A polypeptide for detecting antibodies to bovine dermodenodermatitis virus

Abstract

The application discloses a polypeptide for detecting bovine nodular dermatopathy virus antibody. The polypeptide for detecting bovine nodular dermatopathy virus antibody provided by the application is eA33-1 polypeptide; the eA33-1 is a polypeptide with an amino acid sequence of SEQ ID No. 5. The antibody detection kit prepared by taking BSA-eA33-1 as a coating antigen has the characteristics of high specificity, high sensitivity, high accuracy, simple operation, rapidness and the like, and is suitable for rapid and large-scale screening and detection of bovine nodular dermatopathy infection serum antibody by primary and secondary veterinary departments and entry-exit inspection and quarantine bureaus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a polypeptide for detecting antibodies against bovine nodular dermatitis virus. Background Technology

[0002] Bovine lumpy skin disease (LSD) is primarily transmitted mechanically via arthropod vectors. LSDV, like Goatpox virus (GPV) and Sheeppox virus (SPV), belongs to the family Poxviridae and the genus Capripoxvirus (CaPV). This virus has only one serotype, with the Neethling strain from South Africa as the representative strain. The virus can proliferate and cause cytopathic effects in primary cells such as lamb and calf kidney or testicular cells, sheep embryonic kidney and lung cells, and chicken embryonic fibroblasts, as well as in passaged cells such as bovine kidney cells and African green monkey kidneys. The virus exhibits cell-binding properties; using ultrasound to disrupt cells can release the virus extracellularly.

[0003] The primary natural host of LSDV is cattle, with all breeds susceptible and no clear breed specificity. Rabbits, sheep, goats, giraffes, and black antelopes may also be infected, but there is no evidence that LSDV can infect humans. Clinically and subclinically ill cattle are the main source of transmission, with the movement of live cattle being a significant factor in its widespread spread. LSDV is widely present on the skin, in dermal lesions, in calluses, saliva, nasal discharge, milk, semen, muscles, spleen, and lymph nodes. When ulcers form in the eyes, nose, mouth, rectum, mammary glands, and genitals, the nodules rapidly ulcerate, and the secretions contain viral particles. Infection can occur upon exposure to aerosols produced by infected animals. The incubation period for natural infection is 2–5 weeks. Infected cattle exhibit elevated body temperature, reaching above 41°C, and present with a persistent fever. Initial symptoms include rhinitis, conjunctivitis, and keratitis. On days 4–12 following experimental infection, firm, round, raised nodules, 1–5 cm in diameter, appear on the skin. These nodules are painful to the touch, especially on the head, neck, chest, perineum, udder, and limbs. Located in the epidermis and dermis, these nodules can aggregate into irregular masses, eventually leading to necrosis. Superficial lymph nodes are enlarged, most prominently in the anterior shoulder, groin, anterior thigh, hind limbs, and subauricular region. Nodules and ulcers may also form on the mucous membranes of the eyes, nose, mouth, rectum, udder, and external genitalia. Lactating cows may develop mastitis, resulting in decreased milk production; pregnant cows may experience abortion; bulls may become infertile; and beef cattle production performance declines. Necrotic nodules are susceptible to fly bites, leaving deep cavities on the skin after detachment, rendering the hide unusable. Dissection of the subcutaneous tissue reveals grayish-red serous infiltration. Incision of the nodules reveals caseous, grayish-white necrotic tissue within the cavity, sometimes with pus and blood. The nodules extend deep into the subcutaneous tissue or even to the ribs. Similar nodules may appear on the skin, pharynx, trachea, lungs, rumen, abomasum, and kidneys. In the early stages, eosinophilic inclusion bodies are visible within the damaged cells. A preliminary diagnosis can be made based on these clinical symptoms and pathological changes, but a definitive diagnosis requires further laboratory etiological and serological testing.

[0004] The primary differential diagnosis for LSD is pseudo-LSD caused by bovine herpesvirus type 2 (BoHV-2). This is usually a milder clinical presentation, characterized by superficial nodules, resembling only an early stage of LSD. Intranuclear inclusion bodies and viral syncytia are histopathological features of BoHV-2 infection not seen in LSD. Other differential diagnoses (for skin lesions) include: dermatophytes, dermatophytosis, psoriasis, photosensitivity, actinobacterial infection, urticaria, insect bites, Behringer's filariasis, nocardiosis, demodicosis, onchocerciasis, pseudovaccinia, and cowpox. Differential diagnoses for mucosal lesions include: foot-and-mouth disease, bluetongue, bovine viral diarrhea, malignant catarrhal fever, bovine infectious rhinotracheitis, and bovine epidemic stomatitis.

[0005] Currently, there is a lack of a sufficiently specific method for detecting bovine nodular dermatitis virus antibodies. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide for detecting bovine nodular dermatitis virus antibodies.

[0007] In a first aspect, the present invention claims protection for a polypeptide.

[0008] The polypeptide claimed in this invention is the eA33-1 polypeptide.

[0009] The eA33-1 polypeptide is any of the following:

[0010] (A1) A polypeptide with the amino acid sequence SEQ ID No. 5;

[0011] (A2) A polypeptide whose amino acid sequence is positions 2-33 of SEQ ID No. 5;

[0012] (A3) A polypeptide that can be coupled to a carrier protein is obtained by attaching an amino acid residue to the amino or carboxyl terminus of the polypeptide shown in (A2).

