Recombinant n protein antigen of bovine epizootic hemorrhagic disease virus, preparation method and elisa kit

Abstract

The application provides a bovine red blight virus recombinant N protein antigen and a preparation method and an ELISA kit, relates to the technical field of biotechnology detection, and solves the technical problem that the ELISA detection method in the prior art generally adopts competitive ELISA technical means, the preparation cost is higher, the price of a commercial kit is also relatively expensive, and the development of a beef cattle breeding enterprise in China is not conducive. The amino acid sequence of the bovine red blight virus recombinant N protein antigen is shown in SEQ ID No. 2. The bovine red blight virus recombinant N protein antigen obtained in the application can be specifically combined with the antibody of the bovine red blight virus, and can be used as a candidate antigen for detecting the bovine red blight virus. The ELISA detection kit developed in the application can be used for detecting the antibody of the bovine red blight virus, has the advantages of high specificity, high sensitivity, good repeatability, low cost, and can be used for mass detection.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a recombinant N protein antigen of bovine Akabane disease virus, its preparation method, and an ELISA kit. Background Technology

[0002] Akabane disease (AKAD), also known as Akabane illness, is a viral infectious disease of cattle and sheep characterized by abortion, premature birth, stillbirth, fetal malformations, mummification, and anencephaly with joint flexion and effusion in newborn fetuses (AH syndrome). The causative agent, Akabanevirus (AKAV), is a member of the Peribunyaviridae family, Orthobunyavirus genus, and Simbu serogroup. The virus particles are generally spherical, enveloped, and have spikes, with a diameter of approximately 80–120 nm. The genome is about 12 kb, containing three segments: M, L, and S. The S segment encodes nucleocapsid protein (N) and non-structural proteins (NSs). The nucleotide sequence of this segment is highly conserved, making it suitable as a target for detection methods. This disease poses a significant threat to ruminants such as cattle and sheep, causing severe economic losses worldwide. Currently, there are no commercially available vaccines in my country. The prevention and control of AKAV mainly relies on comprehensive biosafety management, pathogen detection, and elimination.

[0003] The N protein is the nucleocapsid protein of AKAV, and it is also the most abundant and major immunogenic protein in AKAV. This protein is highly conserved among different strains, with an amino acid sequence similarity of 97%–100%. The N protein can induce the body to produce antibodies, making it the preferred target in immunological diagnostic methods. Since there is currently no vaccine for the prevention of Akabane disease in my country, the detection of AKAV-specific antibodies in clinical samples is sufficient to prove the presence or past infection of AKAV in the animal population, providing important reference value in clinical diagnosis. Enzyme-linked immunosorbent assay (ELISA) has many advantages, including convenient and rapid operation, high sensitivity, strong specificity, and the ability to perform large-scale testing, making it easy to promote and use at the grassroots level. It is one of the most commonly used methods in serological testing.

[0004] Among existing methods for detecting bovine AKAV antibodies, the commonly used methods are colloidal gold detection and ELISA detection. However, the applicant has found that the existing technologies have at least the following technical problems:

[0005] ① Existing colloidal gold methods for antibody detection can only qualitatively determine the presence or absence of antibodies, but cannot determine the amount of antibodies present. Furthermore, colloidal gold methods are generally not suitable for large-scale detection, and their sensitivity is also relatively low.

[0006] ② Existing ELISA detection methods generally employ competitive ELISA technology, which has high preparation costs and the price of commercially available kits is also relatively expensive, such as the ID-VET kit sold in France. The Akabane Competition ELISA Kit is priced at 15,000 RMB, with the cost per well alone being 31.25 RMB, which is detrimental to the development of beef cattle farming enterprises in my country. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant N protein antigen of bovine Akabane disease virus, its preparation method, and an ELISA kit, to address the technical problems of existing ELISA detection methods, which generally employ competitive ELISA technology, resulting in high preparation costs and expensive commercial kits, thus hindering the development of beef cattle farming enterprises in my country. The various technical effects of the preferred solutions provided by this invention are detailed below.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The present invention provides a recombinant N protein antigen of bovine red feather disease virus, the amino acid sequence of which is shown in SEQ ID No.2.

[0010] The present invention provides a gene encoding the amino acid sequence of the recombinant N protein antigen of the bovine red feather disease virus, the gene sequence of which is shown in SEQ ID No. 1.

[0011] The method for preparing recombinant N protein antigen of bovine Akabane disease virus provided by the present invention includes the following steps:

[0012] S1. Primer design and synthesis

[0013] Upstream primer AKAV-NF: 5'-CCG GAATTC ATTTTCAACGATGTTC-3';

[0014] Downstream primer AKAV-NR: 5'-CCG CTCGAG TCTGAATACCAAATTGAG-3'; the underlined parts are the added EcoRI and XhoI restriction site sequences, respectively;

[0015] Amplification of S2 and N genes

[0016] RNA was extracted from bovine red feather disease virus and reverse transcribed using reverse transcriptase. This RNA was then used as a template for PCR amplification to obtain the PCR amplification product of the N gene. The sequence was determined by sequencing as shown in SEQ ID No. 1.

[0017] Construction and expression of S3 and N gene prokaryotic expression vectors

[0018] S31. The PCR amplification products of the pET28a vector plasmid and the N gene were digested with enzymes and the products were recovered by gel electrophoresis.

[0019] S32. The digested pET28a vector plasmid and the digested N gene PCR amplification product were ligated using a ligase.

[0020] S33. The ligation product obtained in step S32 is transformed into Top10 competent cells to construct the expression vector pET28a-N; the constructed expression vector pET28a-N plasmid is identified by double digestion with FlyCut EcoRI and FlyCut XhoI and sequencing.

[0021] S34. The successfully constructed expression vector pET28a-N was transformed into Escherichia coli BL21 competent cells. After culture, positive single colonies were picked and expanded to prepare protein samples. The protein samples were detected by SDS-PAGE electrophoresis and analyzed by Coomassie brilliant blue staining. The successfully expressed recombinant N protein was aliquoted and stored for later use.

[0022] Purification and activity identification of S4 and N proteins

[0023] The successfully expressed recombinant N protein obtained in step S34 was purified, the concentration of the purified recombinant N protein was determined, and the activity of the recombinant N protein was identified.

