Goatpox virus specific competitive elisa detection kit and its application

Abstract

The application belongs to the technical field of biological detection, and discloses a capripoxvirus specific competitive ELISA detection kit and application thereof. The application discloses a hybridoma cell strain GTPV-N1P-4H6, and realizes high-efficiency soluble expression of GTPV-N1 based on a prokaryotic expression system. After immunizing mice with GTPV-N1 as an immunogen, a GTPV positive hybridoma cell strain GTPV-N1P-4H6 capable of stably secreting antibodies is successfully prepared and screened after cell fusion. The ELISA titer of the antibody obtained after the monoclonal antibody secreted by the cell strain is purified is 1:64000, the heavy chain of the antibody is IgGa2 subtype, and the light chain is Kappa chain. Based on the antibody, high-efficiency detection of capripoxvirus N1 protein can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a goatpox virus-specific competitive ELISA detection kit and its application. Background Technology

[0002] Goat pox virus ( Goat pox virus GTPV (Goat Poxvirus) belongs to the genus *Gnaphalovirus* of the family Poxviridae. It is a highly contagious DNA virus affecting goats, causing typical symptoms such as fever, skin papules, and scabs in infected animals, leading to significant decline in productivity and economic losses. GTPV is one of the major infectious diseases closely monitored by the World Organisation for Animal Health (WOAH), posing a serious threat to livestock safety in endemic areas. Timely and accurate epidemiological surveillance and diagnosis of GTPV are crucial for developing effective prevention and control strategies and reducing the spread of the disease.

[0003] Currently, diagnostic methods for GTPV mainly include virus isolation, PCR amplification, and serological methods based on antigen or antibody detection. Virus isolation is time-consuming and complex; while molecular detection is sensitive, it requires sophisticated equipment and is unsuitable for large-scale sample screening. In contrast, ELISA technology offers advantages such as high sensitivity, good stability, ease of operation, and suitability for batch testing, making it an important tool for GTPV immunological surveillance. However, the variety of existing commercial ELISA kits is limited, and their specificity, sensitivity, and compatibility still have room for improvement. Therefore, developing a highly specific, highly sensitive ELISA method suitable for rapid detection of large-scale samples is of great significance.

[0004] Monoclonal antibodies (MAbs) are widely used in the development of pathogen diagnostic and immunoassay methods due to their high specificity and stability. The N1 protein of GTPV, as an important structural protein of the virus, possesses good immunogenicity and is an ideal candidate antigen for developing serological detection methods. However, systematic reports on monoclonal antibodies targeting GTPV N1 and competitive ELISA (cELISA) detection methods based on them are still lacking.

[0005] This application is hereby submitted. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. It prepares a monoclonal antibody against the GTPV N1 protein and establishes an antibody-competitive ELISA detection method for GTPV, in order to provide technical support and theoretical basis for the prevention and control of GTPV.

[0007] The first objective of this invention is to provide a hybridoma cell line GTPV-N1P-4H6.

[0008] A second aspect of the present invention is to provide an antibody or antigen-binding fragment thereof against the goatpox virus N1 protein.

[0009] A third aspect of the present invention is to provide a recombinant protein.

[0010] The fourth aspect of this invention aims to provide biological materials related to the antibody or antigen-binding fragment thereof of the second aspect of this invention or the recombinant protein of the third aspect of this invention.

[0011] The fifth aspect of this invention is to provide a coupling material.

[0012] The sixth aspect of this invention aims to provide the application of the antibody or antigen-binding fragment thereof of the second aspect of this invention, the recombinant protein of the third aspect of this invention, the biomaterial of the fourth aspect of this invention, or the conjugate of the fifth aspect of this invention.

[0013] The seventh aspect of this invention aims to provide a product.

[0014] The object of the eighth aspect of the present invention is to provide a method for detecting goatpox virus.

[0015] The object of the ninth aspect of the present invention is to provide a method for preparing the antibody or antigen-binding fragment thereof of the second aspect of the present invention.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a hybridoma cell line GTPV-N1P-4H6, which was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46570, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] In a second aspect, the present invention provides an antibody or antigen-binding fragment thereof against goatpox virus N1 protein, which is secreted by the hybridoma cell line GTPV-N1P-4H6 of the first aspect of the present invention.

[0018] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The heavy chain includes a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively. The light chain includes a light chain variable region, which contains CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 respectively.

[0019] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is any one of a1)-a3): a1) As shown in SEQ ID NO:3; a2) An amino acid sequence with the same or similar function after one or more amino acids have been substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:3. a3) has 90%, 92%, 94%, 96%, 98%, 99% or more sequence identity with SEQ ID NO:3 and has the same or corresponding amino acid sequence.

[0020] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is any one of b1)-b3): b1) As shown in SEQ ID NO:5; b2) Amino acid sequences with the same or similar functions after one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO:5. b3) has an amino acid sequence that is 90%, 92%, 94%, 96%, 98%, 99% or more identical to SEQ ID NO:5 and has the same or similar function.

[0021] Amino acid substitutions within the aforementioned sequence identity range include conserved substitutions, such as substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr.

[0022] In some embodiments of the present invention, based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain. The antibodies disclosed in this invention can be any of the above types or subtypes.

