Senecavirus A swine Fab antibody and competitive ELISA detection method thereof

Abstract

The invention discloses a Seneca virus A swine Fab antibody and a competitive ELISA (enzyme-linked immuno sorbent assay) detection method thereof. Amino acid sequences of a heavy chain variable region and a light chain variable region of a neutralized Fab gene engineering antibody are shown as SEQ ID No.4 and SEQ ID No.7, the antibody and SVA have high affinity, a competitive ELISA detection method is established, and the antibody can be applied to the detection of SVA. Determining that the optimal coating concentration of SVA particles in the ELISA detection method is 1 mu g / mL, the optimal working concentration of a competitive antibody is 0.5 mu g / mL, the optimal dilution ratio of a serum sample to be detected is 1: 10, and the optimal dilution ratio of enzyme-labeled avidin is 1: 30000; when it is determined that the inhibition percentage PI is 47%, the sensitivity and the specificity are the highest and are 96.88% and 100% respectively, and reaction with common major porcine virus positive serum is avoided; the in-batch variation coefficient range is 1.12%-7.34%, and the inter-batch variation coefficient range is 1.19%-11.72%, which shows that the repeatability is good; 224 clinical pig serum samples are detected at the same time by using the method and VNT detection, and the coincidence rate is 93.75%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Seneca virus A porcine-derived Fab antibody and a competitive ELISA detection method thereof. Background Art

[0002] Senecavirus A (SVA) is the only member of the genus Senecavirus in the family Picornaviridae and is the pathogen that causes vesicular lesions in pigs. SVA is a non-enveloped single-stranded positive-sense RNA virus with a genome length of approximately 7.3 kb, which consists of a 5′-untranslated region (UTR) (0.66 kb), an open reading frame (ORF) (6.54 kb), and a 3′-UTR (0.07 kb). The ORF of SVA encodes a large polyprotein precursor, which is then cleaved by proteases to form four structural proteins (VP4, VP2, VP3, and VP1) and eight nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D). The clinical symptoms caused by SVA infection include blisters and ulcers on the nose, oral epithelium and hoof crown, leading to lameness. In young piglets, it can cause lethargy, diarrhea, neurological symptoms and acute death. The mortality rate of piglets aged 0 to 3 days is as high as 40% to 80%, and the mortality rate of piglets aged 4 to 7 days is 0 to 30%. The clinical symptoms caused by SVA infection are similar to those caused by foot-and-mouth disease, swine vesicular disease and vesicular stomatitis, and are difficult to distinguish clinically. Since October 2014, SVA outbreaks have occurred in many countries, and the scope of the epidemic has continued to expand, causing significant economic losses to the pig industry. Therefore, it is necessary to establish a rapid and specific diagnostic method to prevent and control SVA infection.

[0003] At present, the main diagnostic methods for SVA include reverse transcription-PCR (RT-PCR), virus neutralization test (VNT), indirect fluorescent antibody test (IFA), enzyme-linked immunosorbent assay (ELISA), etc. In contrast, the ELISA method is simple, rapid and easy to operate. However, the antibodies used in these ELISA methods are mostly mouse monoclonal antibodies or rabbit polyclonal antibodies. The preparation of rabbit polyclonal antibodies and mouse monoclonal antibodies is not only time-consuming and labor-intensive, requiring the killing of animals, but also the prepared polyclonal antibodies and monoclonal antibodies are rabbit or mouse antibodies, and the epitopes they recognize may be different from the antigenic epitopes recognized by the antibodies of this animal, resulting in the urgent need to improve the specificity and sensitivity of the methods based on this antibody. In recent years, the monoclonal antibody preparation technology based on the combination of single cell technology and flow sorting technology (i.e., single B cell antibody technology) has further optimized the development of antibody preparation technology, making it possible for people to obtain monoclonal antibodies of this animal origin against a certain antigen. The core of this technology is to separate antigen-specific B cells from immune or infected animals (tissues or peripheral blood), and then amplify antibody heavy and light chains from single antibody-secreting B cells through single-cell PCR technology, and then express them in mammalian cells to obtain biologically active monoclonal antibodies. The antibodies obtained by this technology have the characteristics of natural pairing of antibody heavy and light chains, which is crucial for the specificity and affinity of antibodies, and this method has the characteristics of good genetic diversity, high efficiency, and all-natural origin. This technology has been applied to the search for neutralizing antibodies for some important infectious diseases in humans, such as AIDS, influenza, etc.

[0004] Fab antibody is an antigen-binding fragment of immunoglobulin IgG, which consists of a complete light chain (variable region VL and constant region CL) and a partial heavy chain structure (variable region VH and a constant region fragment CH1). The light chain and the heavy chain are connected by a disulfide bond. Its length is equivalent to 1 / 3 of the full-length antibody. It has no Fc segment and is small in size, and is not easy to form a steric hindrance effect. Therefore, compared with full-length IgG, Fab antibody has the advantages of strong tissue penetration, easier to reach the lesion, low immunogenicity, etc., and has no Fc fragment, and will not produce antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Based on the use of single B cell antibody technology to obtain this animal-derived antibody, there is no report on the ELISA antibody detection method for SVA using Fab antibody. Summary of the invention

[0005] The invention aims to provide a Seneca virus A porcine-derived Fab antibody and a competitive ELISA detection method thereof.

