Fusion tandem polypeptide antigen for detecting antibodies against African horse sickness virus

Abstract

The present invention discloses a fusion tandem polypeptide antigen for detecting antibodies against African horse sickness virus. The present invention provides a polypeptide or a set of polypeptides. The polypeptide is eVP7-1; the set of polypeptides consists of eVP2-1, eVP5-1 and eVP7-1 or consists of eVP5-1 and eVP7-1; the eVP2-1 is the polypeptide of SEQ ID No.1; the eVP5-1 is the polypeptide of SEQ ID No.4; the eVP7-1 is the polypeptide of SEQ ID No.7. The antibody detection kit prepared with BSA-eVP2-1 + BSA-eVP5-1 + BSA-eVP7-1 or BSA-eVP5-1 + BSA-eVP7-1 as the coating antigen 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 against African horse sickness virus by grass-roots veterinary departments at all levels and entry-exit inspection and quarantine bureaus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a fusion tandem polypeptide antigen for detecting African horse sickness virus antibodies. Background Art

[0002] African horse sickness (AHS) is an acute or subacute infectious disease mainly caused by Culicoides biting midges, which can cause fever, subcutaneous edema and viremia in equids, and dogs are accidental hosts. African horse sickness virus (AHSV) is a member of the genus Orbivirus in the family Reoviridae. In addition to AHSV, this genus also includes Bluetongue virus (BTV), Epizootic hemorrhagic disease virus (EHDV), Equine encephalosis orbivirus (EEV), etc. African horse sickness is transmitted through insect vectors such as Culicoides, Aedes mosquitoes, and stable flies. Among them, horses are most susceptible, affecting the respiratory and circulatory functions of susceptible horses. The main symptoms are fever, subcutaneous edema and viremia, which can lead to a fatality rate of up to 95%. AHS is a Class I animal disease in China, and the World Organization for Animal Health (WOAH) also lists it as a notifiable animal disease. It is mainly prevalent in sub-Saharan Africa. Currently, 41 countries around the world have reported the epidemic. In recent years, many countries such as Nigeria, Eritrea, Ethiopia, Cameroon, and even Thailand have reported African horse sickness epidemics. Although African horse sickness has not been found in China, its harm is huge and should be highly regarded. The trace of Bluetongue virus was first discovered in the Yunnan border area of China in 1979, and Bluetongue virus was isolated. Subsequently, Bluetongue disease has been reported in some provinces, indicating that China has the ecological and climatic conditions for the transmission and spread of viruses belonging to the same genus as African horse sickness. Therefore, border quarantine should be emphasized and diagnostic technology reserves should be made.

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

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

[0005] To solve the above technical problem, the present invention provides a set of polypeptides for preparing an African horse sickness virus antibody detection reagent or a set of polypeptides for preparing an African horse sickness virus disease diagnosis reagent.

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

[0007] The polypeptide claimed to be protected by the present invention is the eVP7-1 polypeptide.

[0008] The set of polypeptides claimed to be protected by the present invention is set of polypeptides 1 or set of polypeptides 2.

[0009] The set of polypeptides 1 is composed of the eVP2-1 polypeptide, the eVP5-1 polypeptide and the eVP7-1 polypeptide.

[0010] The set of polypeptides 2 is composed of the eVP5-1 polypeptide and the eVP7-1 polypeptide.

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

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

[0013] P12, a polypeptide with the amino acid sequence of positions 2-46 of SEQ ID No.1;

[0014] P13, a polypeptide capable of being coupled with 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 as shown in P21, P22 or P23 below:

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

[0017] P22, a polypeptide with the amino acid sequence of positions 2-38 of SEQ ID No.4;

[0018] P23, a polypeptide capable of being coupled with 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 as shown in P21, P22 or P23 below:

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

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

[0022] P33, a polypeptide capable of being conjugated with a carrier protein obtained by linking 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 position 1 is a linker added for linking with the carrier protein, and the other amino acid residues are derived from the VP2 protein of African horse sickness virus; SEQ ID No.4 consists of 38 amino acid residues. The cysteine residue at position 1 is a linker added for linking with the carrier protein, and the other amino acid residues are derived from the VP5 protein of African horse sickness virus. SEQ ID No.7 consists of 51 amino acid residues. The cysteine residue at position 1 is a linker added for linking with the carrier protein, and the other amino acid residues are derived from the VP7 protein of African horse sickness virus.

[0024] In the set of polypeptides 1, the mass ratio of the eVP2 - 1 polypeptide, the eVP5 - 1 polypeptide, and the eVP7 - 1 polypeptide can be determined by those skilled in the art according to the detection effect of African horse sickness virus antibodies, for example, it can be 3:2:5.

[0025] In the set of polypeptides 2, the mass ratio of the eVP5 - 1 polypeptide and the eVP7 - 1 polypeptide can be determined by those skilled in the art according to the detection effect of African horse sickness virus antibodies, for example, it can be 1:1.

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

[0027] The conjugate claimed by the present invention is an eVP7 - 1 conjugate.

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

[0029] The set of conjugates 1 is composed of an eVP2 - 1 conjugate, an eVP5 - 1 conjugate, and the eVP7 - 1 conjugate.

[0030] The set of conjugates 2 is composed of the eVP5 - 1 conjugate and the eVP7 - 1 conjugate.

