[0024] According to the antibody or antigen-binding fragment thereof of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0025] (a) a chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[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., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) 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 to the sequence set forth in SEQ ID NO: 17;

[0029] and / or,

[0030] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[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., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) 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 to the sequence set forth in SEQ ID NO: 19;

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

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

[0036] In yet another aspect, the present invention further provides a hybridoma cell, the deposit number of the hybridoma cell is CCTCC NO: C202153.

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

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

[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 vector comprising the isolated nucleic acid molecule of the present invention.

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

[0042] Preferably, the kit further comprises a secondary antibody and / or a tertiary antibody;

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

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

[0045] The above-mentioned recombinant antigen protein provided by the present invention can be used for related research on African swine fever, such as pathogenic mechanism, immune response with antibodies, etc.; it can also be used for the development of practical diagnosis, such as ELISA detection kits and detection test strips for African swine fever; and for the development of African swine fever vaccines; and can be applied to the detection of African swine fever by mixed antigens, making the detection more accurate and sensitive.

[0046] In a specific embodiment, the test paper card containing the recombinant antigen protein or antibody of the present invention can specifically detect the antibody of p22 in the blood sample of pigs infected with African swine fever, thereby playing a role in diagnosing African swine fever. The monoclonal antibody test paper card for P22 has a sensitive detection effect and, as a less studied ASFV target, has a high potential research value.

[0047] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0048] For example, through different primer designs, expression vectors, and endonuclease selections, recombinant proteins and immunized animals (including the steps and consumables used in the experimental process can be adjusted to a certain extent) can be obtained hybridoma cells that secrete the same monoclonal antibody.

[0049] ASFV antibody rapid detection test paper cards are prepared using recombinant p2 prokaryotic expression antigens obtained by different methods using different test paper card materials, such as different types of card shells, PVC base plates, absorbent pads, sample pads, NC membranes, fiber membranes, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present application, and together with the specification are used to explain the principles of the present application, and do not constitute improper limitations on the present application.

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

[0052] Figure 2 This is a diagram showing the expression of recombinant plasmids of ASFV p30, p22, and p72 proteins transformed into Escherichia coli after IPTG induction; among them, p30 and p22 are respectively bacterial solution without recombinant plasmid, bacterial solution protein with recombinant plasmid, and Mark from right to left; p72 is respectively Mark, bacterial solution without recombinant plasmid, and bacterial solution protein with recombinant plasmid from right to left;

[0053] Figure 3 is the SDS-PAGE image of ASFV p30, p22, and p72 recombinant proteins after purification;

[0054] Figure 4 It is the result of affinity test of ASFV p30, p22, and p72 antibodies; Figure 4 A is the result of p30(e1)-PET21-PEP8F9G8; Figure 4 B is the result of p22(e1)-PET21-PEP 7B12A1; Figure 4 C is 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 paper card according to the present invention;

[0056] Figure 6 This is a schematic diagram of the principle of the test strip to determine whether a blood sample is negative or positive;

[0057] Figure 7 This is a graph showing the specificity test results of the African swine fever antibody test paper card of the present invention; from left to right 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 sensitivity test results of the African swine fever antibody test paper card of the present invention; from left to right, they are original multiple, 1:50, 1:100, 1:200 and 1:400;

[0059] Fig. 9 This is a graph showing the test results of the African swine fever antibody test paper card of the present invention for repeatability detection;

[0060] Fig.10This is a graph showing the stability test results of the African swine fever antibody test paper card of the present invention;

[0061] Fig.11 This is a comparison test result diagram of similar products of African swine fever antibody detection test paper cards, among which the leftmost one is the test paper card of the present invention.

[0062] Biological Deposit Information

[0063] Hybridoma cell XjZ91 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan 430072, China on March 10, 2021, with the deposit number CCTCC No: C202152;

[0064] Hybridoma cell XjH74 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan 430072, China on March 10, 2021, with the deposit number CCTCC No: C202153;

[0065] Hybridoma cell XjL60 was deposited on March 10, 2021 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, zip code 430072, with the deposit number CCTCC No: C202154. DETAILED DESCRIPTION

[0066] The following description of the application is only for illustrating the various embodiments of the application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive a variety of equivalents, variations and modifications without departing from the scope of the application, and it should be understood that such equivalent embodiments are included within the scope of the invention. All documents cited in this application, including publications, patents and patent applications, are incorporated 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] 1Construction of African swine fever virus p30, p22, p72 recombinant protein expression vector

[0069] 1.1 Based on the data entered in NCBI as a reference, the ASFV antigen recombinant protein gene sequence to be expressed was selected, and the target gene was synthesized based on the public gene fragment (Shanghai Sangon Biotechnology Co., Ltd.) as the PCR template chain:

[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); 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), and the sequence structure is as follows.

