Anti-idiotypic antibody for foot-and-mouth disease virus and its application

Abstract

The present invention discloses an anti-idiotypic antibody for foot-and-mouth disease virus and its application. The amino acid sequence of the anti-idiotypic antibody is shown in SEQ ID No. 1. The present invention utilizes single B cell antibody technology to screen and obtain an anti-idiotypic antibody for foot-and-mouth disease virus, which can truly simulate the FMDV antigen epitope, block the binding of neutralizing antibodies to antigens, and has the characteristics of anti-idiotypic antibody Ab2β. The anti-idiotypic antibody molecule for foot-and-mouth disease virus can induce the body to produce FMDV-specific broad-spectrum antibodies after immunizing animals, and the antibodies produced have binding activity to both type O FMDV and type A FMDV. The anti-idiotypic antibody molecule for foot-and-mouth disease virus provides foot-and-mouth disease virus antigen epitopes and can be used for the design of FMDV broad-spectrum vaccine molecules and the development of new diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-idiotypic antibody for foot-and-mouth disease virus and application thereof. Background Art

[0002] Foot-and-mouth disease virus (FMDV) is a pathogenic microorganism that infects even-toed ungulates. The foot-and-mouth disease (FMD) it causes restricts international trade in animals and animal products, severely impacting the livestock industry. FMDV is classified into seven serotypes, each of which is further divided into distinct topological types, lineages, and genetic clades. Vaccination is the most effective method for protecting livestock from FMDV infection, but cross-protection between FMDV serotypes is generally considered to be lacking. However, expanding the scope of vaccine cross-protection and developing broad-spectrum vaccines presents significant challenges. The identification of broadly neutralizing epitopes across serotypes is fundamental to the design of broad-spectrum vaccine antigen molecules. Anti-idiotypic antibodies (AId or Ab2) are tools that mimic and display broadly conformational neutralizing epitopes. Ab2s, generated against idiotypic epitopes in the variable regions of broadly neutralizing antibodies (Ab1), can be categorized into three types, Ab2α, β, and γ, based on their recognition sites. Ab2β, known as an "internal image" of the antigen, can recognize and mimic the specific region where the antibody (Ab1) binds to the original antigen. Developing novel anti-idiotypic antibody vaccines using Ab2β as an antigen surrogate is an innovative and effective strategy. Nanobodies, found in camelids, offer advantages such as small size, high affinity, excellent specificity, low cost, and ease of mass production, making them an important tool for screening conformational epitopes displayed by Ab2β. Summary of the Invention

[0003] The purpose of the present invention is to provide an anti-idiotypic antibody for foot-and-mouth disease virus and its application.

[0004] The present invention provides an anti-idiotypic antibody of foot-and-mouth disease virus, and an amino acid sequence of ZC3 of the anti-idiotypic antibody of foot-and-mouth disease virus.

[0005] EVQLAESGGGLVQAGGSLRLSCAPSGRTFSTYAMGWYRQAPGKEREFVAAITRSGDSTYYADSVKGRFTISRDNAENTVYLQMNSLKPGDTAVYYCSVGVFEGNGMGYVVNTWGQGTLVTVSS (SEQ ID No. 1).

[0006] The anti-idiotypic antibody ZC3 of foot-and-mouth disease virus consists of four framework regions (FR): FR1, FR2, FR3, and FR4;

[0007] Amino acid sequence of FR1: EVQLAESGG.GLVQAGGSLRLSCAPS (SEQ ID No. 2)

[0008] Amino acid sequence of FR2: MGWYRQAPGKEREFVAA (SEQ ID No. 3)

[0009] Amino acid sequence of FR3: YYADSVKGRFTISRDNAENTVYLQMNSLKPGDTAVYYC (SEQ ID No. 4)

[0010] Amino acid sequence of FR4: WGQGTLVTVSS (SEQ ID No. 5)

[0011] The anti-idiotypic antibody ZC3 against foot-and-mouth disease virus consists of three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3;

[0012] Amino acid sequence of CDR1: GRTFSTYA (SEQ ID No. 6)

[0013] Amino acid sequence of CDR2: ITRSGDST (SEQ ID No. 7)

[0014] The amino acid sequence of CDR3 is: SVGVFEGNGMGYVVNT (SEQ ID No. 8).

