Monoclonal antibody against African swine fever virus I73R protein and its preparation method and application

Abstract

The present invention belongs to the field of veterinary biotechnology, and specifically relates to a method for preparing monoclonal antibodies against the I73R protein of African swine fever virus and its application. The main technology includes using the recombinant I73R protein prepared by prokaryotic expression as an antigen, immunizing BALB / c mice, and then using hybridoma technology, through cell fusion, indirect ELISA method screening, limiting dilution method subcloning, and hybridoma continuous passage culture, to obtain a hybridoma cell line that can stably secrete monoclonal antibodies against the I73R protein of African swine fever virus. The present invention also discloses the sequence of the prepared monoclonal antibody immunogen, the subtype of the monoclonal antibody, the CDR sequence, the antigenic epitope recognized by the monoclonal antibody, the accession number of the hybridoma cell line, and the application of the monoclonal antibody in Western Blot experiments, indirect immunofluorescence experiments, immunoprecipitation experiments, neutralization experiments, and the blocking ELISA method for detecting African swine fever virus antibodies in pig serum.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biotechnology, and specifically relates to a preparation method and application of monoclonal antibodies against the I73R protein of African swine fever virus. Background Art

[0002] African Swine Fever (ASF) is a highly contagious disease caused by the African Swine Fever Virus (ASFV), which mainly infects domestic pigs and wild boars. The clinical features of the disease include high fever, cyanosis of the skin, loss of appetite, difficulty breathing and high mortality. The virus has strong resistance to the external environment and can survive for a long time in low temperature and dry environment, but is sensitive to high temperature and certain disinfectants. There is currently no effective vaccine or specific treatment, which has had a serious impact on the pig industry and international trade.

[0003] ASFV is a large double-stranded DNA virus belonging to the Asfarviridae family. ASFV has a complex icosahedral structure, with a genome length of about 170-190 kb, encoding more than 150 proteins, including structural proteins, non-structural proteins and functional proteins, among which more than half of the protein functions are still unclear.

[0004] The I73R protein is encoded by the I73R gene and is one of the non-structural proteins of ASFV. Its sequence is highly conserved among different genotypes. According to the article "Structural insight into African swine fever virus I73R protein reveals it as a Z-DNA binding protein", the I73R protein begins to be expressed in the very early stage of virus infection and is located in the nucleus at an early stage. As the infection time increases, it gradually moves to the cytoplasm. Crystal structure analysis found that it has a winged helix-turn-helix fold and has Z-DNA binding activity, and is a new member of the Za family.

[0005] Due to the high infectivity of ASF and the lack of effective vaccines and drugs, current prevention and control mainly relies on rapid diagnosis, so early and accurate detection is particularly important. Most of the current detection methods are based on molecular diagnostic technology, selecting highly conserved genes as targets to detect the viral genome; there are fewer detection methods for African swine fever antibodies, and commonly used target proteins include early antigen protein p30, late capsid protein p72, and virulence-related late hemocyte adsorption protein CD2v. During the replication of ASFV, the expression of I73R protein is earlier than the famous early protein p30, and there is currently no detection method for African swine fever virus antibodies based on this protein. Summary of the invention

[0006] Purpose of the invention:

[0007] The purpose of the present invention is to provide a monoclonal antibody against I73R, an antigenic epitope of a new I73R protein and the establishment of an African swine fever virus antibody blocking ELISA method.

[0008] Technical Solution

[0009] A monoclonal antibody against African swine fever virus I73R protein, characterized in that the monoclonal antibody is produced by a hybridoma cell with a deposit number of CCTCC NO: C2024397.

[0010] The monoclonal antibody is characterized in that the amino acid sequences of the heavy chain variable regions VH-CDR1, VH-CDR2 and VH-CDR3 of the monoclonal antibody are shown in SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8 respectively; the light chain variable regions are VL-CDR1, VL-CDR2 and VL-CDR3, and the amino acid sequences of the light chain variable regions are shown in SEQ ID NO.11, SEQ ID NO.13 and SEQID NO.15 respectively.

[0011] The monoclonal antibody is characterized in that the heavy chain of the monoclonal antibody is VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and their amino acid sequences are shown in SEQ ID NO.3-SEQ ID NO.9; the light chain is VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4, and their amino acid sequences are shown in SEQ ID NO.10-SEQ ID NO.16.