[0023] In some embodiments of the present invention, the antibody or its antigen-binding fragment may be selected from IgG, IgA, IgM, IgE and IgD.

[0024] In some embodiments of the present invention, the antibody or its antigen-binding fragment is IgG.

[0025] In some embodiments of the present invention, the antigen-binding fragment comprises (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments connected by disulfide bonds at the hinge region; and (iii) a Fab' fragment, which is essentially a Fab fragment having a portion of the hinge region (see Fundamental). IMMUNOLOGY (edited by Paul, Supplement 3, 1993); (iv) Fv fragments consisting of the VL and VH domains of an antibody single arm; and (v) nanobodies, i.e., heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, these domains can be linked via synthetic linkers using recombinant methods, allowing them to be fabricated into single protein chains where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988), Science, Vol. 242: p. 423). 426 pages; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85: 5879 (Page 5883). Such single-chain antibodies are also intended to be covered within the term "antigen-binding fragment" in antibody terminology.

[0026] In some embodiments of the present invention, the antibody or its antigen-binding fragment can be modified into antibodies adapted to different species, such as humanized antibodies, using conventional methods in the art.

[0027] These antigen-binding fragments can be prepared using conventional techniques known to those skilled in the art, and their use and screening methods are the same as those for intact antibodies.

[0028] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

[0029] Unlike conventional methods that typically rely on single-chain antibody fragments, VHH nanobodies, or heterologous horse antiserum, this invention provides a sequence-validated full-length IgG monoclonal antibody capable of binding to the N1 protein of goatpox virus. Using a full-length IgG antibody provides the advantages of natural Fc-mediated binding (such as FcRn-dependent recycling and extended serum half-life).

[0030] A third aspect of the invention provides a recombinant protein comprising: an antibody or antigen-binding fragment thereof of the second aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0031] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag, HA tag, GST tag, SUMO tag.

[0032] A fourth aspect of the invention provides biological materials relating to the antibody or antigen-binding fragment thereof of the second aspect of the invention or the recombinant protein of the third aspect of the invention; said biological material comprising at least one of c1)-c12): c1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the second aspect of the present invention or a recombinant protein of the third aspect of the present invention; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A carrier containing the nucleic acid molecule described in c1); c4) A carrier containing the expression box described in c2); c5) Transgenic cell lines containing the nucleic acid molecules described in c1); c6) Transgenic cell lines containing the expression cassette described in c2); c7) A transgenic cell line containing the vector described in c3); c8) A transgenic cell line containing the vector described in c4); c9) Microorganisms containing the nucleic acid molecules described in c1); c10) Microorganisms containing the expression cassette described in c2); c11) Microorganisms containing the carrier described in c3); c12) Microorganisms containing the carrier described in c4).

[0033] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0034] In some embodiments of the present invention, the vector may be any suitable recombinant expression vector, including plasmids for amplifying nucleic acid molecules, or viral vectors for transfecting cells, including but not limited to retroviral vectors, DNA vectors, murine leukemia virus vectors, SFG vectors, plasmids, RNA vectors, adenovirus vectors, baculovirus vectors, Epstein-Barr virus vectors, papillomavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, adenovirus-associated vectors, lentiviral vectors, or any combination thereof.

[0035] In some embodiments of the present invention, the transgenic cell line is a host cell that produces the antibody or antigen-binding fragment of the present invention, including but not limited to prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.

[0036] In some embodiments, the transgenic cell line is a human cell, such as CHO cells (including but not limited to CHOS cells, CHO-K1 cells) or HEK293 cells (including but not limited to HEK293A, HEK293T and HEK293FS).

[0037] A fifth aspect of the present invention provides a conjugate comprising at least one of an antibody or an antigen-binding fragment thereof from the second aspect of the present invention or a recombinant protein from the third aspect of the present invention; and a conjugate portion comprising a detectable marker.

[0038] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, enzymes, fluorescent compounds, or any combination thereof.

[0039] In some embodiments of the present invention, the fluorescent compound includes fluorescein, fluorescein isothiocyanate, rhodamine, and 5 dimethylamine l Naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP).

[0040] In some embodiments of the present invention, the enzyme includes horseradish peroxidase, β-carotene peroxidase, etc. Galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody or antigen-binding fragment conjugates with a detectable enzyme, detection can be achieved by adding additional reagents. The enzyme uses these additional reagents to produce a distinguishable reaction product. For example, when horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product.

[0041] In some embodiments of the present invention, the radioactive labeling includes, but is not limited to, the following radioactive isotopes or radioactive nucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0042] In some embodiments of the present invention, the antibody or antigen-binding fragment may also be conjugated to biotin and detected by indirect measurement of binding to avidin or streptavidin.

[0043] The sixth aspect of the present invention provides the use of the hybridoma cell line GTPV-N1P-4H6 of the first aspect of the present invention, the antibody or antigen-binding fragment thereof of the second aspect of the present invention, the recombinant protein of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, or the conjugate of the fifth aspect of the present invention in any one of d1)-d3). d1) Identify goatpox virus; d2) Prepare products for identifying goatpox virus; d3) Prepare products for detecting whether the sample to be tested is infected with goatpox virus; The application described is for in vitro non-disease diagnostic purposes.