[0031] The eVP2 - 1 conjugate is a complete antigen obtained by conjugating 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 conjugating 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 conjugating 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, the eVP5-1 conjugate, and the eVP7-1 conjugate is 3:2:5.

[0035] In a specific embodiment of the present invention, in the set of conjugates 2, the mass ratio of the eVP5-1 conjugate and the eVP7-1 conjugate is 1:1.

[0036] Among them, the carrier protein can be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin, rabbit serum albumin, etc.

[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 kit for detecting antibodies against African horse sickness virus;

[0040] (A2) Preparing a diagnostic antigen for African horse sickness virus.

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

[0042] Furthermore, the kit is used for detecting antibodies against African horse sickness virus; 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 is used as a coating antigen.

[0043] Even further, the kit is a chemiluminescent immunoassay kit; the kit further contains a horseradish peroxidase (HRP)-labeled secondary antibody, a coating buffer, a washing solution, a secondary antibody dilution solution, and / or a calibrator; the secondary antibody is an antibody capable of binding to the antibody against African horse sickness virus.

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

[0045] (B1) A nucleic acid molecule, which is nucleic acid molecule 1; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eVP7-1 polypeptide described in the first aspect above.

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

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

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

[0049] (B5) Transgenic cell line, which is 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) Kit of nucleic acid molecules, which is kit of nucleic acid molecules 1 or kit of nucleic acid molecules 2; the kit of nucleic acid molecules 1 is composed of the nucleic acid molecule 1, nucleic acid molecule 2 and nucleic acid molecule 3 described in (B1); the kit of nucleic acid molecules 2 is composed of the nucleic acid molecule 1 and the nucleic acid molecule 2 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 above; the nucleic acid molecule 3 is a nucleic acid molecule capable of encoding the eVP2-1 polypeptide described in the first aspect above.

[0051] (B7) Kit of expression cassettes, which is kit of expression cassettes 1 or kit of expression cassettes 2; the kit of expression cassettes 1 is composed of the expression cassette 1, expression cassette 2 and expression cassette 3 described in (B2); the kit of expression cassettes 2 is composed of the expression cassette 1 and the expression cassette 2 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) Kit of recombinant vectors, which is kit of recombinant vectors 1 or kit of recombinant vectors 2; the kit of recombinant vectors 1 is composed of the recombinant vector 1, recombinant vector 2 and recombinant vector 3 described in (B3); the kit of recombinant vectors 2 is composed of the recombinant vector 1 and the recombinant vector 2 described in (B3); the recombinant vector 2 is a recombinant vector containing the nucleic acid molecule 2 described in (B6); the recombinant vector 3 is a recombinant vector containing the nucleic acid molecule 3 described in (B6).

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

[0054] (B10) A set of transgenic cell lines, which is the set of transgenic cell lines 1 or the set of transgenic cell lines 2; the set of transgenic cell lines 1 is composed of the transgenic cell line 1, the transgenic cell line 2, and the transgenic cell line 3 described in (B5); the set of transgenic cell lines 2 is composed of the transgenic cell line 1 and the transgenic cell line 2 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 the use of the biological material 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, or 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) Preparing an African horse sickness virus diagnostic antigen.

[0059] In a seventh 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, as an immunogen in the preparation of antibodies against African horse sickness virus.

[0060] Experimental verification shows that conjugates obtained by coupling full-length VP2 recombinant protein of African horse sickness virus or full-length VP5 recombinant protein of African horse sickness virus or full-length VP7 recombinant protein of African horse sickness virus with carrier protein as haptens and used as coating antigens cannot effectively distinguish antibodies against African horse sickness virus from antibodies against equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella. 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 conjugates (BSA-eVP2-1 and / or BSA-eVP5-1 and / or BSA-eVP7-1) obtained by coupling them with carrier protein as coating antigens can effectively distinguish antibodies against equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella, thus improving the specificity of African horse sickness virus antibody detection. Kits for detecting African horse sickness virus antibodies in serum prepared with BSA-eVP2-1 and / or BSA-eVP5-1 and / or BSA-eVP7-1 as coating antigens can accurately distinguish positive sera of antibodies against equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella.

[0061] The total coincidence rate between the CLIA method 1 of the present invention with BSA-eVP2-1 + BSA-eVP5-1 + BSA-eVP7-1 as coating antigen and the serum complement fixation test method for African horse sickness virus is 98.33% (positive coincidence rate is 100%, negative coincidence rate is 96.67%); the total coincidence rate between the CLIA method 4 of the present invention with BSA-eVP5-1 + BSA-eVP7-1 as coating antigen and the serum complement fixation test method for African horse sickness virus is 95.83% (positive coincidence rate is 95%, negative coincidence rate is 96.67%). The total coincidence rate between the control CLIA method 7 of the present invention with BSA-eVP7-1 as coating antigen and the serum complement fixation test method for African horse sickness virus is 80.83% (positive coincidence rate is 80%, negative coincidence rate is 81.67%).