[0013] SEQ ID No. 5 consists of 33 amino acid residues. The cysteine ​​residue at position 1 is a linker arm added for connection with the carrier protein. The other amino acid residues are derived from the bovine nodular dermatovirus A33 protein.

[0014] Secondly, this invention claims protection for a coupling agent.

[0015] The coupling compound claimed in this invention is eA33-1 coupling compound.

[0016] The eA33-1 conjugate is a complete antigen obtained by conjugating the eA33-1 polypeptide and carrier protein as described in the first aspect above.

[0017] The carrier protein may be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin, or rabbit serum albumin, etc.

[0018] In one specific embodiment of the present invention, the carrier protein is bovine serum albumin (BSA).

[0019] Thirdly, the present invention claims protection for any of the following biological materials:

[0020] (B1) Nucleic acid molecule, which is a nucleic acid molecule capable of encoding the polypeptide described in the first aspect above;

[0021] (B2) Expression cassette, which is an expression cassette containing the nucleic acid molecule described in (B1);

[0022] (B3) Recombinant vector, which is a recombinant vector containing the nucleic acid molecules described in (B1);

[0023] (B4) Recombinant bacteria, which are recombinant bacteria containing the nucleic acid molecules described in (B1);

[0024] (B5) A transgenic cell line is a transgenic cell line containing the nucleic acid molecules described in (B1).

[0025] Fourthly, the present invention claims protection for the use of the polypeptide described in the first aspect or the conjugate described in the second aspect above in any of the following:

[0026] (C1) Prepare reagents or kits for detecting bovine nodular dermatosis virus antibodies;

[0027] (C2) Preparation of diagnostic antigen for bovine nodular dermatitis virus.

[0028] Fifthly, the present invention claims a kit containing the polypeptide or conjugate described in the first aspect above or the second aspect above.

[0029] Furthermore, the kit is used to detect bovine nodular dermatitis virus antibodies; in the kit, the polypeptide described in the first aspect above or the conjugate described in the second aspect above is used as the coating antigen.

[0030] Furthermore, the kit may be a chemiluminescent immunoassay kit; the kit may also contain horseradish peroxidase (HRP) labeled secondary antibody, coating buffer, washing solution, secondary antibody diluent and / or calibrators; the secondary antibody is an antibody capable of fighting the bovine nodular dermatosis virus antibody.

[0031] Sixthly, the present invention claims the use of the biological materials described in the third aspect above in the preparation of the polypeptides described in the first aspect above, the conjugates described in the second aspect above, or the kits described in the fifth aspect above.

[0032] Seventhly, the present invention claims protection for the use of the biological material described in the third aspect above in any of the following:

[0033] (C1) Prepare reagents or kits for detecting bovine nodular dermatosis virus antibodies;

[0034] (C2) Preparation of diagnostic antigen for bovine nodular dermatitis virus.

[0035] Eighthly, the present invention claims the use of the polypeptide or conjugate described in the first aspect above as an immunogen in the preparation of bovine nodular dermatosis virus antibodies.

[0036] Experiments have shown that using the full-length A33 recombinant protein of bovine nodular dermatitis as a hapten and conjugating it with a carrier protein to obtain a conjugate (BSA-eA33-1) as a coating agent cannot effectively distinguish between bovine nodular dermatitis antibodies and foot-and-mouth disease virus antibodies. To improve the specificity of bovine nodular dermatitis antibody detection, this invention selects the dominant antigenic epitope eA33 from the full-length A33 protein of bovine nodular dermatitis and conjugates it with a carrier protein to obtain a conjugate (BSA-eA33-1) as a coating agent. This conjugate can effectively distinguish between bovine nodular dermatitis antibodies and antibodies against various viruses such as foot-and-mouth disease, thus improving the specificity of bovine nodular dermatitis antibody detection. A kit for detecting bovine nodular dermatitis antibodies in serum prepared using BSA-eA33-1 as the coating antigen can accurately distinguish bovine nodular dermatitis antibody-positive serum from bovine foot-and-mouth disease virus antibody-positive serum, bovine viral diarrhea virus antibody-positive serum, and bovine infectious rhinotracheitis virus antibody-positive serum. Its accuracy in detecting bovine nodular dermatitis antibody-positive serum is consistent with the results of the traditional gold standard method, the virus neutralization test, and its sensitivity is higher than that of the virus neutralization test.

[0037] The antibody detection kit prepared using BSA-eA33-1 as the coating agent in this invention has high specificity, high sensitivity, high accuracy, and is simple and rapid to operate. It is suitable for rapid and large-scale screening and detection of serum antibodies against bovine nodular skin disease infection in veterinary departments at all levels and entry-exit inspection and quarantine bureaus. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] In the following examples, pET32a(+) is a product of BGI Genomics. The luminol chemiluminescent substrate is a product of Luoyang Modern Biotechnology Research Institute Co., Ltd.; the chemiluminescent ELISA plate is a product of Thermo Fisher Scientific (USA); 10% fetal bovine serum, cell culture medium, and related cell culture reagents are products of Gibco (USA); serum samples with neutralizing titers against bovine nodular dermatosis virus (BND) were preserved by the China Animal Disease Prevention and Control Center (Veterinary Diagnostic Center of the Ministry of Agriculture and Rural Affairs). Negative serum was collected from healthy cattle, and positive serum was collected from clinically ill cattle in the field. Antibody titers were determined using the traditional gold standard method of virus neutralization assay. The chemiluminescent immunoassay analyzer, model HEXU, is a product of Luoyang Modern Biotechnology Co., Ltd.

