Bovine nodular skin disease virus chimeric antigen and application of bovine nodular skin disease virus chimeric antigen in ELISA (enzyme-linked immuno sorbent assay) detection kit

Abstract

The invention relates to the technical field of immunodetection, and discloses a bovine nodular skin disease virus chimeric antigen and application thereof in an ELISA (enzyme-linked immuno sorbent assay) detection kit. According to the chimeric antigen, a gene of a dominant region of a bovine nodular skin disease virus antigen is selected to design a chimeric antigen gene, meanwhile, sequence optimization is performed, a chemical synthesis method is used for synthesizing a coding gene of the chimeric antigen of the bovine nodular skin disease virus, and then the chimeric antigen is obtained in a prokaryotic expression mode. An ELISA antibody detection kit developed on the basis of the chimeric antigen has extremely high sensitivity and specificity, can rapidly and specifically detect bovine nodular skin disease virus antibodies in serum, is suitable for monitoring and checking the nodular skin diseases of cattle in large-scale breeding, and has relatively high practical value.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a bovine lumpy skin disease virus chimeric antigen and its application in an ELISA detection kit. Background Art

[0002] Lumpy skin disease (LSDV) is an acute or subacute infectious disease of cattle caused by the bovine lumpy skin disease virus (LSDV). Cattle of all ages and breeds are susceptible to LSDV, which can be transmitted through mosquito bites or direct contact. Infection manifests as fever, swollen lymph nodes, skin nodules, and, in severe cases, death. The high infectivity and mortality rate of LSDV cause significant economic losses to the livestock industry, and it has been listed as a notifiable major animal disease by the World Organization for Animal Health (OIE). Vaccination and accurate diagnosis are effective means of preventing and controlling LSDV. While vaccines currently offer some protection, more sensitive and specific diagnostic reagents are still needed.

[0003] Lumpy Skin Disease Virus (LSDV) is a double-stranded DNA virus belonging to the Poxviridae family, genus Capripoxvirus. It shares over 90% homology with fellow members of the genus, goatpoxvirus (GTPV) and sheeppoxvirus (SPPV), and vaccine cross-protection among the three viruses is approximately 96%. The LSDV genome is approximately 150 kb, encoding approximately 156 open reading frames. LSDV, GTPV, and SPPV are believed to share highly similar coat proteins and other conserved structural proteins, resulting in high serological cross-reactivity. The commercial ELISA antibody diagnostic kit currently in clinical use is a double-antigen sandwich kit (ID-Vet) based on goatpoxvirus p32. No kit for detecting LSDV p32 antibodies has yet been approved for clinical use. Although goatpoxvirus p32 shares over 97% amino acid homology with LSDV p32, it is still a single antigenic component of goatpoxvirus, which can lead to missed detection.

[0004] The OIE-recommended diagnostic techniques for bovine lumpy skin disease virus include pathogen detection and serological testing. Existing LSD diagnostics primarily rely on virus isolation, RT-PCR, VNT, and ELISA testing. Therefore, the development of an ELISA antibody detection reagent that is easy to prepare, highly sensitive, and specific is particularly urgent. The indirect ELISA method based on chimeric antigens offers the advantages of high specificity and sensitivity. Furthermore, the ELISA method's multi-sample, high-throughput capabilities make it suitable for implementation in large-scale farms. Summary of the Invention

[0005] In view of this, the purpose of the present application is to provide a bovine lumpy skin disease virus chimeric antigen, which has high specificity and sensitivity when used for the detection of bovine lumpy skin disease virus antibodies;

[0006] Another object of the present application is to provide the relevant application of the above-mentioned bovine lumpy skin disease virus chimeric antigen in the preparation of ELISA antibody detection products.

[0007] In order to solve the above-mentioned technical problems / achieve the above-mentioned purposes or at least partially solve the above-mentioned technical problems / achieve the above-mentioned purposes, as the first aspect of the present application, a bovine lumpy skin disease virus chimeric antigen is provided, which is formed by connecting amino acid residues 133-270 of the bovine lumpy skin disease virus LSDV074 protein, amino acid residues 91-182 of the LSDV103 protein, and amino acid residues 847-931 of the LSDV134 protein via a linker protein.

[0008] Optionally, the chimeric antigen is formed by sequentially linking amino acid residues 133-270 of LSDV074 protein, a first linker protein, amino acid residues 91-182 of LSDV103 protein, a second linker protein, and amino acid residues 847-931 of LSDV134 protein.