[0024] The application of the gene shown in SEQ ID No. 1 provided by this invention in the preparation of a bovine Akabane disease antibody detection kit.

[0025] The present invention relates to the application of the recombinant N protein antigen of bovine Akabane virus encoded by the gene shown in SEQ ID No. 2 in the preparation of a bovine Akabane virus antibody detection kit.

[0026] The present invention provides an indirect ELISA kit for detecting bovine Akabane disease virus antibodies, comprising an enzyme-labeled plate coated with the above-mentioned bovine Akabane disease virus recombinant N protein, an enzyme-labeled secondary antibody, a washing solution, a stop solution, a negative control, a positive control, and a result determination standard.

[0027] Furthermore, the method for preparing the ELISA plate containing the recombinant N protein of bovine Akabane disease virus is as follows:

[0028] A1. Coat the recombinant N protein of bovine Akabane disease virus onto an ELISA plate and incubate at 37°C for 2 hours.

[0029] A2. After removing it, shake out the liquid inside the hole, wash with washing solution, and pat the liquid droplets inside the hole dry.

[0030] A3. Add the blocking solution and incubate at 37°C for 2 hours;

[0031] A4. After removing the plate, shake out the liquid in the well, wash with washing solution, pat dry the liquid droplets in the well, and air dry in a 37°C incubator to obtain the ELISA plate of recombinant N protein of bovine Akabane disease virus.

[0032] Furthermore, in step A1, the concentration of the recombinant N protein of bovine Akabane virus used in the ELISA plate for preparing the recombinant N protein of bovine Akabane virus is 7 μg / mL.

[0033] Furthermore, in step A3, the blocking solution is BSA with a mass percentage concentration of 3%-5%, gelatin blocking buffer with a mass percentage concentration of 0.3%, or skim milk powder with a mass percentage concentration of 5%.

[0034] Furthermore, the indirect ELISA kit also includes a sample diluent, the ingredients of which include potassium dihydrogen phosphate, disodium hydrogen phosphate, bovine serum albumin, surfactant, deionized water, and preservative, and the pH of the diluent is 7.4.

[0035] The raw materials for preparing the washing solution include potassium dihydrogen phosphate, disodium hydrogen phosphate, surfactant, and deionized water, and the pH value of the washing solution is 7.4.

[0036] The ingredients for preparing the termination solution include concentrated sulfuric acid and deionized water, and the pH value of the termination solution is 0.4.

[0037] Based on the above technical solution, this embodiment can produce at least the following technical effects:

[0038] (1) The present invention obtains recombinant N protein antigen of bovine Akabane virus by prokaryotic expression, which has been verified to specifically bind to the antibody of bovine Akabane virus and can be used as a candidate antigen for detection of bovine Akabane virus.

[0039] (2) The ELISA detection kit developed in this invention can be used to detect bovine red feather disease virus antibodies. It has the advantages of high specificity, high sensitivity, good repeatability, low cost (calculated based on the price of the reagents in this application, the cost per well is only RMB 8.65, which is about 1 / 4 of the existing imported kits), and can be used for large-scale detection. Attached Figure Description

[0040] Figure 1 The results of PCR amplification of the AKAV N gene;

[0041] Figure 2 PCR identification results for the recombinant expression vector pET28a-N;

[0042] Figure 3 The results of double enzyme digestion identification of the recombinant expression vector pET28a-N;

[0043] Figure 4 The expression results of the recombinant expression vector pET28a-N were detected by SDS-PAGE.

[0044] Figure 5 The results are from Western blot analysis of N protein reactivity. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0046] Example 1:

[0047] Prokaryotic expression, purification and identification of recombinant N protein of bovine Akabane disease virus

[0048] 1. Primer design and synthesis

[0049] Based on the bovine AKAV N protein gene sequence (accession number: KU375444.1) published in GenBank (GenBank: OR791102.1), primers were designed using gene positions 46-727, and the following primers were constructed:

[0050] Upstream primer AKAV-NF: 5'-CCG GAATTC ATTTTCAACGATGTTC-3';

[0051] Downstream primer AKAV-NR: 5'-CCG CTCGAG TCTGAATACCAAATTGAG-3';

[0052] Primers were synthesized by BGI Genomics Co., Ltd. The underlined portion of the upstream primer indicates the added EcoRI restriction site sequence, and the underlined portion of the downstream primer indicates the added XhoI restriction site sequence.

[0053] 2. Amplification of the N gene

[0054] RNA was extracted from bovine Akabane disease virus using the FastPure Viral DNA / RNA Mini Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC311). Reverse transcription was performed using HiScript III RTSuperMix reverse transcriptase (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC323-01). This RNA was then used as a template for PCR amplification. The PCR reaction mixture consisted of 50 μL, including 4 μL of template, 4 μL each of upstream and downstream primers (both at 10 μmol / L), 25 μL of 2×Rapid Taq MaterMix, and 13 μL of ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, followed by 95℃ denaturation for 15 s, 55℃ annealing for 15 s, and 72℃ extension for 15 s, for a total of 37 cycles, with a final extension at 72℃ for 5 min.

[0055] PCR products were subjected to agarose gel electrophoresis. Positive products were recovered using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number DC301-01). The concentration of PCR products was determined using a micro-volume nucleic acid and protein quantification instrument (BioDrop, UK). Figure 1 The image shows the PCR amplification results of the AKAV N gene, where M is the DL 2000 DNA Marker and N is the amplification result of the AKAV N gene, with a size of 682 bp.

[0056] The results showed that a band consistent with the size of the target fragment (682bp) was amplified.

[0057] 3. Construction and expression of the N gene prokaryotic expression vector

[0058] The PCR products of the pET28a vector plasmid and the N gene were digested with FlyCutEcoRI and FlyCutXhoI (Beijing TransGen, catalog numbers JE201-01 and JX201-01), and the products were recovered by gel electrophoresis.

[0059] The two were then ligated using T4 DNA ligase (Takara, catalog number 2011A) at 25°C for 2 hours.

[0060] The ligation product was transformed into Top10 competent cells to construct the expression vector pET28a-N. The constructed pET28a-N plasmid was identified by double digestion with FlyCutEcoRI and FlyCutXhoI and sequencing.

