Bovine viral diarrhea virus multi-epitope fusion protein and application thereof

Abstract

The invention relates to the technical field of bovine viral diarrhea virus detection, in particular to a bovine viral diarrhea virus multi-epitope fusion protein and application thereof, a coding gene of the bovine viral diarrhea virus multi-epitope fusion protein is a nucleotide sequence as shown in SEQ ID NO.5 and corresponds to an amino acid sequence as shown in SEQ ID NO.9, and the coding gene of the bovine viral diarrhea virus multi-epitope fusion protein is an amino acid sequence as shown in SEQ ID NO.9. The recombinant plasmid comprises a coding gene of the bovine viral diarrhea virus multi-epitope fusion protein. The bovine viral diarrhea virus monoclonal antibody is used as a coating antibody, and the bovine viral diarrhea virus multi-epitope fusion protein is used as a marker antigen, so that early infection cases can be found, and a detection result can be provided for health management of cattle herds in time, so that corresponding prevention and control or treatment measures can be taken in time, and the detection efficiency is improved. Different subtypes of BVDVs can be detected more widely, and the applicability to detection of different popular subtypes of BVDVs in different regions is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of bovine viral diarrhea virus detection, and in particular to a bovine viral diarrhea virus multi-epitope fusion protein and a use thereof. Background Art

[0002] Bovine viral diarrhea (BVD) is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). BVDV is one of the most important cattle infectious diseases worldwide. The main clinical manifestations are diarrhea, high fever, mucosal erosion, immune dysfunction, reproductive disorders, etc. The incidence rate of diseased cattle is high and the mortality rate is low. However, after the virus invades the body, it induces immunosuppression and persistent infection, leading to a decrease in the body's immunity; at the same time, BVDV can work together with other pathogens to cause mixed infection or secondary infection, which seriously threatens the health level and production performance of the cattle.

[0003] European countries have achieved certain control effects by adopting comprehensive testing and culling of BVDV-positive cattle, but combined with my country's actual production situation, the use of vaccines to control BVDV is still the best strategy. The positive rate of BVD in my country is gradually increasing. The severe infection situation has put forward higher requirements for the prevention, control and purification of BVD in my country.

[0004] According to the genome and antigenic characteristics, BVDV can be divided into 3 biotypes and multiple biosubtypes. Biotypes are divided into: BVDV-1, BVDV-2 and BVDV-3. Among them, BVDV-1 has at least 23 subtypes, namely: BVDV1a~BVDV1v; BVDV-2 has 3 subtypes, namely: 2a~2d; and BVDV-3 is described as an atypical BVDV, and there are currently no reports on its subtypes. There are 8 main subtypes prevalent in my country, specifically 1a, 1b, 1c, 1d, 1m, 1o, 1p and 1q. Among them, BVDV-1b (31.6%) and BVDV-1m (20.8%) are the most prevalent. The prevalence of many genetic subtype strains has interfered with the prevention and control of BVDV.

[0005] BVDV belongs to the Flaviviridae family and the Pestivirus genus. It is a single-stranded positive-sense RNA virus with an envelope and a genome size of 12.3 kb to 12.5 kb. The open reading frame of BVDV encodes a long polyprotein, which is cleaved into 12 mature viral polypeptides by viral and host proteases, in the following order: p20 (Npro) -p14 (C) -gp48 (Erns) -gp25 (E1) -gp53 (E2) -p7 -p54 (NS2) -p80 (NS3) -p10 (NS4A) -p30 (NS4B) -p58 (NS5A) -p75 (NS5B). Among them, p14 (C), gp48 (Erns), gp25 (E1) and gp53 (E2) are structural proteins, and the remaining 8 are non-structural proteins. Among the four structural proteins, gp48 (Erns), gp25 (E1) and gp53 (E2) are located on the surface of the virus and are envelope proteins; p14 (C) is the nucleocapsid protein.

[0006] At present, the technologies used for BVDV pathogen diagnosis mainly include virus isolation, RT-PCR, etc., and the technologies used for serological diagnosis mainly include serum neutralization test, ELISA method, etc. However, in the commonly used detection methods, virus isolation requires the preparation of bovine monolayer cells, and the separation time is relatively long. Although RT-PCR has a high sensitivity in detecting viruses, it cannot distinguish whether the infected virus is a live virus, and it is easy to report a false positive. In addition, my country already has an inactivated vaccine for bovine viral diarrhea virus. If the cattle have been immunized with the inactivated BVDV vaccine, the detection of antibodies cannot distinguish whether it is produced by the vaccine or by BVDV wild virus infection.