[0009] Further optionally, the chimeric antigen sequence is shown as SEQ ID NO: 1.

[0010] As a second aspect of the present application, a gene encoding the chimeric antigen described in the present application is provided.

[0011] As a third aspect of the present application, provided is the use of the chimeric antigen in the preparation of an immunoassay product for detecting bovine lumpy skin disease virus antibodies.

[0012] Optionally, the immunoassay product is an ELISA antibody detection kit.

[0013] As a fourth aspect of the present application, an ELISA antibody detection kit for detecting bovine lumpy skin disease virus antibodies is provided, comprising the bovine lumpy skin disease virus chimeric antigen described in the present application as a coating antigen.

[0014] Optionally, the ELISA antibody detection kit includes an enzyme labeling plate, an enzyme conjugate, an enzyme conjugate diluent, a washing solution, a substrate, a sample diluent, a stop solution, a negative control and a positive control; the enzyme labeling plate is coated with the bovine lumpy skin disease virus chimeric antigen described in the present application.

[0015] Optionally, the enzyme conjugate is horseradish peroxidase-labeled goat anti-bovine IgG; and the enzyme conjugate diluent is PBST containing casein.

[0016] Optionally, the positive control is the hyperimmune serum prepared after vaccine immunization; the negative control is the serum of healthy bovines.

[0017] Optionally, the sample diluent is PBST containing casein.

[0018] The chimeric antigen described in this application is designed by selecting a gene from a dominant region of the bovine nodular skin disease virus antigen, simultaneously optimizing the sequence and synthesizing the gene encoding the bovine nodular skin disease virus chimeric antigen using chemical synthesis methods, and then obtaining the chimeric antigen through prokaryotic expression. The ELISA antibody detection kit developed based on this chimeric antigen has extremely high sensitivity and specificity, and can quickly and specifically detect bovine nodular skin disease virus antibodies in serum. It is suitable for monitoring and screening for nodular skin disease in cattle herds in large-scale farming and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application.

[0020] Figure 1 Shown is the SDS-PAGE gel image of the LSDV chimeric antigen of the present application. DETAILED DESCRIPTION

[0021] The present application discloses a bovine nodular dermatitis virus chimeric antigen and its application in an ELISA detection kit. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0022] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other in the absence of conflict.

[0023] In the first aspect of the present application, by analyzing and studying the bovine lumpy skin disease virus LSDV074 protein (accession number: NP_150508.1), LSDV103 protein (accession number: NP_150537.1), and LSDV134 protein (accession number: NP_150568.1) on the NCBI website, genes in the antigenic dominant region are selected to design bovine lumpy skin disease virus chimeric antigen genes, and sequence optimization is performed at the same time, and the bovine lumpy skin disease virus chimeric antigen genes are synthesized using chemical synthesis methods, and then the chimeric antigens are obtained by prokaryotic expression.

[0024] Among them, LSDV074 is the p32 protein of bovine nodular dermatitis virus, which is an envelope structural protein with strong immunogenicity and conservatism. The present application selects its antigenic dominant region at amino acids 133-270. The LSDV103 protein is a structural protein that participates in the assembly and maturation of the virus and has high conservatism and immunogenicity. The present application selects its antigenic dominant region at amino acids 91-182. LSDV134 is a non-structural protein that is expressed in the early stages of viral infection and participates in the replication of the virus. It has high conservatism and immunogenicity. The present application selects its antigenic dominant region at amino acids 847-931. The above three antigenic dominant region proteins work together and have the potential to be developed into candidate antigens for diagnostic reagents for early infection of LSDV. The chimeric expression of the three proteins can improve the sensitivity of antibody diagnosis and reduce the probability of missed detection.

[0025] In certain embodiments of the present application, the chimeric antigen is formed by sequentially linking amino acid residues 133-270 of the LSDV074 protein, a first linker protein, amino acid residues 91-182 of the LSDV103 protein, a second linker protein, and amino acid residues 847-931 of the LSDV134 protein. In other embodiments of the present application, the chimeric antigen sequence is as shown in SEQ ID NO: 1, wherein amino acid residues 1-138 are amino acid residues 133-270 of the LSDV074 protein, amino acid residues 139-143 are the first linker protein, amino acid residues 144-235 are amino acid residues 91-182 of the LSDV103 protein, amino acid residues 236-240 are the second linker protein, and amino acid residues 241-325 are amino acid residues 847-931 of the LSDV134 protein. The first adapter protein and the second adapter protein can be independently selected from any suitable adapter protein, such as GGGGS, as shown in SEQ ID NO: 3; in certain embodiments of the present application, the first adapter protein and the second adapter protein are both GGGGS.

