Kit for detecting African horse sickness virus antibodies and polypeptide epitopes thereof

Abstract

The present invention discloses a kit for detecting antibodies to African horse sickness virus and its polypeptide epitopes. The present invention provides a polypeptide or a set of polypeptides, wherein the polypeptide is eVP2-1; the set of polypeptides is composed of eVP2-1 and eVP5-1 or is composed of eVP2-1 and eVP7-1; the eVP2-1 is a polypeptide of SEQ ID No.1; the eVP5-1 is a polypeptide of SEQ ID No.4; the eVP7-1 is a polypeptide of SEQ ID No.7. The antibody detection kit prepared by the present invention using BSA-eVP2-1+BSA-eVP5 or BSA-eVP2-1+BSA-eVP7-1 as a coating source has strong specificity, high sensitivity, high accuracy, simple and rapid operation, and is suitable for rapid and large-scale screening and detection of serum antibodies infected with African horse sickness virus by veterinary departments at all levels and entry-exit inspection and quarantine bureaus at the grassroots level.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for detecting African horse sickness virus antibodies and polypeptide epitopes thereof. Background Art

[0002] African horse sickness (AHS) is an acute or subacute infectious disease transmitted by midges, which mainly causes fever, subcutaneous edema and viremia in equine animals. African horsesickness virus (AHSV) is a member of the genus Orbivirus of the family Reoviridae. In addition to AHSV, this genus also includes Epizootic haemorrhagic disease virus (EHDV), Bluetongue virus (BTV) and Equine encephalosis orbivirus (EEV). African horse sickness virus is transmitted by insect vectors, such as Culicoides, Chelicerae, and Aedes mosquitoes. Horses are the most susceptible, affecting the respiratory and circulatory functions of horses. The main symptoms are fever, subcutaneous edema and viremia, which can lead to a mortality rate of up to 95%.

[0003] African horse sickness virus is divided into 9 serotypes, the virus particle diameter is about 80nm, the genome is a segmented double-stranded RNA, containing 10 segments S1-S10, the genes encode VP1, VP2, VP3, VP4, VP5, VP7, NS1, NS2, NS3 / NS3A, NS4 proteins. Non-structural proteins NS1-NS4 are mainly involved in virus replication, assembly and release in AHSV-infected cells, and play an important role in the virus antagonizing the natural immune response of host cells. Summary of the invention

[0004] A technical problem to be solved by the present invention is how to improve the specificity and sensitivity of African horse sickness virus antibody detection, so as to more accurately diagnose African horse sickness virus disease.

[0005] In order to solve the above technical problems, the present invention provides a set of polypeptides for preparing African horse sickness virus antibody detection reagents or a set of polypeptides for preparing African horse sickness virus disease diagnostic reagents.

[0006] In a first aspect, the present invention claims a polypeptide or a set of polypeptides.

[0007] The polypeptide claimed in the present invention is the eVP2-1 polypeptide.

[0008] The set of polypeptides claimed in the present invention is set of polypeptides 1 or set of polypeptides 2.

[0009] The set of polypeptides 1 consists of the eVP2-1 polypeptide and the eVP5-1 polypeptide.

[0010] The set of polypeptides 2 consists of the eVP2-1 polypeptide and the eVP7-1 polypeptide.

[0011] The eVP2-1 polypeptide is shown as P11, P12 or P13 below:

[0012] P11, a polypeptide having an amino acid sequence of SEQ ID No. 1;

[0013] P12, a polypeptide having an amino acid sequence of positions 2 to 46 of SEQ ID No. 1;

[0014] P13. A polypeptide capable of being coupled to a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P12.

[0015] The eVP5-1 polypeptide is shown as P21, P22 or P23 below:

[0016] P21, a polypeptide having an amino acid sequence of SEQ ID No. 4;

[0017] P22, a polypeptide having an amino acid sequence of positions 2 to 38 of SEQ ID No. 4;

[0018] P23. A polypeptide capable of being coupled to a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P22.

[0019] The eVP7-1 polypeptide is shown as P21, P22 or P23 below:

[0020] P31, a polypeptide having an amino acid sequence of SEQ ID No. 7;

[0021] P32, a polypeptide having an amino acid sequence of positions 2 to 51 of SEQ ID No. 7;

[0022] P33. A polypeptide capable of being coupled to a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P32.

[0023] Among them, SEQ ID No.1 consists of 46 amino acid residues, the cysteine ​​residue at the first position is a linker added for connecting to the carrier protein, and the other amino acid residues are derived from the VP2 protein of the African horse sickness virus; SEQ ID No.4 consists of 38 amino acid residues, the cysteine ​​residue at the first position is a linker added for connecting to the carrier protein, and the other amino acid residues are derived from the VP5 protein of the African horse sickness virus. SEQ ID No.7 consists of 51 amino acid residues, the cysteine ​​residue at the first position is a linker added for connecting to the carrier protein, and the other amino acid residues are derived from the VP7 protein of the African horse sickness virus.

[0024] In the set of polypeptides 1, the mass ratio of the eVP2-1 polypeptide to the eVP5-1 polypeptide can be determined by those skilled in the art based on the detection effect of African horse sickness virus antibodies, such as 1:1.

[0025] In the set of polypeptides 2, the mass ratio of the eVP2-1 polypeptide to the eVP7-1 polypeptide can be determined by those skilled in the art based on the detection effect of African horse sickness virus antibodies, such as 1:1.

[0026] In a second aspect, the present invention claims a conjugate or a set of conjugates.

[0027] The conjugate claimed in the present invention is an eVP2-1 conjugate.

[0028] The set of conjugates claimed in the present invention is set of conjugates 1 or set of conjugates 2.

[0029] The set of conjugates 1 consists of the eVP2-1 conjugate and the eVP5-1 conjugate.

[0030] The set of conjugates 2 consists of the eVP2-1 conjugate and the eVP7-1 conjugate.

[0031] The eVP2-1 conjugate is a complete antigen obtained by coupling the eVP2-1 polypeptide described in the first aspect above with a carrier protein;

[0032] The eVP5-1 conjugate is a complete antigen obtained by coupling the eVP5-1 polypeptide described in the first aspect above with a carrier protein;

[0033] The eVP7-1 conjugate is a complete antigen obtained by coupling the eVP7-1 polypeptide described in the first aspect above with a carrier protein.

[0034] In a specific embodiment of the present invention, in the set of conjugates 1, the mass ratio of the eVP2-1 conjugate to the eVP5-1 conjugate is 1:1.

[0035] In a specific embodiment of the present invention, in the set of conjugates 2, the mass ratio of the eVP2-1 conjugate to the eVP7-1 conjugate is 1:1.

[0036] The carrier protein may be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin or rabbit serum albumin.