[0015] The present invention also provides the use of the anti-idiotypic antibody molecule ZC3 of foot-and-mouth disease virus in the preparation of foot-and-mouth disease virus vaccine. The anti-idiotypic antibody of foot-and-mouth disease virus can be used as an antigen epitope, and simultaneously simulates and displays the conserved epitopes of type O and type A foot-and-mouth disease virus. Immunized animals can induce the body to produce a broad-spectrum antibody response, which can be used for the design of a broad-spectrum vaccine molecule for foot-and-mouth disease virus.

[0016] The anti-idiotypic antibodies of the present invention can also be used to prepare reagents or kits for detecting antibodies to the foot-and-mouth disease virus. The anti-idiotypic antibodies can be used as diagnostic antigens to establish an ELISA method for detecting specific antibodies to the foot-and-mouth disease virus; or combined with the monoclonal antibody pOA-2 to establish a blocking ELISA method for detecting specific antibodies to the foot-and-mouth disease virus.

[0017] The present invention has obtained an anti-idiotypic antibody molecule for foot-and-mouth disease virus that can realistically mimic FMDV antigenic epitopes, blocking the binding of neutralizing antibodies to the antigen, and exhibiting the characteristics of the anti-idiotypic antibody Ab2β. After immunization of animals, this anti-idiotypic antibody molecule can induce the production of broad-spectrum FMDV-specific antibodies, which have binding activity against both type O FMDV and type A FMDV. This anti-idiotypic antibody molecule provides an epitope for the foot-and-mouth disease virus and can be used in the design of broad-spectrum FMDV vaccines and the development of novel diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE was used to examine the expression and purification of the antibody POA-2 and POA-2 scFv;

[0019] Figure 2 Indirect ELISA was used to detect POA-2 specific antibodies in alpaca serum;

[0020] Figure 3 POA-2-specific monoclonal B cells were isolated by flow cytometry;

[0021] Figure 4 VHH gene amplification;

[0022] Figure 5 SDS-PAGE analysis of anti-idiotypic nanobody expression and purification;

[0023] Figure 6 Indirect ELISA was used to detect the reactivity of nanobodies with POA-2 scFV;

[0024] Figure 7 Blocking ELISA was used to detect the blocking effect of nanoantibodies on POA-2 scFV binding to FMDV O / Tibet99;

[0025] Figure 8 Indirect ELISA was used to detect FMDV-specific antibodies in mouse serum. DETAILED DESCRIPTION

[0026] The present invention will be further described below through specific embodiments.

[0027] 293-F cells and FMDV strains (O / Tibet / 99, A / AF72) are all preserved by the National Foot-and-Mouth Disease Reference Laboratory; the recombinant expression plasmids of the heavy chain, light chain and single-chain antibody of the broad-spectrum neutralizing porcine monoclonal antibody POA-2 against foot-and-mouth disease virus types O and A are constructed and preserved by the Host Antiviral Infection and Immunobiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0028] Lymphocyte separation medium (Histopaque-1083, 1.083 g / mL), Freund's complete adjuvant, and incomplete adjuvant were purchased from Sigma-Aldrich. The biotin labeling kit (EZ-Link™ Sulfo-NHS-LC-Biotin) was purchased from Invitrogen. SMM293-TII medium and SMS293-SUPI feed were purchased from Beijing Sino-Bio Science & Technology Co., Ltd. The transfection reagent PEI was purchased from Polysciences. The reverse transcription kit (iScript cDNA Synthesis Kit) was purchased from BIO-RAD. The RNase inhibitor (RNasin® Plus RNase Inhibitor) was purchased from Promega. The PCR amplification enzyme (LA Taq) was purchased from TaKaRa. The horseradish peroxidase (HRP)-conjugated Maus anti-Llama IgG2 / IgG3 antibody was purchased from Antikoerper. Horseradish peroxidase (HRP)-labeled goat-anti-mouse antibody was purchased from GenScript. An endotoxin-free plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd. ISA201 adjuvant was purchased from Sepico (Shanghai) Specialty Chemicals Co., Ltd.