[0026] In a second aspect of the present application, a gene encoding the chimeric antigen described herein is provided. In certain embodiments of the present application, a gene encoding the chimeric antigen sequence shown in SEQ ID NO: 1 is provided, and its sequence is shown in SEQ ID NO: 2.

[0027] In certain embodiments of the present application, the chimeric antigen is prepared by prokaryotic expression of the coding sequence shown in SEQ ID NO: 2, comprising:

[0028] The coding gene is inserted between the multiple cloning sites of the plasmid vector to construct a bovine nodular skin disease virus chimeric antigen gene recombinant expression plasmid, which is then transformed into Escherichia coli competent cells to screen recombinant expression bacteria, and the recombinant expression bacteria are induced by IPTG to express the bovine nodular skin disease virus chimeric antigen protein.

[0029] The plasmid vector can be any suitable commercial plasmid, such as pET21b(+) commercial plasmid. In certain embodiments of the present application, the coding gene is inserted between the NdeI and XhoI recognition sites of the pET21b(+) plasmid.

[0030] In the third aspect of the present application, the chimeric antigen is used to prepare an ELISA antibody detection kit to detect verified bovine foot-and-mouth disease type O antibody-positive serum, bovine viral diarrhea virus antibody-positive serum, bovine rotavirus antibody-positive serum, bovine infectious rhinotracheitis virus antibody-positive serum and bovine nodular skin disease virus antibody-positive serum. The results showed that only the bovine nodular skin disease virus antibody-positive serum tested positive, and the other sera tested negative, indicating that the chimeric antigen described in the present application has a high specificity when used to detect bovine nodular skin disease virus antibodies.

[0031] At the same time, the chimeric antigen was used to prepare an ELISA antibody detection kit to detect bovine nodular skin disease virus antibody-positive serum. The positive serum was diluted to 1:3200 times and could still be detected. However, the ELISA antibody detection kit prepared with LSDV074 amino acid residues 20-270, LSDV103 full-length protein, LSDV132 full-length protein or LSDV134 amino acid residues 786-931 as antigens, when detecting the same bovine nodular skin disease virus antibody-positive serum, the antigen formed by LSDV074 amino acid residues 20-270 and the antigen formed by LSDV134 amino acid residues 786-931 could only detect the positive serum diluted to 1600 times, and the LSDV103 and LSDV132 full-length protein antigens could only detect the positive serum diluted to 800 times. The results show that the chimeric antigen described in the present application has high sensitivity when used to detect bovine nodular skin disease virus antibodies.

[0032] Based on the excellent detection effects in the above two aspects, the present application provides the use of the chimeric antigen in the preparation of an immunoassay product for detecting bovine lumpy skin disease virus antibodies, wherein the immunoassay product is preferably an ELISA antibody detection kit.

[0033] In a fourth aspect of the present application, an ELISA antibody detection kit for detecting bovine lumpy skin disease virus antibodies is provided, comprising the bovine lumpy skin disease virus chimeric antigen described in the present application as a coating antigen.

[0034] In certain embodiments of the present application, the ELISA antibody detection kit includes an ELISA plate, an enzyme conjugate, an enzyme conjugate diluent, a washing solution, a substrate, a sample diluent, a stop solution, a negative control, and a positive control; the ELISA plate is coated with the bovine nodular skin disease virus chimeric antigen described in the present application, and the coating concentration is 1 μg / mL.

[0035] In certain embodiments of the present application, the enzyme conjugate is horseradish peroxidase-labeled goat anti-bovine IgG, and the working concentration of the enzyme conjugate used is preferably 1:20,000; in other embodiments of the present application, the enzyme conjugate diluent is PBST containing casein, for example, PBST containing 1% casein.

[0036] In certain embodiments of the present application, the positive control is a high immune serum prepared after vaccine immunization; the negative control is healthy bovine serum; and the sample diluent is PBST containing casein, such as PBST containing 1% casein.

[0037] In certain embodiments of the present application, the substrate includes substrate A solution: 3,3′,5,5′-tetramethylbenzidine solution and substrate B solution: peroxide solution; the two are preferably mixed in a 1:1 ratio; the stop solution is 2M sulfuric acid solution; and the washing solution is 25-fold concentrated PBST.