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

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

[0064] In the following embodiments, the experimental methods, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0065] pET32a(+) in the following embodiments is a product of BGI; the luminol chemiluminescent substrate is a product of Luoyang Institute of Modern Biotechnology Co., Ltd.; the chemiluminescent enzyme-labeled plate is a product of Thermo Company in the United States; 10% fetal bovine serum, cell culture medium and related cell culture reagents are products of Gibco Company in the United States; African horse sickness virus antibody-negative and positive sample sera with serum neutralization titer background are stored by the China Animal Disease Prevention and Control Center (Veterinary Diagnosis Center of the Ministry of Agriculture and Rural Affairs), from Ingezim Company in Spain, product number: AHSK32. The chemiluminescent immunoassay analyzer, model HEXU, is a product of Luoyang Institute of Modern Biotechnology Co., Ltd.

[0066] The method of African horse sickness virus serum complement fixation test (MCF) in the following embodiments mainly refers to the OIE standard item: CHAPTER 3.6.1 AFRICAN HORSE SICKNESS (INFECTION WITH AFRICAN HORSE SICKNESS VIRUS). In addition, there is also the process of the GB / T 21675-2008 African horse sickness diagnosis technical standard (which has now been replaced by GB / T 21675-2022). Specifically as follows:

[0067] 1 Materials

[0068] 1.1 Barbiturate buffer solution containing 1% gelatin (VBSG).

[0069] 1.2 Serum samples, required to be free of red blood cells and must be heat-inactivated: horse serum is inactivated at 56°C for 30 min.

[0070] 1.3 The antigen is a sucrose-acetone extract of mouse brain infected with AHSV, 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 rabbit serum hyperimmunized with sheep red blood cells (SRBCs), and the SRBCs are obtained by aseptic intravenous blood collection and stored in Alsever's solution.

[0073] 1.6 Hemolysin system (HS) is prepared by diluting hemolysin to contain 2 hemolytic doses, and the standardized concentration of SRBCs after washing with it is 3%.

[0074] 1.7 Control sera: Positive control sera are derived from positive horses confirmed by ELISA or other methods, and negative control sera are derived from healthy antibody-negative horses. Standard sera stored are used in this test.

[0075] 2. Operating method

[0076] The reaction is carried out in a 96-well round-bottom microtiter plate with a final total volume of 100 μL / well. If a macroscopic system is used, it is carried out in a test tube, and carried out at 4 °C for 18 hours.

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

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

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

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

[0081] 2.5 Record the hemolysis of all wells. Read the results with 50% hemolysis as the end point.

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

[0083] When the blank control well shows no hemolysis at all; the complement control well shows complete hemolysis, it indicates that the test results are valid.

[0084] 4. Result determination

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

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

[0087] During the R & D process, the present inventor expressed 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) using pET32a(+). The experimental results showed that chemiluminescent immunoassay methods established 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 could not effectively distinguish antibodies against equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella, with poor specificity. The inventor selected 3 dominant antigenic epitope polypeptides from the full-length VP2 protein of African horse sickness virus (denoted as eVP2-1, eVP2-2, and eVP2-3 respectively), 3 dominant antigenic epitope polypeptides from the full-length VP5 protein of African horse sickness virus (denoted as eVP5-1, eVP5-2, and eVP5-3 respectively), and 3 dominant antigenic epitope polypeptides from the full-length VP7 protein of African horse sickness virus (denoted as eVP7-1, eVP7-2, and eVP7-3 respectively). After coupling with BSA respectively and using them as coating antigens to establish chemiluminescent immunoassay methods, the specificity was significantly improved, and it could effectively distinguish antibodies against African horse sickness virus and foot-and-mouth disease virus, but there were significant differences in sensitivity. The conjugate of eVP2-1 and BSA (denoted as BSA-eVP2-1), the conjugate of eVP5-1 and BSA (denoted as BSA-eVP5-1), and the conjugate of eVP7-1 and BSA (denoted as BSA-eVP7-1) were mixed according to a mass ratio of 3:2:5 to obtain a mixed complete antigen as the coating antigen, and the conjugate of eVP5-1 and BSA (denoted as BSA-eVP5-1) and the conjugate of eVP7-1 and BSA (denoted as BSA-eVP7-1) were mixed according to a mass ratio of 1:1 to obtain a mixed complete antigen as the coating antigen. The chemiluminescent immunoassay methods established had significantly improved specificity and could effectively distinguish antibodies against equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella, and the sensitivity was also significantly improved. The specific experimental methods are as follows:

[0088] I. Preparation of coating antigen

[0089] In this example, the following 16 antigens were prepared:

[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 antigenic epitope polypeptide

[0107] Select dominant antigenic epitope polypeptides from the VP2 protein, VP5 protein and VP7 protein of African horse sickness virus. Beijing Liuhe Huada Gene Technology Co., Ltd. synthesized polypeptides eVP2-1, eVP2-2, eVP2-3, eVP5-1, eVP5-2, eVP5-3, eVP7-1, eVP7-2 and eVP7-3 with cysteine linked to the N-terminus (Table 1), with a purity of greater than 95%, and stored in freeze-dried form.