[0007] In summary, the present application proposes a bovine viral diarrhea virus multi-epitope fusion protein and its use. Summary of the invention

[0008] In view of the problem of low sensitivity in bovine viral diarrhea detection in the background technology, the present invention provides a bovine viral diarrhea virus multi-epitope fusion protein and use thereof.

[0009] In a first aspect, the present application provides a bovine viral diarrhea virus multi-epitope fusion protein, wherein the coding gene of the bovine viral diarrhea virus multi-epitope fusion protein is a nucleotide sequence as shown in SEQ ID NO.5, corresponding to an amino acid sequence as shown in SEQ ID NO.9.

[0010] In a second aspect, the present application provides a recombinant plasmid, wherein the recombinant plasmid comprises the coding gene as described in the first aspect.

[0011] In a third aspect, the present application provides use of the bovine viral diarrhea virus multi-epitope fusion protein as described in the first aspect in detecting bovine viral diarrhea virus antigens.

[0012] In a fourth aspect, the present application provides a monoclonal antibody against bovine viral diarrhea virus, wherein the monoclonal antibody is prepared using the fusion protein described in the first aspect as an immunogen.

[0013] In a fifth aspect, the present application provides a kit for detecting bovine viral diarrhea virus antigen based on a competitive enzyme-linked immunosorbent assay, comprising a coated monoclonal antibody and a labeled antigen; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody described in the fourth aspect; The labeled antigen is the bovine viral diarrhea virus multi-epitope fusion protein described in the first aspect labeled with HRP.

[0014] In a sixth aspect, the present application provides a kit for detecting bovine viral diarrhea virus antigen based on magnetic particle chemiluminescence method, which comprises a coated monoclonal antibody and a labeled antigen; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody described in the fourth aspect; The labeled antigen is the bovine viral diarrhea virus multi-epitope fusion protein described in the first aspect labeled with HRP.

[0015] Compared with the prior art, this application has the following beneficial technical effects: The bovine viral diarrhea virus monoclonal antibody of the present application is used as a coating antibody, and the bovine viral diarrhea virus multi-epitope fusion protein is used as a marker antigen, which is helpful for discovering early cases of infection and can provide test results for the health management of the cattle herd in a timely manner so that corresponding prevention, control or treatment measures can be taken in a timely manner. It can detect different subtypes of BVDV more widely and improve the applicability of detection of BVDV in different regions and with different epidemic subtypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of the Immunomedicine Group's B cell antigen epitope prediction analysis of the gp53 (E2) amino acid sequence of the BVDV-1b CP7 strain; Figure 2 This is the result of SVMTriP's B cell antigen epitope prediction analysis of the gp53 (E2) amino acid sequence of the BVDV-1b CP7 strain; Figure 3 This is the result of the Immunomedicine Group's B cell antigen epitope prediction analysis of the p80 (NS3) amino acid sequence of the BVDV-1b CP7 strain; Figure 4 This is the result of SVMTriP's B cell antigen epitope prediction analysis of the p80 (NS3) amino acid sequence of the BVDV-1b CP7 strain; Figure 5 This is the fragment size diagram of the agarose gel electrophoresis product after PCR amplification of the monoclonal strain according to the universal primer AOX1 of the vector pPICZαA, where M is the molecular weight marker, 1 is the empty vector control, and 2 is the target fragment band; Figure 6 This is the result of identifying the recombinant protein using SDS-PAGE, where 1: protein ruler, 2: empty vector control, 3: target protein band. DETAILED DESCRIPTION

[0017] The present application discloses a bovine viral diarrhea virus multi-epitope fusion protein and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0018] Bovine viral diarrhea virus multi-epitope fusion protein: In the first aspect of the present application, the present application provides a bovine viral diarrhea virus multi-epitope fusion protein, the coding gene of the bovine viral diarrhea virus multi-epitope fusion protein is a nucleotide sequence as shown in SEQ ID NO.5, corresponding to the amino acid sequence as shown in SEQ ID NO.9.

[0019] Recombinant plasmid: In the second aspect of the present application, the present application provides a recombinant plasmid, wherein the recombinant plasmid comprises the coding gene as described in the first aspect.