[0012] The monoclonal antibody is characterized in that the heavy chain of the monoclonal antibody is of IgG2b subtype and the light chain is of kappa type.

[0013] The method for preparing the monoclonal antibody is characterized in that the monoclonal antibody is prepared by the following steps: using the African swine fever virus I73R recombinant protein as an antigen, immunizing BALB / c mice, taking the spleen cells of the immunized mice and fusing them with myeloma cells, obtaining hybridoma cells after subclone screening and indirect ELISA identification, and the monoclonal antibody is produced by the hybridoma cells; wherein the preservation number of the hybridoma cell is CCTCC NO: C2024397, and the amino acid sequence of the African swine fever virus I73R recombinant protein is shown in SEQ ID NO.1.

[0014] The method is characterized in that the monoclonal antibody is obtained by injecting the hybridoma cells into BALB / c mice, collecting ascites 7 to 14 days after the injection, and purifying it with a kit.

[0015] The antigen epitope peptide specifically recognized by the monoclonal antibody is characterized in that the antigen epitope is located at positions 21 to 30 of the I73R protein of the African swine fever virus, and its amino acid sequence is shown in SEQ ID NO.2.

[0016] A method for detecting African swine fever virus, characterized in that the detection method is achieved through the monoclonal antibody.

[0017] An ELISA kit for detecting African swine fever virus, characterized in that the kit comprises the monoclonal antibody.

[0018] The monoclonal antibody is used in the preparation of drugs for preventing or treating African swine fever virus.

[0019] More specifically:

[0020] The accession number of the monoclonal cell is: CCTCC NO: C2024397

[0021] The African swine fever virus I73R recombinant protein selected a full-length 1-72 aa fragment based on the results of antigenicity analysis, and its amino acid sequence is:

[0022] METQKLISMVKEALEKYQYPLTAKNIKVVIQKEHNVVLPTGSINSILYSNSELFEKIDKTNTIYPPLWIRKN, as shown in SEQ ID NO.1.

[0023] The antigen epitope peptide is characterized in that it is located on amino acids 21 to 30 of the I73R protein of the African swine fever virus, and its sequence is:

[0024] 21 LTAKNIKVVI 30 , as shown in SEQ ID NO.2.

[0025] The method is further characterized in that the monoclonal antibody is obtained by injecting the hybridoma cells into BALB / c mice, collecting ascites 7 to 14 days after injection, and purifying it using a Protein A / G kit.

[0026] A method for detecting African swine fever virus, characterized in that the detection method is achieved through the monoclonal antibody.

[0027] An ELISA kit for detecting African swine fever virus, characterized in that the kit comprises the monoclonal antibody.

[0028] Specifically:

[0029] The invention provides a hybridoma cell, and the preservation number of the hybridoma cell I73R-5D5 is CCTCC NO: C2024397.

[0030] The present invention provides a monoclonal antibody against African swine fever virus I73R protein, which is secreted by hybridoma cell I73R-5D5 with a deposit number of CCTCCNO: C2024397. The hybridoma cell provided by the present invention can be stably passaged and continuously secrete the anti-I73R monoclonal antibody.

[0031] The monoclonal antibody provided by the present invention can specifically recognize African swine fever I73R protein in terms of biological function, and can be used for Western blot, immunoprecipitation and indirect immunofluorescence experiments.

[0032] The antigen epitope provided by the present invention is specifically recognized by the monoclonal antibody, and its sequence is highly conserved.

[0033] The present invention also provides the subtype of the monoclonal antibody of the African swine fever virus I73R protein: the heavy chain is IgG2b type, and the light chain is kappa type. The antibody variable region sequence is also provided. The heavy chain is VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and the light chain sequence is VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4 respectively.