[0071] SEQ ID NO: 1

[0072] ATGGATTTTATTTTAAATATATCCATGAAAATGGAGGTCATCTTCAAAACGGATTTAAGATCATCTTCACAAGTTGTGTTTCATGCGGGTAGCTTGTATAATTGGTTTTCTGTTGAGATTATCAATAGCGGTAGAATTGTTACGACCGCTATA AAACATTGCTCAGTACTGTTAAGTATGATATTGTGAAATCTGCTCATATATATGCAGGGCAAGGGTATACTGAACATCAGGCTCAAGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAGGAGACAGAATCCTCAGCATCATCGGAA AGCATTCATGAAAAAAATGATAATGAAACCAATGAATGCACATCCTCCTTTGAAACATTGTTTGAGCAAGAGCCCTCCATCAGAGGAACCTAAAGACTCCAAGCTGTATATGCTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAG GCACCTGATTTTAACAAGGTTATTAGAGCACATAACTTTATTCAAACCATTCATGGAACCCCTCTAAAGGAAGAAGAAAAAGAGGTGGTAAGACTCATGGTCATTAAACTTTTAAAAAAAATAAGCTTTTATCTCACCTACATTTAATGTTT

[0073] SEQ ID NO: 2

[0074] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKY DIVKSAHIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSESIHEKNDNETNECTSSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKNKLLSHLHLMF

[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] TPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNIS DQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST;

[0083] 1.2 Design amplification primers using Primer and DNAStar

[0084] ① Primers for amplifying 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 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 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: DNA template 5 μL, amplification primer mixture (2 μmol / L) 5 μL, 10× PCS buffer 5 μL, 10× dNTPs (2.0 mmol / l) 5 μL (Takara), Taq DNA polymerase (1.5 U (Takara), H 2O 28μL, various components, the total reaction volume is 50μL, incubate at 42℃ for 30-60 minutes, cool on ice, centrifuge for a few seconds, and then perform PCR.

[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, a total of 30 cycles; 72℃ extension for 5 minutes. After the cycle is completed, take out the reaction tube and place it in a 4℃ refrigerator for later use.

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

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

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

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

[0106] The connection system between the target gene and the expression vector: DNA enzyme digestion reaction PET21-PEP 2.5μL, DNA enzyme digestion reaction target gene fragment 9.5μL, 10×T 4 DNA ligase buffer 1.5 μL, T 4 DNA ligase (Takara) 0.5 μL (3 U).

[0107] Ligation reaction conditions: Spin the Eppendorf tube on a table centrifuge for 2 seconds to concentrate the sample, and place the ligated sample in a constant temperature water bath at 16°C overnight. The spectra of the recombinant plasmids p30-PET21-PEP, p22-PET21-PEP, and p72-PET21-PEP are shown in Figure 1.

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

[0109] With CaCl 2The competent E. coli was prepared by solution, and the recombinant expression vectors p30-PET21-PEP, p22-PET21-PEP and p72-PET21-PEP were transformed into the competent E. coli RST, and multiple colonies were picked into 50mL centrifuge tubes and cultured with LB liquid medium at 37℃ for 4h. 5mL was transferred to another centrifuge tube and IPTG (AMRESCO) was added to a final concentration of 1mM to induce expression, and the remaining 15mL bacterial solution was continued to be cultured at 37℃ for 4h. After the bacteria were harvested, 1mL of the induced and uninduced bacterial solutions were taken respectively, and the culture medium was removed by centrifugation. The precipitate was added with 1×Loading and mixed well, and the expression results were observed after SDS-PAGE gel electrophoresis. The electrophoresis results are shown as follows: Figure 2 shown.

[0110] 15 mL of uninduced bacterial solution was added to 1 L of LB liquid medium and cultured with shaking for 4 h. After 4 h, 1 mM IPTG was used to induce expression. After 4 h, the expression was centrifuged at 3800 rpm for 10 min, and the supernatant was discarded, leaving the precipitate.

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

[0112] The precipitated bacteria were ultrasonically disrupted using 50 mL of 8 M urea (Beihua Kaiyuan Company), and then centrifuged at 18,000 rpm for 30 min and the supernatant was retained. The supernatant was purified by column affinity chromatography. After the supernatant passed through the column, the affinity column was gradient eluted using 8 M urea buffer containing 10 mM and 300 mM imidazole. The eluted liquid was sampled for SDS-PAGE detection, and the protein with the highest concentration and purity was selected for PBS dialysis and concentrated with sucrose. The test results are as follows: Figure 3 As shown, the electrophoretic purity of the p30 recombinant protein was 95%, the electrophoretic purity of the p22 recombinant protein was 80%, and the electrophoretic purity of the p72 recombinant protein was 85%.