[0061] The successfully constructed pET28a-N plasmid was transformed into *E. coli* BL21 competent cells. After culture, positive single colonies were picked and expanded. These colonies were then inoculated at a 1% ratio into 200 mL of LB broth containing kanamycin and cultured at 37°C / 220 rpm for approximately 4 hours. OD 600nm When the concentration reaches approximately 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG, Sangon Biotech Shanghai Co., Ltd., catalog number A100487) to a final concentration of 1 mmol / L, and induce at 37℃ and 180 rpm for 5 h. Collect the induced bacterial culture by centrifugation at 8000 rpm / 4℃ for 5 min, resuspend the cells in 10 mL of 1×PBS, and sonicate at 200 W for 10 min on ice. Centrifuge the sonicated suspension at 8000 rpm / 4℃ for 5 min in a refrigerated centrifuge, transfer the supernatant to a new centrifuge tube, and resuspend the pellet in 10 mL of 1×PBS.

[0062] The fragmented precipitate was washed with Wash Buffer 1 (100 mmol / L Tris-HCl, 2 mol / L urea, 10 mL / L Tween-20, pH 7.4) at room temperature for 8 min, then centrifuged at 10,000 rpm for 10 min at 4 °C. The precipitate was collected and washed once with Wash Buffer 2 (50 mmol / L Tris-HCl, 4 mol / L urea, pH 8.0), then centrifuged at 10,000 rpm for 10 min at 4 °C. The precipitate was then washed with sterile physiological saline and centrifuged at 10,000 rpm for 10 min at 4 °C. The precipitate was collected as inclusion bodies and dissolved with denaturing solution (8 mol / L urea) at 4 °C for 10 h. The denatured protein solution was then placed in a dialysis bag and dialyzed for renaturation at 4 °C for 4 h with 100 × volumes of 6 mol / L urea, 4 mol / L urea, 2 mol / L urea, and 1 × PBS solution (pH 7.4), respectively. The supernatant, precipitate, and refolded protein were prepared into protein samples and analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The successfully expressed recombinant N protein was aliquoted and stored at -80°C for later use.

[0063] like Figure 2 The image shows the PCR identification results of the recombinant expression vector pET28a-N, where M is the DL 2000 DNA Marker, lanes 1-6 are the PCR identification results of pET28a-N positive recombinants, and lane 7 is the PCR amplification result of the pET28a empty vector.

[0064] like Figure 3The results of double enzyme digestion identification of the recombinant expression vector pET28a-N are shown, where M is DL5000 DNA Marker; lane 1 is the empty pET28a vector; lane 2 is the recombinant expression vector pET28a-N; lane 3 is the result of double digestion of the empty pET28a vector with EcoRI and XhoI; and lane 4 is the result of double digestion of the recombinant expression vector pET28a-N with EcoRI and XhoI.

[0065] like Figure 4 The image shows the SDS-PAGE results of the recombinant expression vector pET28a-N, where M is a 10-180 kDa protein molecular weight standard marker; lane 1 is the BL21 host bacterial control; lane 2 is the recombinant expression vector control without inducer; lane 3 shows the bacterial cell results 1 h after induction; lane 4 shows the supernatant results 5 h after induction; lane 5 shows the precipitation results 5 h after induction; and lane 6 shows the results after refolding and purification 5 h after induction.

[0066] The amplified product was successfully ligated into the pET28a vector by PCR and double enzyme digestion. Sequencing results confirmed that the fragment had 100% sequence similarity to the N gene of AKAV, confirming the successful construction of the recombinant expression vector pET28a-N. This recombinant expression vector pET28a-N was efficiently expressed after induction with 1 mmol / L IPTG for 5 h.

[0067] 4. Purification and activity identification of N protein

[0068] The refolded protein (successfully expressed recombinant N protein) was centrifuged at 8000 rpm / 4℃ for 15 min, filtered through a 0.45 μm syringe filter, and purified using a Ni-NTA protein purification pre-packed column (Sangon Biotech Shanghai Co., Ltd., catalog number C600332-0001). The washing buffer concentration was 200 mmol / L imidazole, and the elution buffer concentration was 500 mmol / L imidazole. The flow-through, washing, and elution buffers were collected and prepared separately for SDS-PAGE electrophoresis and Coomassie brilliant blue staining to observe the purification effect.

[0069] The concentration of recombinant protein (purified recombinant N protein) was determined using a BCA protein concentration assay kit (Sangon Biotech Shanghai Co., Ltd., catalog number C503061-1250), and the activity of the purified recombinant N protein was then identified by Western blot. The steps are as follows:

[0070] The purified recombinant protein was transferred onto a PVDF membrane (Biosharp, catalog number: BS-PVDF-45) and blocked with 5% BSA at 37°C for 2 hours. Bovine anti-AKAV positive antibody was used as the primary antibody, diluted 1:100, and incubated overnight at 4°C. HRP-labeled rabbit anti-bovine antibody (Beijing Solarbio Science & Technology Co., Ltd., catalog number SE233) was used as the secondary antibody, diluted 1:5000, and incubated at room temperature for 2 hours. The reactivity was then detected using a horseradish catalase DAB colorimetric kit (Sangon Biotech Shanghai Co., Ltd., catalog number C520017).

[0071] like Figure 5 The results of Western blot identification of N protein reactivity are shown, where M is the 10-180 kDa protein molecular weight standard marker; lane 1 shows the hybridization results of recombinant N protein with bovine red feather disease positive serum. Because the protein may undergo spatial folding when binding to the antibody, the band size is slightly larger than the theoretical value.

[0072] The results showed that the recombinant N protein had the ability to bind to bovine AKAV-positive serum, indicating that the expressed protein was reactive.