[0041] The bovine nodular dermatitis virus neutralization test in the following examples was performed according to a method including the following steps:

[0042] (1) Processing of the tested serum

[0043] The serum was inactivated at 58°C for 30 minutes and then serially diluted with serum-free MEM medium for later use.

[0044] (2) Control serum

[0045] Standard negative serum and standard positive serum against bovine nodular dermatosis virus with a background of serum neutralization titer.

[0046] (3) Virus neutralization

[0047] The concentration is 2×10 5 Vero cell suspension (100 μl / well) was seeded into 96-well cell culture plates. The plates were incubated at 37°C for 1-2 days in a 5% CO2 incubator until 70%-80% of the cells formed a monolayer. 50 μl of diluted serum was mixed with an equal volume of a suspension containing 100 TCID50 / 50 μl of bovine nodular dermatitis Xinjiang isolate LSDV / Xinjiang / 2019 (described in the paper "Cloning of P32, GPCR, and RPO30 Genes of my country's First LSDV Isolator and Its Genetic Evolution and Protein Structure Analysis," Lanzhou University, dissertation, DOI: 10.27204 / d.cnki.glzhu.2020.001024, publicly available from the applicant, for replication of this invention only, and not for other uses). The mixture was then incubated at 37°C in a 5% CO2 incubator for 1 hour.

[0048] (4) Cultivation

[0049] One hour after the serum and virus were neutralized, 100 μl of a mixed suspension of virus and serum was added to each well of the cell culture plate, and the plate was incubated at 37°C in a 5% CO2 incubator.

[0050] (5) Result determination

[0051] Preliminary results were assessed 72 hours after inoculation. Wells with specific lesions were discarded, and the culture medium in the other wells was replaced with maintenance medium. The wells were then rotated and cultured for another 7 days before final assessment.

[0052] Judgment criteria: A positive result is defined as the ability to inhibit cytopathic effects (CPE) in 50% or more of the cells. The maximum neutralizing dilution factor of the serum is calculated based on this criterion.

[0053] The bovine nodular dermatitis virus antibody-positive serum in the following examples was tested positive by a virus neutralization test, and the bovine nodular dermatitis virus antibody-negative serum was tested negative by a virus neutralization test.

[0054] Example 1: Chemiluminescent immunoassay for detecting serum antibodies against bovine nodular dermatitis.

[0055] During the research and development process, the full-length A27 protein and the full-length A33 recombinant protein of bovine nodular dermatitis virus (BNDV) were solublely expressed in E. coli BL21(DE3) using pET32a(+). Experimental results showed that the chemiluminescent immunoassay method established using the full-length A27 and A33 recombinant proteins of BNDV as coating antigens could not effectively distinguish between BNDV antibodies and foot-and-mouth disease virus antibodies, exhibiting poor specificity. Furthermore, four dominant antigenic epitope peptides (eA27-1, eA27-2, eA27-3, and eA27-4) were selected from the full-length A27 protein of bovine nodular dermatitis virus (BNDV), and four dominant antigenic epitope peptides (eA33-1, eA33-2, eA33-3, and eA33-4) were selected from the full-length A33 protein of BNDV. These peptides were conjugated with BSA and used as coating antigens to establish a chemiluminescent immunoassay method. The specificity was significantly improved, and it could effectively distinguish between BNDV antibodies and foot-and-mouth disease virus antibodies. However, the sensitivity varied considerably. A chemiluminescent immunoassay method was established by using a mixed complete antigen obtained by mixing eA27-1 conjugate with BSA (BSA-eA27-1) and eA33-1 conjugate with BSA (BSA-eA33-1) at a mass ratio of 1:1 as the coating antigen. The specificity was significantly improved, and it can effectively distinguish between bovine nodular dermatitis antibodies and foot-and-mouth disease virus antibodies. The sensitivity was also significantly improved.

[0056] The specific experimental method is as follows:

[0057] I. Including antigen preparation

[0058] Prepare the following 11 coating antigens:

[0059] (1) BSA-eA27-1+BSA-eA33-1;

[0060] (2) BSA-eA27-1 (eA27-1 conjugate);

[0061] (3) BSA-eA33-1 (eA33-1 conjugate);

[0062] (4) BSA-eA27-2 (eA27-2 conjugate);

[0063] (5) BSA-eA33-2 (eA33-2 conjugate);

[0064] (6) BSA-eA27-3 (eA27-3 conjugate);

[0065] (7) BSA-eA33-3 (eA33-2 coupling compound);

[0066] (8) BSA-eA27-4 (eA27-4 conjugate);

[0067] (9) BSA-eA33-4 (eA33-4 conjugate);

[0068] (10) The full-length A27 protein of bovine nodular dermatitis virus;

[0069] (11) Full-length A33 recombinant protein of bovine nodular dermatitis virus.