[0044] In some embodiments of the present invention, the products described in d2)-d3) include at least one of reagents, detection plates, reagent kits, and detection chips.

[0045] A seventh aspect of the present invention provides a kit comprising at least one of the antibody or antigen-binding fragment thereof of the second aspect of the present invention, the recombinant protein of the third aspect of the present invention, or the conjugate of the fifth aspect of the present invention.

[0046] In some embodiments of the present invention, the kit further includes at least one of the following: antigen, enzyme-labeled secondary antibody, solid-phase carrier, coating solution, washing solution, substrate chromogenic solution, and stop solution.

[0047] In some embodiments of the present invention, the solid support is an enzyme-labeled plate.

[0048] In some embodiments of the present invention, the antigen is coated on the surface of a solid support, and the antigen is an N1 protein.

[0049] In some embodiments of the present invention, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat immunized mouse IgG.

[0050] In some embodiments of the present invention, the colorimetric solution comprises TMB (3,3',5,5'-tetramethylbenzidine).

[0051] In some embodiments of the present invention, the terminating solution is an HCl solution.

[0052] In some embodiments of the present invention, the coating solution is 0.01-0.1 mol / L carbonate buffer (CBS); more specifically, it is 0.04-0.06 mol / L carbonate buffer.

[0053] In some embodiments of the present invention, the washing solution comprises 0.01-0.1 mol / L PBST.

[0054] In some embodiments of the present invention, the kit further includes a blocking solution and a sample diluent.

[0055] In some embodiments of the present invention, the sealing liquid is 4%-6% skim milk.

[0056] In some embodiments of the present invention, the sample diluent is PBS.

[0057] In some embodiments of the present invention, the kit has at least one of the following functions: detecting whether the environment or sample contains goatpox virus, and detecting whether the virus is goatpox virus.

[0058] A seventh aspect of the present invention provides a method for detecting goatpox virus, comprising contacting a sample containing or suspected of containing goatpox virus with an antibody or antigen-binding fragment thereof of the second aspect of the present invention, a recombinant protein of the third aspect of the present invention, or a conjugate of the fifth aspect of the present invention, and detecting the formation of a complex of the antibody or antigen-binding fragment thereof, the recombinant protein, the conjugate, and goatpox virus, the method being used for non-disease diagnostic purposes.

[0059] In some embodiments of the present invention, the method includes the step of using a reagent kit from the sixth aspect of the present invention.

[0060] In some embodiments of the present invention, the method specifically includes the following steps: Coating: Coat the antigen onto the ELISA plate, then wash the plate; Closed; Add the sample to be tested and the antibody of the second aspect of the present invention, incubate, and set up negative and positive controls at the same time, and wash the plate; Secondary antibody reaction: Add enzyme-labeled secondary antibody, incubate, and wash the plate; Color development: Add color developer and develop color; Termination: Add stop solution and measure OD. 450 nm value; Result determination: Calculate the blocking rate (PI) = (OD of negative control well) 450 nm-positive control / serum pore OD to be tested 450 nm) / Negative pore D450 The value is calculated as nm×100%. When the PI of the test sample is ≥40.42%, it is judged as positive. When the PI of the test sample is ≤35.16%, it is judged as negative. When the PI of the test sample is between 35.16% and 40.42%, it is judged as suspicious and needs to be repeated. If the retest is suspicious, it is judged as negative.

[0061] In some embodiments of the present invention, the concentration of the antigen coating is 0.01-0.5 μg / mL; further, 0.05-0.2 μg / mL; and even further, 0.08-0.1 μg / mL.

[0062] In some embodiments of the present invention, the coating conditions are: coating at 3-5°C for 10-15 h; further, coating at 3-5°C for 10-13 h; and even further, coating at 3-4°C for 11-12 h.

[0063] In some embodiments of the present invention, the sealing conditions are 35-38°C for 0.5-3 h; further, 36-37°C for 1-2.5 h; and even further, 37°C for 1.5-2 h.

[0064] In some embodiments of the present invention, the incubation conditions in the sample addition step are: incubation at 35-38°C for 10-50 min; further, incubation at 36-37°C for 20-40 min; and even further, incubation at 37°C for 25-35 min.

[0065] In some embodiments of the present invention, the antibody of the second aspect of the present invention is diluted by a factor of 1: (4000-16000); further by a factor of 1: (5000-12000); and even further by a factor of 1: (7000-9000).

[0066] In some embodiments of the present invention, the dilution factor of the enzyme-labeled secondary antibody is 1:(2500-10000); further, it is 1:(4000-8000); and even further, it is 1:(5000-6000).

[0067] In some embodiments of the present invention, the incubation conditions in the secondary antibody reaction are: incubation at 35-38°C for 10-50 min; further, incubation at 36-37°C for 20-40 min; and even further, incubation at 37°C for 25-35 min.

[0068] In some embodiments of the present invention, the color development time is 10-30 min; further, 10-25 min; and even further, 20-25 min.

[0069] A ninth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the second aspect of the present invention, wherein the hybridoma cell line GTPV-N1P-4H6 of the first aspect of the present invention is inoculated into the peritoneal cavity of mice to prepare ascites, and then the collected ascites is purified to obtain the antibody; or it is obtained by culturing the transgenic cell line or microorganism of the fourth aspect of the present invention.