[0073] Example 2:

[0074] Establishment of an indirect ELISA antibody detection method for bovine red feather disease virus

[0075] 1. Screening of conditions for indirect ELISA antibody detection of bovine red feather disease virus

[0076] 1.1 Determining the optimal antigen coating concentration and optimal serum dilution using checkerboard titration method

[0077] The purified recombinant N protein obtained in Example 1 was coated onto 96-well microplates at different concentrations using carbonate buffer at pH 9.6. A total of 12 concentration gradients of protein solution were prepared, namely 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, and 11 μg / mL. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) served as the negative control (N). Positive serum for bovine Akabane disease was obtained from ID-VET (France), catalog number AKAC-4P, and served as the positive control (P). The negative and positive control sera were diluted 1:50, 1:100, 1:200, and 1:400, respectively. Perform the following steps: Add 100 μL of protein solutions of different concentrations to a 96-well microplate and incubate overnight at 4°C; remove the plate, shake off the liquid, wash 5 times with PBST, and blot dry; add 200 μL of 5% skim milk powder and incubate at 37°C for 2 hours; remove the plate, shake off the liquid, wash 5 times with PBST, and blot dry; add 100 μL of negative and positive control serum at different dilutions to each well and incubate at 37°C for 45 minutes; remove the plate, shake off the liquid, wash 5 times with PBST, and blot dry; add 100 μL of 1:5000 diluted HRP-labeled rabbit anti-bovine serum (Beijing Solarbio Science & Technology Co., Ltd., catalog number SE233) as a secondary antibody and incubate at 37°C for 40 minutes; remove the plate, shake off the liquid, wash 5 times with PBST, and blot dry; add 50 μL of... TMB chromogenic solution (Solebio Biotechnology Co., Ltd., catalog number PR1200), incubate at 37°C in the dark for 15 min, then remove and add 50 μL of stop solution (1M H2SO4) to each well. Measure the OD of each well using a microplate reader within 10 min. 450nm Record the absorbance values ​​and calculate P (positive control OD). 450 ) / N (Negative control OD) 450 The P / N value was used as the criterion for determining the optimal coating concentration and the optimal serum dilution.

[0078] Table 1. Results of screening tests based on antigen coating concentration and serum dilution factor.

[0079]

[0080] The results (Table 1) showed that the P / N ratio was highest at 14.8650 when the concentration of the coating antigen was 7 and 8 μg / mL and the dilution of negative and positive sera was 1:100. Following the principle of conserving coating protein, the optimal coating concentration of N protein was determined to be 7 μg / mL, and the optimal serum dilution was determined to be 1:100.

[0081] 1.2 Screening of protein coating conditions

[0082] The coating conditions for N protein were varied, with three conditions set: 37℃ for 2 hours, 37℃ for 1 hour + 4℃ overnight (12 hours), and 4℃ overnight (12 hours). The N protein coating concentration was 7 μg / mL. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) was used as a negative control (N); bovine Akabane disease positive serum was obtained from ID-VET, France, catalog number AKAC-4P, as a positive control (P). Negative and positive control sera were diluted 1:100, and all other conditions and procedures were the same as in 1.1. The OD of the samples was measured. 450 The value is calculated, and the P / N value is determined.

[0083] Table 2 Results of antigen coating condition screening

[0084] Serum type 37℃ / 2h 37℃ / 1h+4℃ 4℃ Positive control (P) 1.0125 1.0455 1.1632 Negative control (N) 0.1326 0.1411 0.1663 P / N value 7.6207 6.9946 7.4096

[0085] The results (Table 2) show that the N protein had the highest P / N value of 7.6207 when the coating condition was 37℃ for 2 hours. Therefore, the coating condition for the N protein was determined to be 37℃ for 2 hours.

[0086] 1.3 Screening of blocking solutions

[0087] Different types and concentrations of blocking solutions were varied, with four conditions set up: 3% BSA, 5% BSA, 0.3% gelatin blocking buffer (Shanghai Sangon Biotech Co., Ltd., catalog number E661002), and 5% skim milk powder. The N protein coating concentration was 7 μg / mL, and the coating condition was 37℃ for 2 h. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) was used as a negative control (N); bovine Akabane disease positive serum was obtained from ID-VET, France, catalog number AKAC-4P, as a positive control (P). Negative and positive control sera were diluted 1:100, and other conditions and procedures were the same as in 1.1. The OD of the samples was measured. 450 The value is calculated, and the P / N value is determined.

[0088] Table 3. Screening Results of Blocking Fluid

[0089] Serum type 3% BSA 5% BSA 0.3% gelatin 5% skim milk powder Positive control (P) 1.2283 1.2396 1.1967 1.2560 Negative control (N) 0.1560 0.1616 0.1359 0.1090 P / N value 7.8759 7.6732 8.8061 11.5229

[0090] The results (Table 3) show that the P / N value was the highest, at 11.5229, when the blocking solution was 5% skim milk powder. Therefore, 5% skim milk powder was determined as the blocking solution.

[0091] 1.4 Screening of Closure Time

[0092] The blocking time was varied, with three blocking time points: 1 h, 2 h, and 3 h. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2 h. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) was used as a negative control (N); bovine red feather disease positive serum was obtained from ID-VET, France, catalog number AKAC-4P, as a positive control (P). Negative and positive control sera were diluted 1:100. The blocking solution was 5% skim milk powder. All other conditions and procedures were the same as in 1.1. The OD of the samples was measured. 450 The value is calculated, and the P / N value is determined.

[0093] Table 4. Results of Closure Time Screening

[0094] Serum type 37℃ / 1h 37℃ / 2h 37℃ / 3h Positive control (P) 0.9196 0.9584 1.0333 Negative control (N) 0.1467 0.1498 0.2248 P / N value 6.2686 6.3979 4.5965

[0095] The results (Table 4) show that the P / N value is the highest, at 6.3979, when the sealing time is 37℃ for 2 hours. Therefore, the sealing time is determined to be 37℃ for 2 hours.

[0096] 1.5 Screening of primary antibody serum incubation temperature

[0097] The incubation temperature of the primary antibody serum was varied, with two conditions set: 25℃ and 37℃. The N protein coating concentration was 7 μg / mL, and the coating condition was 37℃ for 2 hours. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) served as the negative control (N); bovine Akabane disease positive serum, obtained from ID-VET (France), catalog number AKAC-4P, served as the positive control (P). Negative and positive control sera were diluted 1:100, and the blocking buffer was 5% skim milk powder. The blocking time was 37℃ for 2 hours. All other conditions and procedures were the same as in section 1.1. The OD of the samples was measured. 450nm The value is calculated, and the P / N value is determined.

