Bovine akabane disease virus recombinant N protein antigen, preparation method and ELISA (Enzyme-Linked Immunosorbent Assay) kit

Abstract

The invention provides a bovine akabane disease virus recombinant N protein antigen, a preparation method and an ELISA kit, relates to the field of biotechnology detection, and solves the problems that in the prior art, a competitive ELISA technical means is generally adopted in an ELISA detection method, the preparation cost is high, the price of a commercialized kit is high, and the detection cost is low. The development of beef cattle breeding enterprises in China is not facilitated. The amino acid sequence of the bovine akabane disease virus recombinant N protein antigen is as shown in SEQ ID No.2. The bovine akabane disease virus recombinant N protein antigen obtained in the invention can be specifically combined with an antibody of bovine akabane disease virus through verification, and can be used as a candidate antigen for bovine akabane disease virus detection; the ELISA detection kit developed by the invention can be used for detecting the bovine akabane disease virus antibody, and has the advantages of strong specificity, high sensitivity, good repeatability, low cost, large-scale detection and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biological products, and in particular to a bovine Akabane disease virus recombinant N protein antigen, a preparation method and an ELISA kit. Background Art

[0002] Akabane disease, also known as Akabane Disease (AKAD), is a viral infectious disease of cattle and sheep characterized by miscarriage, premature birth, stillbirth, fetal malformation, mummification, and arthrogryposis anencephaly syndrome (AH syndrome) in newborns. The pathogen of this disease, Akabane virus (AKAV), is a member of the Peribunyaviridae family, Orthobunyavirus genus, and Simbu serogroup. Virus particles are generally spherical, with capsules and fibrils, with a diameter of about 80 to 120 nm, a genome of about 12 kb, and a total of three genomic 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 and suitable as a target for the detection method. The disease can pose a huge threat to ruminants such as cattle and sheep, and has caused serious economic losses worldwide. There is currently no commercial vaccine available in my country. Currently, the prevention and control of AKAV mainly relies on integrated biosafety management and pathogen detection and elimination.

[0003] N protein is the nucleocapsid protein of AKAV, and is also the most abundant protein and the main immunogenic protein in AKAV. This protein is highly conserved among different strains, and the similarity of the amino acid sequence is 97%~100%. N protein can induce the body to produce antibodies, so it is the preferred target in immunological diagnostic methods. Since there is currently no vaccine for Akabane disease prevention in my country, once AKAV-specific antibodies are detected in clinical samples, it can be proved that there is AKAV infection in the animal population or that it has been infected with AKAV, which has important reference value in clinical diagnosis. Enzyme linked immunosorbent assay (ELISA) has many advantages such as convenient and rapid operation, high sensitivity, strong specificity, and large-scale detection. It is easy to promote and use at the grassroots level and is one of the most commonly used methods of serological detection technology.

[0004] Among the existing methods for detecting bovine AKAV antibodies, the commonly used detection methods are: colloidal gold method detection and ELISA method detection, but the applicant has found that the existing technology has at least the following technical problems: ① The existing colloidal gold method for antibody detection can only qualitatively determine the presence of antibodies, but cannot determine the amount of antibodies. In addition, the colloidal gold method is generally not suitable for large-scale detection, and its sensitivity is also low; ② The existing ELISA detection methods generally use competitive ELISA technology, which has a high preparation cost and the price of commercialized test kits is also relatively expensive. For example, the ID Screen® Akabane Competition ELISA Kit sold by the French ID-VET company is priced at RMB 15,000. The cost of a single well of the test kit alone is RMB 31.25, which is not conducive to the development of my country's beef cattle breeding enterprises. Summary of the invention

[0005] The purpose of the present invention is to provide a bovine Akabane disease virus recombinant N protein antigen and a preparation method and an ELISA kit to solve the technical problem that the existing ELISA detection method generally adopts competitive ELISA technical means, has a high preparation cost, and the price of commercial kits is also relatively expensive, which is unfavorable to the development of beef cattle breeding enterprises in my country. The many technical effects that can be produced by the preferred technical scheme among the many technical schemes provided by the present invention are described in detail below.

[0006] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a bovine Akabane disease virus recombinant N protein antigen, whose amino acid sequence is shown in SEQ ID No. 2.

[0007] The gene encoding the amino acid sequence of the bovine Akabane disease virus recombinant N protein antigen provided by the present invention is shown in SEQ ID No. 1.

[0008] The method for preparing the recombinant N protein antigen of bovine Akabane disease virus provided by the present invention comprises 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 added EcoR I and XOt I restriction site sequence; Amplification of S2 and N genes Extract RNA of bovine Akabane disease virus, perform reverse transcription using reverse transcriptase, and use it as a template for PCR amplification to obtain a PCR amplification product of the N gene, and sequence the sequence as shown in SEQ ID No. 1; Construction and expression of prokaryotic expression vectors of S3 and N genes S31, digesting the pET28a vector plasmid and the PCR amplification product of the N gene, and recovering the products by gel respectively; S32, connecting the pET28a vector plasmid after restriction digestion and the PCR amplification product of the N gene after restriction digestion using a ligase; S33, transforming the ligation product obtained in step S32 into Top10 competent cells to construct an expression vector pET28a-N; FlyCut EcoR I and FlyCut Xho I double enzyme digestion and sequencing were used to identify the constructed expression vector pET28a-N plasmid; S34, transforming the successfully constructed expression vector pET28a-N into Escherichia coli BL21 competent cells, picking positive monoclonal colonies for expansion and culture after cultivation, preparing protein samples, and performing SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining analysis on the protein samples; packaging the successfully expressed recombinant N protein for storage and standby use;

[0009] The successfully expressed recombinant N protein obtained in step S34 is purified, the concentration of the purified recombinant N protein is measured, and the activity of the recombinant N protein is identified.