[0038] In certain embodiments of the present application, a method for using the ELISA antibody detection kit is also provided:

[0039] (1) Dilute the serum to be tested with sample diluent at 1:100 and add 100 μL per well to the ELISA plate. At the same time, add 100 μL of negative and positive controls to each well. Seal the ELISA plate with a cover film and incubate at 37°C for 30 min.

[0040] (2) Dilute the washing solution to 1× washing solution, shake and wash the ELISA plate three times, and pat dry.

[0041] (3) Dilute the enzyme conjugate at 1:100 with enzyme conjugate diluent, add 100 μl / well to the ELISA plate, seal the plate with a cover film, and incubate at 37°C for 30 min.

[0042] (4) Wash the ELISA plate three times with 1× washing buffer by shaking and pat dry.

[0043] (5) Mix substrate A solution and substrate B solution at a ratio of 1:1 and add 100 μL / well to the ELISA plate. Seal the plate with a cover film and incubate at 37°C for 10 min. Add 50 μL / well of the stop solution to the plate and immediately read the absorbance at OD 450 nm on a microplate reader.

[0044] (6) Result determination: The result is valid when the positive control OD 450nm value is ≥1.2 and the negative control OD 450nm value is <0.3; when (sample OD 450nm value - negative control OD 450nm value) / (positive control OD 450nm value - negative control OD 450nm value) ≥0.1, it is judged as positive (S / P value ≥0.1); when (sample OD 450nm value - negative control OD 450nm value) / (positive control OD 450nm value - negative control OD 450nm value) <0.1, it is judged as negative (S / P value <0.1).

[0045] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials, except for the differences noted in each group, were kept consistent to ensure comparability. In addition, all materials used in this application can be purchased from commercial sources.

[0046] The following further describes a bovine lumpy skin disease virus chimeric antigen provided in this application and its application in an ELISA detection kit.

[0047] Example 1: Preparation of the bovine lumpy skin disease virus chimeric antigen described in this application

[0048] 1. Construction of the recombinant expression plasmid pET21b-LSDV-NP containing the chimeric antigen gene of bovine lumpy skin disease virus

[0049] Based on the antigenicity analysis of the bovine lumpy skin disease virus LSDV074 protein (accession number: NP_150508.1), LSDV103 protein (accession number: NP_150537.1), and LSDV134 protein (accession number: NP_150568.1) on the NCBI website, genes with antigenic predominance were selected to design a bovine lumpy skin disease virus chimeric antigen gene. Sequence optimization was performed and the bovine lumpy skin disease virus chimeric antigen gene (shown in SEQ ID NO: 2) was synthesized using chemical synthesis methods. Codon optimization and mRNA secondary structure optimization for E. coli were optimized to enhance expression efficiency in E. coli. The pET21b(+) vector was double-digested with NdeI and XhoI. The digestion system consisted of 50 μL of the following: 1 μL each of NdeI and XhoI, 25 μL of the vector fragment, 5 μL of 10X digestion buffer, and 18 μL of ddH2O. The digestion product was recovered by 1% agarose gel electrophoresis using a gel recovery kit. The digested vector was then ligated with the chimeric antigen gene to create a 10 μL ligation system: 7 μL of the chimeric antigen gene fragment, 1 μL of the pET21b vector, 1 μL of T4 DNA ligase, and 1 μL of T4 DNA ligation buffer. The tube was gently tapped, inverted to mix, and then centrifuged. The ligation reaction was allowed to proceed at 16°C for 2 hours. The ligation product was transformed into DH5α competent cells using standard chemical methods. Positive clones were identified by PCR and sequenced. Plasmids that tested positive by sequencing were designated pET21b-LSDV-NP.

[0050] 2. Prokaryotic expression of chimeric antigens

[0051] Transform the pET21b-LSDV-NP recombinant plasmid into BL21(DE3) cells. Aseptically, add 100 μL of the bacterial solution to a LB(Amp+) solid medium plate. Spread the solution evenly and incubate inverted overnight in a 37°C incubator. Identify a single, smooth, round colony for colony PCR to confirm that pET21b-LSDV-NP has been transformed into BL21(DE3). A single positive colony was inoculated into LB(Amp+) liquid medium and cultured overnight at 37°C in a shaker at 210 rpm. The next day, the cells were transferred to 1 L of LB liquid medium and cultured at 37°C in a shaker at 210 rpm for 2-3 hours until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.5 mM and the culture was induced overnight at 20°C at 180 rpm.