[0029] Example 1 Preparation of anti-idiotypic antibody molecules against foot-and-mouth disease virus

[0030] 1. Preparation of broadly neutralizing porcine monoclonal antibodies against foot-and-mouth disease virus types O and A

[0031] The variable region sequence and preparation method of pOA-2, a broadly neutralizing porcine monoclonal antibody against foot-and-mouth disease virus types O and A, are available in CN202410667088.6. The heavy chain variable region (VH) and light chain variable region (VL) of pOA-2 were connected using the flexible linker "GSSSSGSSSSGSSSS." The synthesized gene sequence was optimized with reference to human codons and inserted into the pcDNA3.4 expression vector to produce the single-chain antibody recombinant expression plasmid POA-2 scFV.

[0032] The amino acid sequence of the heavy chain variable region of the monoclonal antibody pOA-2 is:

[0033] MEFRLNWVVLFALLQGVQGEEKVVESGGGLVQPGGSLRLSCVGSGFNFKNYEINWVRQAPGKALEWLAYITQTSDFIYYADSVKGRFTISRDNSRNTAYLQMNNLRTEDTARYFCTRAGLTGCKSRHCMYVWGPGAEVVV (SEQ ID No. 9).

[0034] The amino acid sequence of the light chain variable region of the monoclonal antibody pOA-2 is:

[0035] MAWTVLLIGLLAVGSGVDSQTVIQEPAMSVSLGGTVTLTCGFISGSVTGTNYPSWFQQTPGQPPRLLIYYANSRPTEVPSRFSGAISGNKAALTITGAQAEDEADYFCCLYKTNNNILFGGGTHLTVLGQPKAAPTVNLFPPSSEELGTNK (SEQ ID No. 10).

[0036] Recombinant expression plasmids for the antibody heavy chain, light chain, and single-chain antibody (scFv) were transformed into competent E. coli JM109 cells for amplification. Single positive colonies were selected and grown overnight in Amp-resistant LB medium. They were extracted and purified using an endotoxin-free plasmid extraction kit (TIANGEN). Heavy chain / light chain plasmids were co-transfected at a 2:3 mass ratio, and single-chain antibody (scFv) was transfected separately into 293-F cells using polyethyleneimine (PEI). After 7–8 days of culture, the cell supernatant was harvested and purified by affinity chromatography using an AKTA protein purifier. Antibody expression was verified by SDS-PAGE.

[0037] The POA-2 and POA-2 scFV antibody plasmids were expressed in 293-F cells, purified by affinity chromatography, and the expressed antibodies were verified by SDS-PAGE. Figure 1 ), porcine IgG molecules and single-chain antibody molecules were successfully expressed. The molecular weight of the heavy chain was about 50 KDa, and the molecular weight of the light chain was about 25 KDa. The single-chain antibody had only one band, indicating that the linker connecting the heavy chain and light chain was not broken and was located between 25 and 35 kDa.

[0038] 2. Alpaca Immunity

[0039] Purified POA-2 and POA-2 scFV were emulsified with ISA 201 adjuvant at a 1:1 ratio and monitored the next morning for successful emulsification. A healthy three-year-old male alpaca, designated #1807, was immunized four times with the emulsified antibodies. The first three immunizations were separated by 21 days, and the fourth immunization was performed 5-7 days before flow cytometry sorting of POA-2-specific single B cells. The first immunization was with POA-2, and the second through fourth immunizations were with POA-2 scFV. The immunization dose was 300 µg each, administered intramuscularly in the buttocks. Before each immunization, blood was collected from the jugular vein of the alpaca, incubated at 37°C for 1 hour, then transferred to 4°C overnight. The serum was centrifuged at 3000 rpm for 15 minutes, and stored frozen at -20°C.

[0040] 3. POA-2 specific antibody detection

[0041] Purified POA-2 scFV was used as the coating antigen to detect the antibody titer of alpaca serum. The specific steps are as follows:

[0042] (1) Coating

[0043] POA-2 scFV was diluted to 5 µg / mL in carbonate buffer and coated on the ELISA plate at 100 µL / well at 4°C for 16 h.

[0044] (2) Closed

[0045] Wash the plate five times with PBST and pat dry. Block the plate by adding 200 μL of PBS solution containing 5% sucrose and 1% BSA to each well at 37°C for 1 h. Wash the plate five times with PBST and air dry.

[0046] (3) Incubation with alpaca serum

[0047] The serum to be tested was diluted 2-fold from 1:80 on a serum dilution plate, added to the ELISA plate at 100 μL / well, and incubated at 37°C for 1 h. At the same time, alpaca pre-immune serum was used as a negative control and PBS was used as a blank control.