[0010] The invention provides an application of the gene shown in SEQ ID No. 1 in preparing a kit for detecting antibodies against bovine Akabane disease.

[0011] The present invention provides an application of the amino acid sequence encoded by the gene as shown in SEQ ID No. 2 in preparing a kit for detecting antibodies against bovine Akabane disease.

[0012] The invention provides an indirect ELISA kit for detecting bovine Akabane disease virus antibodies, comprising an ELISA plate coated with the bovine Akabane disease virus recombinant N protein according to claim 1, an enzyme-labeled secondary antibody, a washing solution, a stop solution, a negative control, a positive control and a result judgment standard thereof.

[0013] The preparation method of the ELISA plate of the bovine Akabane disease virus recombinant N protein is as follows: A1. Coat the recombinant N protein of Akabane disease virus onto an ELISA plate and incubate at 37°C for 2 hours; A2. After taking out, shake out the liquid in the hole, wash with detergent, and pat dry the droplets in the hole; A3, add blocking solution and incubate at 37℃ for 2h; A4. After taking out, shake out the liquid in the wells, wash with detergent, pat dry the droplets in the wells, and air-dry in a 37°C incubator to obtain the ELISA plate of bovine Akabane disease virus recombinant N protein.

[0014] Furthermore, in the step A1, the concentration of the recombinant N protein of bovine Akabane disease virus used in the ELISA plate for preparing the recombinant N protein of bovine Akabane disease virus is 7 μg / mL.

[0015] Furthermore, in the 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 skimmed milk powder with a mass percentage concentration of 5%.

[0016] Furthermore, the sample diluent is prepared from raw materials including potassium dihydrogen phosphate, disodium hydrogen phosphate, bovine serum albumin, a surfactant, deionized water, and a preservative, and the pH value of the diluent is 7.4; The raw materials for the washing liquid include potassium dihydrogen phosphate, disodium hydrogen phosphate, a surfactant, and deionized water, and the pH value of the washing liquid is 7.4; The preparation materials of the stop solution include concentrated sulfuric acid and deionized water, and the pH value of the stop solution is 0.4.

[0017] Based on the above technical solution, this embodiment can at least produce the following technical effects: (1) The present invention obtains the recombinant N protein antigen of bovine Akabane disease virus through prokaryotic expression, which has been verified to be able to specifically bind to the antibody of bovine Akabane disease virus and can be used as a candidate antigen for bovine Akabane disease virus detection.

[0018] (2) The ELISA detection kit developed by the present invention can be used for the detection of antibodies to bovine Akabane disease virus and has the advantages of strong specificity, high sensitivity, good repeatability, low cost (calculating 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of PCR amplification of AKAV N gene; Figure 2 This is the PCR identification result of the recombinant expression vector pET28a-N; Figure 3 The results of double enzyme digestion of the recombinant expression vector pET28a-N; Figure 4 The expression results of the recombinant expression vector pET28a-N were detected by SDS-PAGE; Figure 5 This is the result of Western blot N protein reactivity identification. DETAILED DESCRIPTION

[0020] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. Example Prokaryotic expression, purification and identification of recombinant N protein of bovine Akabane disease virus

[0021] 1. Primer design and synthesis According to the gene sequence of bovine AKAV N protein published on GenBank (accession number: KU375444.1) information GenBank: OR791102.1, the 46th to 727th gene was selected to design the vector construction primers, among which: Upstream primer AKAV-NF: 5'-CCG GAATTC ATTTTCAACGATGTTC-3'; Downstream primer AKAV-NR: 5'-CCG CTCGAG TCTGAATACCAAATTGAG-3′; Primers were synthesized by BGI Genomics Co., Ltd. The underlined portion of the upstream primer is added EcoR I restriction site sequence, The underlined part of the downstream primer is added XOt I restriction site sequence.

[0022] 2. Amplification of the N gene The RNA of bovine Akabane disease virus was extracted using the nucleic acid extraction kit FastPure Viral DNA / RNA Mini Kit (Nanjing Novogene Biotech Co., Ltd., Catalog No. RC311), and reverse transcription was performed using reverse transcriptase HiScript III RTSuperMix (Nanjing Novogene Biotech Co., Ltd., Catalog No. RC323-01). The RNA was used as a template for PCR amplification. The PCR reaction system was: 50 μL, including 4 μL of template, 4 μL of upstream primer and downstream primer (both with a concentration of 10 μ mol / L), 25 μL of 2´Rapid Taq Master Mix, and 13 μL of ddH2O; the PCR amplification program was: pre-denaturation at 95°C for 3 min, followed by denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 15 s, for a total of 37 cycles, and finally terminal extension at 72°C for 5 min.

[0023] The PCR products were subjected to agarose gel electrophoresis, and the positive products were recovered using the gel recovery kit FastPure Gel DNA Extraction Mini Kit (Nanjing Novogene Biotech Co., Ltd., Cat. No. DC301-01), and the concentration of the PCR products was determined using a micro-nucleic acid protein quantifier (BioDrop, BioDrop, UK). Figure 1 The figure shows the PCR amplification result of AKAV N gene, where M is DL 2000 DNA Marker and N is the amplification result of AKAV N gene, with a size of 682 bp.