[0098] Table 5. Screening results for primary antibody serum incubation temperature

[0099] Serum type 25℃ 37℃ Positive control (P) 1.0898 1.3884 Negative control (N) 0.1598 0.2411 P / N value 6.8198 5.7586

[0100] The results (Table 5) show that the P / N value was highest at 25℃, reaching 6.8198. Therefore, the optimal incubation temperature for the primary antibody serum was determined to be 25℃.

[0101] 1.6 Screening of primary antibody serum incubation time

[0102] The incubation time of the primary antibody serum was varied, with four conditions set: 15 min, 30 min, 45 min, and 60 min. The N protein coating concentration was 7 μg / mL, and the coating condition was 37℃ for 2 h. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) served as the negative control (N); bovine Akabane disease positive serum was obtained from ID-VET (France), catalog number AKAC-4P, and served as the positive control (P). Negative and positive control sera were diluted 1:100. The blocking buffer was 5% skim milk powder, and the blocking time was 37℃ for 2 h. The primary antibody serum incubation temperature was 25℃. All other conditions and procedures were the same as in section 1.1. The OD of the samples was measured. 450nm The value is calculated, and the P / N value is determined.

[0103] Table 6. Screening results of primary antiserum incubation time

[0104] Serum type 15min 30min 45min 60min Positive control (P) 1.0059 1.1682 1.4695 1.5993 Negative control (N) 0.1579 0.1508 0.1431 0.2383 P / N value 6.3705 7.7482 10.2690 6.7113

[0105] The results (Table 6) showed that the P / N value was highest (10.2690) when the primary antibody serum incubation time was 45 min. Therefore, the primary antibody serum incubation time was determined to be 45 min.

[0106] 1.7 Screening of enzyme-labeled secondary antibody dilutions

[0107] The dilution of the enzyme-labeled secondary antibody was varied, with six dilutions: 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, and 1:8000. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2 hours. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) served as the negative control (N); bovine Akabane disease positive serum, from ID-VET (France), catalog number AKAC-4P, served as the positive control (P). Negative and positive control sera were diluted 1:100. The blocking buffer was 5% skim milk powder, and the blocking time was 37℃ for 2 hours. The primary antibody serum was incubated at 25℃ for 45 minutes. All other conditions and procedures were the same as in section 1.1. The OD of the samples was measured. 450nm The value is calculated, and the P / N value is determined.

[0108] Table 7 Results of Secondary Antibody Dilution Screening

[0109] Serum type 1:3000 1:4000 1:5000 1:6000 1:7000 1:8000 Positive control (P) 2.1348 1.7397 1.4599 1.1214 1.0006 0.9224 Negative control (N) 0.2263 0.186 0.1666 0.1817 0.1607 0.1581 P / N value 9.4335 9.3532 8.7629 6.1717 6.2265 5.8343

[0110] The results (Table 7) show that the P / N value was highest at a dilution of 1:3000 for the enzyme-labeled secondary antibody, reaching 9.4335. Therefore, the optimal dilution for the enzyme-labeled secondary antibody was determined to be 1:3000.

[0111] 1.8 Screening of enzyme-labeled secondary antibody incubation time

[0112] The incubation time of the enzyme-labeled secondary antibody was varied, with four time points: 15 min, 20 min, 30 min, and 40 min. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2 h. Fetal bovine serum (Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500) served as the negative control (N); bovine red feather disease positive serum was obtained from ID-VET (France), catalog number AKAC-4P, and served as the positive control (P). Negative and positive control sera were diluted 1:100. The blocking buffer was 5% skim milk powder, and the blocking time was 37℃ for 2 h. The primary antibody serum was incubated at 25℃ for 45 min, and the enzyme-labeled secondary antibody was diluted 1:3000. All other conditions and procedures were the same as in section 1.1. The OD of the samples was measured. 450nm The value is calculated, and the P / N value is determined.

[0113] Table 8. Screening results of enzyme-labeled secondary antibody incubation time.

[0114] Serum type 15min 20min 30min 40min Positive control (P) 1.0925 1.199 1.4295 1.6268 Negative control (N) 0.1282 0.1215 0.1505 0.1554 P / N value 8.5218 9.8683 9.4983 10.4685

[0115] The results (Table 8) show that the P / N value was highest (10.4685) when the enzyme-labeled secondary antibody was incubated for 40 min. Therefore, the incubation time for the enzyme-labeled secondary antibody was determined to be 40 min.

[0116] Determination of incubation time for 1.9TMB colorimetric solution

[0117] The incubation time of the TMB chromogenic solution was varied, with four time points: 15 min, 20 min, 25 min, and 30 min. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2 h. Negative and positive control sera were diluted 1:100. The blocking buffer was 5% skim milk powder, and the blocking time was 37℃ for 2 h. The primary antibody serum incubation temperature was 25℃ for 45 min, and the enzyme-labeled secondary antibody dilution was 1:3000, with an incubation time of 40 min. All other conditions and procedures were the same as in section 1.1. The OD of the samples was then measured. 450nm The value is calculated, and the P / N value is determined.

[0118] Table 9. Screening Results of Incubation Time for TMB Chromogenic Solution

[0119] Serum type 15min 20min 25min 30min Positive control (P) 1.5830 1.8230 1.7295 1.8169 Negative control (N) 0.1407 0.1390 0.2188 0.2060 P / N value 11.2509 13.1151 7.9045 8.8199

[0120] The results (Table 9) show that the P / N value was highest (13.1151) when the incubation time of the TMB chromogenic solution was 20 min. Therefore, the incubation time of the TMB chromogenic solution was determined to be 20 min.

[0121] 2. Determination of the cut-off value for the indirect ELISA antibody detection method for bovine red feather disease virus.

[0122] In June 2024, 21 serum samples collected from the breeding farm of Guizhou Yellow Cattle Industry Group Qianxi City Co., Ltd. were tested by the Guizhou Provincial Animal Disease Prevention and Control Center. The samples were negative for AKAV nucleic acid and antibodies and were used to determine the cutoff value.