[0034] VH-FR1(SEQ NO.3): QLQESGPGLVKPSQSLSLLTCS VT

[0035] VH-CDR1(SEQ NO.4): GYSITSGYY

[0036] VH-FR2(SEQ NO.5): WNWIRQFPGNKLEWMGY

[0037] VH-CDR2 (SEQ NO.6): IDPANGNT

[0038] VH-FR3(SEQ NO.7): KYDPKFQGKATITADTSSNTA YLQL SS LTSEDTAVYYC

[0039] VH-CDR3(SEQ NO.8): ARWDVNYGFAY

[0040] VH-FR4(SEQ NO.9): WGQGTL

[0041] VL-FR1(SEQ NO.10): DIQMTQTTSSLSASLGDRVTI SCR AS

[0042] VL-CDR1 (SEQ NO.11): QDIDNF

[0043] VL-FR2(SEQ NO.12): LNWFQQKPDGTVKLLIY

[0044] VL-CDR2 (SEQ NO.13) : YTS

[0045] VL-FR3(SEQ NO.14): RLHSGVPSRFSGSGSGTDYSL TIS NL EQEDIATYFC

[0046] VL-CDR3 (SEQ NO.15): QQANTLPWT

[0047] VL-FR4 (SEQ NO.16): FGGGTKLEIK

[0048] The monoclonal antibody provided by the invention has neutralizing activity, can bind to the African swine fever virus, and partially prevent the replication of the African swine fever virus in PAM cells.

[0049] The present invention also provides an anti-African swine fever I73R protein monoclonal antibody for establishing an African swine fever virus antibody detection blocking ELISA method.

[0050] The use of the African swine fever I73R protein monoclonal antibody described in the present invention in the preparation of drugs for preventing or treating African swine fever virus.

[0051] The principle of the African swine fever virus antibody detection blocking ELISA method is:

[0052] The African swine fever virus I73R recombinant protein is coated as an antigen, and then the serum sample to be tested is incubated, followed by the monoclonal antibody and the enzyme-labeled secondary antibody. If the serum sample to be tested is positive (contains antibodies), it will preferentially bind to the coated antigen to block the binding of the antigen to the monoclonal antibody of the present invention, thereby achieving a blocking effect. By calculating the blocking rate, the antibody level in the serum to be tested can be determined to determine whether it is positive. Compared with other ELISAs, this blocking ELISA detection method has higher specificity and sensitivity. Detection formula: Blocking rate = (negative serum OD 450 Value-serum OD to be tested 450 value) / negative serum OD 450 value × 100%‌‌.

[0053] Hybridoma cell line I73R-5D5 was deposited in the China Center for Type Culture Collection on November 25, 2024, with the deposit number CCTCC NO: C2024397, and the deposit address is Wuhan University, Wuhan, China. Beneficial Effects

[0054] The present invention provides a novel anti-African swine fever I73R protein monoclonal antibody and an antigenic epitope recognized by the monoclonal antibody, and the antibody sequence and the antigenic epitope sequence have not been reported in the literature; the monoclonal antibody can be used for African swine fever virus detection in Western blot, immunoprecipitation and indirect immunofluorescence experiments; the African swine fever virus I73R monoclonal antibody provided by the present invention can neutralize the infection of African swine fever virus; the monoclonal antibody can also be used for detecting the level of African swine fever virus antibodies in the serum of pigs infected with African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions implemented in the present disclosure, the following is a brief introduction to the drawings required for use in the implementation. The following drawings only illustrate certain implementations of the present disclosure and should not be considered as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0056] Figure 1This is an SDS-PAGE diagram for the expression verification of the recombinant protein of African swine fever virus I73R (wherein, M: protein marker; 1: pET-28a empty vector; 2: uninduced I73R protein; 3: induced I73R whole bacteria; 4: supernatant after centrifugation after bacterial crushing; 5: precipitate after centrifugation after bacterial crushing). The I73R gene sequence of the African swine fever virus type II CHINA / 2018 / AnhuiXCGQ strain was cloned into the pET-28a prokaryotic expression vector, and the recombinant plasmid was transformed into BL21 Escherichia coli competent cells, and its expression was induced with 1 mM IPTG.

[0057] Figure 2 The SDS-PAGE results for the purification and verification of the African swine fever virus I73R recombinant protein (M: protein marker; 1: unpurified I73R protein; 2: flow-through; 3-6: purified I73R protein). The I73R protein recombinant protein in the supernatant was purified using a His-tagged affinity chromatography column.