[0020] use: In the third aspect of the present application, the present application provides use of the bovine viral diarrhea virus multi-epitope fusion protein as described in the first aspect in detecting bovine viral diarrhea virus antigens.

[0021] Monoclonal Antibodies to Bovine Viral Diarrhea Virus In the fourth aspect of the present application, the present application provides a monoclonal antibody against bovine viral diarrhea virus, wherein the monoclonal antibody is prepared using the fusion protein described in the first aspect as an immunogen.

[0022] Competitive ELISA kit for the detection of bovine viral diarrhea virus antigens: In a fifth aspect of the present application, the present application provides a kit for detecting bovine viral diarrhea virus antigen based on a competitive enzyme-linked immunosorbent assay, comprising a coated monoclonal antibody and a labeled antigen; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody described in the fourth aspect; The labeled antigen is the bovine viral diarrhea virus multi-epitope fusion protein described in the first aspect labeled with HRP.

[0023] Kit for detecting bovine viral diarrhea virus antigen based on magnetic microparticle chemiluminescence method: In a sixth aspect, the present application provides a kit for detecting bovine viral diarrhea virus antigen based on magnetic particle chemiluminescence method, which comprises a coated monoclonal antibody and a labeled antigen; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody described in the fourth aspect; The labeled antigen is the bovine viral diarrhea virus multi-epitope fusion protein described in the first aspect labeled with HRP.

[0024] Example 1: Preparation of coding genes In this embodiment, the preparation method of the coding gene includes the following steps: analyzing the full genome sequence of the typical BVDV-1b type CP7 strain (GenBank: U63479.1) that has been published on GenBank and isolated in my country, selecting protein fragments with relatively concentrated dominant linear antigenic epitopes of gp53 (E2) and p80 (NS3), and connecting them with flexible connecting peptides. The nucleotide sequence corresponding to the recombinant protein fragment is codon-optimized according to the preferences of Escherichia coli, and double enzyme cutting sites are designed at both ends. The optimized nucleotides are synthesized and connected to construct a recombinant plasmid.

[0025] The specific plan is as follows: S101, select the gp53 (E2) amino acid sequence of BVDV-1b CP7 strain, and use the online tool ImmunomedicineGroup: (http: / / imed.med.ucm.es / Tools / antigenic.pl), and SVMTriP (http: / / sysbio.unl.edu / SVMTriP / prediction.php) to predict B cell antigen epitopes. Figure 1 , Figure 2Based on the two prediction results, the amino acid sequence shown in SEQ ID NO.6 and the amino acid sequence shown in SEQ ID NO.7, which have relatively concentrated antigenic epitopes, were selected, corresponding to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2, respectively; the p80 (NS3) amino acid sequence of the BVDV-1b CP7 strain was selected, and the online tool ImmunomedicineGroup: (http: / / imed.med.ucm.es / Tools / antigenic.pl), and SVMTriP (http: / / sysbio.unl.edu / SVMTriP / prediction.php) to predict B cell antigen epitopes. Figure 3 , Figure 4 Combining the two prediction results, the amino acid sequence shown in SEQ ID NO.8 in the antigen epitope set is selected, which corresponds to the nucleotide sequence shown in SEQ ID NO.3; S102, inserting a flexible connecting peptide between the above nucleotide sequences in the order of SEQ ID NO.1-SEQ ID NO.2-SEQ ID NO.3 for connection, wherein the flexible connecting peptide is the nucleotide sequence shown in SEQ ID NO.10, and inserting EcoRⅠ and NotⅠ restriction sites at both ends, wherein EcoRⅠ is the restriction site of the nucleotide sequence shown in SEQ ID NO.11, and NotⅠ is the restriction site of the nucleotide sequence shown in SEQ ID NO.12, to obtain a recombinant nucleotide sequence shown in SEQ ID NO.4; S103, optimizing the nucleotide sequence shown in SEQ ID NO.4 according to the codons preferred by Pichia pastoris to obtain the coding gene of the nucleotide sequence of bovine viral diarrhea virus shown in SEQ ID NO.5, and correspondingly obtaining the amino acid sequence shown in SEQ ID NO.9; Unless otherwise specified, the coding genes of this application were commissioned to be synthesized by General Biotechnology (Anhui) Co., Ltd.