[0052] 3. Purification of chimeric antigens

[0053] After induction of expression, 1L of bacterial solution was centrifuged at 6000r / min for 10 minutes, the supernatant was discarded, the bacteria were collected, and the bacteria were resuspended with 30mL of Tris buffer containing 5mM imidazole, and the E. coli cells were broken with a high-pressure homogenizer. The broken material was centrifuged at 4℃15000r / min for 60 minutes, and the supernatant was filtered with a 0.22μm filter membrane. The recombinant chimeric antigen was purified using a HisTrap HP chromatography column, and impurities were washed with Tris buffer containing 5mM imidazole, and then eluted and purified with Tris buffer containing 50mM, 100mM, and 500mM imidazole respectively. The collected samples were analyzed by SDS-PAGE, and the results are as follows. Figure 1 As shown in the figure, a single band of approximately 38 kD appeared in the gel image after purification. The target protein was concentrated using Merck-Millipore 10 kD concentrators and then the protein concentration was measured by BCA method.

[0054] Example 2: Preparation of an ELISA antibody detection kit based on bovine lumpy skin disease virus chimeric antigen

[0055] 1. Preparation of ELISA Antibody Detection Kit

[0056] An ELISA kit for detecting bovine lumpy skin disease virus antibodies was prepared by the following steps:

[0057] (1) Coating: Coat the ELISA plate with recombinant bovine lumpy dermatosis virus chimeric antigen at a coating concentration of 1 μg / ml, using 100 μL / well. Coat overnight at 4°C. Wash three times with PBST the next day and pat dry.

[0058] (2) Blocking: Block with 1% casein, 200 μL / well, incubate at 37°C for 2 h, wash three times with PBST, spin dry, dry at 37°C for 3 h, and then vacuum seal for later use.

[0059] (3) Positive control: Highly immune serum was prepared from healthy cattle immunized with inactivated bovine nodular dermatitis vaccine and diluted with sample diluent as a positive control.

[0060] (4) Negative control: Take healthy bovine serum as a negative control.

[0061] (5) Enzyme-labeled conjugate: Dilute horseradish peroxidase-labeled goat anti-bovine IgG 200 times as an enzyme conjugate.

[0062] (6) Enzyme conjugate diluent: PBST (0.01 M, pH 7.4) containing 1% casein

[0063] (7) Sample diluent: PBST (0.01M, pH 7.4) containing 1% casein

[0064] (8) Substrate A solution: 3,3′,5,5′-tetramethylbenzidine solution

[0065] (9) Substrate B solution: peroxide solution

[0066] (10) Stop solution: 2M sulfuric acid solution.

[0067] 2. ELISA test method operation steps

[0068] ((1) Dilute the serum to be tested with sample diluent at 1:100 and add 100 μL to each well of the ELISA plate. At the same time, add 100 μL of negative and positive controls to each well. Seal the ELISA plate with a cover film and incubate at 37°C for 30 min.

[0069] (2) Dilute the washing solution to 1× washing solution, shake and wash the ELISA plate three times, and pat dry.

[0070] (3) Dilute the enzyme conjugate at 1:100 with enzyme conjugate diluent, add 100 μl / well to the ELISA plate, seal the plate with a cover film, and incubate at 37°C for 30 min.

[0071] (4) Wash the ELISA plate three times with 1× washing buffer by shaking and pat dry.

[0072] (5) Mix substrate A solution and substrate B solution at a ratio of 1:1 and add 100 μL / well to the ELISA plate. Seal the plate with a cover film and incubate at 37°C for 10 min. Add 50 μL / well of the stop solution to the plate and immediately read the absorbance at OD 450 nm on a microplate reader.

[0073] (6) Result determination: The result is valid when the positive control OD 450nm value is ≥1.2 and the negative control OD 450nm value is <0.3; when (sample OD 450nm value - negative control OD 450nm value) / (positive control OD 450nm value - negative control OD 450nm value) ≥0.1, it is judged as positive (S / P value ≥0.1); when (sample OD 450nm value - negative control OD 450nm value) / (positive control OD 450nm value - negative control OD 450nm value) <0.1, it is judged as negative (S / P value <0.1).

[0074] Example 3: Specificity and sensitivity of ELISA antibody detection kit based on bovine lumpy skin disease virus chimeric antigen

[0075] 1. Specificity detection

[0076] At the same dilution multiple, the prepared ELISA antibody detection kit was used to detect known bovine foot-and-mouth disease type O antibody-positive sera, bovine viral diarrhea virus antibody-positive sera, bovine rotavirus antibody-positive sera, bovine infectious rhinotracheitis virus antibody-positive sera and bovine lumpy skin disease virus antibody-positive sera. The results are shown in Table 1.