[0108] Table 1. Polypeptides

[0109]

[0110]

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

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

[0113] Couple the 9 polypeptides of eVP2-1, eVP2-2, eVP2-3, eVP5-1, eVP5-2, eVP5-3, eVP7-1, eVP7-2 and eVP7-3 in step 1 with BSA respectively to obtain 9 antigens: 1) BSA-eVP2-1 (the conjugate of eVP2-1 and BSA); 2) BSA-eVP2-2 (the conjugate of eVP2-2 and BSA); 3) BSA-eVP2-3 (the conjugate of eVP2-3 and BSA); 4) BSA-eVP5-1 (the conjugate of eVP5-1 and BSA); 5) BSA-eVP5-2 (the conjugate of eVP5-2 and BSA); 6) BSA-eVP5-3 (the conjugate of eVP5-3 and BSA); 7) BSA-eVP7-1 (the conjugate of eVP7-1 and BSA); 8) BSA-eVP7-2 (the conjugate of eVP7-2 and BSA); 9) BSA-eVP7-3 (the conjugate of eVP7-3 and BSA). The specific preparation method is as follows: Use the BSA Tag Conjugation Kit (Readilink TM BSA Conjugation Kit) produced by KPL Company in the United States, product number: 5501, batch number: 148045, and couple the synthesized polypeptide fragments according to the requirements of the instruction manual to prepare the above 9 antigens.

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

[0115] Mix BSA-eVP2-1 and BSA-eVP5-1 according to the mass ratio of 1:1 to obtain the coating antigen BSA-eVP2-1 + BSA-eVP5-1.

[0116] Mix BSA-eVP2-1 and BSA-eVP7-1 according to the mass ratio of 1:1 to obtain the coating antigen BSA-eVP2-1 + BSA-eVP7-1.

[0117] Mix BSA-eVP5-1 and BSA-eVP7-1 at a mass ratio of 1:1 to obtain the coated antigen BSA-eVP5-1 + BSA-eVP7-1.

[0118] 3. Expression of 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

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

[0120] Replace the fragment between the BamH I and XhoI recognition sites (the small fragment between the BamH I recognition site and the XhoI recognition site) of pET32a(+) with the nucleotide sequence from positions 13 to 3218 of GenBank Accession No. KT715602.1 (Update Date is 09-DEC-2015), while keeping the other sequences of pET32a(+) unchanged, to obtain the recombinant expression vector for 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 (the amino acid sequence is shown in SEQ ID No. 10).

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

[0122] Replace the fragment between the BamH I and XhoI recognition sites (the small fragment between the BamH I recognition site and the XhoI recognition site) of pET32a(+) with the nucleotide sequence from positions 20 to 1537 of GenBank Accession No. KM886359.1 (Update Date is 23-SEP-2015), while keeping the other sequences of pET32a(+) unchanged, to obtain the recombinant expression vector for 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 (the amino acid sequence is shown in SEQ ID No. 11).

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

[0124] Replace 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) with the nucleotide sequence at positions 18 - 1067 of GenBank Accession No. FJ011114.1 (Update Date: 21 - MAY - 2009), while keeping the other sequences of pET32a(+) unchanged, to obtain a recombinant expression vector for the full - length VP1 recombinant protein gene of African horse sickness virus, 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 as SEQ ID No. 12).

[0125] (4) Construction of recombinant bacteria

[0126] Separate the three expression vectors pET32a - VP2, pET32a - VP5, and pET32a - VP7 constructed in steps (1) to (3) and transform them into Escherichia coli BL21(DE3) competent cells respectively. Spread them evenly on an LB plate containing ampicillin (50 μg / mL) and culture at 37°C for 16 hours. Culture the single colonies overnight with shaking, extract the plasmids and perform sequencing. Name the recombinant Escherichia coli with the sequencing result showing the presence of pET32a - VP2 as BL21(DE3) - pET32a - VP2, name the recombinant Escherichia coli with the sequencing result showing the presence of pET32a - VP5 as BL21(DE3) - pET32a - VP5, and name the recombinant Escherichia coli with the sequencing result showing the presence of pET32a - VP7 as BL21(DE3) - pET32a - VP7.

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

[0128] Separate the three strains BL21(DE3) - pET32a - VP2, BL21(DE3) - pET32a - VP5, and BL21(DE3) - pET32a - VP7 and inoculate them separately into an LB liquid medium containing 50 μg / ml ampicillin (the medium obtained by adding ampicillin to the LB liquid medium to a concentration of 50 μg / ml), culture at 37°C, and use a Thermo MaxQ6000 type all - temperature oscillator to shake - culture at 200 rpm until OD 600When the value (using LB liquid medium containing 50 μg / ml ampicillin as the blank control) reached 0.6, IPTG was added for induction expression. The induction expression was carried out with 0.75 mM IPTG at 16 °C for 13 h. The fermentation broth after 13 h of IPTG induction expression was taken to collect the cell precipitate. PBS was added to resuspend the precipitate, and it was centrifuged at 8000 rpm / min for 5 min, and the supernatant was discarded. PBS was added to the washed cell precipitate, and the cells were disrupted under high pressure until the bacterial solution was no longer viscous. It was centrifuged at 16000 rpm / min in a 4 °C centrifuge for 30 min, the supernatant was collected, and the precipitate was discarded. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a nickel column pre-equilibrated with Solution 1 (the solute and its concentration are as follows: 20 mM Tris, 150 mM NaCl, the solvent is water, and the pH is 8.0 solution). 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 (the solute and its concentration are as follows: 20 mM Tris, 150 mM NaCl, 50 mM imidazole, the solvent is water, and the pH is 8.0 solution) respectively, and the protein peaks were monitored on the AKTA machine. The target protein hanging on the nickel column was washed with Solution 3 (the solute and its concentration are as follows: 20 mM Tris, 150 mM NaCl, 300 mM imidazole, the solvent is water, and the pH is 8.0 solution), and the elution samples showing the target protein peaks were collected using AKTA and further purified by a Superdex 200 gel column produced by GE Company to obtain the full-length VP2 recombinant protein of African horse sickness virus purified by molecular sieve, the full-length VP5 recombinant protein of African horse sickness virus purified by molecular sieve, and the full-length VP7 recombinant protein of African horse sickness virus purified by molecular sieve respectively.