[0026] Table 1 Sequence information of nucleotide sequences

[0027]

[0028]

[0029]

[0030] Among them, the nucleotide sequence SEQ ID NO.1 is expected to express the amino acid sequence shown in SEQ ID NO.6; The nucleotide sequence SEQ ID NO.2 is expected to express the amino acid sequence shown in SEQ ID NO.7; The nucleotide sequence SEQ ID NO.3 is expected to express the amino acid sequence shown in SEQ ID NO.8; The nucleotide sequence SEQ ID NO.5 is expected to express the amino acid sequence shown in SEQ ID NO.9.

[0031] Table 2 Sequence information of amino acid sequences

[0032] Example 2: Construction of recombinant plasmid In this embodiment, the method for constructing the recombinant plasmid comprises the following steps: S201, culturing the above-mentioned bovine viral diarrhea virus fusion protein gene-positive bacteria and the Escherichia coli species (Zeocin+) containing pPICZαA according to the culturing method of Escherichia coli, and extracting the bovine viral diarrhea virus fusion protein gene-positive plasmid and the pPICZαA plasmid using the TARAKA plasmid extraction kit; S202. PCR reaction was carried out using the bovine viral diarrhea virus fusion protein gene-positive plasmid as a template. The reaction procedure was 95°C for 5 min (95°C for 30 s, 55°C for 1 min, 72°C for 1 min, 30 cycles) and 72°C for 10 min.

[0033] S203, agarose electrophoresis (120v 30min), and recovery of BVD virus fusion protein nucleic acid according to the Tiangen agarose gel DNA recovery kit.

[0034] S204. According to the instructions of the endonuclease (purchased from Quanshijin Biotechnology Co., Ltd.), the bovine viral diarrhea virus fusion protein DNA and pPICZαA plasmid were double-digested. The products were subjected to agarose electrophoresis (120v 30min), and the bovine viral diarrhea virus fusion protein nucleic acid and pPICZαA plasmid were recovered according to the Tiangen agarose gel DNA recovery kit.

[0035] S205. According to the instruction of T4 DNA ligase (purchased from Quanshijin Biotechnology Co., Ltd.), the digested bovine viral diarrhea virus fusion protein nucleic acid and pPICZαA were ligated.

[0036] S206, the ligated product was transferred into DH5α competent cells (full gold) for culture, and the cultured DH5α was coated on LB plates (Zeocin+) and cultured overnight. S207, select monoclonal strains, perform PCR amplification on the monoclonal strains according to the universal primer AOX1 of the vector pPICZαA, and perform agarose gel electrophoresis to confirm that the product fragment size meets the expectations (e.g. Figure 5 ) Example 3: Expression and identification of multi-epitope fusion proteins In this embodiment, the expression and identification of the multi-epitope fusion protein includes the following steps: S301, preparation of competent yeast: ①Cultivate the bacteria in 5 ml YPD medium at 30 degrees overnight.

[0037] ②Take 0.1-0.5 ml of bacterial solution and culture it in 500 ml of fresh YPD culture medium overnight, OD600 = 1.3-1.5.

[0038] ③ Centrifuge at 1500g for 5 minutes at 4 degrees. Resuspend the cells in 500ml of ice-cold sterile water.

[0039] ④ Centrifuge and then resuspend in 250 ml of ice-cold sterile water.

[0040] ⑤ Centrifuge and resuspend in 20 ml of ice-cold sorbitol (1 M).

[0041] ⑥ Centrifuge and resuspend with 1 ml of ice-cold sorbitol. Dispense 1 ml of 100 μl into each tube and store at -80 degrees.

[0042] The recombinant plasmid was linearized, digested at 37°C for 2 h, and inactivated at 65°C for 20 min.

[0043] S302. Electroporation transformation of yeast: Add 15ul of the linearized recombinant plasmid to the prepared 100ul yeast competent cells, mix well, precool on ice for 5min, transfer to a 0.2cm electroporation cup (precooled on ice), place on ice for 10min, put into an electroporator, take out after electroporation, quickly put back on ice and add 1ml of precooled 1M sorbitol, mix well, place on a shaker at 30 degrees for 40min, aspirate 300ul and spread on an MD plate, and culture at 30 degrees for 2 days.

[0044] S303, Mut+ phenotype induced expression experiment ①Pick a single colony and place it in a 250ml shake flask containing 25ml MGY or BMGY medium. Cultivate at 30℃ / 250rpm until OD600 is about 2-6; ② Centrifuge at 1500-3000g for 5 min at room temperature to collect the cells and resuspend them with BMMY to make OD600 around 1.0; ③ Place the obtained bacterial solution in a 1L shake flask, seal it with double gauze, and continue to culture on a shaker at 30℃ / 250rpm; ④ Add 100% methanol to the culture medium every 24 hours to a final concentration of 0.5%; ⑤ After 72 hours, centrifuge the bacterial solution at 12000rpm for 3min and collect the supernatant.

