Reagents for detecting bovine dermodenodosis virus antibodies and polypeptides used therewith

Abstract

The application discloses a reagent for detecting bovine nodular dermatopathy virus antibody and a polypeptide used by the reagent. The polypeptide provided by the application is an eA27-1 polypeptide or a complete polypeptide consisting of the eA27-1 polypeptide and an eA33-1 polypeptide; the eA27-1 is a polypeptide with an amino acid sequence shown in SEQ ID No. 1; and the eA33-1 is a polypeptide with an amino acid sequence shown in SEQ ID No. 5. The antibody detection kit prepared by using the conjugate of the polypeptide of the application as a coating agent has the advantages of high specificity, high sensitivity, high accuracy, simple operation and rapidness, and is suitable for rapid and large-scale screening and detection of bovine nodular dermatopathy infection serum antibody by grass-roots veterinary departments and entry-exit inspection and quarantine bureaus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a reagent for detecting bovine nodular dermatopathy virus antibody and a polypeptide used thereby. BACKGROUND

[0002] Nodular dermatopathy (LSD) first occurred in Zambia in 1929, and then mainly in African countries and regions such as the Sahara and Madagascar, and occurred in Israel in 1989 and 2006, and then spread to countries such as Iran, Pakistan and Saudi Arabia. In 2015, it broke out in Greece and Russia successively, and spread in Central Asia and European countries including Russia, Kazakhstan, Turkey, Syria and Israel, which constitutes a serious threat to China. In the past ten years, the Middle East, Europe and Western Asia have reported the occurrence of LSD (OIE, 2017). The outbreak of nodular dermatopathy tends to occur sporadically, depending on the activity of animals, the immune status and the change of wind and rainfall affecting the population number of medium insects. The main mode of transmission is mechanical transmission by arthropod vectors. On August 12, 2019, the National Foreign Animal Disease Research Center of China Animal Health and Epidemiology Center confirmed that bovine nodular dermatopathy broke out in Yili State, Xinjiang Uygur Autonomous Region, which was the first large-scale outbreak of the disease in China.

[0003] LSDV is a member of the Capripoxvirus genus of the Poxviridae family, which also includes the goatpox virus (GPV) and the sheep pox virus (SPV). The virus particles are brick-shaped or short-cylindrical, consisting of one nucleus, two lateral bodies and two lipid outer membranes, and are about 290 nm x 270 nm in size, which is smaller than other poxviruses. There is only one serotype of the virus, and the representative strain is the Neethling strain from South Africa. The virus can proliferate in primary cells such as lamb kidney or Vero cells, sheep embryo kidney and lung cells, and chicken embryo fibroblasts, as well as in passaged cells such as bovine kidney cells and African green monkey kidney cells, and can cause cytopathic effect. The infected cell culture can be stained with hematoxylin and eosin or hematoxylin and rose bengal, and intracytoplasmic inclusions can be observed. The virus can grow and multiply on the chorioallantoic membrane of chicken embryos, causing pock marks, but the embryos do not die. The virus has a cell-binding property, and ultrasonic waves can be used to destroy the cells to release the virus outside the cells. The virus particles can survive for a long time in an environment with a pH of 6.6-8.6. They can survive in glycerol saline and tissue culture medium at 4℃ for 4-6 months. The virus in the lesion can survive for more than one month after drying, and can survive for several months in a dry stable. The virus is resistant to freeze-thawing, and can remain viable for several years when stored below -20℃, and for 10 years when stored at -80℃. It is sensitive to heat, and can be inactivated at 55℃ for 2 hours or at 65℃ for 30 minutes. It is also sensitive to direct sunlight, acids, bases, and most commonly used disinfectants (alcohol, mercury, iodine tincture, lysol, formalin, and phenol), as well as chloroform and diethyl ether.

[0004] The natural host of LSDV is mainly cattle, and all breeds of cattle are susceptible, with no obvious breed specificity. Rabbits, sheep, goats, giraffes, and black antelopes can also be infected, but there is no evidence that LSDV can infect humans. LSDV can infect all bovine species, with an incidence rate of 5%-85% and a mortality rate of up to 20%. Among them, lactating cows are the most severely affected, resulting in a significant decrease in milk production. Orchitis and testicular atrophy can cause permanent or temporary infertility in bulls, and the virus can be excreted through semen for a long time.

[0005] Clinically and subclinically ill cattle are the main sources of transmission, and the movement of live cattle is an important reason for widespread transmission, such as grazing or trading. LSDV is widely present in the skin, dermal lesions, nodules, saliva, nasal discharge, cow's milk, semen, muscle, spleen, lymph nodes, etc. When ulcers form in the eyes, nose, mouth, rectum, mammary glands, and reproductive organs, the nodules rapidly ulcerate, and the virus particles are present in their secretions. Exposure to aerosols produced by infected animals can also lead to infection.

[0006] The incubation period of natural infection is 2-5 weeks, and that of experimental infection is 4-12 days, usually 7 days. The body temperature of sick cattle rises, which can reach above 41℃, and shows persistent retention fever type. The initial symptoms are rhinitis, conjunctivitis and keratitis. The hard, round, raised, 1-5 cm diameter nodules appear on the skin of the body 4-12 days after experimental infection, and the touch is painful, especially on the head, neck, chest, perineum, udder and limbs. The skin nodules are located in the epidermis and dermis, and can gather into irregular lumps, and finally necrotize. The lymph nodes of the body surface are enlarged, especially the lymph nodes in front of the shoulder, outside of the groin, in front of the thigh, behind the limbs and under the ear. The mucous membranes of the eye, nose, mouth, rectum, udder and external genitalia can also form nodules and ulcers. Dairy cows can have mastitis, and the milk yield decreases. Pregnant cows can have abortion, and bulls can be infertile after the disease. The production performance of beef cattle decreases, and the necrotic nodules are easily bitten by flies, leaving deep holes on the skin after falling off, and the skin cannot be used. The gray-red serous infiltration can be seen in the subcutaneous tissue. The dry cheese-like gray-white necrotic tissue is contained in the cavity of the incision, and some have pus and blood. The nodules can reach the subcutaneous tissue or the rib tissue. Similar nodules can be found on the body surface, pharynx, trachea, lung, rumen, abomasum and kidney surface. At the beginning of the disease, eosinophilic inclusion bodies can be seen in the damaged cells. Preliminary diagnosis can be made according to these clinical symptoms and pathological changes, but further laboratory pathogenic and serological detection is needed for final diagnosis.