[0070] The beneficial effects of this invention are: This invention utilizes a prokaryotic expression system to achieve highly efficient and soluble expression of recombinant N1 protein (rN1P). After immunizing mice with this recombinant protein, cell fusion was performed, and a stable antibody-secreting positive hybridoma cell line, GTPV-N1P-4H6, was successfully prepared and screened. The antibody obtained after purification of the ascites fluid secreted by this cell line showed an ELISA titer of 1:64000, with the heavy chain being IgG1 and the light chain being Kappa. This antibody enables efficient detection of goatpox virus N1 protein.

[0071] This invention provides an antibody against the N1 protein of goat pox virus, which can effectively bind to the N1 protein. A competitive ELISA detection method for GTPV is established based on this antibody, enabling efficient detection of GTPV. This detection method is simple, highly sensitive, specific, and accurate, requires no complex detection equipment, has low operational skill requirements, and is inexpensive, providing technical support for the detection and control of GTPV.

[0072] Specifically, this invention relates to a competitive ELISA detection method based on an antibody-based GTPV N1P antibody with excellent detection performance. This method shows no cross-reactivity with common sheep pathogens (such as foot-and-mouth disease virus, Brucella, and peste des petits ruminants virus), consistent with existing GTPV ELISA research results. Its detection sensitivity reaches 1:128, significantly higher than the sandwich ELISA method (1:32) established by Hurisa et al. based on GTPV 122 protein (WANG WAN-YING, SHI ZHENG-WANG, LUOJUN-CONG, et al. Development of a double-antigen sandwich ELISA for rapid and accurate detection of antibodies against Capripoxvirus[J]. Microbiol Spectr,2025,13(6):e0272924.). This invention found that among samples that were positive by commercial iELISA, 14 were negative by the cELISA established in this invention. This may be related to the fact that monoclonal antibodies recognize a single epitope, have higher specificity, and lower false positives. In summary, the antibody-competitive ELISA detection method based on GTPV N1P protein established in this invention has good repeatability, high sensitivity and strong specificity, which can provide strong technical support for the epidemiological monitoring and prevention and control research of GTPV, reduce economic losses caused by GTPV infection, and promote the sustainable development of goat farming. Attached Figure Description

[0073] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results show the PCR amplification of the N1P gene and the construction and identification of the recombinant plasmid pCold-N1. In Figure A, N1P gene PCR amplification is shown; M: DL2000 maker, 1-2: N1P gene, 3: negative control. In Figure B, the construction and identification of the recombinant plasmid pCold-N1P is shown; M: DL2000 maker, 1-4: pCold-N1P positive plasmid, 5: negative control.

[0074] Figure 2 The results show the expression of N1P protein. In the figure, M: protein marker; 1: pCold empty vector; 2: N1P induced supernatant; 3: N1P uninduced supernatant; 4: N1P induced precipitation; 5: N1P uninduced precipitation.

[0075] Figure 3The results show the purification of N1P protein. In the figure, M: protein marker; 1: supernatant of bacterial culture after sonication in the induction group; 2: protein flow-through; 3: 20 mM imidazole elution; 4: 40 mM imidazole elution; 5: 60 mM imidazole elution; 6: 100 mM imidazole elution; 7: 200 mM imidazole elution; 8: 500 mM imidazole elution; 9: 600 mM imidazole elution.

[0076] Figure 4 The figure shows the Western blot identification results of rN1P protein. In the figure, M: protein marker; 1: rN1P protein.

[0077] Figure 5 The results are for the reactivity verification of monoclonal antibodies 4H6 and GPTV. Detailed Implementation

[0078] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0079] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0080] Cells, viruses, serum, and experimental animals: SP2 / 0 myeloma cells, Escherichia coli BL21 competent cells, plasmid pCold-TF, goat pox vaccine virus, GTPV positive serum, inactivated serum of foot-and-mouth disease type O virus, inactivated serum of Brucella, inactivated serum of peste des petits ruminants virus, and clinical sheep serum were prepared in our laboratory; 8-week-old SPF-grade female BALB / c mice were purchased from Hangzhou Medical College.

[0081] Main reagents: DNA extraction kit purchased from Nanjing Novizan Biotechnology Co., Ltd.; DMEM medium purchased from Gibco; Fetal Bovine Serum purchased from ExCellBio; IPTG, Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT selective medium, and PEG4000 fusion agent purchased from Sigma-Aldrich; TMB chromogenic solution purchased from Shanghai Beyotime Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG and FITC-labeled goat anti-mouse IgG purchased from Wuhan Abclonal Biotechnology Co., Ltd.; Mouse monoclonal antibody (MAb) subclass identification kit purchased from Proteintech Biotechnology Co., Ltd.; Ni-Agarose Resin nickel affinity chromatography column purchased from Thermo Fisher Scientific; Sheep pox virus antibody (GTPV-Ab) enzyme-linked immunosorbent assay kit (BY-QT7164) purchased from Shanghai Baiyi Biotechnology Co., Ltd.