[0045] ⑥ The collected supernatant was purified by nickel ion histidine affinity chromatography to collect the recombinant protein.

[0046] ⑦ SDS-PAGE was used to identify the recombinant protein. It can be seen that the fusion protein expressed by sequences 5 and 7 has an obvious 30Ku protein band, which is the same as the expected size and has a high purity. Figure 6 .

[0047] Example 4: Preparation of monoclonal antibodies In this embodiment, the preparation of monoclonal antibodies specifically includes the following steps: S401. The purified BVDV multi-epitope fusion protein was mixed with an equal volume of Freund's adjuvant and emulsified, and 8-week-old BALB / c mice were immunized by subcutaneous multiple-point injection at a dose of 200 μg / mouse.

[0048] S402. Immunization procedure: After the first immunization, booster immunization was performed every 14 days, for a total of 4 times. (The first immunization used Freund's complete adjuvant, and the subsequent 3 immunizations used Freund's incomplete adjuvant). After 42 days, 200 μg of antigen without adjuvant was directly injected intraperitoneally.

[0049] S403. Three days later, spleen cells of mice were obtained and fused with SP2 / 0 cells. Positive hybridoma cell lines that stably secreted antibodies against BVDV virus multi-epitope fusion proteins were obtained by flow cytometry sorting. Meanwhile, monoclonal cell lines were cryopreserved after amplification and culture.

[0050] Take two 10-week-old BALB / c mice and inject sterilized liquid paraffin (0.5 mL / mouse) intraperitoneally. One week later, inject 5x105 hybridoma cells in the logarithmic growth phase diluted with PBS into the peritoneum. When the abdomen of the mouse is obviously bulging, collect the ascites, centrifuge at 2500g for 10 minutes, and take the supernatant, which is the monoclonal antibody.

[0051] Example 5: Kit for detecting bovine viral diarrhea virus antigen based on competitive enzyme-linked immunosorbent assay In this embodiment, the main components of the kit for detecting bovine viral diarrhea virus antigen based on competitive enzyme-linked immunosorbent assay are: BVDV monoclonal antibody coated plate, horseradish peroxidase-labeled multi-epitope fusion protein, positive quality control, negative quality control, sample diluent, washing solution, chromogenic substrate solution A, chromogenic substrate solution B, and stop solution.

[0052] The method for preparing the BVDV monoclonal antibody coated plate specifically comprises the following steps: S501, Antigen coating: Use CBS buffer (pH=9.6) to dilute the monoclonal antibody to a final concentration of 1μg / mL coating solution, add the coating solution to the ELISA plate at 100μL / well, coat at 4°C for 16h, discard the solution in the wells of the ELISA plate; add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300L / well, wash 5 times, 3min / time; S502, blocking: add OVA blocking solution to the ELISA plate obtained in step S501, 200 μL / well, block at 37°C for 2 hours, discard the solution in the wells of the ELISA plate; add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 L / well, wash 5 times, 3 min / time; obtain a coated ELISA plate.

[0053] The OVA blocking solution is prepared by diluting chicken ovalbumin (OVA) with PBS buffer (pH=7.4), and the concentration of the OVA blocking solution is 2% (mass-to-volume ratio w / v).

[0054] Color development substrate solution A: 13.6g sodium acetate, 1.6g citric acid, 0.3ml 30% hydrogen peroxide, add distilled water to make up to 500ml.

[0055] Color development substrate solution B: 0.2 g disodium ethylenediaminetetraacetate, 0.95 g citric acid, 50 ml glycerol, take 0.15 g TMB and dissolve it in 3 ml DMSO, add distilled water to make up to 500 ml.

[0056] In this embodiment, the working principle of the kit for detecting bovine viral diarrhea virus antigen based on competitive enzyme-linked immunosorbent assay is: a competitive method is used to determine whether a sample contains bovine viral diarrhea virus antigen.