[0048] (4) Incubation with Maus anti-Llama IgG2 / IgG3 HRP

[0049] Wash the plate five times with PBST, pat dry, and add 2.5 µg / mL diluted Maus anti-Llama IgG2 / IgG 3 HRP (100 µL / well) and incubate at 37°C for 30 min.

[0050] (5) Color rendering

[0051] Wash the plate five times with PBST and pat dry. Mix TMB substrate solution A and solution B at a 1:1 ratio, add 100 µL / well of the mixed substrate solution, and incubate at 37°C for 15 minutes for color development.

[0052] (6) Termination and reading

[0053] After the color development reaction is completed, add 100 μL / well of stop solution to terminate the reaction and read the OD value on a microplate reader. 450 nm value. S / CO method statistics, S is the sample OD 450nm value, CO is the CutOff value (positive judgment value), CO=2.1×N (N is the negative control D value).

[0054] In addition to traditional IgG antibodies, camelids also possess novel heavy-chain antibodies, IgG2 and IgG3, which are important sources for nanobodies. Indirect ELISA results (Figure 2) show that the titer of POA-2-specific IgG2 and IgG3 in alpaca serum is 1:2560.

[0055] 4. Isolation of POA-2-specific B cells

[0056] Five days after immunization with POA-2, a broadly neutralizing porcine monoclonal antibody against foot-and-mouth disease virus types O and A, anti-coagulated blood was collected from alpacas. PBMCs were isolated according to the instructions for lymphocyte separation medium. An appropriate amount of 1640 medium was used to resuspend the PBMCs in a centrifuge tube. Biotinylated POA-2 (0.1 µg) was added and incubated on ice for 25 minutes. A FMO control (no biotinylated POA-2) was also prepared. After washing twice, anti-biotin APC and anti-camelid VHH (PE) antibodies were added and incubated on ice for 25 minutes. The cells were then washed and resuspended in 1640 medium for sorting of POA-2-specific B cells. A BD FACSAria II flow cytometer was used. Instrument parameters were adjusted according to the instrument's instructions. The single-cell sorting mode was selected and cells were aliquoted onto a 96-well PCR plate. The plate was adjusted to ensure that cells were accurately centered in the wells before loading. First, monocytes and lymphocytes were circled using the FSC-A and SSC-A settings. Adherent cells were then excluded using the FSC-A and FSC-H settings. Finally, single cells were circled. The sorted POA-2-specific single B cells were aliquoted into 96-well PCR plates pre-loaded with a reverse transcription system.

[0057] Five days after immunization with POA-2, a broadly neutralizing porcine monoclonal antibody against foot-and-mouth disease virus type O and type A, anticoagulated blood was collected from alpacas to isolate PBMCs for staining and flow cytometry sorting. 5 PBMCs were collected to analyze the distribution and proportion of POA-2-specific B cells. First, a P1 gate was drawn based on FSC-A and SSC-A to enclose lymphocytes and monocytes ( Figure 3 A), then draw the P2 gate based on FSC-A and FSC-H to circle out single cells ( Figure 3 B). Select the PE fluorescence channel and draw the P3 gate to circle the IgG2 / IgG3 positive cell population ( Figure 3 C) Based on the FMO control, the P4 gate was used to delineate the POA-2-specific cell population ( Figure 3 D). Flow cytometry results showed that POA-2-specific cells accounted for approximately 0.23% of the total PBMCs. POA-2-specific single B cells within gate P4 were sorted.

[0058] 5. Single-cell cDNA Preparation and Nested PCR Amplification

[0059] Prepare the reverse transcription system using the BIO-RAD reverse transcription kit according to the reagents listed in Table 1. Add the sorted POA-2-specific single B cells and gently shake to mix. Centrifuge at 3000 rpm for 5 minutes at 4°C, then place in a PCR instrument for reverse transcription amplification. Set the PCR program to: 25°C for 5 minutes, 45°C for 45 minutes, and 85°C for 5 minutes. The resulting cDNA is used for subsequent PCR amplification.