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

[0114] The recombinantly expressed p30, p22, and p72 proteins were serologically verified by indirect ELISA. The three antigens were diluted to 1 μg / mL with coating solution, added to the wells of the ELISA plate at 100 μL / well, and incubated at 4°C overnight; the liquid in the well was discarded, washed 3 times with washing solution, 3 minutes each time, and patted dry; 300 μL of blocking solution was added to each well and blocked at 37°C for 1 hour, the liquid in the well was discarded, and the well was patted dry; 100 μL of a primary antibody with a certain dilution (positive serum No. 1-5; negative serum No. #1-#5) was added to each well, incubated at 37°C for 1 hour, the liquid in the well was discarded, washed 3 times with washing solution, 3 minutes each time, and patted dry; 50 μL of 1:10000 dilution of rabbit anti-pig IgG enzyme-labeled secondary antibody (SIGMA) was added to each well, incubated at 37°C for 1 hour, the liquid in the well was discarded, washed 3 times with washing solution, 3 minutes each time, and patted dry; 100 μL of freshly prepared TMB (Biyuntian Company) substrate colorimetric solution was added to each well, incubated at 37°C for 15 minutes; 2 mol / L H 2 SO 4 The reaction was terminated and the OD value was read by a microplate reader. The results are shown in Table 1.

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

[0116]

[0117] From the OD numerical analysis, it can be seen that the above three recombinant expressed proteins can specifically bind to the antibodies in the positive serum infected with African swine fever virus, and do not bind to other antibodies in the negative serum that has not been infected with African swine fever virus.

[0118] 5. Inject p30, p22, and p72 recombinant proteins into mice for animal immunization

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

[0120] ELISA test reagents:

[0121] ①p30 detection coated antigen

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

[0123] ②p22 detection coated antigen

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

[0125] ③p72 detection coating antigen

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

[0127] ④The primary antibody used was the mouse serum 10 days after the third immunization.

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

[0129] Table 2 Indirect ELISA detection of immune effects

[0130]

[0131]

[0132]

[0133] From the OD value results of the test, it can be concluded that the titer of mouse No. 3 immunized with recombinant p30(e1)-PET21-PEP protein reached the requirement, 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 reached the requirement, and the reaction with the tag protein was relatively weak, so mouse No. 4 was selected for fusion; the titers of mice immunized with recombinant p72(e1)-PET21-PEP protein all reached the requirement, but the results of the two antigen reactions were almost the same, so the mice with higher OD values ​​were selected for fusion in turn, starting with No. 4.

[0134] 6. Cell Fusion

[0135] ① Preparation of feeder cells

[0136] BALB / c mice aged 6-10 weeks were killed by pulling their necks, and then immersed in 75% alcohol for disinfection for 3 min. The skin was cut with sterile scissors to expose the peritoneum, and 6 mL of culture medium was injected with a pipette. The cells were repeatedly rinsed, and the rinsing liquid was centrifuged at 1200 rpm for 5 min. The supernatant was discarded and the cells were suspended with 20% calf serum (GIBCO) culture medium to adjust the cell number to 1*10 5 / mL. Take 100 μL and place it in a 96-well plate and incubate it at 37°C.

[0137] ② Preparation of myeloma cells

[0138] 36-48 hours before fusion, expand the myeloma cells. On the day of fusion, use an elbow dropper to gently blow the cells off the bottle wall and collect them in a 50mL centrifuge tube or fusion tube. Centrifuge at 1000r / min for 5-10 minutes, discard the supernatant and add 30mL of incomplete culture medium, centrifuge and wash once. Then resuspend the cells in 10mL of incomplete culture medium and mix well. Take the myeloma cell suspension, add 0.4% trypan blue dye to count the live cells and set aside.

[0139] ③Preparation of spleen cells

[0140] Take the immunized BALB / c mice, kill them by cervical dislocation, soak them in 75% alcohol for 5 minutes, fix them on the culture dish and lift up the skin on the left side of the abdomen to see the spleen. Change to ophthalmic scissors and forceps, use sterile surgical scissors to open the peritoneum in the clean bench, take out the spleen and place it in a dish containing 10mL of incomplete culture medium, wash it gently, and carefully peel off the surrounding connective tissue. Place it on a stainless steel sieve in the dish, grind it into a cell suspension with a syringe needle, and then count it. Allow the spleen cells to enter the incomplete culture medium in the dish. Use a pipette to blow several times to make a single cell suspension. Usually 1×10 per mouse. 8 -2.5×10 8 splenocytes.