[0058] Figure 3 The results of serum antibody titer determination after mice were immunized with African swine fever virus I73R protein. The purified I73R protein was used as an antigen to immunize BALB / c mice, and the serum of the immunized mice was collected 14 days after the second immunization for antibody titer determination.

[0059] Figure 4 The SDS-PAGE results for purification verification of anti-I73R monoclonal antibody (M: protein marker; 1: unpurified antibody; 2: flow-through; 3-11: purified I73R antibody).

[0060] Figure 5 This is the Western blot identification result of anti-I73R monoclonal antibody. PAM cells were infected with African swine fever virus, and an uninfected cell group was set up. Cell samples were collected 24 hours later, and I73R-5D5 antibody was used as the primary antibody to verify its reactivity with I73R protein.

[0061] Figure 6 The immunoprecipitation experiment of anti-I73R monoclonal antibody was verified. PAM cells were infected with African swine fever virus for 24 h (A), or pcDNA4-2×Flag-I73R plasmid was overexpressed in 293T cells for 24 h (B). After collecting the cell lysate, I73R-5D5 antibody was used to verify its binding ability with I73R protein expressed in cells. Blank antibody goat anti-mouse IgG and Flag tag antibody were set as controls.

[0062] Figure 7Indirect immunofluorescence assay for anti-I73R monoclonal antibody identification. PAM cells were infected with African swine fever virus for 24 h, and an uninfected group was set up at the same time. I73R-5D5 was used as the primary antibody to verify whether it could recognize the stereo conformation of I73R protein expressed in PAM cells infected with African swine fever virus.

[0063] Figure 8 The results of antigen epitope identification of the monoclonal antibody against African swine fever virus I73R. A. Five I73R truncated proteins of different lengths were expressed; B. I73R-5D5 was used as the primary antibody and its recognition was verified by WB.

[0064] Fig. 9 This is a conservation analysis of this antigenic epitope in 10 genotypes of ASFV strains.

[0065] Fig.10 The results of the neutralization identification of anti-I73R monoclonal antibodies. After pre-incubation of monoclonal antibodies and African swine fever virus for 1 h at different ratios, the mixture was infected with PAM cells. At the same time, an uninfected group and a group without antibody were set up. Cell samples were collected after 36 h, and then anti-African swine fever p54 and p72 monoclonal antibodies were used as primary antibodies to verify the neutralization effect of the antibody on African swine fever virus infection.

[0066] Fig.11 The results show the specificity of the African swine fever virus blocking ELISA test based on anti-I73R monoclonal antibody. The blocking ELISA method was used to detect the positive sera of pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) to determine the specificity of the method. DETAILED DESCRIPTION

[0067] The following is a further detailed description of the claims of the present disclosure in conjunction with experiments and examples, but does not constitute any limitation to the present disclosure. Any limited modifications made within the scope of protection of the claims of the present disclosure are still within the scope of protection of the claims of the present disclosure. If no specific conditions are specified in the implementation method, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not specified, conventional products can be purchased commercially.

[0068] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0069] Test materials

[0070] African swine fever virus CHINA / 2018 / AnhuiXCGQ strain was from the National African Swine Fever Reference Laboratory, Chinese Center for Animal Health and Epidemiology (Qingdao, Shandong);

[0071] PAM cells (porcine alveolar macrophages) were prepared according to the literature that DDX19A senses viral RNA and mediates NLRP3-dependent inflammasome activation.

[0072] Example 1 Expression of African swine fever I73R protein recombinant protein

[0073] ASFV CHINA / 2018 / AnhuiXCGQ strain (GenBank: MK128995.1) was selected. I73R Sequence, using the ASFV genome as a template, adding the primers in Table 1 to amplify I73R The gene was verified by agarose gel electrophoresis and then recovered. The recovered product and the pET-28a vector were simultaneously purified by restriction endonuclease Bam HI and XOt I was used for double enzyme digestion. After the digestion products were recovered by nucleic acid electrophoresis, they were connected according to the molar ratio of target gene to vector of 2:1. After ligation at 16℃ for 6 h, the ligation products were added to 20 mL of Escherichia coli competent cells DH5a, ice-bathed for 30 min, heat-shocked at 42℃ for 1 min, and ice-bathed again for 2 min; then 200 mL of antibiotic-free LB was added, and cultured at 200 rpm in a 37℃ bacterial incubator for 30 min; the bacterial suspension was evenly spread on an LB agarose plate containing Kan+ resistance and cultured at 37℃ overnight; the next day, a single clone colony was picked and placed in 8 mL of LB containing Kan+ resistance, and cultured at 200 rpm in a 37℃ shaker overnight. Plasmid extraction was performed according to the instructions of the plasmid extraction kit, and the DNA concentration was determined by a spectrophotometer. 200 ng of plasmid was taken and used Bam HI and XOt I was used for double enzyme digestion identification. After verification by nucleic acid electrophoresis, the plasmid with the expected size of the target fragment and vector was selected for sequencing. The plasmid with the correct sequencing was named pET-28a-I73R.