[0024] The results showed that a band consistent with the target fragment size (682 bp) was amplified.

[0025] 3. Construction and expression of N gene prokaryotic expression vector use FlyCut EcoR I. FlyCut Xho I (Beijing Quanshijin, catalog number JE201-01, JX201-01) digested the pET28a vector plasmid and the PCR product of the N gene, and recovered the products by gel respectively; The two were then ligated using T4 DNA ligase (Takara, Cat. No. 2011A) at 25°C for 2 h; The ligation product was transformed into Top10 competent cells to construct the expression vector pET28a-N. FlyCut EcoR I and FlyCut Xho The constructed pET28a-N plasmid was identified by double restriction enzyme digestion and sequencing.

[0026] The successfully constructed pET28a-N plasmid was transformed into E. coli BL21 competent cells, and the positive monoclonal colonies were picked and expanded after cultivation. They were inoculated into 200 mL LB liquid culture medium containing kanamycin at a ratio of 1%, and cultured at 37°C / 220 rpm for about 4 h. The OD 600 nm When the p-value reached about 0.6, add the inducer isopropyl-β-D-thiogalactoside (IPTG, Shanghai Sangon Biotech Co., Ltd., Catalog No. A100487) with a final concentration of 1 mmol / L, and induce at 37°C, 180 rpm for 5 h. Collect the induced bacterial solution by centrifugation at 8 000 rpm / 4°C for 5 min, add 10 mL of 1× PBS to resuspend the bacterial cells, and place them in a cell ultrasonic disruptor in an ice bath at 200 W for 10 min. Place the ultrasonic suspension in a refrigerated centrifuge at 8000 rpm / 4°C for 5 min, transfer the supernatant to a new centrifuge tube, and resuspend the precipitate in 10 mL of 1× PBS.

[0027] The crushed precipitate was washed with washing solution 1 (100 mmol / L Tris-HCl, 2 mol / L urea, 10 mL / L Tween-20, pH 7.4), reacted at room temperature for 8 min, and centrifuged at 4°C, 10000 r / min for 10 min. The precipitate was collected and washed once with washing solution 2 (50 mmol / L Tris-HCl, 4 mol / L urea, pH 8.0), centrifuged at 4°C, 10000 r / min for 10 min, and the precipitate was washed with sterile saline and centrifuged at 4°C, 10000 r / min for 10 min. The precipitate was collected as inclusion bodies, and the inclusion bodies were dissolved with denaturing solution (8 mol / L urea) at 4°C for 10 h. The denatured protein solution was placed in a dialysis bag and dialyzed for 4 h at 4°C with 100× volume of 6 mol / L urea, 4 mol / L urea, 2 mol / L urea, and 1× PBS solution (pH 7.4). The supernatant, precipitate, and refolded protein were prepared into protein samples for SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The successfully expressed recombinant N protein was packaged at -80°C for later use.

[0028] like Figure 2 The figure shows the PCR identification results of the recombinant expression vector pET28a-N, where M is 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.

[0029] like Figure 3 The figure shows the results of double enzyme digestion of the recombinant expression vector pET28a-N, where M is DL 5 000 DNA Marker; lane 1 is the pET28a empty vector; lane 2 is the recombinant expression vector pET28a-N; lane 3 is the result of double enzyme digestion of the pET28a empty vector by EcoR I and Xho I; lane 4 is the result of double enzyme digestion of the recombinant expression vector pET28a-N by EcoR I and Xho I.

[0030] like Figure 4 The figure shows the expression results of the recombinant expression vector pET28a-N detected by SDS-PAGE, wherein M is a 10-180 kDa protein molecular weight standard marker; Lane 1 is the BL21 host bacteria control; Lane 2 is the recombinant expression vector control without the addition of inducer; Lane 3 is the result of the bacteria 1 hour after the induction of the recombinant expression bacteria; Lane 4 is the result of the supernatant 5 hours after the induction of the recombinant expression bacteria; Lane 5 is the result of the precipitate 5 hours after the induction of the recombinant expression bacteria; Lane 6 is the result of the renaturation and purification 5 hours after the induction of the recombinant expression bacteria.

[0031] After PCR and double enzyme digestion identification, the amplified product was successfully connected to the pET28a vector. The sequencing results verified that the fragment had 100% similarity with the N gene sequence of AKAV, confirming that the recombinant expression vector pET28a-N was constructed. The recombinant expression vector pET28a-N was able to express efficiently after 1 mmol / L IPTG induction for 5 hours.

[0032] 4. Purification of N protein and identification of its reaction activity The refolded protein (successfully expressed recombinant N protein) was centrifuged at 8 000 rpm / 4°C for 15 min, filtered through a 0.45 µm syringe filter, and purified using a Ni-NTA protein purification prepacked column (Shanghai Sangon Biotechnology Co., Ltd., Cat. No. C600332-0001). The concentration of the wash buffer was 200 mmol / L imidazole, and the concentration of the elution buffer was 500 mmol / L imidazole. The flow-through, wash, and eluate were collected and sampled for SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to observe the purification effect.