[0141] ④Cell fusion

[0142] 1×10 8 Splenocytes and 1×10 7Myeloma cells SP2 / 0 were mixed in a 50mL fusion tube, and incomplete culture medium was added to 30mL. After thorough mixing, centrifuge at 1000r / min for 5-10 minutes, and the supernatant was aspirated as much as possible. Tap the bottom of the fusion tube lightly on the palm of your hand to loosen and evenly precipitate the cells; add 1mL of preheated 50% PEG (Sigma) within 30s, stir gently while adding, and let it stand for 1min. Add preheated incomplete culture solution to terminate the PEG effect, and add 1mL, 2mL, 3mL, 4mL, 5mL, and 10mL every 2min. Finally, centrifuge at 800rpm for 5 minutes and discard the supernatant. Add 5mL of complete culture medium and gently blow and aspirate the precipitated cells to suspend and mix, and then add complete culture medium to 40-50mL. Aliquot 100μL per well into a 96-well cell culture plate, and then place the culture plate at 37°C and 5% CO 2 Culture in an incubator. After 6 hours, add 50 μL of selection medium to each well. After 3 days, half of the medium is replaced with selection medium. When the cells grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0143] 7. Selection of Hybridoma Cells

[0144] The indirect ELISA method was used to detect the secretion of antibody protein by hybridoma cells. Except for the use of hybridoma cell culture supernatant as the primary antibody, the other reagents used were the same as those used for ELISA serum detection after animal immunization. Antigen was diluted to 10 μg / mL with coating solution, and then added to the wells of the ELISA plate at 100 μL / well, and placed at 4°C overnight or adsorbed at 37°C for 2 hours; the liquid in the well was discarded, and the well was washed with washing solution for 3 times, each time for 3 minutes, and patted dry; 300 μL of blocking solution was added to each well and blocked at 37°C for 1 hour, and the liquid in the well was discarded, and patted dry; 100 μL of the culture supernatant of the hybridoma cells to be tested was added to each well, and positive, negative and blank controls were set up at the same time; after incubation at 37°C for 1 hour, the liquid in the well was discarded, and the well was washed with washing solution for 3 times, each time for 3 minutes, and patted dry; 100 μL of enzyme-labeled secondary antibody was added to each well, and the well was incubated at 37°C for 1 hour, and the liquid in the well was discarded, and the well was washed with washing solution for 3 times, each time for 3 minutes, and patted dry; 100 μL of freshly prepared TMB (Biyuntian Company) substrate colorimetric solution was added to each well, and incubated at 37°C for 15 minutes; 2mol / L H 2 SO 4 The reaction was terminated and the OD value was read by a microplate reader.

[0145] Result determination: P / N ≧ 2.1, or P ≧ N + 3SD is positive. If the negative control well is colorless or nearly colorless, and the positive control well is clearly colored, the result can be directly observed with the naked eye. Select hybridoma cells that express antibody protein positively and do not cross-react with the tag protein for cloning.

[0146] 8. Cloning of hybridoma cells (limiting dilution method)

[0147] Prepare mouse spleen cells as feeder cells. Dilute the hybridoma cells with HT medium containing 20% ​​serum to prepare three different dilutions of hybridoma cell suspensions containing 5, 10, and 20 cells per ml. Add 5×10 4 -1×10 5 Each hybridoma cell suspension was divided into a 96-well plate with 100 μL per well and placed at 37°C and 5% CO. 2 After 6 days of culture in the environment, antibodies can be detected when clones visible to the naked eye appear; observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant for antibody detection, expand the culture of cells in the antibody-positive wells, and freeze them. The results of the subclone ELISA test are shown in Table 3.

[0148] Table 3 ELISA test results

[0149]

[0150]

[0151] From the subclone supernatant test results, it can be seen that the 10 subclone monoclonal antibodies prepared from the three antigens were all positive and did not cross-react 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.

[0152] 9. Identification of Ig class and subclass of monoclonal antibodies

[0153] The ELISA plate was coated with 10 μg / mL antigen, and 50 μL was added to each well at 4°C overnight. 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 set. After washing, 100 μL of HRP-labeled anti-mouse class and subclass Ig antibody reagent was added to each well, and color was developed at 37°C in the dark for 20 minutes; 2 mol / L H 2 SO 4 After the reaction was terminated, the subtype of the antibody was determined based on the color. p30(e1)-PET21-PEP 8F9G8 was detected as IgG1, p22(e1)-PET21-PEP7B12A1 was detected as IgG1, and p72(e1)-PET21-PEP 6D8F2 was detected as IgG1.