[0006] The amino acid sequences of the Seneca virus A porcine Fab antibody, the heavy chain variable region and the light chain variable region of the porcine Fab antibody of the present invention are as follows: Heavy chain variable region (SEQ ID No. 4): EEKLVESGGGLVQPGGSLRLSCVGSGFTFRSTSINWVRQAPGKGLEWLAYIGSSGLGTGYADSVKGRFTISRDNSQNTAYLQINSLRTEDTARYYCARGCGNSAACAALDLWGPGVEVVV Light chain variable region (SEQ ID No. 7): AIQLTQSPASLAASLGDTVSITCRASQSVSSNLDWYQQQPGKAPKLLIYSVSTLQSGVPFRFKGSGSGTDFTLTISGLQAEDVATYYCHQHNSTPYGFGAGTKLELKRADAKPSVFIFPPSKEQLATPTVSVVCLINNFFPREISVKWKVDGVVQSSGHPDSVTEQDSKDSTY The nucleotide sequence encoding the heavy chain variable region gene is shown in SEQ ID NO.2, and the nucleotide sequence encoding the light chain variable region gene is shown in SEQ ID NO.8.

[0007] The amino acid sequence of the heavy chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No. 3; the amino acid sequence of the light chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No. 6.

[0008] The present invention also provides the use of Seneca virus A porcine-derived Fab antibody in preparing a reagent or a kit for detecting Seneca virus.

[0009] The present invention also provides a competitive ELISA detection method based on Seneca virus A porcine Fab antibody, comprising the following steps: (1) Coating: Dilute Seneca virus A antigen to 1 μg / mL with PBS, add 100 μL / well to the ELISA plate, incubate at 4°C overnight, wash the plate three times with PBST, and pat dry; (2) Blocking: Prepare PBST solution containing 1% BSA and 5% sucrose, 100 μL / well, and block at 37°C for 1 h; (3) Detection: Take the test serum, standard negative serum, and standard positive serum diluted 1:10 respectively, mix them with the porcine Fab antibody diluted to 0.5 μg / mL, and transfer them to the ELISA plate, 100 μL / well, incubate at 37℃ for 1 h, wash the plate 3 times with PBST, and pat dry; add enzyme-labeled avidin, 100 μL / well, incubate at 37℃ for 30 min, wash the plate 3 times with PBST, and pat dry; the enzyme-labeled avidin is streptavidin-HRP, and the dilution ratio of streptavidin-HRP is 1:30000; (4) Color development: TMB color development, 100 μL / well, incubate at 37°C in the dark for 15 min; stop and read the absorbance at 450 nm; (5) Determination of results: Calculate the inhibition percentage PI, with 47% PI as the critical value. When the serum PI to be tested is <47%, the test result is negative; when the serum PI to be tested is ≥47%, the test result is negative.

[0010] The invention constructs a porcine Fab antibody with naturally paired light chain and heavy chain, establishes a SVA competitive ELISA detection method by using the antibody, and measures the sensitivity, specificity, repeatability and the conformity rate with the virus neutralization test (VNT). The results showed that a SVA neutralizing Fab genetic engineering antibody was successfully prepared and named as 1M33Fab strain, which had a high affinity with SVA. The optimal coating concentration of SVA particles of C-ELISA method was determined to be 1 μg / mL, the optimal working concentration of competitive antibody Bio-1M33Fab was 0.5 μg / mL, the optimal dilution ratio of serum samples to be tested was 1:10, and the optimal dilution ratio of enzyme-labeled avidin was 1:30 000. When the inhibition percentage PI was 47%, the sensitivity and specificity were the highest, which were 96.88% and 100%, respectively, and it did not react with the positive sera of common major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, and the inter-assay coefficient of variation ranged from 1.19% to 11.72%, indicating good repeatability. A total of 224 clinical porcine serum samples were tested simultaneously using this method and VNT detection, with a compliance rate of 93.75%. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Polyacrylamide gel electrophoresis of Fab antibody; M: protein molecular weight standard A: denaturing polyacrylamide gel electrophoresis of Fab antibody. B: non-denaturing polyacrylamide gel electrophoresis of Fab antibody; Figure 2 The results of indirect immunofluorescence experiment of Fab genetic engineering antibody A: The specific binding result of 1M33Fab and BHK-21 cells infected with SVA HN / 11 / 2017 strain. B: Normal BHK-21 cells without virus infection control; Figure 3 Indirect ELISA was used to detect the reactivity of 1M33Fab antibody with SVA antigen; Figure 4 To determine the sensitivity, specificity and cut-off value; Figure 5 It is a cross-reactivity test; Figure 6These are the results of PCR amplification of the variable region gene of pig IgG antibody; A: electrophoresis result of PCR product of pig IgG heavy chain variable region; B: electrophoresis result of PCR product of pig κ light chain variable region. DETAILED DESCRIPTION

[0012] The present invention will be further described below through specific implementation modes.

[0013] Reagents used in the present invention: The endotoxin-free large-scale plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; SMM293-TⅡ culture medium and M293-SUPI feed were purchased from SinB Biological Co., Ltd.; Polyethylenimine Linear (PEI) MW25000 was purchased from Poiyscience Co., Ltd.; Ni-NTA affinity chromatography medium was purchased from Genscript Co., Ltd.; MEM basal culture medium, trypsin, and double antibody (penicillin-streptomycin solution) were all purchased from Gibco Co.; fetal bovine serum FBS was purchased from Biological Industries Biotechnology Co., Ltd.; long-chain biotin (EZ-LinkTMSulfo-NHS-LC-Biotin) was purchased from Thermo Co.; diethylenimine (BEI), bovine serum albumin (BSA), FITC-labeled rabbit anti-swine IgG antibody, horseradish peroxidase (HRP)-labeled mouse anti-His antibody, streptavidin-HRP, and TMB substrate color developer were all purchased from Sigma Co.