[0070] 1. Dominant antigenic epitope peptides

[0071] Dominant antigenic epitope peptides were selected from the A27 and A33 proteins of bovine nodular dermatovirus. Peptides eA27-1, eA27-2, eA27-3, eA27-4, eA33-1, eA33-2, eA33-3, and eA33-4, with cysteine ​​residues linked to their C-terminus or N-terminus, were synthesized by Beijing Huada Genomics Co., Ltd. (see Table 1 for details). The purity was greater than 95%, and the peptides were lyophilized.

[0072] Table 1. Polypeptides

[0073]

[0074] Note: C* in the sequence is a cysteine ​​residue, which is a linker arm added to the amino terminus of the antigenic epitope polypeptide of the A27 protein or the A33 protein of bovine nodular dermatovirus for linking with the carrier protein; other amino acid residues are derived from the A27 or A33 protein of bovine nodular dermatovirus.

[0075] 2. Preparation of 11 coating antigens

[0076] Ten polypeptides, namely eA27-1, eA27-2, eA27-3, eA27-4, eA33-1, eA33-2, eA33-3, eA33-4, and eA27-full-length and eA33-full-length, were conjugated with BSA to obtain ten coated antigens: (1) BSA-eA27-1 (eA27-1 conjugate with BSA), (2) BSA-eA33-1 (eA33-1 conjugate with BSA), (3) BSA-eA27-2 (eA27-2 conjugate with BSA), (4) BSA-eA33-2 (eA27-2 conjugate with BSA), (5) BSA-eA33-2 (eA27-1 conjugate with BSA), (6) BSA-eA33-2 (eA27-2 conjugate with BSA), (7) BSA-eA33-2 (eA27-1 conjugate with BSA), (8) BSA-eA33-2 (eA27-1 conjugate with BSA), (9) BSA-eA33-2 (eA27-1 conjugate with BSA), (10 ... (5) BSA-eA27-3 (eA27-3 and BSA coupling), (6) BSA-eA33-3 (eA33-3 and BSA coupling), (7) BSA-eA27-4 (eA27-3 and BSA coupling), (8) BSA-eA33-4 (eA33-4 and BSA coupling), (9) BSA-eA27-full-length (eA27-full-length and BSA coupling), (10) BSA-eA33-full-length (eA33-full-length and BSA coupling).

[0077] The coating antigen (11) BSA-eA27-1+BSA-eA33-1 was obtained by mixing BSA-eA27-1 (a conjugate of eA27-1 and BSA) and BSA-eA33-1 (a conjugate of eA33-1 and BSA) in a mass ratio of 1:1.

[0078] The specific preparation methods for coating antigens (1)-(8) and (11) are as follows: The BSA tag-coupled kit (Readilink) produced by KPL Corporation in the United States was used. TM BSA Conjugation Kit (Catalog No.: 5501, Batch No.: 148045) was used to conjugate the synthesized polypeptide fragments according to the instructions to prepare a coated antigen.

[0079] The specific preparation methods for coating antigens (9) and (10) are as follows:

[0080] The full-length A27 protein and the full-length A33 recombinant protein of bovine nodular dermatitis virus were used as coating antigens.

[0081] Step 1: Construct recombinant expression vectors for the full-length A27 protein gene and the full-length A33 protein gene.

[0082] The fragment between the BamHI and XhoI recognition sites of pET32a(+) was replaced by positions 1-388 of the nucleotide sequence (GenBank Accession No. MN901872.1, Update Date 28-OCT-2020), while keeping the other sequences of pET32a(+) unchanged. This yielded a recombinant expression vector for the full-length A27 protein gene of bovine nodular dermatitis virus, named pET32a-flA27. pET32a-flA27 can express a fusion protein containing the full-length A27 protein of bovine nodular dermatitis virus (amino acid sequence 1-129 of GenBank Accession No. MN901872.1, Update Date 28-OCT-2020, i.e., SEQ ID No. 9).

[0083] The fragment between the BamHI and XhoI recognition sites of pET32a(+) was replaced by positions 1-588 of the nucleotide sequence 113441-114031 (Update Date: 20-DEC-2020) of pET32a(+), while keeping the other sequences of pET32a(+) unchanged. This yielded a recombinant expression vector for the full-length A33 protein gene of bovine nodular dermatitis virus, named pET32a-flA33. pET32a-flA33 can express a fusion protein containing the full-length A33 protein of bovine nodular dermatitis virus (amino acid sequence 1-196 of GenBank Accession No. NP_150556.1, Update Date: 20-DEC-2020, i.e., SEQ ID No. 10).

[0084] Step 2: Construction of recombinant strains

[0085] The two expression vectors, pET32a-flA27 and pET32a-flA33, constructed in the first step, were separately transformed into *E. coli* BL21(DE3) competent cells. The cells were evenly spread on LB agar plates containing ampicillin (50 μg / mL) and incubated at 37°C for 16 hours. Single colonies were cultured overnight with shaking, and plasmids were extracted and sequenced. Recombinant *E. coli* containing pET32a-flA27 were named BL21(DE3) / pET32a-flA27, and recombinant *E. coli* containing pET32a-flA33 were named BL21(DE3) / pET32a-flA33.

[0086] Step 3: Soluble expression and purification of the full-length A27 protein of bovine nodular dermatitis virus or the full-length A33 recombinant protein of bovine nodular dermatitis patella.