[0077] Table 1 Specificity test results

[0078] serum samples Chimeric antigen (S / P) Bovine foot-and-mouth disease type O antibody positive serum 0.03 Bovine viral diarrhea virus antibody-positive serum 0.05 Bovine rotavirus antibody-positive serum 0.04 Bovine infectious rhinotracheitis virus antibody-positive serum 0.08 Bovine lumpy skin disease virus antibody negative serum 0.06 Bovine lumpy skin disease virus antibody-positive serum 1.859

[0079] The results showed that the ELISA antibody detection kit prepared based on the chimeric antigen of bovine lumpy skin disease virus only reacted positively with the positive serum of bovine lumpy skin disease virus antibody, and was negative with the positive sera of other bovine disease antibodies, indicating that the ELISA antibody detection kit based on the chimeric antigen of bovine lumpy skin disease virus has good specificity.

[0080] 2. Sensitivity detection

[0081] The known bovine lumpy skin disease virus antibody-positive serum was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, respectively, and the ELISA antibody detection kit prepared based on the chimeric antigen of bovine lumpy skin disease virus of the present application, the ELISA antibody detection kit prepared based on amino acid residues 20-270 of LSDV074, the full-length protein of LSDV103, the amino acid residues 786-931 of LSDV134, and the full-length protein of LSDV132, and the commercial ELISA of goat pox virus p32 were used respectively. The results showed that the ELISA antibody detection kit prepared based on the bovine lumpy skin disease virus chimeric antigen of the present application had the highest sensitivity, reaching 1:3200 times. However, when detecting the same bovine lumpy skin disease virus antibody-positive serum, the antigen formed by the amino acid residues 20-270 of LSDV074 and the antigen formed by the amino acid residues 786-931 of LSDV134 could only detect the positive serum diluted to 1600 times, and the LSDV103 full-length protein antigen and the LSDV132 full-length protein antigen could only detect the positive serum diluted to 800 times.

[0082] At the same time, according to the instructions and criteria of the commercial goatpox virus p32 ELISA kit, positive serum diluted up to 1600 times can be detected (because the criteria of the commercial kit differ from those of the present application, the data are not included in Table 2). The results show that the chimeric antigen described in this application has a high sensitivity for detecting antibodies to bovine lumpy skin disease virus, as shown in Table 2.

[0083] Table 2 Sensitivity of different kits to different dilution multiples of bovine lumpy skin disease virus positive serum

[0084]

[0085]

[0086] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A bovine lumpy skin disease virus chimeric antigen, characterized in that: The protein is formed by connecting the 133-270 amino acid residues of the bovine lumpy skin disease virus LSDV074 protein, the 91-182 amino acid residues of the LSDV103 protein and the 847-931 amino acid residues of the LSDV134 protein via a linker protein.

2. The chimeric antigen according to claim 1, characterized in that It is formed by sequentially linking the amino acid residues 133-270 of the LSDV074 protein, the first linker protein, the amino acid residues 91-182 of the LSDV103 protein, the second linker protein, and the amino acid residues 847-931 of the LSDV134 protein.

3. The chimeric antigen according to claim 1 or 2, characterized in that The sequence is shown in SEQ ID NO:

1.

4. A gene encoding the chimeric antigen according to any one of claims 1 to 3.

5. Use of the chimeric antigen according to claim 1 in the preparation of an immunoassay product for detecting antibodies to bovine lumpy skin disease virus.

6. The use according to claim 3, characterized in that The immunoassay product is an ELISA antibody detection kit.

7. An ELISA antibody detection kit for detecting bovine lumpy skin disease virus antibodies, characterized in that: The invention comprises the bovine lumpy skin disease virus chimeric antigen according to any one of claims 1 to 3 as a coating antigen.

8. The ELISA antibody detection kit according to claim 7, characterized in that The method comprises an enzyme labeling plate, an enzyme conjugate, an enzyme conjugate diluent, a washing solution, a substrate, a sample diluent, a stop solution, a negative control and a positive control; the enzyme labeling plate is coated with the bovine nodular skin disease virus chimeric antigen according to claim 1.

9. The ELISA antibody detection kit according to claim 8, characterized in that The enzyme conjugate is horseradish peroxidase-labeled goat anti-bovine IgG; and the enzyme conjugate diluent is PBST containing casein.

10. The ELISA antibody detection kit according to claim 8, characterized in that The positive control is the high immune serum prepared after vaccine immunization; the negative control is the healthy bovine serum; and the sample diluent is PBST containing casein.

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