[0014] Cells, strains and serum samples used in the present invention: The baby hamster kidney cell line BHK-21, HEK293F suspension cells, SVA (HN / 11 / 2017) strain, SVA positive quality control serum, SVA negative quality control serum, non-immune healthy pig serum (n = 64), SVA positive pig serum (n = 61), porcine reproductive and respiratory syndrome virus (PRRSV) infected pigs (n = 6), porcine circovirus (PCV) infected pigs (n = 6), porcine epidemic diarrhea virus (PEDV) infected pigs (n = 6), classical swine fever virus (CSFV) infected pigs (n = 5), foot-and-mouth disease virus (FMDV) infected pigs (n = 6) and Seneca virus (SVA) infected pig positive serum (n = 6); field serum samples (n = 224) were prepared, identified and preserved by the host antiviral and immunobiology team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0015] Example 1 Preparation of SVA antigen and porcine Fab genetically engineered antibody 1. Preparation of SVA Antigen In order to obtain high-concentration, pure SVA (HN / 11 / 2017) particles, NHK-21 cells were first used to reproduce the virus in large quantities. After repeated freezing and thawing, the virus was added with 0.1 mol / L BEI solution and inactivated at 30°C for 28 h. After centrifugation to remove cell debris, 8% PEG-6000 and 4% NaCl (volume-to-mass ratio) were added, stirred overnight at 2-8°C, and the virus was collected by high-speed centrifuge. PBS was used to prepare 30% sucrose (mass ratio), and 10 mL of 30% sucrose was added to the bottom of the centrifuge tube containing the virus solution using a long needle syringe. The solution was centrifuged at 4°C and 36,000 r / min for 3.5 h, and the supernatant was discarded. 15%, 25%, 35%, and 45% sucrose solutions were prepared, and the precipitate was resuspended in PBS buffer and centrifuged at 4°C and 36,000 r / min for 3.5 h using sucrose continuous density gradient centrifugation. After centrifugation, 0.5 mL fractions were collected from low to high concentrations and measured. OD 260nm Value, Collection OD 260nm Add 4 to 5 tubes of samples at the peak to a 100 kDa ultrafiltration tube, then fill it with PBS buffer and centrifuge at 3,000 r / min at 4°C for 10 min. Use PBS buffer to remove sucrose. After sugar removal, use the BCA method to determine the concentration and add 30% glycerol to store at -80°C.

[0016] SVA virus particles were purified by ultracentrifugation and sucrose continuous density gradient centrifugation, and the concentration was 500μg / mL measured by BCA method.

[0017] Multicolor flow cytometry sorting of SVA-specific single B cells 2.1 Isolation of mononuclear macrophages (PBMCs) Healthy 3-month-old pigs were treated with SVA cytotoxic virus (HN / 11 / 2017 strain, with a virus titer of 1×10 9 TCID 50 / mL) was injected intramuscularly behind the ear, 4mL / head, 2mL on each side. After 21 days, the pigs were immunized again with an inactivated vaccine prepared from the same strain in the same way. 28 days after immunization, the pigs' peripheral blood was collected, and mononuclear macrophages (PBMCs) were isolated from the pigs' peripheral blood using lymphocyte separation fluid (density = 1.077 g / mL). The specific operation was as follows: (1) Balance PBS solution (pH 7.4) and lymphocyte separation solution at room temperature, and transfer 6 mL of lymphocyte separation solution into a 15 mL centrifuge tube; (2) Dilute porcine EDTA anticoagulated blood with PBS solution at a ratio of 1:1, take 8 mL of diluted whole blood and slowly add it to the upper layer of lymphocyte separation solution, and centrifuge at 1200 × g for 30 min; (3) Pipette the milky white layer containing PBMCs and add it to a 15 mL centrifuge tube containing 1 / 2 volume of cell sorting solution (PBS solution containing 1% BSA and 2 mM EDTANa2), and centrifuge at 600 × g for 5 min; (4) Discard the upper layer of liquid, add 1-2 mL of red blood cell lysis buffer, lyse at room temperature for 1-2 min, then add 5 mL of cell sorting buffer, and centrifuge at 250 × g for 10 min; (5) Discard the upper layer of liquid, wash the cells twice with cell sorting solution, centrifuge at 400 × g for 5 min, discard the upper layer of liquid, add cell sorting solution and blow them into single cells. The cells finally obtained are PBMCs.

[0018] 2.2 Flow cytometry staining (1) Take 10 8 The enriched PBMCs were resuspended in 200 μL cell sorting buffer, and 0.5 μg biotinylated SVA virus particle antigen and 2 μg mouse anti-swine IgG-FITC fluorescent antibody (MyBioSource, USA) were added and incubated on ice for 25 min. The same cells were used to set up isotype controls and minus one controls. At the same time, a single positive tube was set up for compensation adjustment.

[0019] (2) Wash the cells twice with cell sorting buffer and centrifuge at 400 × g, 4°C for 5 min.

[0020] (3) Resuspend the cells in 200 μL of cell sorting buffer, add 2 μL of Anti-biotin-APC secondary antibody, and incubate on ice for 20 min.

[0021] (4) Wash the cells twice with cell sorting buffer and centrifuge at 400 × g, 4°C for 5 min.

[0022] (5) Resuspend the cells in 500 μL of cell sorting buffer, keep on ice and avoid light, and prepare to sort the cells.

[0023] 2.3 Isolation of SVA-specific single B cells Use BD FACSAriaⅡu flow sorter to sort SVA-specific single B cells. The instrument setting parameters are: nozzle size: 100 μm; sorting mode: single cell mode; sorting speed: 10,000 cells / second; amplitude: 20psi; oscillation frequency: 30 kHz. After adjusting the above parameter settings, turn off the Sweat button and use Accudrop (Cat. No.: 642412) delay microspheres to calculate the liquid delay time. Adjust the position of the 96-PCR well plate in the sorting chamber until the sorted cells fall accurately into the center of the plate well. After the above settings are completed, put the 96-PCR plate containing 10 μL of lysis solution into the sorting chamber and start loading. Draw a gate to circle out lymphocytes and monocytes, exclude adherent cells according to the FSC-A and FSA-H settings, and circle out single cells on the diagonal line. Then further circle out IgG from it. + Cell population, gate for IgG + SVA + The cells are single B cells specific to SVA.