[0129] Second, chemiluminescent immunoassay was performed using a chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or a chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies

[0130] This example 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 antigen, horseradish peroxidase (HRP)-labeled horse secondary antibody (product number 5220 - 0370, Zhonglianrui (Beijing) Biotechnology Co., Ltd.), coating buffer (product number HC210, Hexu (Zhengzhou) Biotechnology Co., Ltd.), washing solution (product number HX221, Hexu (Zhengzhou) Biotechnology Co., Ltd.), and secondary antibody diluent (product number HM391, Hexu (Zhengzhou) Biotechnology Co., Ltd.). The difference among these 16 kits is only in the coated antigen, and other components are exactly the same.

[0131] These 16 kits are respectively:

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

[0133] The chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being BSA-eVP2-1 in Step 1 is hereinafter referred to as Control Kit 1; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being BSA-eVP2-2 in Step 1 is hereinafter referred to as Control Kit 2; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being BSA-eVP2-3 in Step 1 is hereinafter referred to as Control Kit 3; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being 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 with the coated antigen being 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 with the coated antigen being BSA-eVP5-3 in Step 1 is hereinafter referred to as Control Kit 6; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being BSA-eVP7-1 in Step 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 with the coated antigen being 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 with the coated antigen being BSA-eVP7-3 in Step 1 is hereinafter referred to as Control Kit 9; the chemiluminescent immunoassay kit for diagnosing African horse sickness virus disease or the chemiluminescent immunoassay kit for detecting African horse sickness virus antibodies with the coated antigen being the full-length VP2 recombinant protein of African horse sickness virus in Step 1 is hereinafter referred to as 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 with the coated antigen being the full-length VP5 recombinant protein of African horse sickness virus in Step 1 is hereinafter referred to as 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 with the coated antigen being the full-length VP7 recombinant protein of African horse sickness virus in Step 1 is hereinafter referred to as Control Kit 12.

[0134] (1) Using the kit 1 of the present invention, an optimized experiment was carried out to establish a chemiluminescence immunoassay method with BSA-eVP2-1 + BSA-eVP5-1 + BSA-eVP7-1 as the coating antigen (hereinafter referred to as the present invention CLIA method 1).

[0135] 1. Operating steps

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

[0137] (2) Washing: Pour out the coating original solution in the wells, wash 5 times with washing solution, 3 min each time; pat dry.

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

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

[0140] (5) Sampling: In the serum dilution plate, dilute the test sample with sample diluent (1×PBST) at a ratio of 1:20; take out the coated plate, and set 6 wells of serial calibrators for each experiment. First, add 100 μL of calibrators 1-6 (see below for details) to the serum dilution plate in sequence, and then add 100 μL of the diluted test sample to the remaining wells in sequence. Incubate in a 37°C constant temperature incubator for 30 min (±1 min).

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

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

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

[0144] (9) Add luminol chemiluminescent substrate: Add 100 μL of luminol chemiluminescent substrate to each well, and mix well by shaking; let it stand in the dark at 15°C - 25°C for 5 min, and read the luminescence value with a chemiluminescence analyzer within 15 min after standing.

[0145] 2. Result calculation method

[0146] The NCU (National Clinical Unit), that is, the national clinical unit, is a unit formulated by the National Center for Clinical Laboratory Testing. In this method, this unit assigns a value of 20,000 NCU / ml to the standard positive serum (neutralizing antibody titer is 1:512). A four-parameter calibration curve is established with standard positive serum at 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 realizing semi-quantitative detection. The specific steps are as follows:

[0147] Set calibration products 1 to 6 according to the complement fixation test results for African horse sickness virus antibody-positive sera with serum neutralization titer backgrounds, and the corresponding antibody dose values (0 NCU / mL, 10 NCU / mL, 20 NCU / mL, 40 NCU / mL, 100 NCU / mL, 200 NCU / mL). Taking the luminescence values of calibration products 1 to 6 as the vertical coordinates and the corresponding antibody dose values as the horizontal coordinates, use the ELISA Calc software to draw the four-parameter fitting curve of the calibration products. The four-parameter mode is Y = (a - b) / [1 + (x / c)^b] + d. Among them, a is the estimated value of the upper asymptote of the curve; b is the slope of the curve; c is the dose corresponding to half of the maximum binding; d is the estimated value of the lower asymptote of the curve.

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

[0149] 3. Conditions for the test to be valid and result determination

[0150] Perform four-parameter fitting on the luminescence values of the 6 calibration products and the corresponding antibody dose values, and R 2 ≥0.96, the test is valid. Otherwise, the test is not valid.