[0037] In a specific embodiment of the present invention, the carrier protein is bovine serum albumin (BSA).

[0038] In a third aspect, the present invention claims the use of the polypeptide or set of polypeptides described in the first aspect above or the conjugate or set of conjugates described in the second aspect above in any of the following:

[0039] (A1) preparing a reagent or a kit for detecting antibodies against African horse sickness virus;

[0040] (A2) Preparation of African horse sickness virus diagnostic antigen.

[0041] In a fourth aspect, the present invention claims a kit comprising the polypeptide or set of polypeptides as described in the first aspect above or the set of conjugates as described in the second aspect above.

[0042] Furthermore, the kit is used to detect African horse sickness virus antibodies; in the kit, the polypeptide or set of polypeptides described in the first aspect above or the conjugate or set of conjugates described in the second aspect above are used as coating antigens.

[0043] Furthermore, the kit is a chemiluminescent immunoassay kit; the kit also contains 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 that can resist the African horse sickness virus antibody.

[0044] In a fifth aspect, the present invention claims protection for any of the following biological materials:

[0045] (B1) Nucleic acid molecule, which is nucleic acid molecule 1; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eVP2-1 polypeptide described in the first aspect above.

[0046] (B2) Expression cassette is expression cassette 1; the expression cassette 1 is an expression cassette containing the nucleic acid molecule 1 described in (B1).

[0047] (B3) The recombinant vector is the recombinant vector 1; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 described in (B1).

[0048] (B4) The recombinant bacterium is the recombinant bacterium 1; the recombinant bacterium 1 is a recombinant bacterium containing the nucleic acid molecule 1 described in (B1).

[0049] (B5) The transgenic cell line is the transgenic cell line 1; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 described in (B1).

[0050] (B6) A set of nucleic acid molecules, which is a set of nucleic acid molecules 1 or a set of nucleic acid molecules 2; the set of nucleic acid molecules 1 is composed of nucleic acid molecules 1 and nucleic acid molecules 2 described in (B1); the set of nucleic acid molecules 2 is composed of nucleic acid molecules 1 and nucleic acid molecules 3 described in (B1); the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eVP5-1 polypeptide described in the first aspect of the above text; the nucleic acid molecule 3 is a nucleic acid molecule capable of encoding the eVP7-1 polypeptide described in the first aspect of the above text.

[0051] (B7) A complete set of expression cassettes, which is a complete set of expression cassettes 1 or a complete set of expression cassettes 2; the complete set of expression cassettes 1 is composed of the expression cassettes 1 and 2 described in (B2); the complete set of expression cassettes 2 is composed of the expression cassettes 1 and 3 described in (B2); the expression cassette 2 is an expression cassette containing the nucleic acid molecule 2 described in (B6); the expression cassette 3 is an expression cassette containing the nucleic acid molecule 3 described in (B6).

[0052] (B8) A complete set of recombinant vectors, which is a complete set of recombinant vectors 1 or a complete set of recombinant vectors 2; the complete set of recombinant vectors 1 is composed of the recombinant vectors 1 and the recombinant vectors 2 described in (B3); the complete set of recombinant vectors 2 is composed of the recombinant vectors 1 and the recombinant vectors 3 described in (B3); the recombinant vectors 2 are recombinant vectors containing the nucleic acid molecules 2 described in (B6); the recombinant vectors 3 are recombinant vectors containing the nucleic acid molecules 3 described in (B6).

[0053] (B9) A set of recombinant bacteria is a set of recombinant bacteria 1 or a set of recombinant bacteria 2; the set of recombinant bacteria 1 is composed of the recombinant bacteria 1 and the recombinant bacteria 2 described in (B4); the set of recombinant bacteria 2 is composed of the recombinant bacteria 1 and the recombinant bacteria 3 described in (B4); the recombinant bacteria 2 is a recombinant bacteria containing the nucleic acid molecule 2 described in (B6); the recombinant bacteria 3 is a recombinant bacteria containing the nucleic acid molecule 3 described in (B6).

[0054] (B10) A complete set of transgenic cell lines, which is a complete set of transgenic cell line 1 or a complete set of transgenic cell line 2; the complete set of transgenic cell line 1 is composed of the transgenic cell line 1 and the transgenic cell line 2 described in (B5); the complete set of transgenic cell line 2 is composed of the transgenic cell line 1 and the transgenic cell line 3 described in (B5); the transgenic cell line 2 is a transgenic cell line containing the nucleic acid molecule 2 described in (B6); the transgenic cell line 3 is a transgenic cell line containing the nucleic acid molecule 3 described in (B6).

[0055] In a sixth aspect, the present invention claims protection for the use of the biomaterial described in the fifth aspect above in any of the following:

[0056] (C1) preparing the polypeptide or set of polypeptides described in the first aspect above, the conjugate or set of conjugates described in the second aspect above, or the kit described in the fourth aspect above;

[0057] (C2) preparing a reagent or kit for detecting antibodies against African horse sickness virus;

[0058] (C3) Preparation of African horse sickness virus diagnostic antigen.

[0059] In a seventh aspect, the present invention claims protection for the use of the polypeptide or set of polypeptides described in the first aspect above or the conjugate or set of conjugates described in the second aspect above as an immunogen in the preparation of African horse sickness virus antibodies.

[0060] Experiments have shown that the conjugate obtained by using the full-length VP2 recombinant protein of African horse sickness virus, the full-length VP5 recombinant protein of African horse sickness virus, or the full-length VP7 recombinant protein of African horse sickness virus as a hapten and coupling it with a carrier protein cannot be used as a coating agent to effectively distinguish African horse sickness virus antibodies from equine infectious anemia virus antibodies, equine arteritis virus antibodies, equine herpes virus antibodies, equine influenza virus antibodies, and equine Salmonella antibodies. In order to improve the specificity of African horse sickness virus antibody detection, the present invention selects dominant antigenic epitopes eVP2-1, eVP5-1 and eVP7-1 from the full-length VP2 of African horse sickness virus, the full-length VP5 of African horse sickness virus and the full-length VP7 of African horse sickness virus, respectively, and couples them with carrier proteins to obtain conjugates (BSA-eVP2-1 and / or BSA-eVP5-1 and / or BSA-eVP7-1) as coating agents, which can effectively distinguish equine infectious anemia virus antibodies, equine arteritis virus antibodies, equine herpes virus antibodies, equine influenza virus antibodies, and equine Salmonella antibodies, thereby improving the specificity of African horse sickness virus antibody detection. The kit for detecting African horse sickness virus antibodies in serum prepared using BSA-eVP2-1 and / or BSA-eVP5-1 and / or BSA-eVP7-1 as coating antigens can accurately distinguish sera positive for equine infectious anemia virus antibodies, equine arteritis virus antibodies, equine herpes virus antibodies, equine influenza virus antibodies, and equine Salmonella antibodies.