[0060]

[0061] The alpaca heavy chain antibody VHH gene was amplified using nested PCR. First, the cDNA was used as a template for the first round of amplification, using the Call01F / R primers listed in Table 2. Subsequently, in the second round of amplification, the product from the first round of amplification was used as a template for PCR amplification using the ARMcamel FR1F / FR4R primers listed in Table 2. After two rounds of PCR amplification, the PCR product of the alpaca heavy chain antibody VHH gene was successfully obtained. The nested PCR reaction system and amplification procedure are shown in Tables 3, 4, 5, and 6, respectively. After the second round of PCR amplification, 8 µL of the amplified product was subjected to electrophoresis on a 1.5% agarose gel to analyze the amplification results. The remaining amplified product was sent to Xi'an Sangon Biotechnology Co., Ltd. for DNA sequencing.

[0062]

[0063] Note: Degenerate base annotations in the sequence, W = A or C or T; Y = C or T

[0064] Note: Degenerate base annotation in the sequence, W= A or C or T; Y=C or T

[0065]

[0066]

[0067]

[0068]

[0069] The POA-2 specific B cells were sorted and IgG2 / 3 VHH genes were amplified by reverse transcription and nested PCR. The amplification results showed that ( Figure 4 The VHH gene electrophoresis band was approximately 450 bp, consistent with expectations. The sequencing results were uploaded to the IMGT database for comparison, and seven VHH genes were selected for subsequent expression vector construction.

[0070] 6. Expression plasmid construction and antibody expression and purification

[0071] Based on the sequencing results of the amplified sequences, the signal peptide "MELGLSWVVLAALLQGVQA" was added to the front of the VHH gene that met the criteria. Codon optimization was then performed to improve expression efficiency in 293-F vectors. The optimized gene fragment was inserted into the pcDNA3.4 expression vector using the BamHⅠ and BbVcⅠ restriction sites. To facilitate subsequent purification and identification, a 6×His tag and a Myc tag were added to the end. The recombinant expression plasmid was transformed into competent Escherichia coli JM109 cells for amplification. A single positive colony was picked and grown overnight in Amp-resistant LB medium. The colony was extracted and purified using an endotoxin-free plasmid extraction kit (TIANGEN). The plasmid was transfected into 293-F cells using PEI transfection reagent. After 7-8 days of culture, the cell supernatant was harvested and purified by affinity chromatography using an AKTA protein purifier. Antibody expression was verified by SDS-PAGE.

[0072] Seven antibody expression plasmids were transfected into 293-F cells for expression, and the supernatant was collected. After affinity chromatography purification, the expressed antibodies were verified by SDS-PAGE. The results showed that ( Figure 5 ), a band of the expected size appeared at around 15 kDa, and 7 nanobodies were successfully expressed.

[0073] Example 2 Indirect ELISA to detect the reactivity of anti-idiotypic antibodies

[0074] Purified POA-2 scFV was used as the coating antigen to detect the reactivity of anti-idiotypic nanobodies. The specific steps are as follows:

[0075] (1) Coating

[0076] POA-2 scFV was diluted to 5 µg / mL in carbonate buffer and coated on the ELISA plate at 100 µL / well at 4°C for 16 h.

[0077] (2) Closed

[0078] Wash the plate five times with PBST and pat dry. Block the plate by adding 200 μL of PBS solution containing 5% sucrose and 1% BSA to each well at 37°C for 1 h. Wash the plate five times with PBST and air dry.

[0079] (3) Incubation of anti-idiotypic nanoantibodies

[0080] The antibody to be tested was diluted 2-fold downward at 20 μg / mL on a serum dilution plate, and 100 μL / well was added to the ELISA plate and incubated at 37°C for 1 h. PBS was also set as a negative control.

[0081] (4) Incubate with THE™ c-Myc Antibody

[0082] Wash the plate five times with PBST, pat dry, and incubate with 100 µL / well of THE™ c-Myc Antibody at a dilution of 1:1500 at 37°C for 30 min.

[0083] (5) Color rendering

[0084] Wash the plate five times with PBST and pat dry. Mix TMB substrate solution A and solution B at a 1:1 ratio, add 100 µL / well of the mixed substrate solution, and incubate at 37°C for 15 minutes for color development.