[0151] 4. Determination of the positive and negative critical value

[0152] Perform CLIA detection on 200 African horse sickness virus antibody-negative sera using the above method, and calculate the average value (X) and standard deviation (SD) of the dose values of these 200 African horse sickness virus antibody-negative sera. The average dose value X of the 200 African horse sickness antibody-negative sera is 30.25, and the SD is 3.25. Therefore, the positive and negative critical dose value is 40 NCU / mL. That is, a dose value ≥ 40 NCU / mL is judged as positive; a dose value < 40 NCU / mL is judged as negative.

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

[0154] (3) Using Kit 3 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-1 + BSA-eVP7-1 as the coating antigen was established through optimization experiments (hereinafter referred to as the present invention's CLIA method 3). The steps are the same as in (1). The total mass concentration of the coated BSA-eVP2-1 and BSA-eVP7-1 is 1.0 μg / ml.

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

[0156] (5) Using Control Kit 1 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-1 as the coating antigen was established through optimization experiments (hereinafter referred to as Control CLIA method 1). The steps are the same as in (1). The mass concentration of the coated BSA-eVP2-1 is 1.0 μg / ml.

[0157] (6) Using Control Kit 2 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-2 as the coating antigen was established through optimization experiments (hereinafter referred to as Control CLIA method 2). The steps are the same as in (1). The mass concentration of the coated BSA-eVP2-2 is 1.0 μg / ml.

[0158] (7) Using Control Kit 3 of the present invention, a chemiluminescent immunoassay method with BSA-eVP2-3 as the coating antigen was established through optimization experiments (hereinafter referred to as Control CLIA method 3). The steps are the same as in (1). The mass concentration of the coated BSA-eVP2-3 is 1.0 μg / ml.

[0159] (8) Using Control Kit 4 of the present invention, a chemiluminescent immunoassay method with BSA-eVP5-1 as the coating antigen was established through optimization experiments (hereinafter referred to as Control CLIA method 4). The steps are the same as in (1). The mass concentration of the coated BSA-eVP5-1 is 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 in (I). The mass concentration of the coated BSA-eVP5-2 is 1.0 μg / ml.

[0161] (X) 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 in (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 are the same as in (I). The mass concentration of the coated BSA-eVP7-1 is 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 in (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 in (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 with the full-length VP2 protein as the coating antigen (hereinafter referred to as control CLIA method 10) was established through optimization experiments. The steps are the same as in (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 with the full-length VP5 protein as the coating antigen (hereinafter referred to as control CLIA method 11) was established through optimization experiments. The steps are the same as in (I). The mass concentration of the coated full-length VP5 protein is 1.0 μg / ml.

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

[0168] III. Specificity Test

[0169] The CLIA methods 1-4 of the present invention in Step 2, the control CLIA methods 1-12, positive sera of equine infectious anemia virus, equine arteritis virus, equine herpesvirus, equine influenza virus, and equine salmonella antibody (all positive sera were collected from the sera of clinically ill animals) were compared to observe whether there were cross-reactions with other diseases of the same species. In addition, detection was carried out through the complement fixation test simultaneously (for the specific method, see the previous text).

[0170] The results showed that:

[0171] The detection results of the CLIA methods 1-4 of the present invention showed that the antibody dose values were all less than 40 NCU / ml and were determined to be negative. The specificity was 100%. The neutralization indices of the complement fixation test were all less than the national standard of 1:10, and the results were all negative. For the specific results, see Table 2.

[0172] The detection results of the control CLIA methods 1-12 showed that the antibody dose values of the control CLIA method 1, the control CLIA method 4, and the control CLIA method 7 were all less than 40 NCU / ml and were determined to be negative. The specificity was 100%. The specificities of the others were all less than 100%. For the specific results, see Table 3.

[0173] Table 2. Specificity Test Results of the CLIA Methods 1-4 of the Present Invention and the Complement Fixation Test

[0174]

[0175] Table 3. Specificity Test Results of the Control CLIA Methods 1-12

[0176]

[0177]

[0178] IV. Sensitivity Test

[0179] The positive serum of African horse sickness virus was serially diluted, and the CLIA methods 1-4 of the present invention and serum samples with known serum neutralization titer backgrounds were used for comparative detection respectively to obtain the maximum dilution ratio at the positive critical value. The results showed that the highest dilution multiples for detecting positive sera by the CLIA methods 1-4 of the present invention were all 1:1024 times. The highest dilution multiple for detecting positive sera by the complement fixation test was 1:512. See Table 4.

[0180] Table 4. Sensitivity Results of the CLIA Methods 1-4 of the Present Invention and the Complement Fixation Test

[0181]

[0182] V. Repeatability Test

[0183] Using the CLIA methods 1 - 4 of the present invention, 10 African horse sickness virus antibody - positive sera were respectively detected on the same - batch plates and different - batch plates, with 5 parallel determinations, and the within - batch and between - batch coefficient of variation (CV) were calculated. The results showed that the within - batch repeatability coefficient of variation of the CLIA methods 1 - 4 of the present invention was within 8%, and the between - batch repeatability coefficient of variation was less than 10% (Tables 5 to 8). The results indicated that the African horse sickness virus antibody - positive sera had good repeatability.