[0074] The pET-28a-I73R prokaryotic expression plasmid with successful enzyme digestion and correct sequencing results was transferred into E. coli BL21 competent cells, spread on LB plates, and placed in a 37°C constant temperature incubator for overnight culture. When a single colony grows on the plate, pick a single colony and transfer it to LB medium containing kanamycin. Culture it at 37°C and 250 rpm for 16 h. Take 0.5 mL of the bacterial solution and transfer it to 50 mL of LB medium containing kanamycin. Continue to shake and culture for about 4 h until the OD 600 When the value was between 0.6 and 0.8, 2 mL of bacterial solution was taken as the negative control without induction, and IPTG with a final concentration of 1 mM was added to the remaining bacterial solution to induce expression at 37℃ 250 rpm for 5 h. The induced bacterial solution was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended with 20 mL PBS. 100 mL was mixed with an equal volume of 2×SDS loading buffer as the whole cell lysate sample. The remaining bacterial solution was ultrasonically broken, centrifuged at 4℃ 12000 rpm for 10 min, the supernatant and precipitate were collected respectively, and the precipitate was resuspended with an equal volume of PBS as the supernatant. 100 mL was taken and added with an equal volume of 2×SDS loading buffer as the supernatant and precipitate protein samples, and the samples were boiled at 96℃ for 10 min. Then, a polyacrylamide gel with a separation gel concentration of 15% was prepared, and the expression and solubility of I73R protein were identified by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining.

[0075] result:

[0076] like Figure 1 As shown, the I73R protein expressed after IPTG induction was about 13 KDa and was expressed in both the supernatant and inclusion bodies.

[0077] Example 2 Purification of African swine fever virus I73R recombinant protein

[0078] After centrifugation, the supernatant protein was filtered through a 0.22 mm filter and added to a Ni-NTA affinity column. The protein was incubated at 4°C for 2 h to allow the protein to fully bind to the affinity column. Then, two times the column volume of binding buffer was added to wash away unbound proteins and nonspecific impurities, and the flow-through samples were collected. Different concentrations of elution buffer were added to the purification column, and the eluted proteins were collected. 50 mL of each tube of eluted protein sample was added with an equal volume of 2×SDS loading buffer and placed in a 98°C sample cooker for 10 min. Then, a polyacrylamide gel with a separation gel concentration of 15% was prepared, and the purification of I73R protein was identified by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining.

[0079] result:

[0080] Figure 2 The results showed that the purified protein did not contain other impurities and had a high concentration and purity.

[0081] Example 3 Determination of serum titer after immunization of mice with African swine fever virus I73R recombinant protein

[0082] Blood was collected 2 weeks after the second immunization by tail amputation of mice, and the collected blood was placed in an incubator at 37°C for 1 h, and the upper serum was separated after centrifugation at 1000 rpm for 10 min. The purified I73R recombinant protein in Example 2 was coated at a concentration of 50 ng / well at 4°C overnight, and the serum to be tested was taken as the primary antibody, and multiple dilutions were made from 1:1000 to 1:128000. At the same time, negative serum was set as a control, and the titer of the serum of 4 mice immunized with I73R recombinant protein was detected by indirect ELISA.

[0083] result:

[0084] like Figure 3 As shown, after immunization with I73R recombinant protein, all four mice had high serum antibody titers, about 1:4000, reaching the standard for cell fusion. Mouse No. 2 with the highest titer was selected for the third intraperitoneal immunization, and a subsequent cell fusion experiment was performed one week after immunization.