[0123] Using the ELISA testing conditions determined above, 21 samples were tested, repeated 3 times, totaling...

[0124] Calculate its mean (x) and standard deviation (SD).

[0125] Table 10 Results of three repeated tests of AKAV-negative serum

[0126]

[0127] The test results are shown in Table 10. OD values ​​from three tests of 21 bovine AKAV-negative serum samples were analyzed. 450nm average The values ​​were 0.2684, 0.2811, and 0.2109, respectively, with standard deviations (SD) of 0.0926, 0.0795, and 0.0715. The calculated critical value for the established indirect ELISA antibody detection method was 0.4969.

[0128] 3. Evaluation of the indirect ELISA antibody detection method for bovine Akabane disease virus

[0129] 3.1 Repeatability Test

[0130] Six bovine negative serum samples were collected and tested under the selected optimal testing conditions. Each sample was tested in triplicate, and its OD value was measured. 450nm The values ​​are used to calculate the mean (x) and standard deviation (SD). The intra-batch repeatability of the detection method is determined by the range of variation of the coefficient of variation (standard deviation / mean × 100%).

[0131] Table 11 Results of Intra-Batch Repeatability Tests

[0132]

[0133] The results (Table 11) show that the intra-batch coefficients of variation for the six samples ranged from 0.32% to 4.74%, all less than 5%, indicating good intra-batch reproducibility.

[0134] Six additional bovine negative serum samples were collected and tested in different batches under the selected optimal testing conditions, for a total of three batches. Their OD values ​​were measured. 450nm The mean (x) and standard deviation (SD) of the sample from different batches are calculated, and the inter-batch repeatability of the detection method is determined by the range of variation of the coefficient of variation (standard deviation / mean × 100%).

[0135] Table 12 Results of inter-batch repeatability tests

[0136]

[0137] The results (Table 12) show that the inter-batch coefficients of variation for the six samples ranged from 1.66% to 5.46%, all less than 6%, indicating good inter-batch repeatability.

[0138] 3.2 Specificity test

[0139] The established indirect ELISA antibody detection method for bovine red tongue virus was used to detect positive serum samples from five other bovine pathogens (bovine bluetongue virus, bovine rotavirus, bovine foot-and-mouth disease virus type O, bovine viral diarrhea virus, and bovine mycobacterium paratuberculosis).

[0140] Table 13 Results of Specificity Tests

[0141]

[0142] The test results are shown in Table 13. Positive serum OD values ​​for the five pathogens... 450nm All values ​​were less than the critical value of 0.4969, indicating that the established ELISA method had good specificity.

[0143] 3.3 Sensitivity Test

[0144] The established indirect ELISA antibody detection method for bovine Akabane disease virus was used to detect three bovine Akabane disease positive serum samples from a farm in Guizhou Province. The three serum samples were diluted 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000 and 1:1200, respectively.

[0145] Table 14 Sensitivity Test Results

[0146] Dilution factor Positive serum 1 Positive serum 2 Positive serum 3 1:50 1.8265 1.8651 1.6294 1:100 1.6196 1.3738 1.3425 1:200 1.3018 1.4079 1.133 1:400 0.9127 0.9046 0.5745 1:600 0.9636 0.6974 0.5265 1:800 0.8661 0.5943 0.4592 1:1000 0.7327 0.5143 0.3903 1:1200 0.7226 0.4997 0.6714

[0147] The test results are shown in Table 14. The OD values ​​of the three serum samples were 1:1200. 450nm The values ​​were all greater than the critical value of 0.4969, indicating a positive result, but the OD of positive serum 2 was higher. 450nm The value was 0.4997, which is close to the critical value. Clinical samples are usually diluted between 1:100 and 1:400 during testing. The ELISA detection method established in this patent can still effectively detect samples at a dilution of 1:1200, indicating its high sensitivity.

[0148] 3. Clinical sample testing and comparison with results from commercially available reagent kits from abroad

[0149] Sixty clinical bovine serum samples were collected from a beef cattle farm in Guizhou Province. The samples were analyzed using the established ELISA method and a commercially available reagent kit (ID). Akabane Competition ELISA Kit

[0150] The results of the two tests were compared using the (ID-VET, France, catalog number AKAC-4P) Akabane disease antibody test kit.

[0151] Table 15 Clinical serum test results

[0152]

[0153]

[0154]

[0155] The results (Table 15) show that all 60 samples tested using the method established in this experiment were negative; ID The company's test kit yielded 1 positive sample and 59 negative samples. The concordance rate was 98.3%.

[0156] Example 3: Assembly of the Bovine Akabane Disease Virus Indirect ELISA Antibody Detection Kit

[0157] 3.1 Components of the reagent kit

[0158] 3.1.1 Antigen Pre-coated Plates (96 wells / plate × 1 plate / box). Coat 100 μL of 7 μg / mL N protein onto a 96-well ELISA plate (BioSharp, BS-PS-96WB-100) and incubate at 37°C for 2 h. After removal, discard the liquid from the wells, wash 5 times with washing buffer for 1 min each time, and pat dry the wells. Add 200 μL of 5% skim milk powder (Solepro, D8340) and incubate at 37°C for 2 h. After removal, discard the liquid from the wells, wash 5 times with washing buffer for 1 min each time, pat dry the wells, and air dry at 37°C for 20 minutes. Vacuum package in aluminum foil bags.

[0159] 3.1.2 Serum dilution plates, 96 wells / plate × 1 plate / box. Purchased from Guangzhou Jetech Biofiltration Co., Ltd., the packaging was intact, the labeling was clear, there were no spots, stains, or damage, no obvious color difference, and the bottom and walls of the wells were clean, transparent, and free of foreign matter.

[0160] 3.1.3 Positive control serum 50 μL / vial × 1 vial / box. Purchased from ID-VET, France. OD was measured three times in duplicate. 450nm The average value was above 0.5569. The OD values ​​of the positive control serum were... 450nmThe value was calibrated to the range of 0.5-1.5. The serum was a clear, light yellow liquid, odorless, free of hemolysis or foreign matter, and free from sterile contamination.