[0061] The total conformity rate of the CLIA method 2 of the present invention using the coating antigen of BSA-eVP2-1+BSA-eVP5-1 and the serum complement fixation test method of African horse sickness virus is 95.83% (positive conformity rate is 96.67%, negative conformity rate is 95%); the total conformity rate of the CLIA method 3 of the present invention using the coating antigen of BSA-eVP2-1+BSA-eVP7-1 and the serum complement fixation test method of African horse sickness virus is 95.83% (positive conformity rate is 93.33%, negative conformity rate is 98.33%). The total conformity rate of the control CLIA method 1 of the present invention using the coating antigen of BSA-eVP2-1 and the serum complement fixation test method of African horse sickness virus is 85.83% (positive conformity rate is 83.33%, negative conformity rate is 88.33%).

[0062] The antibody detection kit prepared by the present invention using BSA-eVP2-1+BSA-eVP5-1 or BSA-eVP2-1+BSA-eVP7-1 as the coating agent has strong specificity, high sensitivity, high accuracy, simple and rapid operation, and is suitable for rapid and large-scale screening and detection of serum antibodies infected with African horse sickness virus by veterinary departments at all levels at the grassroots level and entry-exit inspection and quarantine bureaus. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0064] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0065] The pET32a(+) in the following examples is a product of BGI. The luminol chemiluminescent substrate is a product of Luoyang Modern Biotechnology Research Institute Co., Ltd.; the chemiluminescent ELISA plate is a product of Thermo Corporation of the United States; 10% fetal bovine serum, cell culture medium and related cell culture reagents are products of Gibco of the United States; the negative and positive sample serum of African horse sickness virus antibody with serum neutralization titer background is preserved by the China Animal Disease Prevention and Control Center (Veterinary Diagnostic Center of the Ministry of Agriculture and Rural Affairs), a product of Spanish Ingena, item number: AHSK32. The chemiluminescent immunoassay analyzer, model HEXU, is a product of Luoyang Modern Biotechnology Co., Ltd.

[0066] The African horse sickness virus serum complement fixation test (MCF) method in the following examples mainly refers to the OIE standard entry: CHAPTER 3.6.1 AFRICAN HORSE SICKNESS (INFECTION WITH AFRICAN HORSE SICKNESS VIRUS). In addition, there is GB / T 21675-2008 African horse sickness diagnostic technical standard process (now replaced by GB / T21675-2022). The details are as follows:

[0067] 1 Materials

[0068] 1.1 Barbiturate buffered saline (VBSG) containing 1% gelatin.

[0069] 1.2 Serum samples must be free of red blood cells and must be heat-inactivated: horse serum should be inactivated at 56°C for 30 minutes.

[0070] 1.3 The antigen is a sucrose acetone extract of AHSV infected mouse brain, and the control antigen is an uninfected mouse brain extracted in the same way. In the absence of international standard serum, the antigen should be titrated with locally prepared positive control serum, and 4 to 8 units are used in the test.

[0071] 1.4 Complement (C') is normal guinea pig serum.

[0072] 1.5 Hemolysin is made from rabbit serum highly immune to sheep red blood cells (SRBCs). SRBCs are collected from sterile venous blood and stored in Alfred's solution.

[0073] The 1.6 hemolysin system (HS) was prepared by diluting the hemolysin to contain 2 hemolytic doses and using it to a standardized concentration of 3% on washed SRBCs.

[0074] 1.7 Control serum: The positive control serum is derived from a horse that is confirmed positive by ELISA or other methods, and the negative control serum is derived from a healthy horse that is negative for antibodies. The stored standard serum is used in this test.

[0075] 2. Operation method

[0076] Reactions were performed in 96-well round-bottom microtiter plates with a final total volume of 100 μL / well, or in test tubes if using a macrosystem, at 4°C for 18 hours.

[0077] 2.1 All sera, samples and controls were diluted 1 / 5 with VBSG, and 25 μL was added to each serum repeatedly. Each serum was diluted twice, from 1 / 5 to 1 / 180.

[0078] 2.2 Add 25 μL of antigen diluted according to the above steps, add 25 μL of diluted complement, and incubate at 4°C for 18 hours.

[0079] 2.3 Add 25 μL of HS to all wells of the microtiter plate and react at 37°C for 30 minutes.

[0080] 2.4 Centrifuge the microplate at 1000 rpm for 5 min.

[0081] 2.5 Record the hemolysis of all wells and read the results with 50% hemolysis as the endpoint.

[0082] 3. Conditions for the test to be established:

[0083] When the blank control wells are completely non-hemolyzed and the complement control wells are completely hemolyzed, it indicates that the test results are valid.

[0084] 4. Result determination

[0085] The reciprocal of the highest dilution of serum that specifically binds complement to the CF antigen is its titer. A titer of 1 / 10 or higher is positive, and a titer of less than 1 / 10 is negative.

[0086] Example 1: Detection of serum antibodies against African horse sickness virus infection by chemiluminescent immunoassay (CLIA)

[0087] The inventors used pET32a (+) to solublely express the full-length VP2 recombinant protein of African horse sickness virus, the full-length VP5 recombinant protein of African horse sickness virus and the full-length VP7 recombinant protein of African horse sickness virus in Escherichia coli BL21 (DE3) during the research and development process. The experimental results show that the chemiluminescent immunoassay method established by using the full-length VP2 recombinant protein of African horse sickness virus, the full-length VP5 recombinant protein of African horse sickness virus and the full-length VP7 recombinant protein of African horse sickness virus as coating antigens respectively cannot effectively distinguish equine infectious anemia virus antibodies, equine arteritis virus antibodies, equine herpes virus antibodies, equine influenza virus antibodies, and equine Salmonella antibodies, and has poor specificity. The inventor selected 3 dominant antigenic epitope polypeptides (respectively recorded as eVP2-1, eVP2-2, and eVP2-3) from the full-length VP2 protein of African horse sickness virus, selected 3 dominant antigenic epitope polypeptides (respectively recorded as eVP5-1, eVP5-2, and eVP5-3) from the full-length VP5 protein of African horse sickness virus, and selected 3 dominant antigenic epitope polypeptides (respectively recorded as eVP7-1, eVP7-2, and eVP7-3) from the full-length VP7 protein of African horse sickness virus, and respectively coupled with BSA as a chemiluminescent immunoassay method established as a coating antigen, and the specificity was significantly improved, and African horse sickness virus antibodies and foot-and-mouth disease virus antibodies can be effectively distinguished, but the sensitivity difference is large. The conjugate of eVP2-1 and BSA is recorded as BSA-eVP2-1, the conjugate of eVP5-1 and BSA is recorded as BSA-eVP5-1, and the conjugate of eVP7-1 and BSA is recorded as BSA-eVP7-1. The chemiluminescent immunoassay method established by mixing BSA-eVP2-1 and BSA-eVP5-1 at a mass ratio of 1:1 to obtain a mixed complete antigen as a coating antigen, and mixing BSA-eVP2-1 and BSA-eVP7-1 at a mass ratio of 1:1 to obtain a mixed complete antigen as a coating antigen, has significantly improved specificity and can effectively distinguish equine infectious anemia virus antibodies, equine arteritis virus antibodies, equine herpes virus antibodies, equine influenza virus antibodies, and equine Salmonella antibodies, and the sensitivity is also significantly improved. The specific experimental method is as follows:

[0088] 1. Preparation of coating antigen

[0089] The following 16 antigens were prepared in this example:

[0090] 1)BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1;

[0091] 2) BSA-eVP2-1+BSA-eVP5-1;

[0092] 3) BSA-eVP2-1+BSA-eVP7-1;

[0093] 4) BSA-eVP5-1+BSA-eVP7-1;

[0094] 5) BSA-eVP2-1 (eVP2-1 conjugate);

[0095] 6) BSA-eVP2-2 (eVP2-2 conjugate);

[0096] 7) BSA-eVP5-3 (eVP2-3 conjugate);

[0097] 8) BSA-eVP5-1 (eVP5-1 conjugate);

[0098] 9) BSA-eVP5-2 (eVP5-2 conjugate);

[0099] 10) BSA-eVP5-3 (eVP5-3 conjugate);

[0100] 11) BSA-eVP7-1 (eVP7-1 conjugate);

[0101] 12) BSA-eVP7-2 (eVP7-2 conjugate);

[0102] 13) BSA-eVP7-3 (eVP7-3 conjugate);

[0103] 14) Full-length VP2 recombinant protein of African horse sickness virus;

[0104] 15) Full-length VP5 recombinant protein of African horse sickness virus;

[0105] 16) Full-length VP7 recombinant protein of African horse sickness virus.

[0106] 1. Dominant antigen epitope peptide

[0107] Dominant antigenic epitope peptides were selected from the VP2 protein, VP5 protein and VP7 protein of African horse sickness virus, and the peptides eVP2-1, eVP2-2, eVP2-3, eVP5-1, eVP5-2, eVP5-3, eVP7-1, eVP7-2 and eVP7-3 (Table 1) with cysteine ​​linked to the N-terminus were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. The purity of all of them was greater than 95% and they were freeze-dried for storage.

[0108] Table 1. Peptides

[0109]

[0110]

[0111] Note: C* in the sequence is a cysteine ​​residue, which is used to connect to the carrier protein. It is a linker added to the amino terminus of the antigenic epitope polypeptide of the VP2 protein of the African horse sickness virus, the antigenic epitope polypeptide of the VP5 protein, or the antigenic epitope polypeptide of the VP7 protein; other amino acid residues are derived from the African horse sickness virus.

[0112] 2. Preparation of 9 dominant antigens and their combinations

[0113] The nine polypeptides eVP2-1, eVP2-2, eVP2-3, eVP5-1, eVP5-2, eVP5-3, eVP7-1, eVP7-2 and eVP7-3 in step 1 were conjugated with BSA to obtain nine antigens: 1) BSA-eVP2-1 (a conjugate of eVP2-1 and BSA); 2) BSA-eVP2-2 (a conjugate of eVP2-2 and BSA); 3) BSA-eVP2-3 (a conjugate of eVP2-3 and BSA) ; 4) BSA-eVP5-1 (conjugate of eVP5-1 and BSA); 5) BSA-eVP5-2 (conjugate of eVP5-2 and BSA); 6) BSA-eVP5-3 (conjugate of eVP5-3 and BSA); 7) BSA-eVP7-1 (conjugate of eVP7-1 and BSA); 8) BSA-eVP7-2 (conjugate of eVP7-2 and BSA); 9) BSA-eVP7-3 (conjugate of eVP7-3 and BSA). The specific preparation method is as follows: Use the BSA label coupling kit (Readilink) produced by KPL, USA TM BSA Conjugation Kit) catalog number: 5501, batch number: 148045, the synthesized polypeptide fragments were coupled according to the instructions to prepare the above 9 antigens.

[0114] BSA-eVP2-1, BSA-eVP5-1 and BSA-eVP7-1 were mixed in a mass ratio of 3:2:5 to obtain the coating antigen BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1.

[0115] BSA-eVP2-1 and BSA-eVP5-1 were mixed in a mass ratio of 1:1 to obtain the coating antigen BSA-eVP2-1+BSA-eVP5-1.

[0116] BSA-eVP2-1 and BSA-eVP7-1 were mixed in a mass ratio of 1:1 to obtain the coating antigen BSA-eVP2-1+BSA-eVP7-1.

[0117] BSA-eVP5-1 and BSA-eVP7-1 were mixed in a mass ratio of 1:1 to obtain the coating antigen BSA-eVP5-1+BSA-eVP7-1.

[0118] 3. Expression of full-length VP2 recombinant protein of African horse sickness virus, full-length VP5 recombinant protein of African horse sickness virus, and full-length VP7 recombinant protein of African horse sickness virus

[0119] (1) Construction of full-length VP2 recombinant protein gene expression vector

[0120] The fragment between the BamH I and XhoI recognition sites of pET32a(+) (the small fragment between the BamH I recognition site and the XhoI recognition site) was replaced with the nucleotide sequence of GenBank Accession No.KT715602.1 (Update Date is 09-DEC-2015) at position 13-3218, and the other sequences of pET32a(+) were kept unchanged to obtain a recombinant expression vector of the full-length VP5 protein gene of African horse sickness virus, named pET32a-VP2. pET32a-VP2 can express a fusion protein containing the full-length VP2 protein of African horse sickness virus (amino acid sequence as shown in SEQ ID No.10).

[0121] (2) Construction of full-length VP5 recombinant protein gene expression vector

[0122] The fragment between the BamH I and XhoI recognition sites of pET32a(+) (the small fragment between the BamH I recognition site and the XhoI recognition site) was replaced with the nucleotide sequence of GenBank Accession No.KM886359.1 (Update Date is 23-SEP-2015) at position 20-1537, and the other sequences of pET32a(+) were kept unchanged to obtain a recombinant expression vector of the full-length VP5 protein gene of African horse sickness virus, named pET32a-VP5. pET32a-VP5 can express a fusion protein containing the full-length VP5 protein of African horse sickness virus (amino acid sequence as shown in SEQ ID No.11).