[0082] This invention immunizes BALB / c mice with recombinant N1 protein (rN1P) obtained through prokaryotic expression and purification. A stable antibody-secreting hybridoma cell line is prepared and screened using hybridoma technology, leading to the preparation of a specific monoclonal antibody against GTPV N1. Based on this, a competitive ELISA method for detecting sheep pox virus antibodies is established and optimized, using rN1P as the coating antigen and the screened monoclonal antibody as the competing antibody. The reliability and applicability of the method are verified through evaluation of its sensitivity, specificity, repeatability, and compatibility with commercially available kits. The results provide an efficient, economical, and scalable technical means for the rapid detection of GTPV and the immunological monitoring of clinical samples, and have important reference value for the epidemiological investigation and prevention and control strategies of sheep pox.

[0083] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0084] Example 1: Preparation of monoclonal antibody against goatpox virus N1 protein 1. Induced expression, purification and identification of recombinant N1P protein (rN1P) DNA was extracted from the goat pox live vaccine. Based on the N1P sequence (AGZ95457.1) in Genebank, primers were designed and the N1P gene was amplified using snapgene software. The recombinant plasmid pCold-N1P was constructed using homologous recombination technology. The pCold-N1 plasmid was transformed into *E. coli* BL21 competent cells. After incubation at 37°C for 2.5 h, IPTG at a final concentration of 0.25 mmol / L was added, and expression was induced at 16°C. After 16 h, the cells were collected by centrifugation at 10,000 g for 5 min, sonicated, and identified by SDS-PAGE. The recombinant protein was then purified using a Ni-Agarose Resin nickel column. The purified protein was identified by Western blot using HRP-labeled goat anti-mouse IgG antibody (1:5000) as the secondary antibody, incubated at 37°C for 1 h, and then analyzed by colorimetric analysis using ECL chemiluminescence solution (Shanghai Beyotime Biotechnology Co., Ltd., P0018S). The concentration of rN1P was determined using the BCA method.

[0085] Using GTPV cDNA as a template, the N1P gene of GTPV was amplified using primers GTPV-N1P-F (5'- GCTCGGTACCCTCGAGATGGATTCTGATAGTCGAAACGGTTTTATTTT-3', SEQ ID NO:1) and GTPV-N1P-R (5'- CGACAAGCTTGAATTCTTATGGAAAAAATCTACTTTTTATAACAAATAGAAATTCATTCCTAC-3', SEQ ID NO:2) designed using snapgene software. The results are as follows. Figure 1 As shown in Figure A, a band of approximately 723 bp was amplified, consistent with the size of the pre-extracted N1P target band. This band was then recovered via gel extraction and purified. The prokaryotic expression vector pCold-TF was double-digested using EcoRI and XhoI restriction endonucleases, and homologous recombination with the target gene N1P was performed. The extracted plasmid was then used as cDNA for PCR amplification, and the results are shown below. Figure 1 As shown in Figure B, a band of approximately 723 bp was amplified, which is consistent with the size of the pre-extracted N1P target band. The recombinant plasmid with a positive identification result was sent to a sequencing company for sequencing. The sequencing results were compared using Snapgene, and the comparison results showed that it was consistent with the target sequence.

[0086] Recombinant bacteria containing pCold-N1P or the empty vector pCold were induced, with uninduced recombinant plasmids serving as controls. SDS-PAGE was used to detect expression levels. The results showed that the target protein was expressed, with a size of approximately 80 kDa. Figure 2The recombinant protein rN1P was purified using a Ni column, and the flow-through and eluent were collected for SDS-PAGE electrophoresis. The results showed that 60 mmol / L and 100 mmol / L imidazole could wash away impurities, and 500 mmol / L imidazole could elute the target protein with high purity. Figure 3 ).

[0087] rN1P was subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using mouse anti-His tag as the primary antibody (1:1000) and HRP-goat anti-mouse as the secondary antibody (1:2000). The results showed a specific band at approximately 80 kDa. Figure 4 This indicates that the protein has good reactivity.

[0088] 2. Preparation and purification of rN1P protein MAb in mouse ascites fluid Three 6-week-old SPF-grade female BALB / c mice were immunized with purified rN1P. The mice were first immunized via multiple subcutaneous injections into the neck and back, with 100 μL (1 mg / mL) of rN1P emulsified with an equal volume of Freund's complete adjuvant. Thirty days after the first immunization, the mice were immunized a second and third time using the same method, with 100 μL (1 mg / mL) of rN1P emulsified with an equal volume of Freund's incomplete adjuvant, 30 days apart. Seven days after the third immunization, serum was collected via the orbital vein. Using a checkerboard assay, purified rN1P (1 μg / mL) was serially diluted 3 times as the coating antigen, mouse serum (1 μg / mL) diluted 2 times (1:200–1:25600) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (1:5000) was used as the secondary antibody. The serum titer of the immunized mice was detected using indirect ELISA. When the serum titer reached 1:60000, mice were enhanced with intraperitoneal injection of rN1P without adjuvant.

[0089] Purified rN1P was used as an immunogen to immunize mice three times. The serum titer of the immunized mice was detected by indirect ELISA. The results showed that the serum titer of the mice reached 1:64000. The mice were then boosted with immunization. Three days later, all mice were euthanized and their spleen cells were fused with SP2 / 0 hybridoma cells.