[0033] The concentration of the recombinant protein (purified recombinant N protein) was determined by the BCA protein concentration determination kit (Shanghai Sangon Biotechnology Co., Ltd., Cat. No. C503061-1250), and the activity of the purified recombinant N protein was identified by Western blot method. The steps are as follows: The purified recombinant protein was transferred to a PVDF membrane (Biosharp, Catalog No.: BS-PVDF-45), blocked with 5% BSA at 37°C for 2 h, and used as the primary antibody bovine anti-AKAV positive antibody at a dilution of 1:100 and incubated at 4°C overnight. HRP-labeled rabbit anti-bovine antibody (Beijing Solebow Technology Co., Ltd., Catalog No. SE233) was used as the secondary antibody at a dilution of 1:5000 and incubated at room temperature for 2 h. The horseradish peroxidase DAB color development kit (Shanghai Sangon Biotech Co., Ltd., Catalog No. C520017) was then used for color development detection to identify its reactivity.

[0034] like Figure 5 The results of Western blot N protein reactivity identification are shown, where M is the 10-180 kDa protein molecular weight standard marker; Lane 1 is the hybridization result of recombinant N protein and bovine Akabane disease positive serum. Because the protein may fold in space when it binds to the antibody, the band size is slightly larger than the theoretical value.

[0035] The results showed that the recombinant N protein had the ability to bind to bovine AKAV positive serum, indicating that the expressed protein had reactive activity.

[0036] 1.1 Determination of the optimal coating concentration of antigen and the optimal serum dilution by chessboard titration The purified recombinant N protein obtained in Example 1 was coated onto a 96-well ELISA plate at different concentrations. The coating solution was a carbonate buffer solution at pH 9.6. A total of 12 protein solutions with concentration gradients were set, 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 KeyGen Biotech Co., Ltd., catalog number: KGL3006-500) was used as a negative control (N); bovine Akabane disease positive serum from French ID-VET Company, catalog number AKAC-4P, was used as a positive control (P); the negative control and positive control sera were diluted 1:50, 1:100, 1:200 and 1:400, respectively. The following steps were followed: 100 μL of protein solution of different concentrations was added to a 96-well ELISA plate and coated overnight at 4°C; after taking out, the liquid in the well was shaken out, the well was washed 5 times with PBST, and the droplets in the well were patted dry; 200 μL of 5% skimmed milk powder was added and incubated at 37°C for 2 h; after taking out, the liquid in the well was shaken out, the well was washed 5 times with PBST, and the droplets in the well were patted dry; 100 μL of negative control and positive control serum of different dilutions was added to each well and incubated at 37°C for 45 min; after taking out, the liquid in the well was shaken out, the well was washed 5 times with PBST, and the droplets in the well were patted dry; 100 μL of 1:5000 diluted HRP-labeled rabbit anti-bovine serum (Beijing Solebow Technology Co., Ltd., Cat. No. SE233) was added as a secondary antibody and incubated at 37°C for 40 min; after taking out, the liquid in the well was shaken out, the well was washed 5 times with PBST, and the droplets in the well were patted dry; 50 μL TMB colorimetric solution (Solaibao Biotechnology Co., Ltd., catalog number PR1200) was incubated at 37°C in the dark for 15 min. After removal, 50 μL of stop solution (1M H2SO4) was added to each well, and the OD of each well was measured with an ELISA reader within 10 min. 450 nm The absorbance value of the positive control was recorded and P (OD 450 ) / N(negative control OD 450 ) value, with the maximum P / N value as the criterion for determining the optimal coating concentration and the optimal serum dilution.

[0037] Table 1 Results of screening test of antigen coating concentration and serum dilution multiple

[0038] The results (Table 1) showed that when the concentration of the coating antigen was 7 and 8 μg / mL and the dilution of negative and positive serum was 1:100, the P / N ratio was the highest, both of which were 14.8650. According to the principle of saving coating protein, the optimal coating concentration of N protein was determined to be 7 μg / mL and the optimal dilution of serum was 1:100.

[0039] 1.2 Screening of N protein coating conditions The coating conditions of N protein were changed to set three coating conditions: 37℃ 2h, 37℃ 1h + 4℃ overnight (overnight is 12h), 4℃ overnight (overnight is 12h), and N protein coating concentration was 7 μg / mL. Fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as negative control (N); bovine Akabane disease positive serum from French ID-VET Company, Catalog No. AKAC-4P, was used as positive control (P); negative control and positive control serum were diluted at 1:100, and other conditions and steps were the same as 1.1. The OD of the test sample was 450 value and calculate the P / N value.

[0040] Table 2 Results of antigen coating condition screening

[0041] The results (Table 2) showed that when the N protein coating condition was 37℃ for 2h, its P / N value was the highest, which was 7.6207. Therefore, the coating condition of N protein was determined to be 37℃ for 2h.

[0042] 1.3 Screening of blocking solution Different types of blocking solutions or their concentrations were changed, and 4 conditions were set, including 3% BSA, 5% BSA, 0.3% gelatin blocking buffer (Shanghai Sangon Biotechnology Co., Ltd., Catalog No. E661002) and 5% skim milk powder. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2h. Fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as the negative control (N); bovine Akabane disease positive serum from French ID-VET Company, Catalog No. AKAC-4P, was used as the positive control (P); the negative control and positive control serum were diluted at 1:100, and the other conditions and steps were the same as 1.1. The OD of the test sample was 450 value and calculate the P / N value.

[0043] Table 3 Blocking solution screening results

[0044] The results (Table 3) show that when the blocking solution is 5% skim milk powder, the P / N value is the highest, which is 11.5229. Therefore, the blocking solution is determined to be 5% skim milk powder.