[0154] Finally, hybridoma cells that stably expressed ASFV p30, p22, and p72 cloned antibodies were selected, preserved, and sequenced. Among them, the hybridoma cell XjZ91 secreted a monoclonal antibody against the recombinant antigen protein p72, which was deposited in the China Center for Type Culture Collection on March 10, 2021, with the address at Wuhan University, Wuhan, China, zip code 430072, and the deposit number is CCTCC No: C202152.

[0155] The hybridoma cell XjH74 secretes a monoclonal antibody against the recombinant antigen protein p22, which was deposited in the China Center for Type Culture Collection on March 10, 2021, located at Wuhan University, Wuhan, China, with a postal code of 430072, and the deposit number is CCTCCNo: C202153.

[0156] The hybridoma cell XjL60 secretes a monoclonal antibody against the recombinant antigen protein p30, which was deposited in the China Center for Type Culture Collection on March 10, 2021, located at Wuhan University, Wuhan, China, with a postal code of 430072, and the deposit number is CCTCCNo: C202154.

[0157] The heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to the secreted antibody were obtained by sequencing, as shown in Table 4 below.

[0158] Table 4 Antibody variable regions and expressed gene sequences of the present invention

[0159]

[0160]

[0161]

[0162]

[0163] 10. Non-competitive ELISA method to measure antibody affinity

[0164] 10.1 Determination of the optimal coating concentration of antigen

[0165] Dissolve the antigen with coating solution 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, set up internal controls, 3 wells for each concentration, and seal overnight at 4°C; pour out the coating solution, add 200 μl of blocking solution to each well, and block at 37°C for 2 hours; discard the liquid in the plate, wash the plate 3 times with washing solution, 300 μl per well, 5 minutes each time, add 100 μl of 10 mg / L antibody to each well, and incubate at 37°C for 60 minutes; discard the liquid in the plate, wash the plate 3 times with washing solution, 300 μl per well, 5 minutes each time, and add 50 μl of Anti-Pig to each well after patting dry IgG-HRP antibody, antibody dilution ratio 1:3000, sealed and incubated at 37℃ for 30min; shake off the liquid in the plate, wash the plate 3 times with washing solution, 300μl per well, 5min each time, add 100μl TMB solution to each well and color for 15min at room temperature in the dark; add 50μl stop solution to each well, read with an enzyme reader at a wavelength of 450nm, and the results are expressed as OD values.

[0166] 10.2 Non-competitive ELISA test process

[0167] Dissolve the antigen with coating solution 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, set up internal controls, 2 wells for each concentration, and seal overnight at 4°C; pour out the coating solution, add 200 μl of blocking solution to each well, and block at 37°C for 2 h; discard the liquid in the plate, wash the plate 3 times with washing solution, 300 μl per well, 5 min each time, add 100 μl of 10, 5, 2.5, 1.25, 0.625, 0.3125, 1.5625×10 -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 -4mg / L antibody solution, add 100 μl to each well, 2 wells for each concentration, and incubate at 37°C for 60 min; discard the liquid in the plate, wash the plate 3 times with washing solution, 300 μl per well for 5 min each time, pat dry, add 50 μl Anti-Pig IgG-HRP antibody to each well, the antibody dilution ratio is 1:3000, and incubate at 37°C for 30 min; shake off the liquid in the plate, wash the plate 3 times with washing solution, 300 μl per well for 5 min each time, add 100 μl TMB solution to each well and color at room temperature for 15 min in the dark; add 50 μl stop solution to each well, read the results with an enzyme reader at a wavelength of 450 nm, and express the results as OD values.

[0168] The antibody concentration corresponding to half of the maximum OD value (i.e. OD50%) is obtained by plotting. Substitute the formula K=(n-1) / 2(nAb′-Ab) to calculate the affinity constant, where Ab and Ab′ represent the antibody concentration (mol / L) that produces half the absorbance value when the antigen concentration is Ag and Ag′, respectively, and n=Ag / Ag′. Then compare them in pairs. When n=2, 3 K values ​​can be obtained, when n=4, 2 K values ​​can be obtained, and when n=8, 1 K value can be obtained. The average of the 6 K values ​​is obtained as the final result.