[0057] The ELISA plate is coated with a monoclonal antibody of bovine viral diarrhea virus to prepare a stationary phase. At the same time, the sample and HRP-labeled bovine viral diarrhea virus multi-epitope fusion protein are added to the microwells of the coated ELISA plate. The antigen in the sample and the HRP-labeled bovine viral diarrhea virus multi-epitope fusion protein compete with the monoclonal antibody to form an antibody-enzyme antigen complex. After washing, a two-component 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution is added; TMB is converted into blue under the catalysis of HRP, and finally converted into yellow under the action of acid. The depth of color is negatively correlated with the content of bovine viral diarrhea virus antigen in the sample; the absorbance (OD value) is measured at a wavelength of 450nm using an ELISA reader, and the OD value is used to calculate whether the sample contains bovine viral diarrhea virus antigen.

[0058] The working process of the kit for detecting bovine viral diarrhea virus antigen based on enzyme-linked immunosorbent assay specifically includes the following steps: S503, preparation of the sample to be tested: dilute the serum sample to be tested 10 times with PBST buffer (0.05 volume % Tween-20, pH=7.4); specifically: 20 μL of serum sample to be tested, 180 μL of PBST buffer; S504, incubation: add negative control, positive control and sample to be tested to the coated ELISA plate, 100 μL / well, and mix; then add HRP-labeled multi-epitope fusion protein solution, 50 μL / well, and mix; seal the ELISA plate with a sealing film, and incubate at 37°C for 30 min; The HRP-labeled multi-epitope fusion protein solution is prepared by diluting the HRP-labeled multi-epitope fusion protein with PBST buffer (0.05 volume % Tween-20, pH=7.4), with a dilution ratio of 1:4000 (mass to volume ratio w / v); S505, washing: discard the solution in the wells of the ELISA plate, add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / well, wash 5 times, 3 min / time; S506, color development and termination: add two-component TMB color development solution (the volume ratio of color development substrate solution A and color development substrate solution B is 1:1) to the ELISA plate, 100 μL / well, mix well, and color development at 37°C in the dark for 15 minutes; then add 2M concentrated sulfuric acid, 50 μL / well, and gently shake the ELISA plate until the color is uniform; S507, reading: put the ELISA plate into the ELISA instrument, and measure and read the absorbance OD value under the conditions of a test wavelength of 450nm and a reference wavelength of 630nm.

[0059] S508, result determination: The average OD value of the negative control serum is ODNC; the average OD value of the positive control serum is ODPC; the OD value of the serum to be tested is ODS; Threshold calculation of positive control serum inhibition rate, i.e. PIPC = (ODNC-ODPC) / ODNC × 100% The inhibition rate of serum to be tested is PIS = (ODNC-ODS) / ODNC × 100% Test establishment conditions: ODNC>0.5 and PIPC>60%, the test results are valid; otherwise, the test is repeated.

[0060] determination: On the premise that the test is established, if the inhibition rate of the serum to be tested, i.e., PIS, is greater than 50%, the result is considered positive; If PIS ≤50%, the result is considered negative.

[0061] Example 6: Kit for detecting bovine viral diarrhea virus antigen based on magnetic microparticle chemiluminescence method In this embodiment, the working principle of the kit for detecting bovine viral diarrhea virus antigen based on magnetic particle chemiluminescence method is: using the competitive immunological principle combined with magnetic particle separation technology, the sample to be tested is mixed and incubated with biotin-labeled monoclonal antibody, acridinium ester-labeled multi-epitope fusion protein and streptavidin magnetic beads, and the multi-epitope fusion protein competes with the antigen in the sample for the monoclonal antibody to form an immune complex, and after washing to remove unbound impurities, an excitation solution is added to promote luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is inversely proportional to the concentration of bovine viral diarrhea virus antigen, and the RLU=corresponding bovine viral diarrhea virus antigen concentration is output through the built-in standard curve of the instrument.

[0062] In this embodiment, the main components of the kit for detecting bovine viral diarrhea virus antigen based on the magnetic particle chemiluminescence method are: magnetic bead working solution, antibody working solution, acridinium ester marker working solution, pre-excitation solution, excitation solution, calibrator, positive quality control product, and negative quality control product.

[0063] Magnetic bead working solution: prepared from 225 μl of streptavidin magnetic beads and 4275 μl of PBS-BSA solution (pH=7.4).

[0064] Antibody working solution: prepared from 100 μg of biotin-labeled monoclonal antibody and 9900 μl of PBS-BSA solution (pH=7.4).