[0024] Gene amplification of variable region of IgG antibody from single B cells 3.1 Single-cell cDNA molecule preparation After sorting, add 1 μL of stop solution to each well and incubate at room temperature for 2 min to terminate the reaction. Then add 4 μL of SuperScriopt VILO mix solution and 6 μL of DNase / RNase-free water to each well, mix gently, centrifuge at 1500 rpm and 4°C for 5 min; then put the 96-well PCR plate into the PCR instrument for reverse transcription. The reaction conditions are: 25°C, 5 min; 42°C, 120 min; 85°C, 5 min. The obtained cDNA is stored at -20°C for subsequent nested PCR amplification.

[0025] 3.2 Amplification of IgG antibody variable region genes The primers designed and synthesized in Table 1 were used to perform PCR amplification of the IgG heavy chain variable region (VH) gene and the kappa light chain variable region (VL) gene of a single pig B cell by nested PCR method, i.e., two rounds of PCR amplification.

[0026] (1) First round PCR amplification plan First round PCR reaction system: The first round reaction system for amplifying the pig IgG heavy chain variable region (VH) gene and the kappa light chain variable region (VL) gene, except for the primers and templates, the addition of other components was carried out according to Table 2.

[0027] The first round of PCR amplification program was: pre-denaturation at 94°C for 1 min; then denaturation at 98°C for 10 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min, for a total of 30 cycles; and finally extension at 72°C for another 10 min.

[0028] (2) Second round of PCR amplification plan Second round reaction system: Using the first round amplification product as a template, add corresponding upstream and downstream primers to amplify the pig IgG heavy chain variable region (VH) gene and the kappa light chain variable region (VL) gene. The second round PCR reaction system components are as shown in Table 3: The second round of PCR amplification program was as follows: first, pre-denaturation at 94°C for 1 min; then, denaturation at 98°C for 10 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min, for a total of 35 cycles; and finally, extension at 72°C for another 10 min.

[0029] 3.3 PCR product sequencing Take 4 μL of the PCR product from the second round of amplification for agarose gel electrophoresis to observe the amplification results, and perform DNA sequencing on the PCR products of the successfully amplified VH and VL genes. Then use SnapGene software to analyze and compare the sequencing results.

[0030] The nested PCR amplification products were analyzed by agarose gel electrophoresis. Figure 6 As shown, the pig IgG heavy chain has a clear visible band between 450 bp and 600 bp ( Figure 6 A); A clear band appears at about 500 bp in the variable region of the porcine κ light chain ( Figure 6 B). The sequencing results were compared in the BLAST database, confirming that the sequence obtained by nested PCR amplification was the gene sequence of the variable region of the porcine IgG antibody.

[0031] Construction of porcine Fab gene engineering antibody expression vector The signal peptide "MEFRLNW0VVLFALLQGVQG" sequence was introduced at the front end of the amplified porcine IgG antibody VH gene, and inserted into the CH-pcDNA3.4 vector containing the IgG heavy chain CH1 constant region through the two restriction sites of Not I and BbvCI; the signal peptide "MRAPMHLLGLLLLWVPGARS" sequence was introduced at the front end of the κ light chain VL gene, and inserted into the CL-pcDNA3.4 vector containing the IgGκ light chain constant region through the two restriction sites of Nhe I and BbvCI. The heavy chain constant region of the IgG antibody contains a 6×His tag at the end. Sequence synthesis and vector construction were entrusted to Jinweizhi Biotechnology Co., Ltd. Endotoxin-free plasmids were prepared according to the instructions of the endotoxin-free large-scale plasmid kit to obtain the heavy chain plasmid CH-pcDNA3.4-1M33Fab and the light chain plasmid CL-pcDNA3.4-1M33L.

[0032] Preparation of porcine Fab genetically engineered antibodies First, endotoxin-free plasmids were prepared according to the instructions of the commercial endotoxin-free plasmid extraction kit; 20 μg of heavy chain plasmid (CH-pcDNA3.4-1M33Fab) and 20 μg of corresponding light chain plasmid (CL-pcDNA3.4-1M33L) (heavy chain and light chain in a ratio of 1:1) were mixed and added to a 5 mL sterile centrifuge tube, and 2 mL of DPBS was added to dilute the plasmid; 15 μL of transfection reagent PEI (2 mg / mL) was added to another new 2 mL centrifuge tube, and 2 mL of DPBS was added to dilute the transfection reagent, inverted to mix, and allowed to stand at room temperature for 10-15 min; the mixture was slowly added dropwise to a 2×10 6 cells / mL of HEK293F cells. After 7-9 days of continuous culture, the cell supernatant was collected, filtered with a 0.45 μm filter, and purified using Ni-NTA filler. The eluted antibody was dialyzed in PBS at 4°C, pH=8.0, and the dialysate was replaced every 2 hours for a total of three times. The dialyzed antibody was embedded in PEG-8000 solid particles, placed at 4°C for concentration, and the concentration was measured. Finally, SDS-PAGE was run for verification and stained with Coomassie Brilliant Blue.

[0033] 20 μg of heavy chain plasmid and corresponding light chain plasmid (heavy chain and light chain ratio 1:1) were co-transfected into HEK293F cells for expression. Cell supernatant was collected 9 days after transfection and analyzed by polyacrylamide gel electrophoresis after protein purification. The results are as follows: Figure 1As shown, the Fab genetic engineering antibody showed a target band at 24 kDa under denaturing conditions and a target band at 50 kDa under non-denaturing conditions. The results were consistent with expectations, indicating that the porcine Fab antibody molecule was successfully expressed and purified and named 1M33Fab.