[0007] The main differential diagnosis is bovine herpesvirus 2 (BoHV-2) causing pseudolumpy skin disease. This is usually a milder clinical condition characterized by superficial nodules and only resembles the early stages 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: dermatosis, dermatophytosis, bovine dandruff, photosensitivity, actinobacillosis, urticaria, insect bites, bertiella infection, nocardiosis, demodicosis, dracunculosis, pseudocowpox and cowpox. Differential diagnoses for mucosal lesions include: foot-and-mouth disease, bluetongue, bovine viral diarrhea, malignant catarrhal fever, contagious bovine rhinotracheitis and bovine epidemic stomatitis. SUMMARY

[0008] One of the technical problems to be solved by the present application is how to improve the sensitivity and specificity of the antibody detection of bovine nodular skin disease virus, so as to more accurately diagnose bovine nodular skin disease.

[0009] In order to solve the above technical problem, the present application provides a set of polypeptides for preparing a bovine nodular skin disease virus antibody detection reagent or a set of polypeptides for preparing a bovine nodular skin disease virus diagnosis reagent.

[0010] In a first aspect, the present application claims a polypeptide or a set of polypeptides.

[0011] The polypeptide claimed in the present application is an eA27-1 polypeptide.

[0012] The set of polypeptides claimed in the present application consists of the eA27-1 polypeptide and the eA33-1 polypeptide.

[0013] The eA27-1 polypeptide is as shown in P11, P12 or P13 below:

[0014] P11, a polypeptide with an amino acid sequence of SEQ ID No. 1,

[0015] P12, a polypeptide with an amino acid sequence of positions 2-28 of SEQ ID No. 1,

[0016] P13, a polypeptide capable of coupling with a carrier protein obtained by connecting an amino acid residue at the amino terminal or carboxyl terminal of the polypeptide shown in P12.

[0017] The eA33-1 polypeptide is as shown in P21, P22 or P23 below:

[0018] P21, a polypeptide with an amino acid sequence of SEQ ID No. 5,

[0019] P22, a polypeptide with an amino acid sequence of positions 2-33 of SEQ ID No. 5,

[0020] P23, a polypeptide capable of coupling with a carrier protein obtained by connecting an amino acid residue at the amino terminal or carboxyl terminal of the polypeptide shown in P22.

[0021] wherein SEQ ID No. 1 consists of 28 amino acid residues, the cysteine residue at position 1 is a connecting arm added for connection with a carrier protein, and the other amino acid residues are derived from the A27 protein of bovine dermatophilus; and SEQ ID No. 5 consists of 33 amino acid residues, the cysteine residue at position 1 is a connecting arm added for connection with a carrier protein, and the other amino acid residues are derived from the A33 protein of bovine dermatophilus.

[0022] In a second aspect, the present application claims a conjugate or a set of conjugates.

[0023] The conjugate claimed in the present application is an eA27-1 conjugate.

[0024] The set of conjugates claimed in the present application consists of the eA27-1 conjugate and the eA33-1 conjugate.

[0025] The eA27-1 conjugate is a complete antigen obtained by coupling the eA27-1 polypeptide described in the first aspect above with a carrier protein; and the eA33-1 conjugate is a complete antigen obtained by coupling the eA33-1 polypeptide described in the first aspect above with a carrier protein.

[0026] In one embodiment of the present application, the mass ratio of the eA27-1 conjugate and the eA33-1 conjugate in the set of conjugates is 1:1.

[0027] The carrier protein can be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin, or rabbit serum albumin, etc.

[0028] In one embodiment of the present application, the carrier protein is bovine serum albumin (BSA).

[0029] In a third aspect, the present application claims the polypeptide or set of polypeptides of the first aspect above or the conjugate or set of conjugates of the second aspect above for use in any one of the following:

[0030] (A1) for preparing a reagent or kit for detecting antibodies against bovine dermatophilus virus;

[0031] (A2) for preparing a diagnostic antigen of bovine dermatophilus virus.

[0032] In a fourth aspect, the present application claims a kit comprising the polypeptide or set of polypeptides of the first aspect above or the conjugate or set of conjugates of the second aspect above.

[0033] Further, the kit is used for detecting antibodies against bovine dermatophilus virus; in the kit, the polypeptide or set of polypeptides of the first aspect above or the conjugate or set of conjugates of the second aspect above is used as a coating antigen.

[0034] Still further, the kit can be a chemiluminescence immunoassay kit; the kit can further comprise a horseradish peroxidase (HRP)-labeled secondary antibody, a coating buffer, a washing solution, a secondary antibody diluent, and / or a calibrator; the secondary antibody is an antibody capable of being against the antibodies against bovine dermatophilus virus.