[0123] (3) Construction of full-length VP7 recombinant protein gene expression vector

[0124] The fragment between the BamH I and XhoI recognition sites of pET32a(+) (the small fragment between the BamH I recognition site and the XhoI recognition site) was replaced with the nucleotide sequence of GenBank Accession No. FJ011114.1 (Update Date is 21-MAY-2009) at position 18-1067, and the other sequences of pET32a(+) were kept unchanged to obtain a recombinant expression vector of the full-length VP1 recombinant protein gene of African horse sickness virus, which was named pET32a-VP7. pET32a-VP7 can express a fusion protein containing the full-length VP7 protein of African horse sickness virus (the amino acid sequence is shown in SEQ ID No. 12).

[0125] (4) Construction of recombinant bacteria

[0126] The three expression vectors pET32a-VP2, pET32a-VP5 and pET32a-VP7 constructed in steps (1) to (3) were transformed into Escherichia coli BL21 (DE3) competent cells separately. They were evenly spread on LB plates containing ampicillin (50 μg / mL) and cultured at 37°C for 16 hours. Single colonies were shaken and cultured overnight, and plasmids were extracted for sequencing. The recombinant Escherichia coli containing pET32a-VP2 according to the sequencing results was named BL21 (DE3) -pET32a-VP2, the recombinant Escherichia coli containing pET32a-VP5 according to the sequencing results was named BL21 (DE3) -pET32a-VP5, and the recombinant Escherichia coli containing pET32a-VP7 according to the sequencing results was named BL21 (DE3) -pET32a-VP7.

[0127] (5) Soluble expression of full-length VP2 recombinant protein of African horse sickness virus, full-length VP5 recombinant protein of African horse sickness virus, or full-length VP7 recombinant protein of African horse sickness virus

[0128] The three strains BL21(DE3)-pET32a-VP2, BL21(DE3)-pET32a-VP5, and BL21(DE3)-pET32a-VP7 were inoculated into LB liquid medium containing 50 μg / ml ampicillin (a medium obtained by adding ampicillin to the LB liquid medium until the concentration of ampicillin is 50 μg / ml) and cultured at 37°C with a Thermo MaxQ6000 full-temperature oscillator at 200 rpm until 0 D 600When the value (LB liquid medium containing 50μg / ml ampicillin as blank control) reaches 0.6, add IPTG to induce expression. The induced expression is induced at 16℃ for 13h with 0.75mM IPTG. Take the fermentation liquid after 13h of IPTG induction expression to collect the bacterial precipitate. Add PBS to resuspend the precipitate, centrifuge at 8000rpm / min for 5min, and discard the supernatant. Add PBS to the washed bacterial precipitate, crush the bacteria under high pressure, lyse until the bacterial solution is no longer viscous, centrifuge at 16000rpm / min in a 4℃ centrifuge for 30min, collect the supernatant, and discard the precipitate. Filter the supernatant with a 0.22μm filter membrane and load it onto a nickel column that has been equilibrated with solution 1 (solutes and their concentrations are as follows: 20mM Tris, 150mM NaCl, solvent is water, pH8.0 solution). The nickel column was connected to an AKTA machine, and the impurity protein in the nickel column was washed with 10 column volumes of solution 1 and 10 column volumes of solution 2 (solutes and their concentrations were as follows: 20mM Tris, 150mM NaCl, 50mM imidazole, the solvent was water, and the pH was 8.0 solution), 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 were as follows: 20mM Tris, 150mM NaCl, 300mM imidazole, the solvent was water, and the pH was 8.0 solution), and the eluted sample with the target protein peak was collected using AKTA, and further purified by molecular sieve using a Superdex200 gel column produced by GE, to obtain molecular sieve-purified full-length VP2 recombinant protein of African horse sickness virus, molecular sieve-purified full-length VP5 recombinant protein of African horse sickness virus, and molecular sieve-purified full-length VP7 recombinant protein of African horse sickness virus.

[0129] 2. Perform chemiluminescent immunoassay using the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies

[0130] This embodiment provides 16 chemiluminescent immunoassay kits for diagnosing African horse sickness virus disease or chemiluminescent immunoassay kits for detecting African horse sickness virus antibodies. These 16 kits all include coated antigens, horseradish peroxidase (HRP) labeled horse secondary antibodies (article number 5220-0370, Zhonglianrui (Beijing) Biotechnology Co., Ltd.), coating buffer (article number HC210, Hexu (Zhengzhou) Biotechnology Co., Ltd.), washing solution (article number HX221, Hexu (Zhengzhou) Biotechnology Co., Ltd.), and secondary antibody diluent (article number HM391, Hexu (Zhengzhou) Biotechnology Co., Ltd.). The difference between these 16 kits is only that the coated antigens are different, and the other components are exactly the same.

[0131] The 16 kits are:

[0132] A chemiluminescent immunoassay kit for diagnosing African horse sickness virus or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies, wherein the coating antigen is BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1 in step 1, hereinafter referred to as Kit 1 of the present invention; a chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies, wherein the coating antigen is BSA-eVP2-1+BSA-eVP5-1 in step 1, hereinafter referred to as Kit 2 of the present invention; a chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies, wherein the coating antigen is BSA-eVP2-1+BSA-eVP7-1 in step 1, hereinafter referred to as Kit 3 of the present invention; a chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies, wherein the coating antigen is BSA-eVP5-1+BSA-eVP7-1 in step 1, hereinafter referred to as Kit 4 of the present invention.

[0133] The coating antigen is the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP2-1 in step 1, hereinafter referred to as control kit 1; the coating antigen is the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP2-2 in step 1, hereinafter referred to as control kit 2; the coating antigen is the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP2-3 in step 1, hereinafter referred to as control kit 3; the coating antigen is The chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP5-1 in step 1 is hereinafter referred to as control kit 4; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP5-2 in step 1 is hereinafter referred to as control kit 5; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP5-3 in step 1 is hereinafter referred to as control kit 6; the coating antigen is B in step 1 The chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of SA-eVP7-1 is hereinafter referred to as control kit 7; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP7-2 in step 1 is hereinafter referred to as control kit 8; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies of BSA-eVP7-3 in step 1 is hereinafter referred to as control kit 9; the coating antigen is African horse sickness virus in step 1 The chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies is the full-length VP2 recombinant protein of the African horse sickness virus in step 1, hereinafter referred to as the control kit 10; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies is the coating antigen of the full-length VP5 recombinant protein of the African horse sickness virus in step 1, hereinafter referred to as the control kit 11; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies is the coating antigen of the full-length VP7 recombinant protein of the African horse sickness virus in step 1, hereinafter referred to as the control kit 12.

[0134] (i) Using the kit 1 of the present invention, a chemiluminescent immunoassay method using BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1 as coating antigens (hereinafter referred to as CLIA method 1 of the present invention) was established through optimization experiments.