[0045] 1.4 Screening of closed time The blocking solution time was changed, and three blocking time points were set at 1h, 2h, and 3h, respectively. The N protein coating concentration was 7 μg / mL, and the coating conditions were 37℃ for 2h. Fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as the negative control (N); bovine Akabane disease positive serum from French ID-VET Company, Catalog No. AKAC-4P, was used as the positive control (P); the negative control and positive control serum were diluted at 1:100, and the blocking solution was 5% skim milk powder. Other conditions and steps were the same as 1.1. The OD of the test sample was 450 value and calculate the P / N value.

[0046] Table 4 Closure time screening results

[0047] The results (Table 4) showed that when the blocking time was 37℃, 2h, the P / N value was the highest, which was 6.3979. Therefore, the blocking time was determined to be 37℃, 2h.

[0048] 1.5 Screening of primary antibody serum incubation temperature The incubation temperature of the primary antibody serum was changed, with two conditions of 25°C and 37°C. The N protein coating concentration was 7 μg / mL, the coating condition was 37°C for 2h, and fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as the negative control (N); the positive serum of Akabane disease from ID-VET, France, Catalog No. AKAC-4P, was used as the positive control (P); the negative control and positive control serum were diluted 1:100, the blocking solution was 5% skim milk powder, and the blocking time was 37°C for 2h. The other conditions and steps were the same as 1.1, and the OD of the sample was detected. 450nm value and calculate the P / N value.

[0049] Table 5 Primary antibody serum incubation temperature screening results

[0050] The results (Table 5) showed that when the primary antiserum incubation temperature was 25°C, its P / N value was the highest, which was 6.8198. Therefore, the primary antiserum incubation temperature was determined to be 25°C.

[0051] 1.6 Screening of primary antiserum incubation time The incubation time of the primary antibody serum was changed, with four conditions of 15min, 30min, 45min and 60min. The N protein coating concentration was 7 μg / mL, the coating conditions were 37℃ for 2h, and fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., catalog number: KGL3006-500) was used as the negative control (N); the positive serum of bovine Akabane disease came from the French ID-VET company, catalog number AKAC-4P, as the positive control (P); the negative control and positive control serum were diluted at 1:100, the blocking solution was 5% skim milk powder, the blocking time was 37℃ for 2h, and the primary antibody serum incubation temperature was 25℃. Other conditions and steps were the same as 1.1, and the OD of the test sample was 450nm value and calculate the P / N value.

[0052] Table 6 Primary antibody serum incubation time screening results

[0053] The results (Table 6) showed that when the primary antibody serum incubation time was 45 min, the P / N value was the highest, which was 10.2690. Therefore, the primary antibody serum incubation time was determined to be 45 min.

[0054] 1.7 Screening of enzyme-labeled secondary antibody dilution The dilution of the enzyme-labeled secondary antibody was changed, and 6 dilutions were set, namely 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 2h. Fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as the negative control (N); the positive serum for Akabane disease of cattle was from ID-VET, France, Catalog No. AKAC-4P, as the positive control (P); the negative control and positive control serum were diluted at 1:100, the blocking solution was 5% skim milk powder, the blocking time was 37℃ for 2h, the primary antibody serum incubation temperature was 25℃, and the incubation time was 45min. Other conditions and steps were the same as 1.1, and the OD of the sample was detected. 450nm value and calculate the P / N value.

[0055] Table 7 Secondary antibody dilution screening results

[0056] The results (Table 7) show that when the dilution of the enzyme-labeled secondary antibody is 1:3000, its P / N value is the highest, which is 9.4335. Therefore, the dilution of the enzyme-labeled secondary antibody is determined to be 1:3000.

[0057] 1.8 Screening of enzyme-labeled secondary antibody incubation time The incubation time of the enzyme-labeled secondary antibody was changed, and 4 time points were set at 15min, 20min, 30min and 40min. The N protein coating concentration was 7 μg / mL, the coating conditions were 37℃ for 2h, and fetal bovine serum (Jiangsu KeyGen Biotech Co., Ltd., Catalog No.: KGL3006-500) was used as the negative control (N); the positive serum of bovine Akabane disease came from the French ID-VET company, Catalog No. AKAC-4P, as the positive control (P); the negative control and positive control serum were diluted at 1:100, the blocking solution was 5% skim milk powder, the blocking time was 37℃ for 2h, the primary antibody serum incubation temperature was 25℃, the incubation time was 45min, and the dilution of the enzyme-labeled secondary antibody was 1:3000. Other conditions and steps were the same as 1.1, and the OD of the test sample was 450nm value and calculate the P / N value.

[0058] Table 8 Screening results of enzyme-labeled secondary antibody incubation time

[0059] The results (Table 8) show that when the enzyme-labeled secondary antibody incubation time is 40 minutes, the P / N value is the highest, which is 10.4685. Therefore, the incubation time of the enzyme-labeled secondary antibody is determined to be 40 minutes.

[0060] 1.9 Determination of TMB colorimetric solution incubation time Change the incubation time of TMB colorimetric solution, set 4 time points of 15min, 20min, 25min and 30min. The N protein coating concentration is 7 μg / mL, the coating conditions are 37℃ 2h, the negative control and positive control serum are diluted 1:100, the blocking solution is 5% skim milk powder, the blocking time is 37℃ 2h, the primary antibody serum incubation temperature is 25℃, the incubation time is 45min, the enzyme-labeled secondary antibody dilution is 1:3000, and the incubation time is 40min. Other conditions and steps are the same as 1.1, and the OD of the sample is detected. 450nm value and calculate the P / N value.