[0034] Nucleotide sequence of the porcine Fab antibody heavy chain (SEQ ID No. 1): ATGGAGTTTCGGCTGAACTGGGTGGTCTTGTTTGCTCTCTTACAAGGTGTCCAGGGTGAGGAGAAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGAAGTACCTCCATTAACTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGCTGGCATATATTGGTAGCAGTGGACTTGGCACCGGCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCCAGAACACGGCCTATCTGCAAATCAACAGCCTGAGAACCGAAGACACGGCCCGCTATTACTGTGCACGCGGTTGCGGTAATAGTGCCG CATGTGCTCTGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGCCCCAAAGACTGCCCCTTCCGTGTACCCACTGGCTCCTTGCGGAAGGGATACTAGCGGCCCAAATGTCGCTCTGGGCTGTCTGGCCAGCAGCTACTTCCCAGAGCCAGTGACAATGACATGGAACAGCGGAGCT CTGACATCCGGCGTGCACACTTTTCCAAGCGTGCTGCAACCAAGCGGACTGTACTCTCTGAGCTCCATGGTGACAGTCCCAGCCAGCTCCCTCTCCAGCAAGAGCTACACATGCAACGTGAATCACCCAGCCACTACAACTAAGGTCGACAAGAGGGTGGGCGGACACCACCACCACCCATATTGA Nucleotide sequence of the variable region of the heavy chain of the porcine Fab antibody (SEQ ID No. 2): GAGGAGAAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGAAGTACCTCCATTAACTGGGTCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTGGCATATATTGGTAGCAGTGGACTTGGCACCGGCT ACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCCAGAACACGGCCTATCTGCAAATCAACAGCCTGAGAACCGAAGACACGGCCCGCTATTACTGTGCACGCGGTTGCGGTAATAGTGCCGCATGTGCTCTGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTG Amino acid sequence of the porcine Fab antibody heavy chain (SEQ ID No. 3): MEFRLNWVVLFALLQGVQGEEKLVESGGGLVQPGGSLRLSCVGSGFTFRSTSINWVRQAPGKGLEWLAYIGSSGLGTGYADSVKGRFTISRDNSQNTAYLQINSLRTEDTARYYCARGCGNSA ACALDLWGPGVEVVVSSAPKTAPSVYPLAPCGRDTSGPNVALGCLASSYFPEPVTMTWNSGALTSGVHTFPSVLQPSGLYSLSSMVTVPASSLSSKSYTCNVNHPATTTKVDKRVGGHHHHHH Amino acid sequence of the variable region of the heavy chain of porcine Fab antibody (SEQ ID No. 4): EEKLVESGGGLVQPGGSLRLSCVGSGFTFRSTSINWVRQAPGKGLEWLAYIGSSGLGTGYADSVKGRFTISRDNSQNTAYLQINSLRTEDTARYYCARGCGNSAACAALDLWGPGVEVVV Nucleotide sequence of porcine Fab antibody light chain (SEQ ID No. 5): ATGAGGGCCCCCATGCACCTCCTTGGCCTCCTGCTGCTCTGGGTCCCA-GGTGCCAGGAGTGCCATCCAGCTGACCCAGTCTCCAGCCTCCCTGGCTGCATCTCTCGGAGACACGGTCTCCATCACTTGCCGGGCCAGTCAGAGTGTTAGCAGTAATTTAGACTGGTATCAACAACAACCAGGGAAGGCTCCTAAACTCTTGATCTATTCAGTTTCCACTTTGCAAAGTGGGGTCCCATTCCGGTTCAAGGGCAGTGGATCTGGCACCGATTTCACCCTCACCATCAGTGGCCTGCAGGCTGAAGATGTTGCAACTTATTACTGTCACCAGCATAACAGTACACCGTATGGTTTCGGCGCGGGGACCAAGCTGGAGCTCAAACGGGCTGATGCCAAGCCATCCGTCTTCATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGGTGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAAGTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCCACGTCACAGTACCTAAGTCATAATTTATATTCCTGTGAGGTCACCCACAAGACCCTGGCCTCCCCTCTGGTCACAAGCTTCAACAGGAACGAGTGTGAGGCTTAG Amino acid sequence of porcine-derived Fab antibody light chain (SEQ ID No. 6): MRAPMHLLGLLLLWVPGARSAIQLTQSPASLAASLGDTVSITCRASQSVSSNLDWYQQQPGKAPKLLIYSVSTLQSGVPFRFKGSGSGTDFTLTISGLQAEDVATYYCHQHNSTPYGF GAGTKLELKRADAKPSVFIFPPSKEQLATPTVSVVCLINNFFPREISVKWKVDGVVQSSGHPDSVTEQDSKDSTYSLSSTLSLPTSQYLSHNLYSCEVTHKTLASPLVTSFNRNECEA Amino acid sequence of the light chain variable region of porcine Fab antibody (SEQ ID No. 7): AIQLTQSPASLAASLGDTVSITCRASQSVSSNLDWYQQQPGKAPKLLIYSVSTLQSGVPFRFKGSGSGTDFTLTISGLQAEDVATYYCHQHNSTPYGFGAGTKLELKRADAKPSVFIFPPSKEQLATPTVSVVCLINNFFPREISVKWKVDGVVQSSGHPDSVTEQDSKDSTY Nucleotide sequence of the light chain variable region of the porcine Fab antibody (SEQ ID No. 8): GCCATCCAGCTGACCCAGTCTCCAGCCTCCCTGGCTGCATCTCTCGGAGACACGGTCTCCATCACTTGCCGGGCCAGTCAGAGTGTTAGCAGTAATTTAGACTGGTATCAACAACAACCAGGGAAGGCT CCTAAACTCTTGATCTATTCAGTTTCCACTTTGCAAAGTGGGGTCCCATTCCGGTTCAAGGGCAGTGGATCTGGCACCGATTTCACCCTCACCATCAGTGGCCTGCAGGCTGAAGATGTTGCAACTTATT ACTGTCACCAGCATAACAGTACACCGTATGGTTTCGGCGCGGGGACCAAGCTGGAGCTCAAACGGGCTGATGCCAAGCCATCCGTCTTCATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGT CTCTGTGGTGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAAGTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC Example 2 Activity detection of SVA porcine Fab genetic engineering antibody 1. Indirect immunofluorescence assay (IFA) The SVA HN / 11 / 2017 strain was inoculated into a monolayer of BHK-21 cells; incubated in a cell culture incubator at 37°C for 8 h; a mixture of methanol and acetone (1:1) was used as a fixative and fixed at room temperature for 20 min; rinsed with PBS three times and permeabilized with 0.1% TritonX-100 for 10 min; rinsed with PBS three times and blocked with blocking solution (1% BSA + 0.1% Triton X-100); rinsed with PBS three times and added purified Fab antibody and incubated at 37°C for 1 h; rinsed with PBS three times and added FITC-labeled rabbit anti-swine IgG antibody (dilution ratio of 1:400) as a secondary antibody and incubated at 37°C for 1 h; discarded the secondary antibody and rinsed with PBS five times. In a light-proof environment, the fluorescence signal was observed using a fluorescence microscope and photographed and recorded.