[0169] 10.3 Results

[0170] 10.3.1 Optimal Antigen Coating Concentration

[0171] p30(e1)-PET21-PEP, p22(e1)-PET21-PEP, and p72(e1)-PET21-PEP antigens were added at 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, and 0.3125 mg / L, respectively. The 6 serial dilution concentrations were coated on the ELISA plate, and the final OD450 values ​​were displayed. The p30(e1)-PET21-PEP values ​​were 2.656, 2.396, 1.903, 1.779, 1.257, and 0.724, respectively; the p22(e1)-PET21-PEP values ​​were 2.474, 2.123, 1.602, 1.344, 0.619, and 0.489, respectively; the p72(e1)-PET21-PEP values ​​were 2.785, 2.644, 2.404, 1.899, 1.206, and 0.680, respectively; () OD 450 The value between 0.15 and 2.5 was used as the coating concentration. The coating concentration of the antigen under the dilution gradient of this series had a good linear relationship with the OD value. For 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 ELISA plate coating, and the antigen-antibody reaction curve was determined.

[0172] 10.3.2 Antibody affinity test

[0173] The experimental results are shown in Tables 5 to 7. Figure 4 As shown. From the experimental results, we can know that:

[0174] The concentration of p30(e1)-PET21-PEP 8F9G8 at OD50% of the 2.5 μg coating curve was 13 μg / L (0.87×10 - 10 mol / L), the concentration at OD50% of the 1.25 μg coating curve was 23 μg / L (1.53×10 -10 mol / L), and the concentration at OD50% of the 0.625 μg coating curve was 55 μg / L (3.67×10 -10 mol / L), the concentration at OD50% of the 0.3125μg coating curve was 60μg / L (4×10 -10 mol / L). Substituting into the formula K = (n-1) / 2(nAb′-Ab), the affinity constant is calculated to be 1.256×10 9 L / mol.

[0175] The concentration of p22(e1)-PET21-PEP 7B12A1 at OD50% of the 2.5 μg coating curve was 21 μg / L (1.4×10 - 10 mol / L), the concentration at OD50% of the 1.25 μg coating curve was 38 μg / L (2.53×10 -10 mol / L), and the concentration at OD50% of the 0.625 μg coating curve was 46 μg / L (3.07×10 -10 mol / L), and the concentration at OD50% of the 0.3125 μg coating curve was 52 μg / L (3.47×10 -10 mol / L). Substituting into the formula K = (n-1) / 2(nAb′-Ab), the affinity constant was calculated to be 1.347×10 9 L / mol.

[0176] The concentration of p72(e1)-PET21-PEP 6D8F2 at OD50% of the 2.5 μg coating curve was 55 μg / L (3.67×10 - 10 mol / L), the concentration at OD50% of the 1.25 μg coating curve was 60 μg / L (4×10 -10 mol / L), and the concentration at OD50% of the 0.625 μg coating curve was 69 μg / L (4.6×10 -10 mol / L), the concentration at OD50% of the 0.3125μg coating curve was 90μg / L (6×10 -10mol / L). Substituting into the formula K = (n-1) / 2(nAb′-Ab), the affinity constant was calculated to be 8.914×10 8 L / mol.

[0177] Table 5 P30 ELISA test results

[0178]

[0179] Table 6 P22 ELISA test results

[0180]

[0181] Table 7P72 ELISA test results

[0182]

[0183] Example 2 Preparation of a test paper card for specific detection of African swine fever antibodies

[0184] Preparation of ASFV antibody rapid detection test paper card, the specific structure is as follows Figure 5 As shown, it includes: a bottom plate A, a 25mm long solid phase nitrocellulose membrane (chromatographic membrane) 3 is set in the middle of the bottom plate A and adhered to a 60mm wide hard plastic bottom plate 1, an absorbent filter paper (absorbent pad) 2 is adhered with a length of about 18mm and overlaps with the solid phase nitrocellulose membrane 3 by 2mm, a 10mm long gold label pad 4 overlaps with the solid phase nitrocellulose membrane 3 by 2mm, a glass cellulose membrane sample pad 5 is about 15mm long and overlaps with 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.

[0185] A quality control line 7 and a detection line 8 are provided on the solid phase nitrocellulose membrane (chromatographic membrane) 3; the detection line 8 is arranged close to the sample pad 5, and is formed by spraying mouse anti-pig IgG antibody; the quality control line 7 is arranged close to the absorbent pad 4, and is formed by spraying sheep anti-mouse IgG antibody.

[0186] The gold label pad 4 is sprayed with the antigen protein P22 prepared in Example 1 combined with colloidal gold, and ASFV monoclonal antibody detection can be performed.