[0035] In a fifth aspect, the present application claims any one of the following biological materials:

[0036] (B1) a nucleic acid molecule, which is nucleic acid molecule 1 or nucleic acid molecule 2; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eA27-1 polypeptide of the first aspect above; the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eA33-1 polypeptide of the first aspect above;

[0037] (B2) an expression cassette, which is expression cassette 1 or expression cassette 2; the expression cassette 1 is an expression cassette comprising the nucleic acid molecule 1 of (B1); the expression cassette 2 is an expression cassette comprising the nucleic acid molecule 2 of (B1);

[0038] (B3) a recombinant vector, which is a recombinant vector 1 or a recombinant vector 2; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 in (B1); the recombinant vector 2 is a recombinant vector containing the nucleic acid molecule 2 in (B1);

[0039] (B4) a recombinant bacterium, which is a recombinant bacterium 1 or a recombinant bacterium 2; the recombinant bacterium 1 is a recombinant bacterium containing the nucleic acid molecule 1 in (B1); the recombinant bacterium 2 is a recombinant bacterium containing the nucleic acid molecule 2 in (B1);

[0040] (B5) a transgenic cell line, which is a transgenic cell line 1 or a transgenic cell line 2; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 in (B1); the transgenic cell line 2 is a transgenic cell line containing the nucleic acid molecule 2 in (B1);

[0041] (B6) a nucleic acid molecule set, which consists of the nucleic acid molecule 1 and the nucleic acid molecule 2 in (B1);

[0042] (B7) an expression cassette set, which consists of the expression cassette 1 and the expression cassette 2 in (B2);

[0043] (B8) a recombinant vector set, which consists of the recombinant vector 1 and the recombinant vector 2 in (B3);

[0044] (B9) a recombinant bacterium set, which consists of the recombinant bacterium 1 and the recombinant bacterium 2 in (B4);

[0045] (B10) a transgenic cell line set, which consists of the transgenic cell line 1 and the transgenic cell line 2 in (B5).

[0046] In a sixth aspect, the present application claims the use of the biological material as described in the fifth aspect above in any one of the following:

[0047] (C1) preparing the polypeptide or polypeptide set as described in the first aspect above or the conjugate or conjugate set as described in the second aspect above or the kit as described in the third aspect above;

[0048] (A1) preparing a reagent or kit for detecting antibody against bovine dermatophilus;

[0049] (A2) preparing a diagnostic antigen of bovine dermatophilus.

[0050] In a seventh aspect, the present application claims the use of the polypeptide or polypeptide set as described in the first aspect above or the conjugate or conjugate set as described in the second aspect above as immunogen in preparing antibody against bovine dermatophilus.

[0051] The experiment proves that the conjugate obtained by coupling the full-length A27 recombinant protein of bovine nodular dermatitis or the full-length A33 recombinant protein of bovine nodular dermatitis as a hapten with a carrier protein cannot effectively distinguish bovine nodular dermatitis antibodies and foot-and-mouth disease virus antibodies. In order to improve the specificity of bovine nodular dermatitis antibody detection, the present application selects the dominant antigen epitope eA27 and eA33 from the full-length A27 of bovine nodular dermatitis and the full-length A33 of bovine nodular dermatitis, respectively, and couples them with a carrier protein to obtain conjugates (BSA-eA27-1 and / or BSA-eA33-1) as coating agents, which can effectively distinguish bovine nodular dermatitis antibodies and various virus antibodies such as foot-and-mouth disease, thereby improving the specificity of bovine nodular dermatitis antibody detection. The kit for detecting bovine nodular dermatitis antibodies in the detection serum prepared by using BSA-eA27-1 and / or BSA-eA33-1 as coating antigens 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 nasal airway inflammation virus antibody positive serum, and the accuracy of the kit for detecting bovine nodular dermatitis antibody positive serum is consistent with the result of the traditional gold standard method of virus neutralization test, and the sensitivity is higher than that of the virus neutralization test.

[0052] The antibody detection kit prepared by using BSA-eA27-1 and / or BSA-eA33-1 as coating agents has high specificity, high sensitivity, high accuracy, simple operation and rapidness, and is suitable for rapid and large-scale screening and detection of bovine nodular dermatitis infection serum antibodies by primary and secondary veterinary departments and entry and exit inspection and quarantine bureaus. DETAILED DESCRIPTION

[0053] The present application will be further described in detail below in combination with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0054] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0055] The pET32a(+) in the following examples is a product of Huada Gene Co., Ltd. Luminol chemiluminescent substrate is purchased from Luoyang Modern Biotechnology Research Institute Co., Ltd.; chemiluminescent enzyme-labeled plate is purchased from Thermo, USA; 10% fetal bovine serum, cell culture medium and related cell culture reagents are purchased from Gibco, USA; bovine nodular dermatopathy virus antibody negative and positive sample serum with serum neutralization titer background are preserved by China Animal Disease Prevention and Control Center (Veterinary Diagnostic Center of the Ministry of Agriculture and Rural Affairs) (negative serum is collected from healthy cattle, positive serum is collected from cattle with clinical symptoms in the field, and antibody titer is identified by using traditional gold standard method virus neutralization test). Chemiluminescence immunoassay instrument, model HEXU, is purchased from Luoyang Modern Biotechnology Co., Ltd.

[0056] The bovine nodular dermatopathy virus neutralization test method in the following examples is as follows:

[0057] (1) Serum treatment

[0058] The serum is inactivated at 58°C for 30 minutes, and gradient dilution is performed with serum-free MEM medium for standby.

[0059] (2) Control serum

[0060] The standard negative serum and the standard positive serum against bovine nodular dermatopathy virus with serum neutralization titer background are preserved by China Animal Disease Prevention and Control Center (Veterinary Diagnostic Center of the Ministry of Agriculture and Rural Affairs).