[0035] IFA test results Figure 2As shown in the figure, the Fab genetic engineering antibody 1M33Fab can specifically bind to BHK-21 cells infected with the SVA HN / 11 / 2017 strain, and obvious green fluorescence appears, while the uninfected control cells do not produce visible green fluorescence. This result shows that a porcine monoclonal genetic engineering antibody that can specifically react with SVA has been successfully obtained.

[0036] Indirect ELISA The purified SVA (HN / 11 / 2017 strain) antigen was diluted to 2 μg / mL with PBS and coated on the ELISA plate overnight at 4°C; the plate was blocked with 1% BSA at 37°C for 1 h and dried; the antibody to be tested was serially diluted and added to the ELISA plate and incubated at 37°C for 1 h; HRP-labeled mouse anti-His antibody was added and incubated at 37°C for 1 h; TMB colorimetric solution was added and color was developed at room temperature for 10 min; stop solution was added to stop color development, and the absorbance at 450 nm was measured using an ELISA reader ( OD 450nm ). Calculate the average value of the negative control group, and then multiply the average value by 2.1 to get the Cutoff (CO) value; according to the Cutoff value, the wells with a value greater than this value are judged as positive wells, and those with a value less than this value are judged as negative wells.

[0037] Indirect ELISA was used to identify whether the antibody specifically bound to the antigen. Figure 3 As shown, antibody 1M33Fab can specifically bind to SVA antigen, and the binding of the antibody to SVA antigen is positively correlated with the concentration of the antibody.

[0038] Virus neutralization assay The virus neutralization experiment was performed on the Fab genetically engineered antibodies screened using the SVA HN / 11 / 2017 strain. First, the antibody to be tested was diluted 2-fold and added to a 96-well plate at a volume of 50 μL / well. Generally, two replicates were set, and then the antibody was diluted with 50 μL of 100 TCID 50 The mixture was incubated at 37°C for 1 h. 50 Then, 100 μL of 5×10 4 The complete medium of BHK-21 cells was used, and normal cells were set as controls. The 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for 72 h to observe the cytopathic effect. The minimum antibody concentration that can cause 50% of the cells to become cytopathic (i.e., half effective inhibitory concentration, IC 50 ) indicates the ability of antibodies to neutralize viruses, in μg / mL. 50A value of 50 μg / mL was used as the critical value for neutralization, and ≥50 μg / mL was determined as non-neutralizing activity; <50 μg / mL was determined as having neutralizing activity.

[0039] A micro-virus neutralization test was performed on BHK21 cells to test whether this antibody has the activity to neutralize the SVA HN / 11 / 2017 strain. The results showed that the porcine Fab genetic engineering antibody 1M33Fab has the ability to neutralize the HN / 11 / 2017 strain and is a SVA neutralizing antibody. Its IC 50 It is 1.26 μg / mL.

[0040] Western Blot (WB) assay About 1 μg of SVA HN / 11 / 2017 strain antigen was taken for SDS-PAGE electrophoresis, and then the separated protein bands were transferred to nitrocellulose membrane, blocked with TBST buffer containing 5% skim milk powder for 2 h, washed, and diluted with TBST buffer containing 5% skim milk powder to the working concentration (5 μg / mL), incubated at room temperature for 2 h, washed with TBST, and HRP-labeled anti-His tag antibody (1:4 000) was added and incubated at room temperature for 1 h. After washing with TBST, ECL chemiluminescent substrate was added for exposure imaging.

[0041] The purified virus particles of SVA HN / 11 / 2017 strain were used as antigens for SDS-PAGE electrophoresis and then transferred to the membrane. The WB results showed that the porcine Fab genetically engineered antibody did not show any reaction bands, while the SVA positive serum showed clearly visible reaction bands, indicating that the obtained Fab genetically engineered antibody did not recognize linear epitopes but recognized conformational epitopes.

[0042] Example 3 Establishment and optimization of competitive ELISA detection method 1. Biotinylated competing antibody Equilibrate biotin to room temperature in advance, centrifuge briefly, add 180 μL of enzyme-free water to 0.1 mg of biotin, dissolve thoroughly, and centrifuge briefly. Dilute the purified antibody to 100 μg / mL, add 60 μL of biotin solution per mL, mix well by pipetting, and incubate in an ice bath for 4 h. Add the biotin-labeled antibody to a dialysis bag and dialyze with PBS solution, changing it every 2 h for a total of four times to remove free biotin molecules, and store at 4 °C for later use.