[0087] The two strains, BL21(DE3) / pET32a-flA27 and BL21(DE3) / pET32a-flA33, were separately inoculated into LB liquid medium containing 50 μg / ml ampicillin (the medium was prepared by adding ampicillin to LB liquid medium to a concentration of 50 μg / ml). The cultures were incubated at 37°C with shaking at 200 rpm using a Thermo Max Q6000 full-temperature shaker until 0D. 600When the pH value (using LB liquid medium containing 50 μg / ml ampicillin as a blank control) reached 0.6, IPTG was added for induction of expression. This induction was performed using 0.75 mM IPTG at 16℃ for 13 h. The fermentation broth after 13 h of IPTG induction was collected to form a cell pellet. The pellet was resuspended in PBS, centrifuged at 8000 rpm / min for 5 min, and the supernatant was discarded. PBS was added to the washed cell pellet, and the cells were autoclaved until the lysate was no longer viscous. The pellet was then centrifuged at 16000 rpm / min for 30 min at 4℃, and the supernatant was collected, discarding the pellet. The supernatant was filtered through a 0.22 μm filter and loaded onto a nickel column pre-equilibrated with solution 1 (solute and concentration as follows: 20 mM Tris, 150 mM NaCl, solvent: water, pH 8.0). The nickel column was connected to the AKTA instrument, and impurity proteins in the nickel column were washed with 10 column volumes of Solution 1 and 10 column volumes of Solution 2 (solute and concentration as follows: 20 mM Tris, 150 mM NaCl, 50 mM imidazole, solvent: water, pH 8.0 solution), and the protein peak was monitored on the AKTA instrument. The target protein adhering to the nickel column was washed with Solution 3 (solute and concentration as follows: 20 mM Tris, 150 mM NaCl, 300 mM imidazole, solvent: water, pH 8.0 solution), and the eluted sample showing the target protein peak was collected using the AKTA instrument. The sample was further purified by molecular sieve using a GE Superdex 200 gel column to obtain the full-length A27 protein and the full-length A33 recombinant protein of bovine nodular dermatitis virus, respectively, purified by molecular sieve.

[0088] II. Chemiluminescent immunoassay using a chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus or a chemiluminescent immunoassay kit for detecting bovine nodular dermatitis virus antibodies.

[0089] This embodiment provides 11 chemiluminescent immunoassay kits for diagnosing bovine nodular dermatosis virus (BNDV) or for detecting BNDV antibodies. All 11 kits include a coating antigen, horseradish peroxidase (HRP)-labeled bovine secondary antibody (catalog number HM390, Hexu (Zhengzhou) Biotechnology Co., Ltd.), coating buffer (catalog number HC210, Hexu (Zhengzhou) Biotechnology Co., Ltd.), washing buffer (catalog number HX221, Hexu (Zhengzhou) Biotechnology Co., Ltd.), and secondary antibody dilution buffer (catalog number HM391, Hexu (Zhengzhou) Biotechnology Co., Ltd.). The only difference between these 11 kits is the coating antigen; all other components are identical.

[0090] These 11 kits are, respectively, a chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus (BNV) or a chemiluminescent immunoassay kit for detecting BNV antibodies, with the antigen coated as BSA-eA27-1+BSA-eA33-1 from step one, hereinafter referred to as Kit 1 of the present invention; a chemiluminescent immunoassay kit for diagnosing BNV or detecting BNV antibodies, with the antigen coated as BSA-eA27-1 from step one, hereinafter referred to as Kit 2 of the present invention; and a chemiluminescent immunoassay kit for diagnosing BNV or detecting BNV antibodies, with the antigen coated as BSA-eA33-1 from step one, hereinafter referred to as Kit 3 of the present invention. The following kits are used for diagnosing bovine nodular dermatitis virus (BNV) or detecting BNV antibodies: Kit 1 (coated with BSA-eA27-2 from step one); Kit 2 (coated with BSA-eA33-2 from step one); Kit 3 (coated with BSA-eA27-3 from step one); Kit 4 (coated with BSA-eA33-3 from step one); and Kit 5 (coated with BSA-eA33-2 from step one). The following kits are provided: 5. A chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus (BSA-eA27-4) or for detecting bovine nodular dermatitis virus antibodies, hereinafter referred to as Control Kit 5; 6. A chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus or for detecting bovine nodular dermatitis virus antibodies, coated with BSA-eA33-4 antigen from step 1; 7. A chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus or for detecting bovine nodular dermatitis virus antibodies, coated with the full-length A27 protein of bovine nodular dermatitis virus from step 1; 8. A chemiluminescent immunoassay kit for diagnosing bovine nodular dermatitis virus or for detecting bovine nodular dermatitis virus antibodies, coated with the full-length A33 protein of bovine nodular dermatitis virus from step 1.

[0091] (i) Using the kit 1 of the present invention, a chemiluminescent immunoassay method with BSA-eA27-1+BSA-eA33-1 as the coating antigen was established through optimized experiments (hereinafter referred to as CLIA method 1 of the present invention), the coating antigen, horseradish peroxidase (HRP) labeled secondary antibody, and secondary antibody dilution solution were prepared.