[0187] The above-mentioned test paper for rapid detection of ASFV antibodies is prepared by the following steps:

[0188] 1. Preparation of Colloidal Gold Labeled Antigen

[0189] 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 are about 15 nm in diameter. 2 CO 3 Adjusting the pH of colloidal gold to 8.5 to obtain gold sol;

[0190] The prepared recombinant antigen protein P22 was added to the gold sol at a labeling ratio of 1:1000, and the mixture was slowly stirred for 10 minutes; 20% PEG10000 was added to a final concentration of 0.05%, and the mixture was centrifuged at 4°C and 1500rpm for 20 minutes to remove unbound colloidal gold particles; the mixture was centrifuged at 4°C and 15000rpm for 1 hour, and the supernatant was removed to obtain a preliminarily purified colloidal gold-labeled antigen mixture; and the mixture was separated and purified by propylene dextran S-400 column chromatography to obtain colloidal gold-labeled antigen;

[0191] 2. Test Strip Preparation and Assembly

[0192] Preparation of gold-labeled pad 4: dilute the obtained colloidal gold-labeled antigen to 1 mg / mL at 1:100, spray evenly on the glass fiber membrane at 1 μL / cm, dry at 56°C for 1 h to obtain gold-labeled pad 4; vacuum seal, dry and store for later use;

[0193] Preparation of sample pad 5: After fully soaking glass wool in a buffer solution, drying at 56°C for 1 hour to obtain sample pad 5; vacuum sealing, drying at 4°C for storage; the buffer solution is 0.01 mol / L PBS, 0.1% Triton×100 or 4% Tween-20, 0.01% sodium azide;

[0194] Preparation of chromatographic membrane 3: Pump 2 mg / mL mouse anti-swine IgG antibody into sample pool A of XYZ3000 three-dimensional membrane sprayer, and pump 2 mg / mL goat anti-mouse IgG antibody into sample pool B; set the parameters to 50 dots / mL / cm; spray mouse anti-swine IgG antibody detection line and goat anti-mouse IgG antibody quality control line onto nitrocellulose membrane, with the two imprinting lines parallel and the distance between them being about 0.5 cm; dry the nitrocellulose membrane in a drying oven at 42°C to obtain the chromatographic membrane; vacuum seal, dry and store for later use;

[0195] Assembly and cutting: Paste the chromatographic membrane 3 on the bottom plate 1; paste the absorbent pad 2 on one end of the chromatographic membrane 3, the absorbent pad 2 is cut from absorbent filter paper; the absorbent pad 2 overlaps the chromatographic membrane 3 by 2mm; then paste the gold label pad 4 on the other end of the chromatographic membrane 3, the gold label pad 4 overlaps the chromatographic membrane 3 by 2mm; finally paste the sample pad 5, the overlap size of the sample pad 5 and the gold label pad 4 is 3mm; put the assembled test paper into a cutting machine, cut it into test paper strips B with a width of 3mm, and then encapsulate the plastic mold 6 on the surface; the test paper strip can also be placed in a card shell to make a test card.

[0196] Example 3 African swine fever antibody test paper card specific detection

[0197] The recombinant antigen protein p22 was used to test the ASFV antibody according to the test paper card prepared above. The sera to be tested were: ASFV antibody pig positive serum, pig 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 (PEDV) positive serum. The detection method is as follows:

[0198] 1. Place the test card flat on a dry surface.

[0199] 2. Use a disposable capillary to draw 10 μL of the liquid to be tested, then move the capillary to the top of the sample loading hole (S end) of the test paper card and squeeze to load the sample.

[0200] 3. Vertically add 2 drops of buffer into the sample well.

[0201] 4. Set the timer to 15 minutes and determine the test result based on the result determination method.

[0202] The judgment results may be as follows, and the principle diagram is as follows Figure 6 As shown:

[0203] ① Positive result: Two red lines, that is, one red reaction line appears on the test line (T) and the quality control line (C), indicating that the antibody concentration in the sample is equal to or higher than the detection limit;

[0204] ② Negative result: Only one red reaction line appears on the quality control line (C), indicating that the antibody concentration in the sample is lower than the detection limit;

[0205] ③ Invalid result: No red reaction line appears on the quality control line (C), and the test is invalid. When an invalid result appears, please confirm whether the test method is correct and retest with a new test strip card, paying special attention to whether the sample volume is sufficient;

[0206] Specific test results such as Figure 7 As shown, it can be seen that the ASFV positive serum has two dark reaction bands at the detection line (T line) and the quality control line (C line) at the same time, and the result is judged to be positive; the swine 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 (PEDV) positive serum only have color at the quality control line (C line), showing a negative reaction; the test results show that the test paper card prepared as described above can accurately detect ASFV positive serum without being interfered by other virus positive serum, with high specificity and no cross reaction.