[0135] 1. Operation steps

[0136] (1) Coating: Dilute the concentration of BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1 in step 1 (the total mass concentration of BSA-eVP2-1, BSA-eVP5-1, and BSA-eVP7-1) with coating buffer to 1.0 μg / ml to obtain a coating antigen solution. Coat the experimental wells with the coating buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C for 16 h.

[0137] (2) Washing: Pour off the original coating solution in the wells, wash five times with washing solution, 3 minutes each time; pat dry.

[0138] (3) Blocking: Add 1% BSA blocking solution, 350 μL / well, and incubate at 37°C for 2 h.

[0139] (4) Washing: Same as step (2).

[0140] (5) Sample addition: Dilute the sample to be tested in a serum dilution plate with sample diluent (1×PBST) at a ratio of 1:20; remove the coated plate, and set up 6 wells of a series of calibrators for each experiment. First, add 100 μL of calibrators 1 to 6 (see below for details) to the serum dilution plate in sequence, and then add 100 μL of the diluted sample to be tested to each well in sequence, and incubate in a 37°C constant temperature incubator for 30 min (±1 min).

[0141] (6) Washing: Same as step (2).

[0142] (7) Add 100 μL of horseradish peroxidase (HRP)-labeled secondary antibody diluted with secondary antibody diluent to each well and vortex to mix. Transfer 100 μL of each well to the corresponding well of the coated plate and incubate in a 37°C constant temperature incubator for 30 min (± 1 min).

[0143] (8) Washing: Same as step (2).

[0144] (9) Add luminol chemiluminescent substrate: Add 100 μL of luminol chemiluminescent substrate to each well and vortex to mix; incubate at 15°C to 25°C in the dark for 5 min and read the luminescence value using a chemiluminescence meter within 15 min after incubation.

[0145] 2. Result calculation method

[0146] NCU (National clinical unit) is a unit established by the National Clinical Inspection Center. In this method, the unit is used to assign a value of 20,000 NCU / ml to the standard positive serum (neutralizing antibody titer is 1:512). The four-parameter calibration curve is established with standard positive serum of different dilution multiples. The antibody dose value (NCU / ml) of the sample is converted from the luminescence value of the test sample and the calibration curve, thereby achieving semi-quantitative detection. The details are as follows:

[0147] The positive serum of African horse sickness virus antibody with serum neutralization titer background was set as calibrators 1 to 6 according to the complement fixation test results, and the corresponding antibody dose values ​​were (0NCU / mL, 10NCU / mL, 20NCU / mL, 40NCU / mL, 100NCU / mL, 200NCU / mL). The luminescence values ​​of calibrators 1 to 6 were used as the ordinate, and the corresponding antibody dose values ​​were used as the abscissa. The four-parameter fitting curve of the calibrator was drawn by ELISACalc software, and the four-parameter model was Y=(ab) / [1+(x / c)*b]+d. Among them, a is the asymptote estimate on the curve; b is the slope of the curve; c is the dose corresponding to half of the maximum binding; d is the asymptote estimate under the curve.

[0148] Substituting the luminescence value of the sample into the calibration curve for calculation, the content of African horse sickness virus antibodies in the sample can be obtained.

[0149] 3. Test establishment conditions and result judgment

[0150] The luminescence values ​​of the six calibrators were fitted with the corresponding antibody dose values ​​using four parameters, R 2 If ≥0.96, the test is established. Otherwise, the test is not established.

[0151] 4. Determination of positive and negative critical values

[0152] 200 samples of African horse sickness virus antibody negative serum were subjected to CLIA test using the above method, and the mean (X) and standard deviation (SD) of the dose values ​​of the 200 samples of African horse sickness virus antibody negative serum were calculated. The mean dose value X of the 200 samples of African horse sickness virus antibody negative serum was 30.25, and the SD was 3.25, so the positive and negative critical dose value was The dose value is 40 NCU / mL. That is, a dose value ≥ 40 NCU / mL is considered positive; a dose value < 40 NCU / mL is considered negative.

[0153] (II) Using the kit 2 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as CLIA method 2 of the present invention) was established through optimization experiments with BSA-eVP2-1+BSA-eVP5-1 as the coating antigen. The steps were the same as those of (I). The total mass concentration of the coated BSA-eVP2-1 and BSA-eVP5-1 was 1.0 μg / ml.

[0154] (III) Using the kit 3 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as CLIA method 3 of the present invention) was established through optimization experiments using BSA-eVP2-1+BSA-eVP7-1 as the coating antigen. The steps were the same as those of (I). The total mass concentration of the coated BSA-eVP2-1 and BSA-eVP7-1 was 1.0 μg / ml.

[0155] (IV) Using the kit 4 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as CLIA method 4 of the present invention) was established through optimization experiments with BSA-eVP5-1+BSA-eVP7-1 as the coating antigen. The steps were the same as those of (I). The total mass concentration of the coated BSA-eVP5-1 and BSA-eVP7-1 was 1.0 μg / ml.

[0156] (V) Using the control kit 1 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-1 as the coating antigen (hereinafter referred to as control CLIA method 1) was established through optimization experiments. The steps were the same as those in (I). The mass concentration of the coated BSA-eVP2-1 was 1.0 μg / ml.

[0157] (VI) Using the control kit 2 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-2 as the coating antigen (hereinafter referred to as control CLIA method 2) was established through optimization experiments. The steps were the same as those in (I). The mass concentration of the coated BSA-eVP2-2 was 1.0 μg / ml.

[0158] (VII) Using the control kit 3 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-3 as the coating antigen (hereinafter referred to as control CLIA method 3) was established through optimization experiments. The steps were the same as (I). The mass concentration of the coated BSA-eVP2-3 was 1.0 μg / ml.

[0159] (VIII) Using the control kit 4 of the present invention, a chemiluminescent immunoassay method with BSA-eVP5-1 as the coating antigen (hereinafter referred to as control CLIA method 4) was established through optimization experiments. The steps were the same as those in (I). The mass concentration of the coated BSA-eVP5-1 was 1.0 μg / ml.

[0160] (IX) Using the control kit 5 of the present invention, a chemiluminescent immunoassay method with BSA-eVP5-2 as the coating antigen (hereinafter referred to as control CLIA method 5) was established through optimization experiments. The steps are the same as (I). The mass concentration of the coated BSA-eVP5-2 is 1.0 μg / ml.

[0161] (J) Using the control kit 6 of the present invention, a chemiluminescent immunoassay method with BSA-eVP5-3 as the coating antigen (hereinafter referred to as control CLIA method 6) was established through optimization experiments. The steps are the same as (I). The mass concentration of the coated BSA-eVP5-3 is 1.0 μg / ml.

