Monoclonal antibody against VP4 protein of avian polyomavirus and its application

Abstract

The present application discloses a monoclonal antibody that binds to the APV structural protein VP4, a nucleic acid molecule encoding the monoclonal antibody, a kit for detecting and / or diagnosing avian polyomavirus infection, and the use of the antibody in a kit for diagnosing APV. The monoclonal antibody of the present application has high affinity and high sensitivity, and is of great significance for the future IFA diagnosis, detection and treatment of APV.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and specifically to monoclonal antibodies against avian polyomavirus VP4 protein and their application in avian polyomavirus infection detection. Background Art

[0002] Avian polyomavirus disease, also known as budgerigar fledgling disease (BFD) in the early days, is highly pathogenic to chicks. Infecting adult birds can cause chronic symptoms in adult birds, including feather abnormalities, lack of feathers on the back and abdomen, and sarcomas on the head and neck. The disease has no strict host specificity and can infect a variety of poultry, including chickens, parrots, pigeons, ducks, etc. There are no feasible prevention and control measures for the disease.

[0003] APV is a circular double-stranded DNA virus with a size of about 4981bp. The virus particles are icosahedral symmetrical spherical particles with a diameter of about 45nm and no capsule. In 2017, the International Committee on Taxonomy of Viruses (ICTV) classified it as Polyomaviridae, genus Gammapolyomavirus. The main protein components of APV are the large T antigen and small t antigen proteins encoded in the early region, and the VP1, VP2, VP3, VP4 structural proteins and VP4 variant VP4-delta encoded in the late region. Compared with mammalian polyomaviruses, VP4 and VP4-delta are specific proteins that only exist in APV. In addition, VP4 is crucial in APV infection and is believed to be related to viral genome packaging and cell apoptosis in cell culture.

[0004] Studies have shown that deleting or inactivating VP4 can reduce the virulence of APV. Therefore, in-depth research on the functions and characteristics of the VP4 gene will help explore the pathogenic mechanism of avian polyomavirus. This study prepared monoclonal antibodies against the APV structural protein VP4, which can provide an important theoretical basis for the establishment of APV diagnostic methods and vaccine research. Summary of the invention

[0005] In this field, obtaining VP4 protein monoclonal antibodies with good reactivity can lay the foundation for detecting avian polyomavirus, establishing rapid detection methods and vaccine development.

[0006] Previously, Zhang Zhi and others invented an avian polyomavirus detection kit, which used specific primers to synthesize specific nucleic acid probes labeled with digoxigenin through PCR. Through dot molecular hybridization, the specific probe detected the presence of avian polyomavirus nucleic acid in diseased samples. Li Yang and others invented a parrot multiple virus detection kit that can detect psittacine beak and feather disease (PBFD), fowl adenovirus (FAdV) and APV infection, but both are time-consuming and complex, and the detection can only be completed in 24h to 36h. The fluorescent quantitative PCR detection method of Liu Yan, Yan Jiajia and others requires a high level of technical skills of the operator. As an immunological detection method based on antibody-antigen reaction, IFA has strong specificity, high sensitivity, and convenient operation, and is widely used in clinical diagnosis and biological research.

[0007] Therefore, it is necessary to provide a VP4 protein monoclonal antibody with good reactivity and apply it to future IFA-based APV rapid diagnosis and detection methods to develop APV rapid detection kits.

[0008] The technical problem to be solved by the present application is to provide a monoclonal antibody binding to the APV structural protein VP4 and a preparation method thereof in view of the deficiencies in the prior art.

[0009] The technical problem that this application also solves is to provide the application of the above-mentioned monoclonal antibody. Specifically, this application involves the following contents:

[0010] 1. A monoclonal antibody that binds to the APV structural protein VP4, comprising three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0011] The amino acid sequence of CDR-H1 is shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5;

[0012] The amino acid sequence of CDR-H2 is shown in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8;

[0013] The amino acid sequence of CDR-H3 is shown in SEQ ID NO.9 or SEQ ID NO.10 or SEQ ID NO.11;

[0014] The amino acid sequence of CDR-L1 is shown in SEQ ID NO.12 or SEQ ID NO.13 or SEQ ID NO.14;

[0015] The amino acid sequence of CDR-L2 is shown in SEQ ID NO.15 or SEQ ID NO.16 or SEQ ID NO.17;

[0016] The amino acid sequence of CDR-L3 is shown in SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20.

[0017] 2. The antibody according to item 1, wherein CDR-H1 has the amino acid sequence shown in SEQ ID NO.3, the CDR-H2 has the amino acid sequence shown in SEQ ID NO.6, the CDR-H3 has the amino acid sequence shown in SEQ ID NO.9, the CDR-L1 has the amino acid sequence shown in SEQ ID NO.12, the CDR-L2 has the amino acid sequence shown in SEQ ID NO.15, and the CDR-L3 has the amino acid sequence shown in SEQ ID NO.18.

[0018] 3. According to the antibody of item 1, the CDR-H1 has the amino acid sequence shown in SEQ ID NO.4, the CDR-H2 has the amino acid sequence shown in SEQ ID NO.7, the CDR-H3 has the amino acid sequence shown in SEQ ID NO.10, the CDR-L1 has the amino acid sequence shown in SEQ ID NO.13, the CDR-L2 has the amino acid sequence shown in SEQ ID NO.16, and the CDR-L3 has the amino acid sequence shown in SEQ ID NO.19.