[0061] (3) Virus neutralization

[0062] Vero cell suspension with a concentration of 2×10 5 μl / 50μl is inoculated into a 96-well cell plate, 100μl / well. Incubate in a 37°C 5% CO2 incubator for 1-2 days until 70%-80% of the cells form a monolayer. Mix 50μl of the diluted serum to be tested with an equal volume of bovine nodular dermatopathy virus Xinjiang isolate LSDV / Xinjiang / 2019 strain (recorded in the article "Genetic Evolution Relationship and Protein Structure Analysis of P32, GPCR and RPO30 Genes of the First LSDV Isolate in China", Lanzhou University, Dissertation, DOI: 10.27204 / d.cnki.glzhu.2020.001024", which can be obtained from the applicant and can only be used for repeating the experiments of the present application, and cannot be used for other purposes) suspension containing 100 TCID50 / 50μl, and place in a 37°C 5% CO2 incubator for 1 hour.

[0063] (4) Incubation

[0064] After 1 hour of serum and virus neutralization, 100 μl of the mixed suspension of virus and serum was added to the neutralization wells of the cell culture plate, and the plate was incubated at 37°C in a 5% CO2 incubator.

[0065] (5) Result determination

[0066] The initial determination of the result was performed 72 hours after inoculation, the wells with specific lesions were discarded, the culture solution of the other wells was replaced with maintenance solution, and the rotation culture was continued for 7 days to perform the final determination.

[0067] Determination criteria: those capable of inhibiting 50% or more of the cell from exhibiting cytopathic effect (CPE) were determined as positive. The maximum neutralization dilution fold of the serum was calculated according to this criterion.

[0068] The bovine dermatophilus antibody positive serum in the following examples was serum determined as positive by the virus neutralization test, and the bovine dermatophilus antibody negative serum was serum determined as negative by the virus neutralization test.

[0069] Example 1: Detection of bovine dermatophilus infection serum antibody by chemiluminescence immunoassay

[0070] The present inventors used pET32a(+) to solubly express the full-length A27 protein of bovine dermatophilus and the full-length A33 recombinant protein of bovine dermatophilus in E. coli BL21(DE3) during the development process. The experimental results showed that the chemiluminescence immunoassay method established by using the full-length A27 protein of bovine dermatophilus and the full-length A33 recombinant protein of bovine dermatophilus as coating antigens respectively could not effectively distinguish bovine dermatophilus antibody and foot-and-mouth disease virus antibody, and the specificity was poor. The present inventors further selected four dominant antigen epitope polypeptides (eA27-1, eA27-2, eA27-3 and eA27-4) from the full-length A27 protein of bovine dermatophilus, and four dominant antigen epitope polypeptides (eA33-1, eA33-2, eA33-3 and eA33-4) from the full-length A33 protein of bovine dermatophilus, and after coupling with BSA respectively as coating antigens, the specificity of the chemiluminescence immunoassay method established by using the same was significantly improved, which could effectively distinguish bovine dermatophilus antibody and foot-and-mouth disease virus antibody, but the sensitivity difference was large. The mixed complete antigen obtained by mixing the coupling product of eA27-1 and BSA (BSA-eA27-1) and the coupling product of eA33-1 and BSA (BSA-eA33-1) according to a mass ratio of 1:1 as coating antigen, the specificity of the chemiluminescence immunoassay method established by using the same was significantly improved, which could effectively distinguish bovine dermatophilus antibody and foot-and-mouth disease virus antibody, and the sensitivity was also significantly improved. The specific experimental method is as follows:

[0071] I. Preparation of antigens

[0072] The following 11 kinds of coated antigens were prepared in this example:

[0073] (1) BSA-eA27-1 + BSA-eA33-1,

[0074] (2) BSA-eA27-1 (eA27-1 conjugate),

[0075] (3) BSA-eA33-1 (eA33-1 conjugate),

[0076] (4) BSA-eA27-2 (eA27-2 conjugate),

[0077] (5) BSA-eA33-2 (eA33-2 conjugate),

[0078] (6) BSA-eA27-3 (eA27-3 conjugate),

[0079] (7) BSA-eA33-3 (eA33-2 conjugate),

[0080] (8) BSA-eA27-4 (eA27-4 conjugate),

[0081] (9) BSA-eA33-4 (eA33-4 conjugate),

[0082] (10) Full-length A27 protein of bovine dermatophilus,

[0083] (11) Full-length A33 recombinant protein of bovine dermatophilus.

[0084] 1. Dominant antigen epitope polypeptide

[0085] The dominant antigen epitope polypeptide was selected from the A27 protein and the A33 protein of bovine dermatophilus, and the polypeptides eA27-1, eA27-2, eA27-3, eA27-4, eA33-1, eA33-2, eA33-3, and eA33-4 with a cysteine connected at the C-terminal or N-terminal were synthesized by Beijing Lihe Huada Gene Technology Co., Ltd. (Table 1). The purity is greater than 95%, and it is stored by freeze-drying.

[0086] Table 1, Polypeptide

[0087]

[0088]

[0089] 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.

[0090] 2. Preparation of 11 coating antigens

[0091] 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).

[0092] 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.

[0093] The specific preparation methods for coating antigens (1)-(8) and (11) are as follows: The BSA tag-coupled kit (Readilink) manufactured 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 the coated antigen.

[0094] The specific preparation methods for coating antigens (9) and (10) are as follows:

[0095] The full-length A27 protein and the full-length A33 recombinant protein of bovine nodular dermatitis virus were used as coating antigens.

[0096] Step 1: Construction of recombinant expression vectors for the full-length A27 protein gene and the full-length A33 protein gene

[0097] 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).

[0098] 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).

[0099] Step 2: Construction of recombinant strains

[0100] The two expression vectors pET32a-flA27 and pET32a-flA33 constructed in the first step were separately transformed into E. coli BL21(DE3) competent cells. They were uniformly coated on LB plates containing ampicillin (50 μg / mL) and incubated at 37°C for 16 hours. Single colonies were cultured overnight, and plasmids were extracted for sequencing. The recombinant E. coli containing pET32a-flA27 was named BL21(DE3) / pET32a-flA27, and the recombinant E. coli containing pET32a-flA33 was named BL21(DE3) / pET32a-flA33.