[0162] (XI) Using the control kit 7 of the present invention, a chemiluminescent immunoassay method with BSA-eVP7-1 as the coating antigen (hereinafter referred to as control CLIA method 7) was established through optimization experiments. The steps were the same as (I). The mass concentration of the coated BSA-eVP7-1 was 1.0 μg / ml.

[0163] (XII) Using the control kit 8 of the present invention, a chemiluminescent immunoassay method with BSA-eVP7-2 as the coating antigen (hereinafter referred to as control CLIA method 8) was established through optimization experiments. The steps are the same as (I). The mass concentration of the coated BSA-eVP7-2 is 1.0 μg / ml.

[0164] (XIII) Using the control kit 9 ​​of the present invention, a chemiluminescent immunoassay method with BSA-eVP7-3 as the coating antigen (hereinafter referred to as control CLIA method 9) was established through optimization experiments. The steps are the same as (I). The mass concentration of the coated BSA-eVP7-3 is 1.0 μg / ml.

[0165] (XIV) Using the control kit 10 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as control CLIA method 10) was established through optimization experiments using the full-length VP2 protein as the coating antigen. The steps are the same as (I). The mass concentration of the coated full-length VP2 protein is 1.0 μg / ml.

[0166] (XV) Using the control kit 11 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as control CLIA method 11) was established after optimization experiments using the full-length VP5 protein as the coating antigen. The steps were the same as (I). The mass concentration of the coated full-length VP5 protein was 1.0 μg / ml.

[0167] (XVI) Using the control kit 12 of the present invention, a chemiluminescent immunoassay method (hereinafter referred to as control CLIA method 12) was established after optimization experiments using the full-length VP7 protein as the coating antigen. The steps were the same as (I). The mass concentration of the coated full-length VP7 protein was 1.0 μg / ml.

[0168] Specificity Test

[0169] The CLIA method 1-4 of the present invention and the control CLIA method 1-12 of step 2 are compared with the positive serum of equine infectious anemia virus antibody, equine arteritis virus antibody, equine herpes virus antibody, equine influenza virus antibody, and equine Salmonella antibody (all positive sera are collected from the sera of clinically ill animals) to observe whether there is a cross reaction with other diseases of the same species. In addition, the complement fixation test is also used for detection (see the above for the specific method).

[0170] The results show:

[0171] The test results of CLIA methods 1-4 of the present invention show that the antibody dosage values ​​are all less than 40 NCU / ml, which are judged as negative. The specificity is 100%. The neutralization index of the complement fixation test is less than the national standard of 1:10, and the results are all negative. See Table 2 for specific results.

[0172] The test results of CLIA methods 1-12 showed that the antibody dosage values ​​of CLIA method 1, CLIA method 4, and CLIA method 7 were all less than 40 NCU / ml, and were judged as negative. The specificity was 100%. The other specificities were all less than 100%. See Table 3 for specific results.

[0173] Table 2. CLIA methods 1-4 and complement fixation test specificity test results of the present invention

[0174]

[0175]

[0176] Table 3. Results of specificity tests of CLIA methods 1-12

[0177]

[0178]

[0179] 4. Sensitivity test

[0180] The serum positive for African horse sickness virus antibody was diluted in multiples, and compared and tested using CLIA methods 1-4 of the present invention and serum samples with known serum neutralization titer background, respectively, to obtain the maximum dilution at the positive critical value. The results showed that the highest dilution multiple of positive serum detected by CLIA methods 1-4 of the present invention was 1:1024. The highest dilution multiple of positive serum detected by complement fixation test results was 1:512. See Table 4.

[0181] Table 4. CLIA method 1-4 and complement fixation test sensitivity results of the present invention

[0182]

[0183] 5. Repeatability test

[0184] The CLIA method 1-4 of the present invention was used to detect 10 African horse sickness virus antibody positive sera on the same batch plate and different batch plates, respectively, and the parallel determination was performed 5 times, and the intra-batch and inter-batch coefficients of variation (CV) were calculated. The results showed that the intra-batch repeat coefficients of variation of the CLIA method 1-4 of the present invention were within 8%, and the inter-batch repeat coefficients of variation were less than 10% (Tables 5 to 8). The results showed that the African horse sickness virus antibody positive sera had good repeatability.

[0185] Table 5. Repeatability test of CLIA method 1 of the present invention

[0186]

[0187] Table 6. Repeatability test of CLIA method 2 of the present invention

[0188]

[0189] Table 7. Repeatability test of CLIA method 3 of the present invention

[0190]

[0191] Table 8. Repeatability test of CLIA method 4 of the present invention

[0192]

[0193] VI. Compliance test

[0194] Using bovine serum stored in the China Animal Disease Prevention and Control Center (Veterinary Diagnosis Center of the Ministry of Agriculture and Rural Affairs), 60 horse sera with a serum neutralization titer background of positive African horse sickness virus antibody and 60 horse sera with a serum neutralization titer background of negative African horse sickness virus antibody were selected. These 120 horse sera were tested using CLIA methods 1-4 of the present invention and control CLIA methods 1-12, and their compliance rates with the complement fixation test method were calculated.

[0195] The results show that for the 160 portions of bovine sera, the total conformity rate between the CLIA method 1 of the present invention and the complement fixation test method is 98.33% (the positive conformity rate is 100%, and the negative conformity rate is 96.67%), the total conformity rate between the CLIA method 2 of the present invention and the African horse sickness complement fixation test method is 95.83% (the positive conformity rate is 96.67%, and the negative conformity rate is 95%), the total conformity rate between the CLIA method 3 of the present invention and the African horse sickness complement fixation test method is 95.83% (the positive conformity rate is 93.33%, and the negative conformity rate is 98.33%), and the total conformity rate between the CLIA method 4 of the present invention and the African horse sickness complement fixation test method is 95.83% (the positive conformity rate is 95%, and the negative conformity rate is 96.67%).