[0065] Acridinium ester label working solution: prepared by 1.0 μg of acridinium ester labeled multi-epitope fusion protein and 9999 μl of PBS-BSA solution (pH=7.4).

[0066] Pre-stimulation solution: 0.1 mol / L nitric acid; 0.13 mol / L urea peroxide.

[0067] Stimulating solution: 0.4 mol / L sodium hydroxide, 0.02 mol / L Triton X-100, and normal saline.

[0068] The working process of detecting bovine viral diarrhea virus antigen based on magnetic particle chemiluminescence method specifically includes the following steps: The sample (10 μL) and antigen working solution (50 μl) + magnetic bead working solution (20 μl) + acridinium ester marker working solution (50 μl) were reacted at 37°C for 15 min, washed with 0.1 mol / L PBST buffer solution, and pre-excitation solution (100 μl) and excitation solution (100 μl) were added to detect the luminescence value.

[0069] The sensitivity of the enzyme-linked immunosorbent assay of the present application, the magnetic microparticle chemiluminescence method of the present application, a domestic Elisa kit, and a domestic colloidal gold kit for detecting bovine viral diarrhea virus antigens was compared. The results of the assay are shown in Table 1.

[0070] The sensitivity comparison of the enzyme-linked immunosorbent assay of the present application, the magnetic microparticle chemiluminescence method of the present application, a domestic Elisa kit, and a domestic colloidal gold kit is shown in Table 3: Table 3

[0071] It can be seen from Table 3 that the sensitivity of the enzyme-linked immunosorbent assay and the magnetic particle chemiluminescence assay of the present application are much higher than the sensitivity of a domestic Elisa kit and a domestic colloidal gold kit.

[0072] When detecting bovine viral diarrhea virus (BVDV) antigens, the kit is based on magnetic microparticle chemiluminescence, a detection technology that has high sensitivity. Compared with traditional detection methods, it can detect lower concentrations of viral antigens, which helps to detect infection cases early. This kit overcomes this problem and can accurately distinguish between vaccine immunity and wild virus infection, which is crucial for accurately assessing the infection status of cattle herds.

[0073] The detection process based on magnetic microparticle chemiluminescence is relatively efficient. The entire detection process, including the reaction of samples and reagents, washing, luminescence excitation and luminescence value detection, can be completed in a relatively short time. Compared with traditional methods of virus isolation, which require the preparation of bovine monolayer cells and take a long time to separate, this kit greatly shortens the detection cycle and can provide test results for the health management of cattle in a timely manner, so that corresponding prevention, control or treatment measures can be taken in a timely manner.

[0074] During the construction of this kit, the full genome sequence of the typical BVDV-1b CP7 strain isolated in my country on GenBank was analyzed to construct a multi-epitope fusion protein operation, which took into account the genetic diversity of the virus to a certain extent. This enables the kit to detect different subtypes of BVDV more widely, improves the applicability of the kit for detecting different regions and different epidemic subtypes of BVDV, and is conducive to the comprehensive prevention and control of the spread of BVDV in cattle herds in my country.

[0075] The above specific embodiments are only several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A bovine viral diarrhea virus multi-epitope fusion protein, characterized in that: The coding gene of the bovine viral diarrhea virus multi-epitope fusion protein is a nucleotide sequence as shown in SEQ ID NO.5, corresponding to the amino acid sequence as shown in SEQ ID NO.

9.

2. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the coding gene as claimed in claim 1.

3. Use of the bovine viral diarrhea virus multi-epitope fusion protein as claimed in claim 1 in detecting bovine viral diarrhea virus antigens.

4. A monoclonal antibody against bovine viral diarrhea virus, characterized in that: The monoclonal antibody is prepared using the fusion protein according to claim 1 as an immunogen.

5. A kit for detecting bovine viral diarrhea virus antigen based on competitive enzyme-linked immunosorbent assay, characterized in that: It contains coated monoclonal antibodies and labeled antigens; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody according to claim 4; The labeled antigen is the bovine viral diarrhea virus multi-epitope fusion protein according to claim 1 labeled with HRP.

6. A kit for detecting bovine viral diarrhea virus antigen based on magnetic particle chemiluminescence method, characterized in that: It contains coated monoclonal antibodies and labeled antigens; The coated monoclonal antibody is the bovine viral diarrhea virus monoclonal antibody according to claim 4; The labeled antigen is the multi-epitope fusion protein of bovine viral diarrhea virus according to claim 1 labeled with an acridinium ester.

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