[0101] Third step: soluble expression and purification of full-length A27 protein of bovine papular dermatosis virus or full-length A33 recombinant protein of bovine papular dermatosis virus

[0102] 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 obtained by adding ampicillin to LB liquid medium to a concentration of 50 μg / ml) and cultured at 37°C using a Thermo Max Q6000 type full-temperature shaker at 200 rpm until the OD 600When the value (blank control with LB liquid medium containing 50 μg / ml ampicillin) reached 0.6, IPTG was added to induce expression. The induction was performed with 0.75 mM IPTG at 16°C for 13 h. The bacterial pellet was collected from the fermentation broth after 13 h of IPTG induction. The pellet was resuspended in PBS, centrifuged at 8000 rpm / min for 5 min, and the supernatant was discarded. The washed bacterial pellet was resuspended in PBS, and the bacteria were broken by high pressure. The lysate was centrifuged at 16000 rpm / min for 30 min at 4°C, and the supernatant was collected and the pellet was discarded. The supernatant was filtered through a 0.22 μm filter and loaded onto a nickel column previously equilibrated with solution 1 (solutes and their concentrations: 20 mM Tris, 150 mM NaCl, solvent: water, pH 8.0). The nickel column was connected to an AKTA machine, and the impurity proteins in the nickel column were washed with 10 column volumes of solution 1 and 10 column volumes of solution 2 (solutes and their concentrations: 20 mM Tris, 150 mM NaCl, 50 mM imidazole, solvent: water, pH 8.0), and the protein peak was monitored on the AKTA machine. The target protein hanging on the nickel column was washed with solution 3 (solutes and their concentrations: 20 mM Tris, 150 mM NaCl, 300 mM imidazole, solvent: water, pH 8.0), and the elution sample containing the target protein peak was collected using the AKTA. The full-length A27 protein of bovine dermatosis virus and the full-length A33 recombinant protein of bovine dermatosis virus were further purified by molecular sieving using a Superdex 200 gel column produced by GE.

[0103] II. Chemiluminescence immunoassay using the chemiluminescence immunoassay kit for diagnosing bovine dermatosis virus or the chemiluminescence immunoassay kit for detecting bovine dermatosis virus antibodies

[0104] This example provides 11 kinds of chemiluminescence immunoassay kits for diagnosing bovine dermatosis virus or detecting bovine dermatosis virus antibodies. The 11 kits all include a coating antigen, a horseradish peroxidase (HRP) labeled bovine secondary antibody (item number HM390, Hua Xu (Zhengzhou) Biotechnology Co., Ltd.), a coating buffer (item number HC210, Hua Xu (Zhengzhou) Biotechnology Co., Ltd.), a washing solution (item number HX221, Hua Xu (Zhengzhou) Biotechnology Co., Ltd.), and a secondary antibody diluent (item number HM391, Hua Xu (Zhengzhou) Biotechnology Co., Ltd.). The only difference between the 11 kits is the coating antigen, and the other components are exactly the same.

[0105] The 11 kinds of kits are respectively diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA27-1+BSA-eA33-1 of step one as coating antigen, hereinafter referred to as kit 1 of the present application; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA27-1 of step one as coating antigen, hereinafter referred to as kit 2 of the present application; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA33-1 of step one as coating antigen, hereinafter referred to as kit 3 of the present application. Diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA27-2 of step one as coating antigen, hereinafter referred to as control kit 1; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA33-2 of step one as coating antigen, hereinafter referred to as control kit 2; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA27-3 of step one as coating antigen, hereinafter referred to as control kit 3; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA33-3 of step one as coating antigen, hereinafter referred to as control kit 4; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA27-4 of step one as coating antigen, hereinafter referred to as control kit 5; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with BSA-eA33-4 of step one as coating antigen, hereinafter referred to as control kit 6; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with full-length A27 protein of bovine dermatophilosis virus of step one as coating antigen, hereinafter referred to as control kit 7; diagnostic bovine dermatophilosis virus chemiluminescence immunoassay kit or detection bovine dermatophilosis virus antibody chemiluminescence immunoassay kit with full-length A33 protein of bovine dermatophilosis virus of step one as coating antigen, hereinafter referred to as control kit 8.

[0106] (1) Using the kit 1 of the present application, an optimized experiment was performed to establish a chemiluminescence immunoassay method (hereinafter referred to as the CLIA method 1 of the present application) using BSA-eA27-1+BSA-eA33-1 as the coating antigen, coating antigen, horseradish peroxidase (HRP) labeled secondary antibody, and secondary antibody diluent.

[0107] 1. Operation steps

[0108] (1) Coating: dilute the BSA-eA27-1+BSA-eA33-1 in step (1) to a concentration (total mass concentration of BSA-eA27-1 and BSA-eA33-1) of 1.0 μg / ml with the coating buffer to obtain a coating antigen solution, coat the experimental wells with the coating buffer, 100 μL / well to an enzyme-labeled plate, and incubate at 4°C for 16 h.

[0109] (2) Washing: pour out the coating antigen solution in the wells, wash with the washing solution for 5 times, 3 min each time, and pat dry.

[0110] (3) Blocking: add 1% BSA blocking solution, 350 μL / well, and incubate at 37°C for 2 h.

[0111] (4) Washing: synchronize with step (2).

[0112] (5) Sample addition: in the serum dilution plate, dilute the sample to be tested at a ratio of 1:20 with the sample diluent (1×PBST); take out the coating plate, and set up 6 wells of calibration samples for each experiment. First, add 100 μL of calibration samples 1-6 to the serum dilution plate in turn, and then add 100 μL of the diluted sample to be tested to each well in turn, and incubate in a 37°C constant temperature incubator for 30 min (±1 min).