[0061] Table 9 TMB colorimetric solution incubation time screening results

[0062] The results (Table 9) show that when the TMB colorimetric solution incubation time is 20 minutes, the P / N value is the highest, which is 13.1151. Therefore, the TMB colorimetric solution incubation time is determined to be 20 minutes.

[0063] 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, and their AKAV nucleic acid and antibodies were negative for determining the critical value.

[0064] The 21 samples were tested using the ELISA test conditions determined above, repeated 3 times, and the mean (x) and standard deviation (SD) were calculated. The critical value (cut-off value) = `x +3 SD.

[0065] Table 10 Results of three repeated tests of AKAV negative serum

[0066] The test results are shown in Table 10. The OD values ​​of the three tests of 21 bovine AKAV negative sera were 450nm The mean values ​​(`x) were 0.2684, 0.2811 and 0.2109, and the standard deviations (SD) were 0.0926, 0.0795 and 0.0715, respectively. After calculation, the critical value of the established indirect ELISA antibody detection method was 0.4969.

[0067] 3.1 Repeatability test Take 6 negative bovine serum samples and test them according to the best test conditions. Do 3 replicates for each sample and measure the OD 450nm The mean value (x) and standard deviation (SD) were calculated, and the intra-batch repeatability of the detection method was determined by the variation range of the coefficient of variation (standard deviation / mean value × 100%).

[0068] Table 11 Intra-batch repeatability test results

[0069] The results showed (Table 11) that the intra-batch coefficient of variation of the six samples was between 0.32% and 4.74%, all less than 5%, indicating good intra-batch repeatability.

[0070] Another 6 negative bovine serum samples were taken and tested in different batches according to the best test conditions screened out. A total of 3 batches were tested and their OD 450nm The mean value (x) and standard deviation (SD) of the samples in different batches were calculated, and the inter-batch repeatability of the detection method was determined by the variation range of the coefficient of variation (standard deviation / mean value × 100%).

[0071] Table 12 Results of inter-batch repeatability test

[0072] The results showed (Table 12) that the inter-batch coefficients of variation of the six samples were between 1.66% and 5.46%, all less than 6%, indicating good inter-batch repeatability.

[0073] 3.2 Specificity test The established indirect ELISA method for Akabane disease virus was used to detect positive sera from five other bovine pathogens (bovine bluetongue virus, bovine rotavirus, bovine foot-and-mouth disease virus type O, bovine viral diarrhea virus and Mycobacterium paratuberculosis).

[0074] Table 13 Specificity test results

[0075] The test results are shown in Table 13. The positive serum OD of the five pathogens 450nm The values ​​were all less than the critical value of 0.4969, and all were negative, indicating that the established ELISA method had good specificity.

[0076] 3.3 Sensitivity test The established indirect ELISA antibody detection method for bovine Akabane disease virus was used to detect three bovine Akabane disease-positive sera from a farm in Guizhou Province. The three sera were diluted 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000 and 1:1200 respectively.

[0077] Table 14 Sensitivity test results

[0078] The test results are shown in Table 14. When the three sera were diluted to 1:1200, the OD 450nm The values ​​were all greater than the critical value of 0.4969, which was still positive, but the OD of positive serum 2 450nm The value is 0.4997, which is close to the critical value. Usually, the dilution multiple of clinical samples is between 1:100-1:400 during testing. The ELISA detection method established in this patent can still effectively detect when the dilution is 1:1200, indicating that it has high sensitivity.

[0079] 60 clinical bovine serum samples were collected from a beef cattle farm in Guizhou Province and tested using the established ELISA method and the commercialized Akabane disease antibody detection kit ID Screen® Akabane Competition ELISA Kit (ID-VET, France, Cat. No. AKAC-4P), and the consistency of the test results was compared.

[0080] Table 15 Clinical serum test results

[0081]

[0082]

[0083] The results (Table 15) showed that all 60 samples tested by the method established in this experiment were negative, while the results of the ID Screen® kit were 1 positive sample and 59 negative samples. The coincidence rate between the two was 98.3%.

[0084] 3.1.1 Antigen pre-coated plate 96 wells / plate × 1 plate / box. Coat 100 μL of N protein at a concentration of 7 μg / mL onto a 96-well ELISA plate (biosharp, BS-PS-96WB-100), incubate at 37°C for 2 h; shake out the liquid in the wells after taking out, wash 5 times with washing solution, 1 min each time, and pat dry the droplets in the wells; add 200 μL of 5% skim milk powder (Solabo, D8340), incubate at 37°C for 2 h; shake out the liquid in the wells after taking out, wash 5 times with washing solution, 1 min each time, pat dry the droplets in the wells, and air-dry at 37°C for 20 minutes. Vacuum pack in aluminum foil bag.

[0085] 3.1.2 Serum dilution plate 96 wells / plate × 1 plate / box. Purchased from Guangzhou Jiete Biofiltration Co., Ltd., with intact packaging, clear labels, no spots, stains, no damage, no obvious color difference, clean and transparent well bottom and well wall, and no foreign matter.

[0086] 3.1.3 Positive control serum 50 μL / tube × 1 tube / box. Purchased from ID-VET, France. OD was measured three times. 450 nm The average value was above 0.5569. 450nm The value is calibrated to the range of 0.5-1.5. The serum is a clear, light yellow liquid, odorless, free of hemolysis or foreign matter, and free of sterile contamination.