[0092] 1. Operating Steps

[0093] (1) Coating: Dilute the concentration of BSA-eA27-1+BSA-eA33-1 in step one (total mass concentration of BSA-eA27-1 and BSA-eA33-1) with coating buffer to 1.0 μg / ml to obtain the coated antigen solution. Coat the experimental wells with the coating buffer, add 100 μL / well to the microplate, and incubate at 4℃ for 16 h.

[0094] (2) Washing: Pour off the original coating solution in the well, wash with washing solution 5 times, 3 minutes each time; pat dry.

[0095] (3) Blocking: Add 1% BSA blocking solution, 350 μL / well, and incubate at 37°C for 2 h.

[0096] (4) Washing: Same as step (2).

[0097] (5) Sample addition: Dilute the test sample in the serum dilution plate with sample diluent (1×PBST) at a ratio of 1:20; remove the coated plate, and set up 6 wells for each experiment with a series of calibrators. First, add 100 μL of calibrators 1 to 6 to the serum dilution plate in sequence, and add 100 μL of the diluted test sample to each of the remaining wells in sequence. Incubate at 37℃ for 30 min (±1 min).

[0098] (6) Washing: Same as step (2).

[0099] (7) Add 100 μL of horseradish peroxidase (HRP)-labeled secondary antibody diluted with secondary antibody dilution buffer to each well and vortex to mix. Transfer 100 μL from each well to the corresponding well of the coated plate; incubate at 37°C for 30 min (±1 min).

[0100] (8) Washing: Same as step (2).

[0101] (9) Add luminol chemiluminescent substrate: Add 100 μL of luminol chemiluminescent substrate to each well and shake to mix; let stand in the dark for 5 min at 15℃~25℃, and read the luminescence value with a chemiluminescence analyzer within 15 min after standing.

[0102] 2. Result Calculation Method

[0103] NCU (National Clinical Unit) refers to a unit designated by the National Center for Clinical Laboratories. In this method, the standard positive serum (neutralizing antibody titer of 1:512) is assigned a value of 20000 NCU / ml by this unit. A four-parameter calibration curve is established using standard positive serum at different dilution ratios. The antibody dose value (NCU / ml) of the sample is calculated from the luminescence value of the test sample and the calibration curve, thereby achieving semi-quantitative detection.

[0104] Bovine nodular dermatitis virus antibody-positive sera with neutralizing titers were used to create calibrators 1-6 based on the virus neutralization test results, corresponding to antibody dose values ​​(0 NCU / mL, 10 NCU / mL, 20 NCU / mL, 50 NCU / mL, 100 NCU / mL, 200 NCU / mL). A four-parameter fitting curve for the calibrators was plotted using ELISACalc software, with the luminescence values ​​of calibrators 1 to 6 on the ordinate and the corresponding antibody dose values ​​on the abscissa. The four-parameter model is Y = (ab) / [1 + (x / c)*b] + d. Here, a is the estimated asymptote on the curve; b is the slope of the curve; c is the dose corresponding to half of the maximum binding; and d is the estimated asymptote below the curve.

[0105] By substituting the luminescence value of the sample into the calibration curve, the content of bovine nodular dermatitis virus antibody in the sample can be obtained.

[0106] 3. Conditions for the validity of the experiment and judgment of the results

[0107] The luminescence values ​​of the six calibrators were fitted with the corresponding antibody dose values ​​using a four-parameter method, R0. 2 If the value is ≥0.99, the experiment is valid. Otherwise, the experiment is invalid.

[0108] 4. Determination of the critical values ​​for positive and negative sex

[0109] Two hundred bovine nodular dermatitis virus antibody-negative sera were subjected to CLIA testing using the above method. The mean (X) and standard deviation (SD) of the dose values ​​of these 200 bovine nodular dermatitis virus antibody-negative sera were calculated. The mean dose value X of the 200 bovine nodular dermatitis virus antibody-negative sera was 28.59, and the SD was 3.64. Therefore, the critical dose value for positive and negative results was... The value was 39.51 NCU / mL. A dose value ≥ 39.51 NCU / mL is considered positive; a dose value < 39.51 NCU / mL is considered negative.

[0110] (II) Using the kit 2 of the present invention, a chemiluminescent immunoassay method with BSA-eA27-1 as the coating antigen was established through optimized experiments (hereinafter referred to as CLIA method 2 of the present invention). The steps are the same as in (I).

[0111] (III) Using the kit 3 of the present invention, a chemiluminescent immunoassay method with BSA-eA33-1 as the coating antigen was established through optimized experiments (hereinafter referred to as CLIA method 3 of the present invention). The steps are the same as in (I).

[0112] (iv) Using the control kit 1 of the present invention, a chemiluminescent immunoassay method with BSA-eA27-2 as the coating antigen was established through optimized experiments (hereinafter referred to as control CLIA method 1). The steps are the same as in (i).

[0113] (V) Using the control kit 2 of the present invention, a chemiluminescent immunoassay method with BSA-eA33-2 as the coating antigen (hereinafter referred to as control CLIA method 2) was established through optimized experiments. The steps are the same as in (I).

[0114] (vi) Using the control kit 3 of the present invention, a chemiluminescent immunoassay method with BSA-eA27-3 as the coating antigen was established through optimized experiments (hereinafter referred to as control CLIA method 3). The steps are the same as in (i).

[0115] (vii) Using the control kit 4 of the present invention, a chemiluminescent immunoassay method with BSA-eA33-3 as the coating antigen was established through optimization experiments (hereinafter referred to as control CLIA method 4). The steps are the same as in (i).