[0090] 3. Cell fusion and monoclonal antibody preparation Three days after booster immunization, all mice were euthanized. Spleen cells from the immunized mice were harvested under aseptic conditions and fused with SP2 / 0 cells in logarithmic growth phase using the PEG method (spleen cells and SP2 / 0 hybridoma cells were fused at a ratio of 5:1). The fused cells were then placed in 10 wells of a 96-well plate and aseptically isolated. The supernatant from the fused hybridoma cells was screened using the aforementioned indirect ELISA method, and positive hybridoma cells were subcloned three times consecutively using limiting dilution. When the positive rate of a single hybridoma cell reached 100%, the positive hybridoma cells (4H6) secreting the rN1P monoclonal antibody were obtained.

[0091] After expanding the culture of hybridoma cells 4H6, take 10 5 -10 6 Positive hybridoma cells were injected intraperitoneally into the peritoneal cavity of 10-week-old SPF-grade BALB / c mice sensitized with liquid paraffin to prepare mouse ascites containing rN1P monoclonal antibody 4H6. The reactivity of rN1P monoclonal antibody 4H6 mouse ascites with GTPV was detected using indirect immunofluorescence assay (IFA). rN1P monoclonal antibody 4H6 was purified using saturated ammonium sulfate precipitation.

[0092] Hybridoma cell 4H6 (accession name GTPV-N1P-4H6) was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46570, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0093] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4H6 is shown in SEQ ID NO:3, and the nucleotide sequence encoding its heavy chain variable region is shown in SEQ ID NO:4. The amino acid sequence of the light chain variable region of monoclonal antibody 4H6 is shown in SEQ ID NO:5, and the nucleotide sequence encoding its light chain variable region is shown in SEQ ID NO:6.

[0094] EVKLEESGGGLVQPGGSMKLSCAASGFTLS DAWMD WVRQSPEKGLEWVA EIRSKAKNHATYYTESVKG RFTISRDDSKSSVYLQMNALRAEDTGIYYCSY LGY WGQGTTLTVSS (SEQ ID NO:3), where the underlined portion represents three CDR areas.

[0095] GAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACCTTGAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAGGCTAAGAATCATGCAACATACTATACTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACGCCTTAAGAGCTGAAGACACTGGCATTTATTACTGTTCCTATCTAGGCTACTGGGGCCAAGGCACTACTCTCACAGTCTCCTCA (SEQ ID NO:4).

[0096] QIVLTQSPTVMSASPGEKVTITC SATSSVNYIH WFQQKPGTSPKLWIY STSNLAS GVPTRFSGSGSGTSYSLTISRMEAEDAASYYC QQRTSYPLT FGAGTKLDLK (SEQ ID NO:5), wherein the underlined parts are three CDR regions.

[0097] CAAATTGTTCTCACCCAGTCTCCAACAGTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCACCTCAAGTGTAAATTACATACACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTACTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCTCTTATTACTGCCAGCAAAGGACTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGACCTGAAA (SEQ ID NO:6).

[0098] The reactivity of monoclonal antibody 4H6 in mouse ascites fluid and cells with GTPV (vaccine virus) was identified using indirect immunofluorescence assay (IFA). The results showed that the prepared monoclonal antibody 4H6 specifically reacted with GTPV (vaccine virus), exhibiting strong green fluorescence, while Vero cells, used as a negative control, showed no green fluorescence. Figure 5 ).

[0099] Example 2: Determination of the titer and subclass identification of monoclonal antibody 4H6 1. Determination of monoclonal antibody titer The titer of rN1P monoclonal antibody 4H6 was determined by indirect ELISA: using a checkerboard method, purified rN1P diluted 3 times as the coating antigen (1 μg / mL), monoclonal antibody 4H6 diluted 2 times (1:200-1:25600) as the primary antibody, and HRP-labeled goat anti-mouse IgG (1:5000) as the secondary antibody, the titer of monoclonal antibody 4H6 was detected by indirect ELISA.

[0100] The titer of monoclonal antibody 4H6 was determined using indirect ELISA, and the results showed that the titer of monoclonal antibody 4H6 reached 1:64000.

[0101] 2. Identification of Monoclonal Antibody Subclasses Monoclonal antibodies were subclassed according to the instructions of the mouse subtype identification kit (Shanghai Beyotime Biotechnology Co., Ltd., PC169).

[0102] The results showed that the heavy chain of the monoclonal antibody 4H6 was IgG1, and the light chain was Kappa type (Table 1).