[0113] (6) Washing: synchronize with step (2).

[0114] (7) Add 100 μL of horseradish peroxidase (HRP) labeled secondary antibody diluted with the secondary antibody diluent to each well, shake to mix. Take 100 μL from each well and transfer to the corresponding well of the coating plate; incubate in a 37°C constant temperature incubator for 30 min (±1 min).

[0115] (8) Washing: synchronize with step (2).

[0116] (9) Add 100 μL of luminol chemiluminescence substrate to each well, shake to mix; avoid light and stand for 5 min at 15-25°C, and read the luminescence value within 15 min after standing with a chemiluminescence instrument.

[0117] 2. Result calculation method

[0118] NCU (National clinical unit) is a unit established by the national clinical inspection center. In the method, the unit is assigned a value of 20000 NCU / ml for the standard positive serum (neutralizing antibody titer of 1:512). A four-parameter method calibration curve is established using standard positive serum with different dilution ratios. The antibody dose value (NCU / ml) of the sample is calculated from the luminescence value of the sample and the calibration curve, thereby realizing semi-quantitative detection.

[0119] The bovine nodular dermatopathy virus antibody positive serum with a serum neutralization titer background is set as calibration 1-6 according to the virus neutralization test results, and the corresponding antibody dose values (0 NCU / mL, 10 NCU / mL, 20 NCU / mL, 50 NCU / mL, 100 NCU / mL, 200 NCU / mL) are set. The luminescence values of the calibration 1 to calibration 6 are taken as the ordinate, and the corresponding antibody dose values are taken as the abscissa. The four-parameter fitting curve of the calibration is drawn by ELISA Calc software, and the four-parameter mode is Y = (a-b) / [1+(x / c)*b]+d. Wherein, a is the asymptote value of the curve; b is the slope of the curve; c is the corresponding dose when the maximum binding is half; d is the asymptote value of the curve.

[0120] The luminescence value of the sample is substituted into the calibration curve to calculate the content of bovine nodular dermatopathy virus antibody in the sample.

[0121] 3. Test establishment condition and result determination

[0122] The luminescence values of the six calibrations are four-parameter fitted with the corresponding antibody dose values, R 2 ≥0.99, the test is established. Otherwise, the test is not established.

[0123] 4. Determination of positive and negative critical values

[0124] 200 bovine nodular dermatopathy virus antibody negative sera are detected by CLIA using the above method, and the average value (X) and the standard deviation (SD) of the dose value of the 200 bovine nodular dermatopathy virus antibody negative sera are calculated. The average dose value X of the 200 bovine nodular dermatopathy virus antibody negative sera is 28.59, and the SD is 3.64, so the positive and negative critical dose value is 39.51 NCU / mL. That is, the dose value ≥39.51 NCU / mL is judged as positive; and the dose value <39.51 NCU / mL is judged as negative.

[0125] (2) The kit 2 of the application is used to establish a chemiluminescence immunoassay method (hereinafter referred to as the CLIA method 2 of the application) with BSA-eA27-1 as the coated antigen through optimization experiments. The steps are the same as (1).

[0126] (Three) By using the kit 3 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the CLIA method 3 of the present application) using BSA-eA33-1 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0127] (Four) By using the control kit 1 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 1) using BSA-eA27-2 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0128] (Five) By using the control kit 2 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 2) using BSA-eA33-2 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0129] (Six) By using the control kit 3 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 3) using BSA-eA27-3 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0130] (Seven) By using the control kit 4 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 4) using BSA-eA33-3 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0131] (Eight) By using the control kit 5 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 5) using BSA-eA27-4 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0132] (Nine) By using the control kit 6 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 6) using BSA-eA33-4 as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0133] (Ten) By using the control kit 7 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 7) using full-length A27 protein as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0134] (Eleven) By using the control kit 8 of the present application, a chemiluminescent immunoassay method (hereinafter referred to as the control CLIA method 8) using full-length A33 protein as a coating antigen was established through optimization experiments. The steps are the same as (One).

[0135] Three, Specificity Test

[0136] The CLIA method 1-3 of step two, the control CLIA method 1-8 and the bovine foot-and-mouth disease virus antibody positive serum, the bovine virus diarrhea virus antibody positive serum and the bovine infectious nasal rhinotracheitis virus antibody positive serum (various positive sera are collected from the serum of clinically ill animals) are compared to observe whether there is cross reaction with other diseases of the same species. In addition, the virus neutralization test is also used for detection (for specific method, refer to the foregoing).

[0137] The results show that the antibody dose values of the CLIA method 1-3 and the control CLIA method 1-8 are less than 40 NCU / ml, which is determined as negative. The specificity is 100%. The neutralization indexes of the virus neutralization test are less than 1:5, which is the national standard, and the results are all negative. The specific results are shown in Table 1 and Table 2.

[0138] Table 1, the specificity test results of the method 1-3 and the virus neutralization test of the application

[0139]

[0140] Table 2, the specificity test results of the control method 1-8

[0141]

[0142]

[0143] Four, sensitivity test

[0144] The bovine nodular dermatosis virus antibody positive serum is diluted by multiple, and the CLIA method 1-3 of the application and the serum sample with known serum neutralization titer background are used for comparison and detection to obtain the maximum dilution degree when the positive critical value. The results show that the highest dilution multiple of the positive serum detected by the CLIA method 1-3 of the application is 1:1024. The highest dilution multiple of the positive serum detected by the virus neutralization test is 1:512. See Table 3.