[0043] Establishment of competitive ELISA detection method The purified SVA antigen was diluted with PBS to the optimal coating concentration, and 100 μL / well was added to the ELISA plate, and it was allowed to stand overnight at 4°C. The plate was washed 3 times with PBST and patted dry. A PBST solution containing 1% BSA and 5% sucrose was prepared at 100 μL / well and blocked at 37°C for 1 hour. 60 μL of diluted serum (serum to be tested, standard negative and standard positive serum) and 60 μL of competitive antibody diluted to the optimal working concentration were added to the serum dilution plate, mixed and transferred to the ELISA plate at 100 μL / well, incubated at 37°C for 1 hour, washed 3 times with PBST and patted dry. Enzyme-labeled avidin was added at 100 μL / well, incubated at 37°C for 30 min, washed 3 times with PBST and patted dry. TMB color development was performed at 100 μL / well and incubated at 37°C in the dark for 15 min. The reaction was terminated and the 450 μL reading was read. The absorbance value at nm is expressed as inhibition percentage (PI), PI=[1-(absorbance value of the sample to be tested / absorbance value of the blank control)×100%].

[0044] Optimization of competitive ELISA assay 3.1 Determination of the optimal working concentration of competitive antibodies and the optimal dilution of serum After purification, the SVA (HN / 11 / 2017 strain) antigen was diluted to 0.5 μg / mL with PBS and coated. The biotin-labeled antibody was diluted to 2, 1, 0.5, and 0.25 μg / mL with PBS. The negative and positive quality control sera were diluted in a gradient of 1:5-1:640. The absorbance at 450 nm was measured with an ELISA reader, and the N / P value was calculated (N: negative control OD 450nm Value, P: positive control OD 450nm value), and it is best when the N / P value is the largest.

[0045] Measured by square matrix titration OD 450nm The values ​​and N / P values ​​are shown in Table 4. When the concentration of the competing antibody Bio-1M33Fab is 0.5 μg / mL and the serum dilution ratio is 1:10, the N / P value is the largest. Therefore, the optimal working concentration of Bio-1M33Fab is determined to be 0.5 μg / mL and the optimal serum dilution ratio is 1:10.

[0046] Determination of the Optimal Coating Concentration and the Optimal Dilution Ratio of Streptavidin-HRP The SVA antigen coating concentration was selected as 2, 1, 0.5, and 0.25 μg / mL, and streptavidin-HRP was diluted in a gradient of 1:15,000, 1:20,000, 1:25,000, 1:30,000, 1:35,000, 1:40,000, and 1:45,000. The negative and positive quality control sera were diluted at the optimal dilution. OD 450nm value and calculate the N / P value. The best value is when the N / P value is the largest.

[0047] Matrix titration was used to determine the optimal coating concentration of SVA and the optimal dilution ratio of streptavidin-HRP. OD 450nm The values ​​and N / P values ​​are shown in Table 5. When the SVA coating concentration is 1 μg / mL and the streptavidin-HRP is diluted at 1:30 000, the N / P value is the largest. Therefore, it can be determined that the optimal SVA coating concentration is 1 μg / mL and the optimal dilution ratio of streptavidin-HRP is 1:30000.

[0048] Determination of sensitivity, specificity and cut-off values The method was used to detect 64 sera from non-immune healthy pigs and 61 sera from SVA-positive pigs. The PI values ​​were calculated and the ROC curve analysis was performed to determine the sensitivity, specificity and critical value of the method.

[0049] This method was used to detect 64 non-immune healthy pig sera and 61 SVA-positive pig sera, and the PI values ​​were calculated. ROC curve analysis was performed to determine the sensitivity, specificity and critical value. The results are shown in Figure 4 As shown. When the critical value was 47%, the sensitivity and specificity were the highest, 96.88% and 100%, respectively. The area under the ROC curve was 0.9 992, P < 0.0 001. Therefore, the critical value of PI was set at 47%. That is, serum samples with PI < 47% were considered negative, while serum samples with PI ≥ 47% were considered positive.

[0050] Cross-reactivity test This method was used to detect positive sera from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (n = 6), porcine circovirus (PCV) (n = 6), porcine epidemic diarrhea virus (PEDV) (n = 6), classical swine fever virus (CSFV) (n = 5), foot-and-mouth disease virus (FMDV) (n = 6) and Seneca virus (SVA). The test results were statistically analyzed to determine whether the method had cross-reactivity.

[0051] To evaluate the cross-reactivity of the method, positive sera from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (n = 6), porcine circovirus (PCV) (n = 6), porcine epidemic diarrhea virus (PEDV) (n = 6), classical swine fever virus (CSFV) (n = 5), and foot-and-mouth disease virus (FMDV) (n = 5) were tested. Figure 5 As shown, except for the SVA-positive serum, all sera were negative with PI values ​​lower than 47%, i.e., the method was specific for detecting SVA-specific antibodies and had no cross-reactivity with sera infected with other viruses.

[0052] Repeatability test Intra-batch repeatability test: This method was used to test 8 serum samples, and the test was repeated 3 times at different time periods using C-ELISA plates prepared from the same batch. Inter-batch repeatability test: The same 8 serum samples were tested 3 times using C-ELISA plates prepared from different batches at the same time period. The intra-batch and inter-batch coefficients of variation of this method were evaluated.

[0053] In order to evaluate the repeatability of this method, C-ELISA reaction plates coated with the same and different batches were used, and 8 serum samples were tested for repeatability. The intra-batch and inter-batch coefficients of variation (CV) were calculated. The results are shown in Table 6. The intra-batch coefficient of variation of the 8 serum samples ranged from 1.12% to 7.34%, and the inter-batch coefficient of variation ranged from 1.19% to 11.72%, indicating that this method has high repeatability.