[0196] The total conformity rate of the CLIA method 1 test results of the present invention with the African horse sickness complement fixation test method is 85.83% (positive conformity rate is 83.33%, negative conformity rate is 88.33%), the total conformity rate of the CLIA method 2 test results of the present invention with the African horse sickness complement fixation test method is 85% (positive conformity rate is 83.33%, negative conformity rate is 86.67%), the total conformity rate of the CLIA method 3 test results of the present invention with the African horse sickness complement fixation test method is 80% (positive conformity rate is 81.67%, negative conformity rate is 78.33%), The total conformity rate of the CLIA method 4 test results of the present invention with the African horse sickness complement fixation test method is 76.67% (the positive conformity rate is 78.33%, and the negative conformity rate is 75%), the total conformity rate of the CLIA method 5 test results of the present invention with the African horse sickness complement fixation test method is 76.67% (the positive conformity rate is 73.33%, and the negative conformity rate is 80%), and the total conformity rate of the CLIA method 6 test results of the present invention with the African horse sickness complement fixation test method is 68.33% (the positive conformity rate is 68.33%, and the negative conformity rate is 68.33%). The total conformity rate of the CLIA method 7 test results of the present invention with the African horse sickness complement fixation test method is 80.83% (positive conformity rate is 80%, negative conformity rate is 81.67%), the total conformity rate of the CLIA method 8 test results of the present invention with the African horse sickness complement fixation test method is 73.33% (positive conformity rate is 75%, negative conformity rate is 71.67%), the total conformity rate of the CLIA method 9 test results of the present invention with the African horse sickness complement fixation test method is 73.33% (positive conformity rate is 71.67%, negative conformity rate is 75%), the present invention The total conformity rate of the CLIA method 10 test results and the African horse sickness complement fixation test method was 76.67% (the positive conformity rate was 73.33%, and the negative conformity rate was 80%), the total conformity rate of the CLIA method 11 test results of the present invention and the African horse sickness complement fixation test method was 80.83% (the positive conformity rate was 75%, and the negative conformity rate was 86.67%), and the total conformity rate of the CLIA method 12 test results of the present invention and the African horse sickness complement fixation test method was 80.83% (the positive conformity rate was 76.67%, and the negative conformity rate was 86.67%).

[0197] Please refer to Table 9 to Table 24 for details.

[0198] The above results indicate that the overall agreement rate of the chemiluminescent immunoassay kit for diagnosing African horse sickness virus antibodies or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies prepared using BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1, BSA-eVP2-1+BSA-eVP5-1, BSA-eVP2-1+BSA-eVP7-1, and BSA-eVP5-1+BSA-eVP7-1 as coating antigens was significantly higher than that of the chemiluminescent immunoassay kit for diagnosing African horse sickness virus antibodies and the complement fixation test method using BSA-eVP2-1+BSA-eVP5-1+BSA-eVP7-1 as coating antigens. -1, BSA-eVP2-2, BSA-eVP2-3, BSA-eVP5-1, BSA-eVP5-2, BSA-eVP5-3, BSA-eVP7-1, BSA-eVP7-2, BSA-eVP7-3, full-length VP2 protein of African horse sickness virus, full-length VP5 protein of African horse sickness virus and full-length VP7 protein of African horse sickness virus are prepared as coating antigens, and a chemiluminescent immunoassay kit for diagnosing African horse sickness virus antibodies or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies.

[0199] Table 9. CLIA method 1 of the present invention for testing bovine serum samples

[0200]

[0201] Table 10. CLIA method 2 of the present invention for testing bovine serum samples

[0202]

[0203] Table 11. CLIA method 3 of the present invention for testing bovine serum samples

[0204]

[0205] Table 12. CLIA method of the present invention for testing bovine serum samples 4 results

[0206]

[0207]

[0208] Table 13. Results of CLIA method 1 on bovine serum samples

[0209]

[0210] Table 14. Results of CLIA method 2 on bovine serum samples

[0211]

[0212] Table 15. Comparison of CLIA method 3 test results for bovine serum samples

[0213]

[0214] Table 16. Results of the CLIA method 4 on bovine serum samples

[0215]

[0216] Table 17. Results of CLIA method 5 on bovine serum samples

[0217]

[0218] Table 18. Results of CLIA method 6 on bovine serum samples

[0219]

[0220] Table 19. Results of CLIA method 7 on bovine serum samples

[0221]

[0222] Table 20. Results of CLIA method 8 on bovine serum samples

[0223]

[0224] Table 21. Results of CLIA method 9 on bovine serum samples

[0225]

[0226]

[0227] Table 22. Results of CLIA method 10 for bovine serum samples

[0228]

[0229] Table 23. Results of CLIA method 11 on bovine serum samples

[0230]

[0231] Table 24. Results of CLIA method 12 on bovine serum samples

[0232]

[0233] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A complete set of conjugates, characterized in that: The set of conjugates is set of conjugates 1 or set of conjugates 2; The set of conjugates 1 consists of eVP2-1 conjugate and eVP5-1 conjugate; The set of conjugates 2 consists of eVP2-1 conjugate and eVP7-1 conjugate; The eVP2-1 conjugate is a complete antigen obtained by coupling the eVP2-1 polypeptide and the carrier protein; The eVP5-1 conjugate is a complete antigen obtained by coupling the eVP5-1 polypeptide and the carrier protein; The eVP7-1 conjugate is a complete antigen obtained by coupling the eVP7-1 polypeptide and the carrier protein; The eVP2-1 polypeptide is shown as P11, P12 or P13 below: P11, a polypeptide having an amino acid sequence of SEQ ID No. 1; P12, a polypeptide having an amino acid sequence of positions 2 to 46 of SEQ ID No. 1; P13, a polypeptide capable of coupling with a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P12; The eVP5-1 polypeptide is shown as P21, P22 or P23 below: P21, a polypeptide having an amino acid sequence of SEQ ID No. 4; P22, a polypeptide having an amino acid sequence of positions 2 to 38 of SEQ ID No. 4; P23, a polypeptide capable of coupling with a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P22; The eVP7-1 polypeptide is shown as P21, P22 or P23 below: P31, a polypeptide having an amino acid sequence of SEQ ID No. 7; P32, a polypeptide having an amino acid sequence of positions 2 to 51 of SEQ ID No. 7; P33. A polypeptide capable of coupling with a carrier protein obtained by connecting amino acid residues to the amino terminus or carboxyl terminus of the polypeptide shown in P32.

2. The set of conjugates according to claim 1, characterized in that: In the set of conjugates 1, the mass ratio of the eVP2-1 conjugate to the eVP5-1 conjugate is 1:

1.

3. The set of conjugates according to claim 1, characterized in that: In the set of conjugates 2, the mass ratio of the eVP2-1 conjugate to the eVP7-1 conjugate is 1:

1.

4. Use of the set of conjugates according to any one of claims 1 to 3 in any of the following: (A1) Preparing a reagent or kit for detecting antibodies against African horse sickness virus; (A2) Preparation of African horse sickness virus diagnostic antigen.

5. A kit comprising a set of conjugates according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that: The kit is used for detecting African horse sickness virus antibodies; in the kit, the set of conjugates described in any one of claims 1 to 3 is used as a coating antigen.

7. The kit according to claim 6, characterized in that: The kit is a chemiluminescent immunoassay kit; the kit also contains horseradish peroxidase-labeled secondary antibody, coating buffer, washing solution, secondary antibody diluent and / or calibration substance; the secondary antibody is an antibody capable of resisting the African horse sickness virus antibody.

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