[0087] 3.1.4 Negative control serum 50 μL / tube × 1 tube / box. Purchased from Jiangsu KeyGen Biotech Co., Ltd., catalog number: KGL3006-500. Measure OD three times 450 nm The average value was below 0.1836. 450nm The value is calibrated to the range of 0.18-0. The serum comes from Uruguay, a mad cow disease-free area. Mycoplasma detection and virus screening are both negative, without sterile contamination. The serum is a clear, light yellow liquid, odorless, without hemolysis or foreign matter.

[0088] 3.1.5 Enzyme-labeled secondary antibody 10 μL / tube × 1 tube / box. Purchased from Beijing Solebow Technology Co., Ltd., item number SE233, rabbit anti-bovine IgG-HRP. Diluted with pH 7.4 PBST, dilution multiple 1:3000.

[0089] 3.1.6 Sample diluent 30mL / bottle × 1 bottle / box. Preparation method is as follows: 1.724g potassium dihydrogen phosphate, 7.667g disodium hydrogen phosphate, 1.0g bovine serum albumin, add deionized water to 800mL, after fully dissolved, add 0.5mL Tween-20, 200μL Proclin 300 preservative, adjust pH to 7.4, and make up to 1000mL. After filtration and sterilization, aseptically quantitatively package and label.

[0090] 3.1.7 10× concentrated detergent 30mL / bottle × 1 bottle / box. Weigh 17.24g potassium dihydrogen phosphate and 76.67g disodium hydrogen phosphate, add deionized water to 800mL, and after fully dissolving, add 5.0mL Tween-20, adjust the pH value to 7.4, and make up to 1000mL. Sterilize at 121℃ for 20 minutes, then quantitatively package and label.

[0091] 3.1.8 TMB colorimetric solution 10mL / bottle × 1 bottle / box. Purchased from Beijing Solebow Technology Co., Ltd., item number PR1200, single-component TMB colorimetric solution. This reagent is generally colorless in appearance, and the OD value is less than 0.04 when detected at 650nm. Store the brown reagent bottle away from light.

[0092] 3.1.9 Stop solution 10mL / bottle × 1 bottle / box. Preparation method: 98% analytical grade concentrated sulfuric acid 108.7mL, add deionized water to 800mL, adjust pH to 0.4, and make up to 1000mL. Quantitatively package and label.

[0093] 3.1.10 Instruction manual: 1 copy / box.

[0094] 3.2 The test kit should be packaged in a suitable outer packaging box and labeled (with identification name, batch number, production date, shelf life and production unit information, etc.).

[0095]

Name of veterinary drug

[0096] (1) 1 plate of antigen-coated plate (96 wells / plate) (2) 1 serum dilution plate (96 wells / plate) (3) 1 vial of positive control serum (50 μL / vial) (4) 1 negative control serum tube (50 μL / tube) (5) 1 vial of enzyme-labeled secondary antibody (10 μL / vial) (6) 1 bottle of sample diluent (30 mL / bottle) (7) 10× concentrated detergent 1 bottle (30mL / bottle) (8) 1 bottle of TMB colorimetric solution (10 mL / bottle) (9) 1 bottle of stop solution (10 mL / bottle) (10) 1 instruction manual

[0097] Used to detect antibodies to Akabane disease virus N protein in bovine serum, suitable for detecting antibodies to Akabane disease virus in bovine serum samples.

[0098] 1 Usage 1.1 Sample preparation Take whole blood from cattle, wait for the blood to coagulate, centrifuge at 4000r / min for 10 minutes, collect the supernatant for later use; you can also collect blood, wait for the serum to precipitate naturally after coagulation, and aspirate the serum for later use. The serum should be clear and free of hemolysis.

[0099] 1.2 Preparation of washing solution: 10× concentrated washing solution is restored to room temperature and shaken thoroughly. If there are still crystals, heat them in a 37℃ water bath to dissolve them, then dilute them 10 times with deionized water for later use.

[0100] 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 serum sample to 198 μL sample diluent).

[0101] 1.4 Operation steps 1.4.1 Adding samples Take the antigen-coated plate and add 100 μL of the diluted test serum, negative control serum and positive control serum to the corresponding wells, 2 wells for negative and 2 wells for positive control serum. Use a pipette to gently pipette and mix.

[0102] 1.4.2 Incubate in a 25°C incubator for 45 minutes.

[0103] 1.4.3 Washing: 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 using an automatic plate washer.

[0104] 1.4.4 Add enzyme-labeled secondary antibody Dilute the enzyme-labeled secondary antibody 1:3000 with diluent (prepare immediately before use), and add 100 μL of the diluted enzyme-labeled secondary antibody to each well.

[0105] 1.4.5 Incubate in a 37°C incubator for 40 minutes.

[0106] 1.4.6 Washing is the same as 1.4.3.

[0107] 1.4.7 Color development: Add 100 μL TMB color development solution to each well and allow the color development to proceed in a dark incubator at 37°C for 20 minutes.

[0108] 1.4.8 Stop Add 50 μL of stop solution to each well and measure the results within 10 minutes.

[0109] 1.4.9 Reading Read the absorbance of each well at 450 nm on a microplate reader.

[0110] 2 Judgment 2.1 The test conditions and results meet the following conditions: Average OD of positive control 450nm ≥0.50, average OD of negative control 450nm <0.18, to be effective.