[0145] Table 3, the sensitivity results of the CLIA method 1-3 and the neutralization test of the application

[0146]

[0147] Five, repeatability test

[0148] The 10 portions of bovine nodular skin disease virus antibody positive serum are respectively detected on the same batch plate and different batch plates by the method 1-3 of the application, parallel determination is carried out for 5 times, and the batch and batch variation coefficients (CV) are calculated. The results show that the batch repeat variation coefficient of the CLIA method 1-3 of the application is within 8%, and the batch repeat variation coefficient is less than 10% (Tables 4-6). The results show that the bovine nodular skin disease virus antibody positive serum has good repeatability.

[0149] Table 4, repeatability test of the CLIA method 1 of the application

[0150]

[0151] Table 5, repeatability test of the CLIA method 2 of the application

[0152]

[0153] Table 6, repeatability test of the CLIA method 3 of the application

[0154]

[0155]

[0156] Six, conformity test

[0157] The 80 portions of bovine serum with bovine nodular skin disease virus antibody positive serum neutralization titer background and the 80 portions of bovine serum with bovine nodular skin disease virus antibody negative serum neutralization titer background are selected from the bovine serum preserved by the China Animal Disease Control Center (Ministry of Agriculture and Rural Affairs Veterinary Diagnosis Center). The 160 portions of bovine serum are respectively detected by the CLIA method 1-3 of the application and the control CLIA method 1-8, and the coincidence rate with the neutralization test method is calculated.

[0158] The results show that the total coincidence rate of the 160 samples of bovine serum, the CLIA method 1 of the application and the neutralization test method is 98.75% (positive coincidence rate is 100%, negative coincidence rate is 97.5%), the total coincidence rate of the CLIA method 2 of the application and the bovine nodular skin disease virus neutralization test method is 95.625% (positive coincidence rate is 93.75%, negative coincidence rate is 97.5%), the total coincidence rate of the CLIA method 3 of the application and the bovine nodular skin disease virus neutralization test method is 96.25% (positive coincidence rate is 92.5%, negative coincidence rate is 100%). The total coincidence rate of the detection result of the control CLIA method 1 of the application and the bovine nodular skin disease virus neutralization test method is 85.625% (positive coincidence rate is 85%, negative coincidence rate is 86.25%), the total coincidence rate of the detection result of the control CLIA method 2 of the application and the bovine nodular skin disease virus neutralization test method is 78.125% (positive coincidence rate is 81.25%, negative coincidence rate is 75%), the total coincidence rate of the detection result of the control CLIA method 3 of the application and the bovine nodular skin disease virus neutralization test method is 75.625% (positive coincidence rate is 80%, negative coincidence rate is 71.25%), the total coincidence rate of the detection result of the control CLIA method 4 of the application and the bovine nodular skin disease virus neutralization test method is 78.125% (positive coincidence rate is 100%, negative coincidence rate is 56.25%), the total coincidence rate of the detection result of the control CLIA method 5 of the application and the bovine nodular skin disease virus neutralization test method is 79.375% (positive coincidence rate is 80%, negative coincidence rate is 78.75%), the total coincidence rate of the detection result of the control CLIA method 6 of the application and the bovine nodular skin disease virus neutralization test method is 81.25% (positive coincidence rate is 88.75%, negative coincidence rate is 73.75%), the total coincidence rate of the detection result of the control CLIA method 7 of the application and the bovine nodular skin disease virus neutralization test method is 85.625% (positive coincidence rate is 100%, negative coincidence rate is 71.25%), the total coincidence rate of the detection result of the control CLIA method 8 of the application and the bovine nodular skin disease virus neutralization test method is 85.625% (positive coincidence rate is 97.5%, negative coincidence rate is 73.75%). For details, see Tables 7 to 16.

[0159] The above results show that the total coincidence rate of the diagnostic bovine dermatophilus virus antibody chemiluminescence immunoassay kit or the chemiluminescence immunoassay kit for detecting bovine dermatophilus virus antibody prepared by taking BSA-eA27-1+BSA-eA33-1, BSA-eA27-1 and BSA-eA33-1 as the coating antigen, respectively, and the neutralization test method is significantly higher than that of the diagnostic bovine dermatophilus virus antibody chemiluminescence immunoassay kit or the chemiluminescence immunoassay kit for detecting bovine dermatophilus virus antibody prepared by taking 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 dermatophilus virus and the full-length A33 protein of bovine dermatophilus virus as the coating antigen, respectively.

[0160] Table 7, detection results of bovine serum samples by CLIA method 1 of the present application

[0161]

[0162] Table 8, detection results of bovine serum samples by CLIA method 2 of the present application

[0163]

[0164] Table 9, detection results of bovine serum samples by CLIA method 3 of the present application

[0165]

[0166] Table 10, detection results of bovine serum samples by control CLIA method 1

[0167]

[0168]

[0169] Table 11, detection results of bovine serum samples by control CLIA method 2

[0170]

[0171] Table 12, detection results of bovine serum samples by control CLIA method 3

[0172]

[0173] Table 13, detection results of bovine serum samples by control CLIA method 4

[0174]

[0175] Table 14, detection results of bovine serum samples by control CLIA method 5

[0176]

[0177]

[0178] Table 15. Results of detection of bovine serum samples by Control CLIA Method 6

[0179]

[0180] Table 16. Results of detection of bovine serum samples by Control CLIA Method 7

[0181]

[0182] Table 17. Results of detection of bovine serum samples by Control CLIA Method 8

[0183]

[0184] The application has been described in detail. Within the scope of equivalents, concentrations and conditions, the application can be practiced in a wider range without departing from the spirit and scope of the application, and without unnecessary experiments. Although specific examples have been given, it should be understood that further modifications can be made to the application. In general, the application is intended to include all modifications, uses or improvements of the application falling within the scope of the application as defined by the following claims. Some basic features can be applied within the scope of the following claims.