[0054] Comparison of test results of competitive ELISA and VNT field samples This method and VNT were used to detect 224 field samples. The test results of this method and VNT were compared, and the consistency rate of the two methods was calculated.

[0055] To verify the applicability of this method, 224 clinical pig serum samples collected from Gansu Province in 2024 were tested by C-ELISA and VNT. As shown in Table 7, 182 sera were negative and 42 were positive by VNT, while 172 sera were negative and 52 were positive by C-ELISA. 12 sera were positive by C-ELISA and negative by VNT, and 2 were negative by C-ELISA and positive by VNT. The results showed that the consistency rate between C-ELISA and VNT was about 93.75% (210 / 224).

[0056] The heavy chain plasmid and the corresponding light chain plasmid were co-transfected into HEK293F cells for expression, and a SVA-specific porcine Fab genetically engineered antibody 1M33Fab was obtained. In order to evaluate the reactivity of the porcine Fab genetically engineered antibody 1M33Fab to SVA, the antibody 1M33Fab was subjected to indirect ELISA, immunofluorescence test (IFA), virus neutralization test (VNT), and Western blot analysis. The results of indirect ELISA showed that the antibody 1M33Fab reacted with the purified SVA antigen in a dose-dependent manner. The results of IFA showed that the antibody 1M33Fab could specifically react with the SVA antigen in SVA-infected BHK-21 cells, while the Western blotting results showed that the antibody did not react with the SVA antigen, indicating that this antibody recognizes the conformational epitope of the viral capsid. In addition, the antibody was subjected to an in vitro neutralization test, and the results showed that this antibody could effectively neutralize SVA in vitro, and its IC 50 The results showed that the optimal coating concentration of SVA particles was 1 μg / mL, the optimal working concentration of the competitive antibody Bio-1M33Fab was 0.5 μg / mL, the optimal dilution ratio of the serum sample to be tested was 1:10, and the optimal dilution ratio of the enzyme-labeled avidin was 1:30 000. When the inhibition percentage PI was 47%, the sensitivity and specificity were the highest, which were 96.88% and 100%, respectively, and it did not react with the common positive sera of major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, and the inter-assay coefficient of variation ranged from 1.19% to 11.72%, indicating that the method had good repeatability. To further evaluate the performance of the method, 224 clinical porcine serum samples collected from Gansu Province in 2024 were tested using C-ELISA, and the results were compared with the VNT results. The concordance rate between C-ELISA and VNT was approximately 93.7% (210 / 224), indicating that C-ELISA and VNT have strong consistency in clinical diagnostic performance. Therefore, the C-ELISA developed in this study is comparable to VNT for SVA antibody detection and can replace VNT for large-scale screening because it is faster, simpler, and less expensive to perform.

[0057] The present invention relates to the establishment and application of a SVA competitive ELISA detection method based on Fab antibodies, which provides powerful technical support for the epidemiological monitoring and prevention and control of SVA.

Claims

1. A Seneca virus A porcine Fab antibody, characterized in that: The amino acid sequences of the heavy chain variable region and light chain variable region of the porcine Fab antibody are as follows: Heavy chain variable region: EEKLVESGGGLVQPGGSLRLSCVGSGFTFRSTSINWVRQAPGKGLEWLAYIGSSGLGTGYADSVKGRFTISRDNSQNTAYLQINSLRTEDTARYYCARGCGNSAACAALDLWGPGVEVVV Light chain variable region: AIQLTQSPASLAASLGDTVSITCRASQSVSSNLDWYQQQPGKAPKLLIYSVSTLQSGVPFRFKGSGSGTDFTLTISGLQAEDVATYYCHQHNSTPYGFGAGTKLELKRADAKPSVFIFPPSKEQLATPTVSVVCLINNFFPREISVKWKVDGVVQSSGHPDSVTEQDSKDSTY.

2. The Seneca virus A porcine Fab antibody according to claim 1, characterized in that: The nucleotide sequence encoding the heavy chain variable region gene is shown in SEQ ID NO.2, and the nucleotide sequence encoding the light chain variable region gene is shown in SEQ ID NO.

8.

3. The Seneca virus A porcine Fab antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No. 3; the amino acid sequence of the light chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No.

6.

4. Use of the Seneca virus A porcine-derived Fab antibody according to any one of claims 1 to 3 in the preparation of a reagent or kit for detecting Seneca virus.

5. A competitive ELISA detection method based on the Seneca virus A porcine Fab antibody according to claim 1, comprising the following steps: (1) Coating: Dilute Seneca virus A antigen to 1 μg / mL with PBS, add 100 μL / well to the ELISA plate, incubate at 4°C overnight, wash the plate three times with PBST, and pat dry; (2) Blocking: Prepare PBST solution containing 1% BSA and 5% sucrose, 100 μL / well, and block at 37°C for 1 h; (3) Detection: Take the test serum, standard negative serum, and standard positive serum diluted 1:10, respectively, and mix them with the porcine Fab antibody diluted to 0.5 μg / mL, then transfer them to the ELISA plate, 100 μL / well, incubate at 37°C for 1 h, wash the plate 3 times with PBST, and pat dry; Add enzyme-labeled avidin, 100 μL / well, incubate at 37°C for 30 min, wash the plate 3 times with PBST, and pat dry; the enzyme-labeled avidin is streptavidin-HRP, and the dilution ratio of streptavidin-HRP is 1:30000; (4) Color development: TMB color development, 100 μL / well, incubate at 37°C in the dark for 15 min; stop and read the absorbance at 450 nm; (5) Determination of results: Calculate the inhibition percentage PI, with 47% PI as the critical value. When the serum PI to be tested is <47%, the test result is negative; when the serum PI to be tested is ≥47%, the test result is negative.

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