[0116] (viii) Using the control kit 5 of the present invention, a chemiluminescent immunoassay method with BSA-eA27-4 as the coating antigen was established through optimization experiments (hereinafter referred to as control CLIA method 5). The steps are the same as in (i).

[0117] (ix) Using the control kit 6 of the present invention, a chemiluminescent immunoassay method with BSA-eA33-4 as the coating antigen (hereinafter referred to as control CLIA method 6) was established through optimized experiments. The steps are the same as in (i).

[0118] (x) Using the control kit 7 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as control CLIA method 7) was established through optimized experiments using full-length A27 protein as the coating antigen. The steps are the same as in (i).

[0119] (xi) Using the control kit 8 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as control CLIA method 8) was established through optimized experiments using full-length A33 protein as the coating antigen. The steps are the same as in (i).

[0120] III. Specificity Test

[0121] The CLIA methods 1-3 of this invention (step two), control CLIA methods 1-8, and positive serum samples of bovine foot-and-mouth disease virus antibodies, bovine viral diarrhea virus antibodies, and bovine infectious rhinotracheitis virus antibodies (all positive serum samples were collected from clinically ill animals) were compared to observe whether there was any cross-reactivity with other diseases of the same species. In addition, a virus neutralization test was performed simultaneously (see above for specific methods).

[0122] The results showed that the antibody dose values ​​of CLIA methods 1-3 and control CLIA methods 1-8 of this invention were all less than 39.51 NCU / ml, and were therefore negative. The specificity was 100%. The neutralization index of the virus neutralization test was less than the national standard of 1:5, and the results were all negative. See Tables 1 and 2 for detailed results.

[0123] Table 1. Results of the specificity test of methods 1-3 of the present invention with virus neutralization test.

[0124]

[0125] Table 2. Results of specificity tests for control methods 1-8

[0126]

[0127] IV. Sensitivity Test

[0128] Serum samples positive for bovine nodular dermatitis virus antibodies were serially diluted and compared with serum samples from known neutralizing titers using CLIA methods 1-3 of this invention. The maximum dilution at the positive cutoff value was determined. Results showed that the highest dilution for detecting positive serum samples using CLIA methods 1-3 of this invention was 1:1024. The highest dilution for detecting positive serum samples using the virus neutralization test was 1:512. (See Table 3).

[0129] Table 3. Sensitivity results of CLIA methods 1-3 and neutralization tests of the present invention

[0130]

[0131]

[0132] V. Repeatability Test

[0133] Ten positive serum samples for bovine nodular dermatitis virus antibodies were tested using methods 1-3 of this invention on both same-batch and different-batch plates, with five parallel tests performed. Intra-batch and inter-batch coefficients of variation (CV) were calculated. The results showed that the intra-batch repeatability CV of methods 1-3 of this invention was within 8%, and the inter-batch repeatability CV was less than 10% (Tables 4 to 6). These results indicate that positive serum samples for bovine nodular dermatitis virus antibodies exhibit good repeatability.

[0134] Table 4. Repeatability test of CLIA method 1 of the present invention

[0135]

[0136] Table 5. Repeatability test of CLIA method 2 of the present invention

[0137]

[0138]

[0139] Table 6. Repeatability Tests of CLIA Method 3 of the Present Invention

[0140]

[0141] VI. Conformity Test

[0142] Bovine serum samples preserved by the China Animal Disease Prevention and Control Center (Veterinary Diagnostic Center of the Ministry of Agriculture and Rural Affairs) were used to select 80 bovine serum samples with a background neutralizing titer of positive for bovine nodular dermatophyte virus (BND) antibodies and 80 bovine serum samples with a background neutralizing titer of negative for BND antibodies. These 160 bovine serum samples were tested using CLIA methods 1-3 and control CLIA methods 1-8 of this invention, and the concordance rate with the neutralization test methods was calculated.

[0143] The results showed that among the 160 bovine serum samples, the overall concordance rate between CLIA Method 1 of the present invention and the neutralization test method was 98.75% (positive concordance rate 100%, negative concordance rate 97.5%), the overall concordance rate between CLIA Method 2 of the present invention and the bovine nodular dermatitis virus neutralization test method was 95.625% (positive concordance rate 93.75%, negative concordance rate 97.5%), and the overall concordance rate between CLIA Method 3 of the present invention and the bovine nodular dermatitis virus neutralization test method was 96.25% (positive concordance rate 92.5%, negative concordance rate 100%). The overall concordance rate between the results of CLIA method 1 and the neutralization test for bovine nodular dermatitis virus (BND) of this invention was 85.625% (positive concordance rate 85%, negative concordance rate 86.25%). The overall concordance rate between the results of CLIA method 2 and the neutralization test for BND was 78.125% (positive concordance rate 81.25%, negative concordance rate 75%). The overall concordance rate between the results of CLIA method 3 and the neutralization test for BND was 75.625% (positive concordance rate 80%, negative concordance rate 71.25%). The overall concordance rate between the results of CLIA method 4 and the neutralization test for BND was 78.125% (positive concordance rate 100%, negative concordance rate 56.25%). The overall concordance rate between the results of CLIA method 5 (as described in this invention) and the neutralization test for bovine nodular dermatitis virus was 79.375% (positive concordance rate 80%, negative concordance rate 78.75%). The overall concordance rate between the results of CLIA method 6 (as described in this invention) and the neutralization test for bovine nodular dermatitis virus was 81.25% (positive concordance rate 88.75%, negative concordance rate 73.75%). The overall concordance rate between the results of CLIA method 7 (as described in this invention) and the neutralization test for bovine nodular dermatitis virus was 85.625% (positive concordance rate 100%, negative concordance rate 71.25%). The overall concordance rate between the results of CLIA method 8 (as described in this invention) and the neutralization test for bovine nodular dermatitis virus was 85.625% (positive concordance rate 97.5%, negative concordance rate 73.75%). See Tables 7 to 16 for details.