[0103] Table 1. Results of Monoclonal Antibody Subclass Identification

[0104] Example 3: Establishment of a Competitive ELISA Detection Method A competitive ELISA assay for detecting goat pox virus includes the following steps: (1) Antigen coating: dilute rN1P protein (prepared from Example 1) with coating buffer (0.05 mol / L CBS) to 0.1 μg / mL, add to ELISA plate (100 μL per well), and coat at 4°C for 12 h; (2) Blocking: After coating, discard the coating solution, wash the plate 3 times with PBST, add 100 μL of blocking solution (5% skim milk powder) to each well, and block at 37℃ for 2 h. Remove the microplate, discard the inner solution, wash the plate 3 times with PBST, and pat dry with absorbent paper; (3) Sample addition: Add the serum to be tested (serum dilution ratio of 1:32, diluent PBS, 100 μL per well) and diluted monoclonal antibody 4H6 (prepared from Example 1, dilution ratio of 1:8000, diluent PBS, 100 μL per well) to the microplate, incubate at 37°C for 30 min, and set up negative control (negative serum), positive control (positive serum) and blank control (PBS). After discarding the liquid in the plate, wash 3 times with PBST and pat dry with absorbent paper. (4) Secondary antibody reaction: HRP-labeled goat anti-mouse IgG was diluted 1:5000 with PBS and added to the microplate (100 μL per well) and incubated at 37°C for 0.5 h; (5) Color development: Remove the microplate, discard the inner solution, wash the plate 3 times with PBST, add 100 μL of TMB color development solution to each well, and develop the color at room temperature for 20 min; (6) Termination: Add 50 μL of XM HCl solution to each well to terminate the reaction. Immediately take a reading at 450 nm on the microplate reader. Repeat the experiment 6 times.

[0105] Calculate the blocking rate (PI) = (OD of negative control wells) 450 nm-positive control / serum pore OD to be tested 450 nm) / Negative pore D450 nm×100%.

[0106] Calculate the mean blocking rate and standard deviation of the negative serum blocking rate. The positive cutoff value = mean negative serum blocking rate ± 3 × standard deviation of the negative serum blocking rate; the negative cutoff value = mean negative serum blocking rate ± 2 × standard deviation of the negative serum blocking rate. When the sample blocking rate is ≥ the positive cutoff value, the result is considered positive; when the sample blocking rate is ≤ the negative cutoff value, the result is considered negative. That is, when the sample PI ≥ 40.42%, it is considered positive; when the sample PI ≤ 35.16%, it is considered negative; and when the sample PI is between 35.16% and 40.42%, it is considered suspicious and requires repeat testing. If the repeat test is suspicious, it is considered negative.

[0107] Example 4: Condition Optimization of Competitive ELISA Detection Methods This embodiment optimizes the detection conditions of the competitive ELISA detection method constructed in Example 3, as follows: Purified rN1P was used as the coating antigen, and the screened monoclonal antibody 4H6 was used as the competitive antibody. The optimal antigen coating concentration and monoclonal antibody dilution were determined by checkerboard titration. The coating conditions (0.05 mol / L pH 9.6 carbonate buffer (CBS) or PBS as the coating solution, overnight coating at 4 ℃) were evaluated using cELISA. Other parameters included blocking buffer (5% skim milk or 5% BSA), blocking time (1, 1.5, 2 h, or 2.5 h), sheep negative / positive serum dilution (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, catalog number 1:1600), serum-monoclonal antibody competition time (30 min, 1 h, 2 h), HRP-goat anti-mouse IgG dilution (1:2500, 1:5000, 1:10000, or 1:20000), and TMB color development time (10 min, 15 h, 1:2500, 1:5000, 1:10000, or 1:20000). Screening was conducted at 1 min, 20 min, and 25 min, and OD values ​​were measured respectively. 450 The nm value was used to calculate the blocking rate (PI). The optimal reaction conditions were determined based on the highest PI.

[0108] Using a checkerboard matrix assay with rN1P protein as the coating antigen and purified monoclonal antibody 4H6 (initial concentration 2 mg / mL) as the primary antibody, the optimal working conditions (PI) were observed when the antigen coating concentration was 0.1 μg / mL and the monoclonal antibody dilution was 1:8000. Based on these results, other conditions for the competitive ELISA were optimized to obtain the following optimal reaction conditions: Coating conditions: antigen coated with CBS at 4℃ for 12 h; Blocking conditions: blocking with 5% skim milk powder for 2 h; Serum dilution: 1:32; Competition time between serum and monoclonal antibody 4H6: 30 min; HRP-goat anti-mouse IgG dilution: 1:5000; TMB color development time: 20 min (Table 2).

[0109] Table 2. Optimization results of various reaction conditions for the competitive ELISA method.

[0110] Further determination of the positive and negative thresholds was performed, and the optimized competitive ELISA detection method described above was used to detect 50 sheep negative serum samples, measuring OD values. 450 nm value, calculate the blocking rate (PI) as shown in Table 3, negative critical value = +2s = 35.16%, positive critical value = +3s=40.42%, meaning that when PI≤35.16%, it is considered negative, and when PI≥40.42%, it is considered positive.

[0111] Table 3. Determination of Criteria for Competitive ELISA Methods

[0112] Example 5 Repeatability Test This embodiment examines the repeatability of the competitive ELISA detection method constructed in Example 3 by performing intra-batch and inter-batch reproducibility tests, as detailed below: Four samples with clear backgrounds were tested using the competitive ELISA detection method described in Example 3. Intra-assay and inter-assay repeatability tests were performed, and the PI was calculated. The coefficients of variation (CV) were calculated for both intra-assay and inter-assay tests. The results showed that the intra-assay CV was 4.7%, the inter-assay CV was 7.4%, and both were <10% (Table 4), indicating that the competitive ELISA detection method constructed in Example 3 has good intra-assay and inter-assay repeatability.