[0161] 3.1.4 Negative control serum 50 μL / vial × 1 vial / box. Purchased from Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGL3006-500. OD was measured three times in duplicate. 450nm The average value was below 0.1836. The OD values ​​of the negative control serum were... 450nm The values ​​were calibrated to the range of 0.18-0. The serum was from Uruguay, a region free of BSE. Mycoplasma testing and virus screening were both negative. The serum was sterile and clear, a light yellow liquid, odorless, and free of hemolysis or foreign matter.

[0162] 3.1.5 Enzyme-labeled secondary antibody 10μL / vial × 1 vial / box. Purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number SE233, rabbit anti-bovine IgG-HRP. Diluted using pH 7.4 PBST, at a dilution factor of 1:3000.

[0163] 3.1.6 Sample diluent: 30 mL / bottle × 1 bottle / box. Preparation method: Dissolve 1.724 g potassium dihydrogen phosphate, 7.667 g disodium hydrogen phosphate, and 1.0 g bovine serum albumin in deionized water to a final volume of 800 mL. Add 0.5 mL Tween-20 and 200 μL Proclin 300 preservative. Adjust the pH to 7.4 and bring the volume to a final volume of 1000 mL. Filter sterilize, aseptically dispense quantitatively, and label.

[0164] 3.1.710× Concentrated Washing Solution 30mL / bottle × 1 bottle / box. Weigh 17.24g of potassium dihydrogen phosphate and 76.67g of disodium hydrogen phosphate, add deionized water to 800mL, dissolve thoroughly, add 5.0mL of Tween-20, adjust the pH to 7.4, and bring the volume to 1000mL. Sterilize at 121℃ for 20 minutes, then dispense quantitatively and label.

[0165] 3.1.8 TMB colorimetric reagent 10mL / bottle × 1 bottle / box. Purchased from Beijing Solarbio Science & Technology Co., Ltd., product number PR1200, is a single-component TMB colorimetric reagent. This reagent is generally colorless in appearance, and the OD value at 650nm is less than 0.04. Store in a brown reagent bottle protected from light.

[0166] 3.1.9 Termination solution 10mL / bottle × 1 bottle / box. Preparation method: Add 108.7mL of 98% analytical grade concentrated sulfuric acid to 800mL with deionized water, adjust the pH to 0.4, and bring the volume to 1000mL. Quantitatively dispense and label.

[0167] 3.1.10 One instruction manual per box.

[0168] 3.2 The reagent kit shall be packaged in a suitable outer box and labeled with information such as name, batch number, production date, shelf life and manufacturer information.

[0169] 3.3 Specification Design Draft

[0170] Instructions for use of the Bovine Akabane Virus Indirect ELISA Antibody Detection Kit

[0171] [Veterinary Drug Name] Veterinary Use

[0172] Generic name: Bovine Akabane Disease Virus Indirect ELISA Antibody Detection Kit

[0173]

Main Ingredients and Content

[0174] (1) One antigen-coated plate (96 wells / plate)

[0175] (2) Serum dilution plate (96 wells / plate)

[0176] (3) One vial of positive control serum (50 μL / vial)

[0177] (4) One vial of negative control serum (50 μL / vial)

[0178] (5) One vial of enzyme-labeled secondary antibody (10 μL / vial)

[0179] (6) One bottle of sample diluent (30 mL / bottle)

[0180] (7) 1 bottle of 10× concentrated detergent (30mL / bottle)

[0181] (8) One bottle of TMB colorimetric solution (10mL / bottle)

[0182] (9) One bottle of stop solution (10mL / bottle)

[0183] (10) One instruction manual

[0184]

Functions and Uses

[0185] Antibodies used to detect Akabane virus N protein in bovine serum are suitable for detecting antibodies against Akabane virus in bovine serum samples.

[0186]

Usage and Judgment

[0187] 1. Usage

[0188] 1.1 Sample Preparation: Collect whole bovine blood. After the blood has coagulated, centrifuge at 4000 rpm for 10 minutes and collect the supernatant for later use. Alternatively, blood can be collected, and serum can be extracted naturally after coagulation. The serum should be clear and free of hemolysis.

[0189] 1.2 Preparation of Washing Solution: After the 10× concentrated washing solution has been brought to room temperature, shake it thoroughly. If crystals still remain, dissolve them by heating in a 37°C water bath, and then dilute it 10 times with deionized water for later use.

[0190] 1.3 Sample dilution on a serum dilution plate: Dilute the serum to be tested and the negative and positive control sera at a volume ratio of 1:100 (recommended dilution method: add 2 μL of serum sample to 198 μL of sample dilution solution).

[0191] 1.4 Operating Procedures

[0192] 1.4.1 Adding samples: Take the antigen-coated plate and add 100 μL of diluted serum to be tested, negative control serum and positive control serum to the corresponding wells. There are 2 wells for each of the negative and positive control serums. Gently pipette to mix.

[0193] 1.4.2 Incubate at 25°C for 45 minutes.

[0194] 1.4.3 Wash and discard the reaction solution. Add 200 μL of diluted washing solution to each well, let it stand at room temperature for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat 5 times; or wash 5 times with an automatic plate washer.

[0195] 1.4.4 Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody 1:3000 with diluent (prepare fresh for immediate use), and add 100 μL of the diluted enzyme-labeled secondary antibody to each well.

[0196] 1.4.5 Incubate at 37°C for 40 minutes.

[0197] 1.4.6 Washing is the same as 1.4.3.

[0198] 1.4.7 For color development, add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 20 minutes.

[0199] 1.4.8 Add 50 μL of stop solution to each well and measure the results within 10 minutes.

[0200] 1.4.9 Reading: Read the absorbance at 450 nm wavelength for each well using an ELISA reader.

[0201] 2judgment

[0202] 2.1 The experimental conditions for success are met simultaneously: the average OD of the positive control is... 450nm ≥0.50, mean OD of negative control 450nm A value less than 0.18 is considered valid.

[0203] 2.2 Determination of OD in the serum to be tested 450nm <0.4969, indicating a negative result for bovine red feather disease antibody; serum OD to be tested450nm A value ≥0.4969 indicates a positive result for bovine red feather disease antibodies.