[0144] The above results indicate that the overall concordance rate between the chemiluminescent immunoassay kits for diagnosing bovine nodular dermatitis virus antibodies or for detecting bovine nodular dermatitis virus antibodies prepared using BSA-eA27-1+BSA-eA33-1, BSA-eA27-1, and BSA-eA33-1 as coating antigens and the neutralization assay method was significantly higher than that of the chemiluminescent immunoassay kits for diagnosing bovine nodular dermatitis virus antibodies or for detecting bovine nodular dermatitis virus antibodies prepared using BSA-eA27-2, BSA-eA27-3, BSA-eA27-4, BSA-eA33-2, BSA-eA33-3, BSA-eA33-4, the full-length A27 protein of bovine nodular dermatitis virus, and the full-length A33 protein of bovine nodular dermatitis virus as coating antigens.

[0145] Table 7. Detection results of bovine serum samples using CLIA method 1 of the present invention.

[0146]

[0147] Table 8. Detection results of bovine serum samples using CLIA method 2 of the present invention.

[0148]

[0149] Table 9. Detection results of bovine serum samples using CLIA method 3 of the present invention.

[0150]

[0151] Table 10. Detection results of bovine serum samples compared with CLIA method 1

[0152]

[0153] Table 11. Results of bovine serum sample detection compared to CLIA Method 2

[0154]

[0155] Table 12. Detection results of bovine serum samples compared with CLIA method 3.

[0156]

[0157] Table 13. Results of bovine serum sample detection compared to CLIA method 4

[0158]

[0159] Table 14. Detection results of bovine serum samples compared with CLIA method 5

[0160]

[0161] Table 15. Detection results of bovine serum samples compared with CLIA method 6

[0162]

[0163] Table 16. Detection results of bovine serum samples compared with CLIA method 7

[0164]

[0165] Table 17. Detection results of bovine serum samples compared with CLIA method 8

[0166]

[0167]

[0168] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A polypeptide, characterized in that: The polypeptide is either (A1) or (A2): (A1) A polypeptide with the amino acid sequence SEQ ID No. 5; (A2) A polypeptide whose amino acid sequence is the second to third position of SEQ ID No.

5.

2. A polypeptide, characterized in that: The polypeptide is as follows (A3): (A3) A polypeptide that can be coupled to a carrier protein by attaching a cysteine ​​residue to the amino or carboxyl terminus of the polypeptide described in claim 1 (A2).

3. A coupling agent, characterized in that: The conjugate is a complete antigen obtained by conjugating the polypeptide and carrier protein as described in claim 1 or 2.

4. Any of the following biological materials: (B1) A nucleic acid molecule capable of encoding the polypeptide of claim 1 or 2; (B2) Expression cassette, which is an expression cassette containing the nucleic acid molecule described in (B1); (B3) Recombinant vector, which is a recombinant vector containing the nucleic acid molecule described in (B1); (B4) Recombinant bacteria, which are recombinant bacteria containing the nucleic acid molecules described in (B1); (B5) A transgenic cell line is a transgenic cell line containing the nucleic acid molecules described in (B1).

5. The use of the polypeptide of claim 1 or 2 or the conjugate of claim 3 in any of the following: (C1) Preparation of reagents or kits for detecting bovine nodular dermatosis virus antibodies; (C2) Preparation of diagnostic antigen for bovine nodular dermatitis virus.

6. A kit containing the polypeptide of claim 1 or 2 or the conjugate of claim 3.

7. The reagent kit according to claim 5, characterized in that: The kit is used to detect bovine nodular dermatitis virus antibodies; in the kit, the polypeptide of claim 1 or 2 or the conjugate of claim 3 is used as the coating antigen.

8. The kit according to claim 6 or 7, characterized in that: The kit is a chemiluminescent immunoassay kit; the kit also contains horseradish peroxidase (HRP) labeled secondary antibody, coating buffer, washing solution, secondary antibody dilution solution and / or calibrators; the secondary antibody is an antibody capable of binding to the bovine nodular dermatosis virus antibody.

9. The use of the biomaterial of claim 4 in the preparation of the polypeptide of claim 1 or 2, the conjugate of claim 3, or the kit of any one of claims 6-8.

10. The use of the biomaterial of claim 4 in any of the following: (C1) Preparation of reagents or kits for detecting bovine nodular dermatosis virus antibodies; (C2) Preparation of diagnostic antigen for bovine nodular dermatitis virus.

11. The use of the polypeptide of claim 1 or 2 or the conjugate of claim 3 as an immunogen in the preparation of bovine nodular dermatosis virus antibodies.

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