[0111] 2.2 Determination of serum OD 450nm <0.4969, judged as negative for Akabane disease antibody; serum OD to be tested 450nm ≥0.4969, it is judged as positive for bovine Akabane disease antibody.

[0112] 1 Before use, all reagents in the kit except the enzyme-labeled secondary antibody should be equilibrated to room temperature before use. If there are crystals, be sure to heat them to completely dissolve them. Mix the liquid reagents before use. To ensure accurate and reliable results, the operating environment temperature should be controlled at 20±5℃.

[0113] 2 The micropipette used for sample addition should be calibrated regularly to avoid large errors affecting the test results.

[0114] 3. Ensure the quality of serum samples, avoid using severely hemolyzed samples and lipid samples; avoid repeated freezing and thawing of serum samples; serum samples should be stored at 2-8°C for no more than 5 days, and should be kept below -20°C for long-term storage; if serum samples appear turbid or flocculent, centrifuge and precipitate before taking the supernatant for testing.

[0115] 4 Avoid mutual contamination of reagents and samples during use. The kit should be used within the validity period and avoid mixing kits from different batches.

[0116] 5. Unused ELISA plates should be placed back into the sealed bag together with the desiccant and stored at 2-8°C.

[0117] 6 When conducting multiple ELISA reaction plate tests, the reaction plates cannot be stacked and should be laid flat in a 37°C incubator to avoid uneven heating of the reaction plates.

[0118] 7 Do not expose TMB color developing solution to strong light and avoid contact with oxidants.

[0119] 8 Stop solution is corrosive, avoid contact with eyes and skin.

[0120] 9 Strictly follow the instructions and control the reaction time and temperature of each step.

[0121] 10. Comply with laboratory biosafety regulations and all waste should be disposed of harmlessly in accordance with regulations.

[0122] 96 wells / block, 1 block / box

[0123] Store at 2~8℃, valid for 6 months.

[0124]

Approval No.

Manufacturer

Claims

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

2.

2. The gene encoding the amino acid sequence of the recombinant N protein antigen of bovine Akabane disease virus according to 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: The following steps are involved: 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 added EcoR I and XOt I restriction site sequence; Amplification of S2 and N genes Extract RNA of bovine Akabane disease virus, perform reverse transcription using reverse transcriptase, and use it as a template for PCR amplification to obtain a PCR amplification product of the N gene, and sequence the sequence as shown in SEQ ID No. 1; Construction and expression of prokaryotic expression vectors of S3 and N genes S31, digesting the pET28a vector plasmid and the PCR amplification product of the N gene, and recovering the products by gel respectively; S32, connecting the pET28a vector plasmid after restriction digestion and the PCR amplification product of the N gene after restriction digestion using a ligase; S33, transforming 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, transforming the successfully constructed expression vector pET28a-N into Escherichia coli BL21 competent cells, picking positive monoclonal colonies for expansion and culture after cultivation, preparing protein samples, and performing SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining analysis on the protein samples; packaging the successfully expressed recombinant N protein for storage and standby use; Purification and Reaction Activity Identification of S4 and N Proteins The successfully expressed recombinant N protein obtained in step S34 is purified, the concentration of the purified recombinant N protein is measured, and the activity of the recombinant N protein is identified.

4. Use of the gene shown in SEQ ID No. 1 in the preparation of a kit for detecting antibodies against bovine Akabane disease.

5. Use of the amino acid sequence encoded by the gene as shown in SEQ ID No. 2 in the preparation of a kit for detecting antibodies against bovine Akabane disease.

6. An indirect ELISA kit for detecting antibodies to bovine Akabane disease virus, characterized in that: The method comprises an enzyme-labeled plate coated with the bovine Akabane disease virus recombinant N protein according to claim 1, an enzyme-labeled secondary antibody, a washing solution, a stop solution, a negative control, a positive control and a result judgment standard thereof.

7. The preparation method of the ELISA plate of the recombinant N protein of bovine Akabane disease virus is as follows: A1. Coat the recombinant N protein of Akabane disease virus onto an ELISA plate and incubate at 37°C for 2 hours; A2. After taking out, shake out the liquid in the hole, wash with detergent, and pat dry the droplets in the hole; A3, add blocking solution and incubate at 37℃ for 2h; A4. After taking out, shake out the liquid in the wells, wash with detergent, pat dry the droplets in the wells, and air-dry in a 37°C incubator to obtain the ELISA plate of bovine Akabane disease virus recombinant N protein.

8. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 5, characterized in that: In the step A1, the concentration of the recombinant N protein of bovine Akabane disease virus used in the ELISA plate for preparing the recombinant N protein of bovine Akabane disease virus is 7 μg / mL.

9. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 5, characterized in that: In the step A3, the blocking solution is 3%-5% BSA by mass, 0.3% gelatin blocking buffer by mass, or 5% skimmed milk powder by mass.

10. The indirect ELISA kit for detecting bovine Akabane disease virus antibodies according to claim 5, characterized in that: The sample diluent is prepared by using raw materials including potassium dihydrogen phosphate, disodium hydrogen phosphate, bovine serum albumin, a surfactant, deionized water, and a preservative, and the pH value of the diluent is 7.4; The raw materials for the washing liquid include potassium dihydrogen phosphate, disodium hydrogen phosphate, a surfactant, and deionized water, and the pH value of the washing liquid is 7.4; The preparation materials of the stop solution include concentrated sulfuric acid and deionized water, and the pH value of the stop solution is 0.4.

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