[0184] Table 5. Repeatability Test of CLIA Method 1 of the Present Invention

[0185]

[0186] Table 6. Repeatability Test of CLIA Method 2 of the Present Invention

[0187]

[0188] Table 7. Repeatability Test of CLIA Method 3 of the Present Invention

[0189]

[0190] Table 8. Repeatability Test of CLIA Method 4 of the Present Invention

[0191]

[0192] VI. Compliance Test

[0193] Using bovine sera stored in the China Animal Disease Control and Prevention Center (Veterinary Diagnosis Center of the Ministry of Agriculture and Rural Affairs), 60 horse sera with a serum neutralization titer background positive for African horse sickness virus antibody and 60 horse sera with a serum neutralization titer background negative for African horse sickness virus antibody were selected. These 120 horse sera were respectively detected using the CLIA methods 1 - 4 of the present invention and the control CLIA methods 1 - 12, and the coincidence rate with the complement fixation test method was calculated.

[0194] The results showed that: for the 160 bovine sera, the overall coincidence rate between the CLIA method 1 of the present invention and the complement fixation test method was 98.33% (the positive coincidence rate was 100% and the negative coincidence rate was 96.67%); the overall coincidence rate between the CLIA method 2 of the present invention and the African horse sickness complement fixation test method was 95.83% (the positive coincidence rate was 96.67% and the negative coincidence rate was 95%); the overall coincidence rate between the CLIA method 3 of the present invention and the African horse sickness complement fixation test method was 95.83% (the positive coincidence rate was 93.33% and the negative coincidence rate was 98.33%); the overall coincidence rate between the CLIA method 4 of the present invention and the African horse sickness complement fixation test method was 95.83% (the positive coincidence rate was 95% and the negative coincidence rate was 96.67%).

[0195] The overall coincidence rate of the detection results of the present invention compared with the CLIA method 1 and the African horse sickness complement fixation test method is 85.83% (the positive coincidence rate is 83.33%, and the negative coincidence rate is 88.33%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 2 and the African horse sickness complement fixation test method is 85% (the positive coincidence rate is 83.33%, and the negative coincidence rate is 86.67%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 3 and the African horse sickness complement fixation test method is 80% (the positive coincidence rate is 81.67%, and the negative coincidence rate is 78.33%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 4 and the African horse sickness complement fixation test method is 76.67% (the positive coincidence rate is 78.33%, and the negative coincidence rate is 75%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 5 and the African horse sickness complement fixation test method is 76.67% (the positive coincidence rate is 73.33%, and the negative coincidence rate is 80%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 6 and the African horse sickness complement fixation test method is 68.33% (the positive coincidence rate is 68.33%, and the negative coincidence rate is 68.33%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 7 and the African horse sickness complement fixation test method is 80.83% (the positive coincidence rate is 80%, and the negative coincidence rate is 81.67%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 8 and the African horse sickness complement fixation test method is 73.33% (the positive coincidence rate is 75%, and the negative coincidence rate is 71.67%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 9 and the African horse sickness complement fixation test method is 73.33% (the positive coincidence rate is 71.67%, and the negative coincidence rate is 75%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 10 and the African horse sickness complement fixation test method is 76.67% (the positive coincidence rate is 73.33%, and the negative coincidence rate is 80%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 11 and the African horse sickness complement fixation test method is 80.83% (the positive coincidence rate is 75%, and the negative coincidence rate is 86.67%). The overall coincidence rate of the detection results of the present invention compared with the CLIA method 12 and the African horse sickness complement fixation test method is 80.83% (the positive coincidence rate is 76.67%, and the negative coincidence rate is 86.67%).

[0196] For details, please refer to Tables 9 to 24.

[0197] The above results indicate that the total coincidence rates of the chemiluminescence immunoassay kits for diagnosing African horse sickness virus antibodies or the chemiluminescence immunoassay kits for detecting African horse sickness virus antibodies prepared by 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 respectively are significantly higher than those of the chemiluminescence immunoassay kits for diagnosing African horse sickness virus antibodies or the chemiluminescence immunoassay kits for detecting African horse sickness virus antibodies prepared by using BSA-eVP2-1, BSA-eVP2-2, BSA-eVP2-3, BSA-eVP5-1, BSA-eVP5-2, BSA-eVP5-3, BSA-eVP7-1, BSA-eVP7-2, BSA-eVP7-3, the full-length VP2 protein of African horse sickness virus, the full-length VP5 protein of African horse sickness virus, and the full-length VP7 protein of African horse sickness virus as coating antigens respectively.

[0198] Table 9. Detection results of bovine serum samples by CLIA method 1 of the present invention

[0199]

[0200] Table 10. Detection results of bovine serum samples by CLIA method 2 of the present invention

[0201]

[0202] Table 11. Detection results of bovine serum samples by CLIA method 3 of the present invention

[0203]

[0204] Table 12. Detection results of bovine serum samples by CLIA method 4 of the present invention

[0205]

[0206]

[0207] Table 13. Detection results of bovine serum samples by control CLIA method 1

[0208]

[0209] Table 14. Detection results of bovine serum samples by control CLIA method 2

[0210]

[0211] Table 15. Detection results of bovine serum samples by control CLIA method 3

[0212]

[0213] Table 16. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 4

[0214]

[0215] Table 17. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 5

[0216]

[0217]

[0218] Table 18. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 6

[0219]

[0220] Table 19. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 7

[0221]

[0222] Table 20. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 8

[0223]

[0224] Table 21. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 9

[0225]

[0226] Table 22. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 10

[0227]

[0228] Table 23. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 11

[0229]

[0230] Table 24. Detection Results of Bovine Serum Samples by Comparing with CLIA Method 12

[0231]

[0232] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art.