[0085] Example 4 Preparation and purification of anti-I73R monoclonal antibody

[0086] The spleen cells of mouse No. 1 were mixed with myeloma cells SP2 / 0 at a ratio of 10:1, and PEG2000 was added to chemically fuse them. The fused cells were cultured in HAT selection medium for 10 d. The I73R protein purified in Example 2 was used as the coating antigen, and the supernatant secreted by the fused hybridoma cells was used as the primary antibody. The antibody content in the supernatant was detected by indirect ELISA to screen positive clones. The cells in the positive wells were resuspended, and subclone screening was performed 5 times by limiting dilution method, and finally a monoclonal hybridoma cell line that can secrete anti-I73R antibodies was obtained, which was named cell line I73R-5D5.

[0087] result:

[0088] Take 1×10 6 5D5 hybridoma cells were injected into the peritoneal cavity of BALB / c mice. About 10 days later, when the mouse abdomen was observed to be swollen, the mouse ascites was aspirated with a syringe, filtered and purified through a Protein A / G purification column. The resulting monoclonal antibody was named I73R-5D5. Figure 4 shown.

[0089] Example 5 Identification of subtypes of anti-I73R monoclonal antibody 5D5

[0090] The subtype of the I73R protein monoclonal antibody 5D5 strain was identified using a mouse monoclonal antibody subtype identification kit. The identification results showed that the heavy chain of the I73R-5D5 monoclonal antibody was IgG2b type and the light chain was κ chain as shown in Table 2.

[0091] Example 6 Western Blot Identification of I73R-5D5 Monoclonal Antibody

[0092] PAM cells were cultured at 2 × 10 6 / well were inoculated in a 6-well plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. When the cells attached to the wall and grew into a monolayer, they were inoculated with 1 MOI of African swine fever virus CHINA / 2018 / AnhuiXCGQ strain, and an uninfected group was set up at the same time and placed in a 37°C, 5% CO2 incubator for 36 hours. The cells were washed with PBS to remove the virus and culture medium mixture in the culture supernatant, and after the liquid was dried, 300 mL / well of 2×SDS sample buffer was added, and heated in a 98°C metal bath for 10 min for Western Blot detection.

[0093] result:

[0094] like Figure 5 As shown, the prepared I73R-5D5 monoclonal antibody can specifically bind to the I73R protein expressed in virus-infected cells and can be used in Western Blot experiments with good specificity.

[0095] Example 7 Identification of IP Reactivity of I73R-5D5 Monoclonal Antibody

[0096] 6 × 10 6PAM cells were placed in a cell culture incubator at 37°C and 5% CO2 overnight. When the cells attached to the wall and grew into a monolayer, they were inoculated with 1 MOI of African swine fever virus CHINA / 2018 / AnhuiXCGQ strain and continued to be placed in a 37°C and 5% CO2 incubator for 24 hours. The cells were washed with PBS to remove the virus and culture medium mixture in the culture supernatant. After the liquid was dried, 800 mL of cell lysis buffer was added. After lysis on ice for 3 min, the cell lysate was collected. The cells were disrupted with an ultrasonic disruptor, 1 s on, 0.5 s off, and ultrasonic for 30 s. The disrupted cell lysate was centrifuged at 8000 rpm for 10 min. The supernatant after centrifugation was separated, and 30 mL was mixed with an equal volume of 2×SDS sample buffer as the whole cell lysate sample. The remaining cell lysate was divided into two parts, and 15 mL of Protein A / G magnetic beads and 1 mg of I73R-5D5 monoclonal antibody or goat anti-mouse IgG antibody were added to each part, and incubated at 4°C overnight. The next day, the cell lysate-antibody mixture was discarded, and the magnetic beads were washed 10 times with cell lysate to remove non-specifically bound antigens and antibodies and unbound proteins. Finally, the antigen-antibody complex attached to the magnetic beads was eluted with 50 mL of 2×SDS sample buffer, and after heating in a 98°C metal bath for 10 min, Western Blot detection was performed.