[0207] Example 4: Sensitivity test of African swine fever antibody test paper card

[0208] Using pig negative serum as a control, the 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 paper cards prepared above.

[0209] Specific test results such as Figure 8 As shown, it can be seen that when detecting ASFV antibody-positive serum with dilution multiples of 1:50, 1:100 and 1:200, two reaction bands appeared at the detection line and quality control line of the prepared test strip at the same time, and the result was judged to be positive; when the sample with a dilution multiple of 400 was detected, only one reaction band appeared at the quality control line, and the result was judged to be negative; this shows that the sensitivity of the test paper card prepared as described above for ASFV positive serum is a dilution multiple of 400, showing excellent sensitivity.

[0210] Example 5 Repeatability test of African swine fever antibody test paper card

[0211] The standard positive serum was diluted to three concentrations, high, medium and low. Each serum gradient was tested three times in parallel using the test paper cards prepared in the same batch. The color development of the C line and T line was observed. The color development was observed with the naked eye to see if it was uniform. Fig. 9 As shown, the colorimetric results of the tests were consistent and had good repeatability.

[0212] Example 6 African swine fever antibody test paper card stability test

[0213] The ASFV antibody test paper prepared above was placed at a constant temperature of 37°C for 0, 1, 5, 11, 14, 15, and 16 days, and then the negative control serum and the ASFV positive standard were tested at high and low concentrations, respectively. The test was repeated 3 times to observe the test results. Fig.10 As shown, the test results show that the test paper card has good stability.

[0214] Example 7 African swine fever antibody test paper card compliance rate detection

[0215] The ELISA kits available on the market were purchased and tested on 52 clinically positive and 100 clinically negative samples. At the same time, the ASFV antibody test paper card prepared by the present invention was used for comparative testing, and the compliance rate between the two methods was calculated to determine the accuracy of the test paper card. The compliance rate calculation method is shown in Table 8 below:

[0216] Table 8 Calculation method of compliance rate

[0217]

[0218] Positive coincidence rate calculation formula = 100% [a / (a+c)]

[0219] Negative coincidence rate calculation formula = 100% [d / (b+d)]

[0220] Total compliance calculation formula = 100% [(a+d) / n]

[0221] The compliance test results are shown in Table 9.

[0222] Table 9 Test paper card compliance test results

[0223]

[0224] The positive compliance rate was 96.15%, the negative compliance rate was 100%, and the total compliance rate was 98.68%.

[0225] Example 8 Comparison test of similar products of African swine fever antibody detection test paper card

[0226] The ASFV antibody test paper card prepared above was used to compare and test four samples of pig serum samples (sample #2, sample #3, sample #4, and sample #5) that were positive for ELISA. The results are as follows: Fig.11 As shown, the left side of the figure is the test paper card product developed by this patent, which has a detection compliance rate of 100% for four clinical positive samples, and the T line color is more obvious than the other two products, indicating that the test paper card of the present invention has higher sensitivity.

[0227] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A recombinant antigen protein p22 of African swine fever virus, wherein the recombinant antigen protein comprises the sequence shown in SEQ ID NO:

4.

2. An isolated nucleic acid molecule, characterized in that The isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:

3.

3. An antibody or antigen-binding fragment thereof to 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, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

19.

4. A hybridoma cell, characterized in that: The hybridoma cell is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C202153.

5. A monoclonal antibody against the recombinant antigen protein p22 of African swine fever virus, wherein the monoclonal antibody is secreted and produced by the hybridoma cell according to claim 4.

6. An isolated nucleic acid molecule, characterized in that A heavy chain variable region and / or a light chain variable region encoding the antibody or antigen-binding fragment thereof according to claim 3; the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO: 18, and the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:

20.

7. A carrier, characterized in that Comprising the isolated nucleic acid molecule of claim 6.

8. A kit for detecting African swine fever virus, characterized in that: It comprises the recombinant antigen protein according to claim 1, or the antibody or antigen-binding fragment thereof according to claim 3.

9. The kit according to claim 8, characterized in that The kit further comprises a secondary antibody and / or a tertiary antibody; Preferably, the antibody comprises a detectable label; Preferably, the kit is a colloidal gold kit, and the recombinant antigen protein according to claim 1, or the antibody or antigen-binding fragment thereof according to claim 3 is loaded on a test strip of the colloidal gold kit.

10. Use of the recombinant antigen protein according to claim 1, or the antibody or antigen-binding fragment thereof according to claim 3 in the preparation of a kit for detecting African swine fever virus.

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