[0113] Table 4. Repeatability test results of competing ELISA detection methods

[0114] Example 6 Specificity Test This embodiment examines the specificity of the competitive ELISA detection method constructed in Example 3, as detailed below: The competitive ELISA detection method in Example 3 was used to detect GTPV positive serum, bovine type O foot-and-mouth disease inactivated positive serum, brucellosis inactivated positive serum, and peste des petits ruminants inactivated positive serum.

[0115] The results are shown in Table 5. The GTPV positive serum was positive, while the detection results of inactivated serum for foot-and-mouth disease type O, brucellosis, and peste des petits ruminants were all negative. The above results indicate that the competitive ELISA detection method constructed in Example 3 of this invention has high specificity.

[0116] Table 5 Specificity tests of competing ELISA detection methods

[0117] Example 7 Sensitivity of competing ELISA detection methods This embodiment examines the sensitivity of the competitive ELISA detection method constructed in Example 3, as detailed below: The competitive ELISA detection method of Example 3 was used to detect GTPV positive serum at dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256, respectively. The PI value was calculated to evaluate the sensitivity of the method.

[0118] The results showed that the serum was still positive at a dilution of 1:128, indicating that the established competitive ELISA detection method had good sensitivity (Table 6).

[0119] Table 6. Sensitivity of competing ELISA detection methods

[0120] Example 8: Clinical Sample Testing and Comparison of Conformity Rates Two hundred clinical sheep serum samples (34 positive and 174 negative) collected from different sheep farms were tested using the competitive ELISA detection method constructed in Example 3 and a commercial kit (sheep pox virus antibody (GTPV-Ab) enzyme-linked immunosorbent assay kit). The positive rate and concordance rate were calculated.

[0121] The results showed that the detection results of the competing ELISA detection method in Example 3 had a 100% concordance rate. The detection results of the purchased commercial kit had a 90% concordance rate.

[0122] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A hybridoma cell line GTPV-N1P-4H6 was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46570, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. An antibody or antigen-binding fragment thereof against goatpox virus N1 protein, secreted by the hybridoma cell line GTPV-N1P-4H6 as described in claim 1; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The heavy chain includes a heavy chain variable region containing CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively. The light chain includes a light chain variable region, which contains CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 respectively.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region is any one of a1)-a3): a1) As shown in SEQ ID NO:3; a2) An amino acid sequence with the same or similar function after one or more amino acids have been substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:

3. a3) An amino acid sequence that has 90% or more sequence identity with SEQ ID NO:3 and has the same or similar function; Preferably, the amino acid sequence of the light chain variable region is any one of b1)-b3): b1) As shown in SEQ ID NO:5; b2) Amino acid sequences with the same or similar functions after one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO:

5. b3) An amino acid sequence that has 90% or more sequence identity with SEQ ID NO:5 and has the same or similar function.

4. The antibody or its antigen-binding fragment according to claim 2 or 3, characterized in that, The antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, or scFv.

5. A recombinant protein, comprising: The antibody or its antigen-binding fragment and tag sequence as described in any one of claims 2-4.

6. A biological material relating to the antibody or antigen-binding fragment thereof of any one of claims 2-4 or the recombinant protein of claim 5, said biological material comprising at least one of c1)-c12): c1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 2-4 or the recombinant protein as described in claim 5; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A carrier containing the nucleic acid molecule described in c1); c4) A carrier containing the expression box described in c2); c5) Transgenic cell lines containing the nucleic acid molecules described in c1); c6) Transgenic cell lines containing the expression cassette described in c2); c7) A transgenic cell line containing the vector described in c3); c8) A transgenic cell line containing the vector described in c4); c9) Microorganisms containing the nucleic acid molecules described in c1); c10) Microorganisms containing the expression cassette described in c2); c11) Microorganisms containing the carrier described in c3); c12) Microorganisms containing the carrier described in c4).

7. A conjugate comprising: at least one of the antibody or antigen-binding fragment thereof as described in any one of claims 2-4 and the recombinant protein as described in claim 5; And a coupling portion, which includes a detectable marker.

8. The use of the hybridoma cell line GTPV-N1P-4H6 of claim 1, the antibody or antigen-binding fragment thereof of any one of claims 2-4, the recombinant protein of claim 5, the biomaterial of claim 6, or the conjugate of claim 7 in any one of d1)-d3); d1) Identify goatpox virus; d2) Prepare products for identifying goatpox virus; d3) Prepare products for detecting whether the sample to be tested is infected with goatpox virus; The application described is for in vitro non-disease diagnostic purposes.

9. A kit comprising at least one of the antibody or antigen-binding fragment thereof according to any one of claims 2-4, the recombinant protein according to claim 5, or the conjugate according to claim 7; Preferably, the kit further includes at least one of the following: antigen, enzyme-labeled secondary antibody, solid-phase carrier, coating solution, washing solution, substrate chromogenic solution, and stop solution.

10. A method for detecting goatpox virus, comprising contacting a sample containing or suspected of containing goatpox virus with an antibody or antigen-binding fragment thereof as described in any one of claims 2-4, a recombinant protein as described in claim 5, or a conjugate as described in claim 7, and detecting the formation of a complex of the antibody or antigen-binding fragment thereof, the recombinant protein, the conjugate, and goatpox virus, the method being used for in vitro non-disease diagnostic purposes.

Images