[0085] (6) Termination and reading

[0086] After the color development reaction is completed, add 100 μL / well of stop solution to terminate the reaction and read the OD value on a microplate reader. 450 nm value. S / CO method statistics, S is the sample OD 450 nm value, CO is the CutOff value (positive judgment value), CO=2.1×N (N is the negative control D value).

[0087] Indirect ELISA results showed that ( Figure 6 ), among the POA-2-specific antibodies sorted by flow cytometry, only one strain, ZC3, showed good reactivity to POA-2scFV, while the other antibodies had no reactivity.

[0088] Example 3 Blocking ELISA to detect the effect of anti-idiotypic antibodies blocking POA-2

[0089] The bovine monoclonal antibody E32 from our laboratory was used as the capture antibody to indirectly coat FMDV O / Tibet / 99, and the biotinylated antibody POA-2 scFV was used as the detection antibody to test the blocking effect of the anti-idiotypic nanobody. First, the optimal dilution ratio of the biotinylated antibody POA-2 scFV was titrated downwards by 1:1000 using indirect ELISA. 450 The dilution ratio when nm is about 2.0 is used as the dilution ratio for blocking ELISA. The steps of blocking ELISA are as follows:

[0090] (1) Coated capture antibody E32

[0091] E32 was diluted to 0.5 µg / mL in carbonate buffer and coated on the ELISA plate at 100 µL / well at 4°C for 16 h.

[0092] (2) Capture antigen

[0093] Wash the plate five times with PBST and pat dry. Dilute FMDV O / Tibet / 99 at 1 µg / mL and add 100 µL / well to the plate. Capture the plate at room temperature for 2 h.

[0094] (3) Closed

[0095] Wash the plate five times with PBST and pat dry. Block the plate by adding 200 μL of PBS solution containing 5% sucrose and 1% BSA to each well at 37°C for 1 h. Wash the plate five times with PBST and air dry.

[0096] (4) Co-incubation of anti-idiotypic nanoantibodies and biotin-labeled antibodies

[0097] The test antibody was diluted in two-fold increments at 20 µg / mL on a serum dilution plate. Titrated biotinylated POA-2 scFV was added, mixed, and incubated on the serum dilution plate at 37°C for 1 hour. 200 µL of the mixture was added to the ELISA plate at each well and incubated at 37°C for 1 hour. PBS was also used as a negative control.

[0098] (5) Incubation with secondary antibody

[0099] Wash the plate five times with PBST and pat dry. Dilute HRP-labeled streptavidin antibody at a dilution of 1:30,000, add 100 µL / well, and incubate at 37°C for 15 min.

[0100] (6) Color rendering

[0101] Wash the plate five times with PBST and pat dry. Mix TMB substrate solution A and solution B at a 1:1 ratio, add 100 µL / well of the mixed substrate solution, and incubate at 37°C for 15 minutes for color development.

[0102] (7) Termination and reading

[0103] After the color development reaction is completed, add 100 μL / well of stop solution to terminate the reaction and read the OD value on a microplate reader. 450 nm value. Blocking rate calculation method: 100% × (negative control OD 450 nm-OD of the sample to be tested 450 nm) / negative control OD 450 nm.

[0104] Blocking ELISA results showed that ( Figure 7 Of the seven nanoantibodies, only ZC3 could block the binding of POA-2 scFV to FMDV / Tibet / 99, and exhibited a strong blocking effect. The blocking rate reached 50% at a concentration of 0.025 µg / mL. The remaining antibodies had no ability to block the binding of POA-2 scFV to FMDV antigens.

[0105] Example 4 Animal experiments to evaluate its immunogenicity

[0106] 1 Mouse immunization procedure

[0107] To further validate the immunogenicity of anti-idiotypic nanobodies, animal experiments were conducted. The resulting nanobody, designated ZC3, was used to immunize 15 6-8 week-old Balb / c female mice, along with a PBS negative control. The primary immunization was performed with Freund's complete adjuvant, while the secondary and tertiary immunizations were emulsified with Freund's incomplete adjuvant. Immunizations were separated by 21 days, with a 50 µg dose. Eyeballs were enucleated and blood was collected 21 days after the first, second, and third immunizations. Five mice were sacrificed. After the blood was allowed to rest overnight at 4°C, it was centrifuged at 3000 rpm for 8 minutes to separate the serum.