Claims

1. A polypeptide, characterized in that: The polypeptide is an eA27-1 polypeptide; The eA27-1 polypeptide is as shown in P11 or P12 below: P11, a polypeptide with an amino acid sequence of SEQ ID No. 1; P12, a polypeptide with an amino acid sequence of positions 2-28 of SEQ ID No.

1.

2. A polypeptide combination, characterized by: The polypeptide combination consists of an eA27-1 polypeptide and an eA33-1 polypeptide; The eA27-1 polypeptide is as shown in P11 or P12 below: P11, a polypeptide with an amino acid sequence of SEQ ID No. 1; P12, a polypeptide with an amino acid sequence of positions 2-28 of SEQ ID No.

1. The eA33-1 polypeptide is as shown in P21 or P22 below: P21, a polypeptide with an amino acid sequence of SEQ ID No. 5; P22, a polypeptide with an amino acid sequence of positions 2-33 of SEQ ID No.

5.

3. Conjugate, characterized in that: The conjugate is an eA27-1 conjugate; The eA27-1 conjugate is a complete antigen obtained by conjugating the eA27-1 polypeptide in claim 1 with a carrier protein.

4. A combination of conjugates characterized in that: The conjugate combination consists of an eA27-1 conjugate and an eA33-1 conjugate; The eA27-1 conjugate is a complete antigen obtained by conjugating the eA27-1 polypeptide in claim 2 with a carrier protein; the eA33-1 conjugate is a complete antigen obtained by conjugating the eA33-1 polypeptide in claim 2 with a carrier protein.

5. The conjugate combination of claim 4, wherein: In the conjugate combination, the mass ratio of the eA27-1 conjugate to the eA33-1 conjugate is 1:

1.

6. Use of the polypeptide in claim 1 or the polypeptide combination in claim 2 or the conjugate in claim 3 or the conjugate combination in claim 4 or 5 in any one of the following: (A1) preparation of a reagent for detecting bovine dermatophilus antibody; (A2) preparation of bovine dermatophilus diagnostic antigen.

7. A kit containing the polypeptide in claim 1 or the polypeptide combination in claim 2 or the conjugate in claim 3 or the conjugate combination in claim 4 or 5.

8. The kit of claim 7, wherein: The kit is used for detecting bovine dermatophilus antibody; in the kit, the polypeptide in claim 1 or the polypeptide combination in claim 2 or the conjugate in claim 3 or the conjugate combination in claim 4 or 5 is used as a coating antigen.

9. The kit of claim 7 or 8, wherein: The kit is a chemiluminescence immunoassay kit; the kit further contains horseradish peroxidase-labeled secondary antibody, coating buffer, washing solution, secondary antibody diluent and / or calibrant; the secondary antibody is an antibody capable of binding to the bovine dermatophilus antibody.

10. A nucleic acid molecule, characterized in that: The nucleic acid molecule is a nucleic acid molecule capable of encoding the eA27-1 polypeptide in claim 1.

11. An expression cassette characterized in that: The expression cassette is an expression cassette containing the nucleic acid molecule in claim 10.

12. A recombinant vector characterized in that: The recombinant vector is a recombinant vector containing the nucleic acid molecule in claim 10.

13. A recombinant bacterium, characterized in that: The recombinant bacteria are recombinant bacteria containing the nucleic acid molecule in claim 10.

14. A transgenic cell line, characterized by: The transgenic cell line is a transgenic cell line containing the nucleic acid molecule in claim 10.

15. A combination of nucleic acid molecules, characterized in that: The nucleic acid molecule combination consists of nucleic acid molecule 1 and nucleic acid molecule 2; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eA27-1 polypeptide as described in claim 2; the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eA33-1 polypeptide as described in claim 2.

16. A combination of expression cassettes, characterized in that: The expression cassette combination consists of expression cassette 1 and expression cassette 2; the expression cassette 1 is an expression cassette containing the nucleic acid molecule 1 as described in claim 15; the expression cassette 2 is an expression cassette containing the nucleic acid molecule 2 as described in claim 15.

17. A recombinant vector combination characterized in that: The recombinant vector combination consists of recombinant vector 1 and recombinant vector 2; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 as described in claim 15; the recombinant vector 2 is a recombinant vector containing the nucleic acid molecule 2 as described in claim 15.

18. A combination of recombinant bacteria, characterized in that: The recombinant bacteria combination consists of recombinant bacteria 1 and recombinant bacteria 2; the recombinant bacteria 1 is a recombinant bacteria containing the nucleic acid molecule 1 as described in claim 15; the recombinant bacteria 2 is a recombinant bacteria containing the nucleic acid molecule 2 as described in claim 15.

19. A combination of transgenic cell lines, characterized in that: The transgenic cell line combination consists of transgenic cell line 1 and transgenic cell line 2; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 as described in claim 15; the transgenic cell line 2 is a transgenic cell line containing the nucleic acid molecule 2 as described in claim 15.

20. Use of the nucleic acid molecule of claim 10 or the expression cassette of claim 11 or the recombinant vector of claim 12 or the recombinant bacteria of claim 13 or the transgenic cell line of claim 14 or the nucleic acid molecule combination of claim 15 or the expression cassette combination of claim 16 or the recombinant vector combination of claim 17 or the recombinant bacteria combination of claim 18 or the transgenic cell line combination of claim 19 in any of the following: (C1) preparing the polypeptide of claim 1 or the polypeptide combination of claim 2 or the conjugate of claim 3 or the conjugate combination of claim 4 or 5 or the kit of any one of claims 7-9; (C2) preparing a reagent for detecting antibody against bovine dermatophilus; (C3) preparing a diagnostic antigen for bovine dermatophilus.

Images