Claims

1. A polypeptide or a set of polypeptides, characterized in that: The polypeptide is an eVP7-1 polypeptide; The set of polypeptides is set of polypeptides 1 or set of polypeptides 2; The set of polypeptides 1 consists of eVP2-1 polypeptide, eVP5-1 polypeptide and the eVP7-1 polypeptide; The set of polypeptides 2 consists of the eVP5-1 polypeptide and the eVP7-1 polypeptide; 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 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.

2. A conjugate or a set of conjugates, characterized in that: The conjugate is an eVP7-1 conjugate; The set of conjugates is set of conjugates 1 or set of conjugates 2; The set of conjugates 1 consists of eVP2-1 conjugate, eVP5-1 conjugate and the eVP7-1 conjugate; The set of conjugates 2 consists of the eVP5-1 conjugate and the eVP7-1 conjugate; The eVP2-1 conjugate is a complete antigen obtained by coupling the eVP2-1 polypeptide described in claim 1 with a carrier protein; The eVP5-1 conjugate is a complete antigen obtained by coupling the eVP5-1 polypeptide described in claim 1 with a carrier protein; The eVP7-1 conjugate is a complete antigen obtained by coupling the eVP7-1 polypeptide described in claim 1 and a carrier protein.

3. The set of conjugates according to claim 2, characterized in that: In the set of conjugates 1, the mass ratio of the eVP2-1 conjugate, the eVP5-1 conjugate and the eVP7-1 conjugate is 3:2:5; and / or In the set of conjugates 2, the mass ratio of the eVP5-1 conjugate to the eVP7-1 conjugate is 1:

1.

4. Use of the polypeptide or set of polypeptides according to claim 1 or the conjugate or set of conjugates according to claim 2 or 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 the polypeptide or set of polypeptides according to claim 1 or the set of conjugates according to claim 2 or 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 polypeptide or set of polypeptides described in claim 1 or the conjugate or set of conjugates described in claim 2 or 3 are used as coating antigens.

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.

8. Any of the following biological materials: (B1) a nucleic acid molecule, which is a nucleic acid molecule 1; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eVP7-1 polypeptide of claim 1; (B2) expression cassette, which is expression cassette 1; the expression cassette 1 is an expression cassette containing the nucleic acid molecule 1 described in (B1); (B3) a recombinant vector, which is a recombinant vector 1; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 described in (B1); (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); (B5) A transgenic cell line, which is a transgenic cell line 1; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 described in (B1); (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 consists of the nucleic acid molecules 1, 2 and 3 described in (B1); the set of nucleic acid molecules 2 consists of the nucleic acid molecules 1 and 2 described in (B1); the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eVP5-1 polypeptide described in claim 1; the nucleic acid molecule 3 is a nucleic acid molecule capable of encoding the eVP2-1 polypeptide described in claim 1; (B7) A set of expression cassettes, which is a set of expression cassettes 1 or a set of expression cassettes 2; the set of expression cassettes 1 is composed of the expression cassettes 1, 2 and 3 in (B2); the set of expression cassettes 2 is composed of the expression cassettes 1 and 2 in (B2); the expression cassette 2 is an expression cassette containing the nucleic acid molecule 2 in (B6); the expression cassette 3 is an expression cassette containing the nucleic acid molecule 3 in (B6); (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, 2 and 3 described in (B3); the complete set of recombinant vectors 2 is composed of the recombinant vectors 1 and 2 described in (B3); the recombinant vectors 2 are recombinant vectors containing the nucleic acid molecule 2 described in (B6); the recombinant vectors 3 are recombinant vectors containing the nucleic acid molecule 3 described in (B6); (B9) A set of recombinant bacteria, which 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, recombinant bacteria 2 and recombinant bacteria 3 described in (B4); the set of recombinant bacteria 2 is composed of the recombinant bacteria 1 and the recombinant bacteria 2 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); (B10) A set of transgenic cell lines, which is a set of transgenic cell line 1 or a set of transgenic cell line 2; the set of transgenic cell line 1 is composed of the transgenic cell line 1, transgenic cell line 2 and transgenic cell line 3 described in (B5); the set of transgenic cell line 2 is composed of the transgenic cell line 1 and the transgenic cell line 2 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).

9. Use of the biomaterial according to claim 8 in any of the following: (C1) preparing the polypeptide or set of polypeptides according to claim 1, the conjugate or set of conjugates according to claim 2 or 3, or the kit according to any one of claims 4 to 7; (C2) Preparation of reagents or kits for detecting antibodies against African horse sickness virus; (C3) Preparation of African horse sickness virus diagnostic antigen.

10. Use of the polypeptide or set of polypeptides according to claim 1 or the conjugate or set of conjugates according to claim 2 or 3 as an immunogen in the preparation of antibodies against African horse sickness virus.

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