[0108] 2. Detection of FMDV-specific antibodies in mouse serum by indirect ELISA

[0109] FMDV O / Tibet99 and A / F72 were used as coating antigens to detect FMDV-specific antibodies in mouse serum. The specific steps are as follows:

[0110] (1) Coating

[0111] FMDV O / Tibet / 99 and A / AF72 were diluted to 1 μg / mL in carbonate buffer and coated on ELISA plates at a rate of 100 μL / well at 4°C for 16 h.

[0112] (2) Closed

[0113] Wash the plate five times with PBST and pat dry. Block the plate by adding 200 μL of PBS solution containing 5% sucrose and 1% BSA to each well at 37°C for 1 h. Wash the plate five times with PBST and air dry.

[0114] (3) Incubation with mouse serum

[0115] The serum to be tested was diluted 2-fold from 1:20 on a serum dilution plate, added to the ELISA plate at 100 μL / well, and incubated at 37°C for 1 h. At the same time, mouse pre-immune serum was set as a negative control and PBS was set as a blank control.

[0116] (4) Incubation with Goat-anti-Mouse HRP

[0117] Wash the plate five times with PBST, pat dry, and incubate with Goat-anti-Mouse HRP diluted 1:10,000, 100 µL / well, at 37°C for 1 h.

[0118] (5) Color rendering

[0119] Wash the plate five times with PBST and pat dry. Mix TMB substrate solution A and solution B at a 1:1 ratio, add 100 µL / well of the mixed substrate solution, and incubate at 37°C for 15 minutes for color development.

[0120] (6) Termination and reading

[0121] After the color development reaction is completed, add 100 μL / well of stop solution to terminate the reaction and read the OD value on a microplate reader. 450 nm value. S / CO method statistics, S is the sample OD 450 nm value, CO is the Cut Off value (positive judgment value), CO=2.1×N (N is the negative control D value).

[0122] After immunizing Balb / c mice three times with ZC3, the eyeballs were removed and blood was collected to separate the serum. The serum of mice was tested for specific antibody responses to type O FMDV (O / Tibet / 99 strain) and type A FMDV (A / AF72 strain) using indirect ELISA. Figure 8As shown, mice produced specific antibodies that bind to FMDV, with better reactivity to FMDV A / AF72 than O / Tibet / 99. Therefore, ZC3 is considered an anti-idiotypic antibody molecule that authentically mimics FMDV antigenic epitopes and can induce a broad-spectrum FMDV antibody response in immunized animals.

Claims

1. An anti-idiotypic antibody against foot-and-mouth disease virus, characterized in that: The anti-idiotypic antibody of foot-and-mouth disease virus is a nanobody, comprising three complementary determining regions CDR1, CDR2, and CDR3; Amino acid sequence of CDR1: GRTFSTYA Amino acid sequence of CDR2: ITRSGDST The amino acid sequence of CDR3 is: SVGVFEGNGMGYVVNT.

2. The anti-idiotypic antibody for foot-and-mouth disease virus according to claim 1, characterized in that: The anti-idiotypic antibody of foot-and-mouth disease virus further comprises four framework regions FR1, FR2, FR3, and FR4; Amino acid sequence of FR1: EVQLAESGGGLVQAGGSLRLSCAPS Amino acid sequence of FR2: MGWYRQAPGKEREFVAA Amino acid sequence of FR3: YYADSVKGRFTISRDNAENTVYLQMNSLKPGDTAVYYC The amino acid sequence of FR4 is: WGQGTLVTVSS.

3. The anti-idiotypic antibody for foot-and-mouth disease virus according to claim 1, characterized in that: The amino acid sequence of the anti-idiotypic antibody of foot-and-mouth disease virus is: EVQLAESGGGLVQAGGSLRLSCAPSGRTFSTYAMGWYRQAPGKEREFVAAITRSGDSTYYADSVKGRFTISRDNAENTVYLQMNSLKPGDTAVYYCSVGVFEGNGMGYVVNTWGQGTLVTVSS.

4. Use of the anti-idiotypic antibody against foot-and-mouth disease virus according to any one of claims 1 to 3 in the preparation of a broad-spectrum vaccine against foot-and-mouth disease virus.

5. Use of the anti-idiotypic antibody against foot-and-mouth disease virus according to any one of claims 1 to 3 in the preparation of a reagent or kit for detecting foot-and-mouth disease virus antibodies.

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