[0097] 1.6×10 6293T cells were cultured overnight in a cell culture incubator at 37°C and 5% CO2. When the cells attached to the wall and grew into a monolayer, 3 mg of pcDNA4-2×Flag-I73R plasmid was transfected in each culture dish and continued to be placed in a 37°C and 5% CO2 incubator for 24 hours. After discarding the supernatant, wash the cells twice with PBS. After absorbing the PBS, add 800 mL of cell lysis buffer to each culture dish and place it on ice for 3 min. Disrupt the cells with an ultrasonic disruptor, 1 son, 0.5 s off, and ultrasonic for 30 s. Centrifuge the disrupted cell lysate at 8000 rpm for 10 min. Separate the supernatant after centrifugation, take 30 mL and mix it with an equal volume of 2×SDS sample buffer as the whole cell lysate sample. Divide the remaining cell lysate into two parts, add 15 mL of Protein A / G magnetic beads and 1 mg of I73R-5D5 monoclonal antibody or goat anti-mouse IgG antibody to each part, and set the Flag antibody group as a positive control, and incubate it at 4°C overnight. The next day, discard the cell-antibody mixture, wash the magnetic beads 10 times with cell lysis buffer to remove non-specifically bound antigens and antibodies and unbound proteins. Finally, elute the antigen-antibody complex attached to the magnetic beads with 50 mL of 2×SDS sample buffer, and perform Western Blot detection after heating in a 98°C metal bath for 10 min.

[0098] result:

[0099] Figure 6 The prepared I73R-5D5 monoclonal antibody showed good IP reactivity and could infect virus-infected cells ( Figure 6 A) and overexpressing cells ( Figure 6 The I73R protein in B) is precipitated and can be used for immunoprecipitation and co-immunoprecipitation experiments with good specificity.

[0100] Example 8 IFA Identification of I73R-5D5 Monoclonal Antibody

[0101] PAM cells were cultured at 2 × 10 6 / well was added to a 6-well plate with a slide, and placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. When the cells adhered to the wall and grew into a monolayer, the African swine fever virus CHINA / 2018 / AnhuiXCGQ strain was inoculated with 1 MOI, and an uninfected group was set up at the same time, and incubated at 37°C and 5% CO2 for 36 hours. The cells were washed with PBS and then fixed with paraformaldehyde fixative at 37°C for 30 min. The fixative was discarded, and after washing with PBS three times, a PBS blocking solution containing 3% BSA was added, and the cells were blocked at 37°C for 30 min. The blocking solution was discarded, and the purified 5D5 monoclonal antibody was added at a final concentration of 10 mg / mL, and incubated at 37°C for 1 h. The primary antibody was recovered, and the cells were washed with PBS three times, each time on a shaker for 10 minutes. FITC-labeled goat anti-mouse IgG was added at a dilution of 1:500 and incubated at 37°C for 30 min. Discard the secondary antibody, wash the cells 3 times with PBS, and shake on a shaker for 10 min each time. Add DAPI solution for visualization of cell nuclei, seal the slides and observe under a fluorescence microscope.

[0102] result:

[0103] like Figure 7 As shown in the figure, in ASFV-infected cells, the prepared anti-I73R monoclonal antibody 5D5 can bind to ASFV-infected cells and produce specific fluorescence, while no specific fluorescence was found in the cells of the uninfected group. This result shows that the I73R-5D5 antibody can bind to the three-dimensional conformation of the I73R protein and can be used in subsequent IFA experiments with good specificity.

[0104] Example 9 Identification of the Antigenic Epitope Recognized by the Anti-I73R Monoclonal Antibody

[0105] Based on the full-length sequence of I73R protein shown in SEQ ID NO.1, five truncated I73R recombinant fragments were designed: I73R-N1 (1-55aa), I73R-N2 (1-40aa), I73R-C1 (11-72aa), I73R-C2 (21-72aa) and I73R-C3 (31-72aa).

[0106] result:

[0107] Figure 8 A in the figure shows the schematic diagram of the design of I73R truncation and the identified epitopes. The five truncation gene fragments were cloned into the pET-28a vector, and the five recombinant truncation proteins were induced to express by the E. coli expression system; the expression of truncation proteins was detected by Western blot experiments using His tag antibodies and I73R-5D5 antibodies;

[0108] like Figure 8 As shown in B, all truncated proteins can be recognized by His antibodies, and I73R-5D5 monoclonal antibody can recognize the expression of the remaining truncations except I73R-C3, indicating that the antigen epitope recognized by I73R-5D5 is located at I73R (21-31aa).