[0204]

Precautions

[0205] 1. Before use, all reagents in the kit, except for the enzyme-labeled secondary antibody, should be equilibrated to room temperature. If crystals have precipitated, be sure to heat them to completely dissolve them. Liquid reagents should be mixed well before use. To ensure accurate and reliable results, the operating environment temperature should be controlled at 20±5℃.

[0206] 2. Micropipettes used for sample addition should be calibrated regularly to avoid excessive errors affecting test results.

[0207] 3. Ensure the quality of serum samples and avoid using severely hemolyzed or lipophilic samples; avoid repeated freeze-thaw cycles of serum samples; serum samples should not be stored at 2–8°C for more than 5 days, and for long-term storage, they should be stored below -20°C; if serum samples are turbid or contain flocculent matter, they should be centrifuged and the supernatant should be collected for testing.

[0208] 4. Avoid cross-contamination between reagents and samples during use. The kit should be used within its expiration date; avoid mixing kits from different batches.

[0209] 5. Unused ELISA plates should be returned to the sealed bag along with the desiccant and stored at 2–8°C.

[0210] 6. When performing multiple ELISA test plates, the plates should not be stacked but laid flat in a 37°C incubator to avoid uneven heating of the plates.

[0211] Do not expose 7TMB developer to strong light or allow it to come into contact with oxidants.

[0212] 8. The stop solution is corrosive; avoid contact with eyes and skin.

[0213] 9. Strictly follow the instructions and control the reaction time and temperature at each step.

[0214] 10. Adhere to laboratory biosafety protocols, and all waste must be disposed of in accordance with regulations to ensure its safety.

[0215]

Specification

[0216] 96 holes / piece, 1 piece / box

[0217] Storage and Shelf Life

[0218] Store at 2–8℃. Shelf life is 6 months.

[0219] Approval Number: None available.

[0220] [Manufacturer] None available.

[0221] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A recombinant N protein antigen of bovine red feather disease virus, characterized in that, Its amino acid sequence is shown in SEQ ID No.

2.

2. A gene encoding the amino acid sequence of the recombinant N protein antigen of bovine Akabane disease virus as described in claim 1, characterized in that, The gene sequence is shown in SEQ ID No.

1.

3. The method for preparing the recombinant N protein antigen of bovine Akabane disease virus according to claim 1, characterized in that, Includes the following steps: S1. Primer design and synthesis Upstream primer AKAV-NF: 5'-CCG GAATTC ATTTTCAACGATGTTC-3'; Downstream primer AKAV-NR: 5'-CCG CTCGAG TCTGAATACCAAATTGAG-3'; the underlined parts are the additions. EcoR I and Xho I restriction site sequence; Amplification of S2 and N genes RNA was extracted from bovine red feather disease virus and reverse transcribed using reverse transcriptase. This RNA was then used as a template for PCR amplification to obtain the PCR amplification product of the N gene. The sequence was determined by sequencing as shown in SEQ ID No.

1. Construction and expression of S3 and N gene prokaryotic expression vectors S31. The PCR amplification products of the pET28a vector plasmid and the N gene were digested with enzymes and the products were recovered by gel electrophoresis. S32. The digested pET28a vector plasmid and the digested N gene PCR amplification product were ligated using a ligase. S33. Transform the ligation product obtained in step S32 into Top10 competent cells to construct the expression vector pET28a-N; pass FlyCut EcoR I and FlyCut Xho I. Double enzyme digestion and sequencing were used to identify the constructed expression vector pET28a-N plasmid; S34. The successfully constructed expression vector pET28a-N was transformed into Escherichia coli BL21 competent cells. After culture, positive single colonies were picked and expanded to prepare protein samples. The protein samples were detected by SDS-PAGE electrophoresis and analyzed by Coomassie brilliant blue staining. The successfully expressed recombinant N protein was aliquoted and stored for later use. Purification and activity identification of S4 and N proteins The successfully expressed recombinant N protein obtained in step S34 was purified, the concentration of the purified recombinant N protein was determined, and the activity of the recombinant N protein was identified.

4. Application of the gene shown in SEQ ID No. 1 in the preparation of a bovine Akabane disease antibody detection kit.

5. Application of the recombinant N protein antigen of bovine Akabane virus encoded by the gene shown in SEQ ID No. 2 in the preparation of bovine Akabane virus antibody detection kit.

6. An indirect ELISA kit for detecting bovine Akabane disease virus antibodies, characterized in that, The enzyme-labeled plate coated with the recombinant N protein of bovine red feather disease virus as described in claim 1, the enzyme-labeled secondary antibody, the washing solution, the stop solution, the negative control, the positive control, and the result determination criteria are included.

7. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 6, characterized in that, The method for preparing the ELISA plate containing the recombinant N protein of bovine Akabane disease virus is as follows: A1. Coat the recombinant N protein of bovine Akabane disease virus onto an ELISA plate and incubate at 37°C for 2 hours; A2. After removing it, shake out the liquid inside the hole, wash with washing solution, and pat the liquid droplets inside the hole dry. A3. Add the blocking solution and incubate at 37°C for 2 hours; A4. After removing the plate, shake out the liquid in the well, wash with washing solution, pat dry the liquid droplets in the well, and air dry in a 37°C incubator to obtain the ELISA plate of recombinant N protein of bovine Akabane disease virus.

8. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 7, characterized in that, In step A1, the concentration of recombinant N protein of bovine Akabane virus used in the ELISA plate for preparing the recombinant N protein of bovine Akabane virus is 7 μg / mL.

9. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 7, characterized in that, In step A3, the blocking solution is BSA with a mass percentage concentration of 3%-5%, gelatin blocking buffer with a mass percentage concentration of 0.3%, or skim milk powder with a mass percentage concentration of 5%.

10. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 6, characterized in that, The indirect ELISA kit also includes a sample diluent, the ingredients of which include potassium dihydrogen phosphate, disodium hydrogen phosphate, bovine serum albumin, surfactant, deionized water, and preservative, and the pH of the diluent is 7.

4. The raw materials for preparing the washing solution include potassium dihydrogen phosphate, disodium hydrogen phosphate, surfactant, and deionized water, and the pH value of the washing solution is 7.

4. The ingredients for preparing the termination solution include concentrated sulfuric acid and deionized water, and the pH value of the termination solution is 0.4.

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