[0109] like Fig. 9 As shown, the antigen epitope sequence in different genotype strains was compared, and the sequence was highly conserved among different strains.

[0110] Example 10 Identification of the neutralizing activity of anti-I73R monoclonal antibodies

[0111] PAM cells were cultured at 4 × 10 5 / well was added to a 12-well plate and placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. The purified monoclonal antibody in Example 4 was mixed with 1 MOI of African swine fever virus CHINA / 2018 / AnhuiXCGQ strain in different proportions and placed in a 37°C incubator for pre-incubation for 1 h, and then the virus-antibody mixture was added to the PAM cells that had grown into a monolayer and placed in a 37°C, 5% CO2 incubator for 36 hours. The cell supernatant was discarded, and the cells were washed with PBS to remove the virus, antibody and culture medium mixture, and PBS was discarded. 200 mL / well of 2×SDS sample buffer was added, and Western Blot detection was performed after heating in a 98°C metal bath for 10 min.

[0112] result:

[0113] like Fig.10 As shown, the I73R-5D5 monoclonal antibody has neutralizing ability, neutralizes part of the activity of African swine fever virus, and partially inhibits the infection of African swine fever virus.

[0114] Example 11 Preliminary establishment of an ELISA antibody detection method based on anti-I73R monoclonal antibody

[0115] The purified I73R recombinant protein in Example 2 was coated on the ELISA plate at 4°C overnight, and the coating solution was discarded and blocked with 3% skim milk at 37°C for 1 h. Then, 10-fold diluted African swine fever negative and positive sera to be tested were added and incubated for 1 h. Then, 600 mg / mL I73R-5D5 monoclonal antibody (1:16000-1:128000 dilution) and enzyme-labeled secondary antibody were added, and OD was measured after color development. 450 The numerical value of .

[0116]

[0117] result:

[0118] Table 3 shows that compared with the control group, the I73R-5D5 monoclonal antibody can be used in the blocking ELISA experiment for African swine fever virus antibody detection, with a maximum blocking rate of about 40%.

[0119] Example 12 Investigation of the specificity of anti-I73R monoclonal antibodies

[0120] The specificity of the African swine fever virus blocking ELISA test based on anti-I73R monoclonal antibody was determined by detecting positive sera for African swine fever virus (ASFV), pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV).

[0121] result:

[0122] like Fig.11 As shown, only the inhibition rate of ASFV was higher than 50%, while the inhibition rate of other viruses was lower than 15%, indicating that this method is specific for African swine fever virus (ASFV).

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against African swine fever virus I73R protein, characterized in that: The monoclonal antibody is produced by a hybridoma cell with a deposit number of CCTCC NO: C2024397.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of the heavy chain variable regions VH-CDR1, VH-CDR2 and VH-CDR3 of the monoclonal antibody are shown in SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8 respectively; the light chain variable regions are VL-CDR1, VL-CDR2 and VL-CDR3, and the amino acid sequences of them are shown in SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.15 respectively.

3. The monoclonal antibody according to claim 1, characterized in that The heavy chains of the monoclonal antibody are VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and their amino acid sequences are shown in SEQ ID NO.3 to SEQ ID NO.9, respectively; the light chains are VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4, and their amino acid sequences are shown in SEQ ID NO.10 to SEQ ID NO.16, respectively.

4. The monoclonal antibody according to any one of claims 1 to 3, characterized in that The heavy chain of the monoclonal antibody is of IgG2b subtype, and the light chain is of kappa type.

5. An antigen epitope peptide specifically recognized by the monoclonal antibody according to any one of claims 1 to 4, characterized in that: The antigen epitope is located at positions 21 to 30 of the I73R protein of the African swine fever virus, and its amino acid sequence is shown in SEQ ID NO.

2.

6. Use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of a reagent for detecting African swine fever virus.

7. An ELISA kit for detecting African swine fever virus, characterized in that: The kit comprises the monoclonal antibody according to any one of claims 1 to 4.

8. Use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating African swine fever virus.

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