[0019] 4. The antibody according to item 1, wherein the CDR-H1 has the amino acid sequence shown in SEQ ID NO.5, the CDR-H2 has the amino acid sequence shown in SEQ ID NO.8, the CDR-H3 has the amino acid sequence shown in SEQ ID NO.11, the CDR-L1 has the amino acid sequence shown in SEQ ID NO.14, the CDR-L2 has the amino acid sequence shown in SEQ ID NO.17, and the CDR-L3 has the amino acid sequence shown in SEQ ID NO.20.

[0020] 5. The monoclonal antibody according to any one of items 1 to 4, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0021] The amino acid sequence of HCVR is as shown in SEQ ID NO.21 or SEQ ID NO.22 or SEQ ID NO.23, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO.21 or SEQ ID NO.22 or SEQ ID NO.23;

[0022] The amino acid sequence of LCVR is shown in SEQ ID NO.24 or SEQ ID NO.25 or SEQ ID NO.26, or is an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO.24 or SEQ ID NO.25 or SEQ ID NO.26.

[0023] 6. The monoclonal antibody according to item 5, wherein the antibody heavy chain variable region HCVR has the amino acid sequence as shown in SEQ ID NO.21, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO.21;

[0024] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.24, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.24.

[0025] 7. The monoclonal antibody according to item 5, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as shown in SEQ ID NO. 22, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO. 22;

[0026] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.25, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.25.

[0027] 8. The monoclonal antibody according to item 5, wherein the antibody heavy chain variable region HCVR has an amino acid sequence as shown in SEQ ID NO. 23, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO. 23;

[0028] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.26, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.25.

[0029] 9. A nucleic acid molecule encoding the monoclonal antibody according to any one of items 1 to 8.

[0030] 10. Use of the monoclonal antibody according to any one of items 1 to 8 or the antibody encoded by the nucleic acid molecule according to item 9 in the preparation of a reagent for detecting and / or diagnosing animals infected with avian polyomavirus.

[0031] 11. A kit for detecting and / or diagnosing avian polyomavirus infection, comprising: the monoclonal antibody described in any one of Items 1 to 8 or the antibody encoded by the nucleic acid molecule described in Item 9, preferably the kit is a kit based on indirect immunofluorescence detection method.

[0032] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0033] (1) The monoclonal antibody obtained in the present application can be used to detect avian polyomavirus, has strong specificity, high antibody titer and affinity index, and specifically binds to CEF cells infected with APV and produces a fluorescent reaction, while CEF cells not infected with the virus do not show green fluorescence.

[0034] (2) The present application has obtained a monoclonal antibody against APV, which provides an important theoretical basis for the establishment of APV diagnostic methods and vaccine research. It is also of great significance for the future IFA diagnosis and detection of APV and the development of APV rapid detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0036] Figure 1 Schematic diagram of the PCR results of APVVP4 gene.

[0037] Figure 2A Schematic diagram of small-scale protein expression gel.

[0038] Figure 2B Schematic diagram of the gel image showing large-scale protein expression.

[0039] Figure 3 Schematic diagram of protein purification gel.

[0040] Figure 4 Schematic diagram of the serum titer results of immunized mice.

[0041] Figure 5 Schematic diagram of the monoclonal antibody SDS-PAGE purity test gel.

[0042] Figure 6 Schematic diagram of monoclonal antibody affinity constant and titer test results.

[0043] Figure 7 Schematic diagram of Western blot identification of antibodies.

[0044] Figure 8 It is a schematic diagram of the IFA test results (200×) of VP4 monoclonal antibody (the scale bar in the lower right corner is 200).

[0045] Fig. 9 Schematic diagram of the indirect immunofluorescence test results (200×) of VP4 protein monoclonal antibodies at different dilutions.

[0046] Fig.10 Schematic diagram of the indirect immunofluorescence test results (200×) of FITC-goat anti-mouse IgG at different dilutions.

[0047] Fig.11 Schematic diagram for determining the incubation time of VP4 protein monoclonal antibody (200×).

[0048] Fig.12 Schematic diagram of the results of indirect immunofluorescence specificity test. DETAILED DESCRIPTION

[0049] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0050] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0051] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0052] Generally speaking, the terms used in this specification have the following meanings.

[0053] In this specification, the term "monoclonal antibody" means an antibody obtained from a colony of substantially homologous antibodies, except possible variant antibodies (e.g., containing naturally occurring mutations or produced in the production process of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of monoclonal antibody products is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of transgenic animals comprising human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0054] In this specification, the term "hybridoma cell" refers to a cell formed by fusion of a myeloma cell and a B lymphocyte in the process of preparing a monoclonal antibody, and is generally prepared by culturing tumor cells.

[0055] In this specification, the terms "heavy chain" ("heavy chain, H"), "light chain" ("light chain, L"), "heavy chain constant region" ("CH"), "light chain constant region" ("LH"), "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "Framework Region" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, and different types of antibodies can be formed through different combinations. For example, a conventional IgG antibody is a tetramer composed of two light chains and two heavy chains. Among them, VL and VH are composed of different CDRs and FRs. CDR contains residues that contact the antigen and determine the antigen specificity of VL and VH. FR is used to maintain the variable region structure and determine the position of the CDR loop. Usually, the heavy chain variable region and the heavy chain constant region constitute a complete heavy chain, while the light chain variable region and the light chain constant region constitute a complete light chain.

[0056] In this specification, "PEG method" refers to a method of inducing cell fusion using a chemical inducer, polyethylene glycol (PEG). Cell fusion, also known as somatic cell hybridization, refers to the use of artificial methods to fuse two or more somatic cells into heterokaryotic cells. Subsequently, the heterokaryons enter mitosis synchronously, the nuclear membrane collapses, and the genes from the two parent cells combine to form a hybrid cell containing only one cell nucleus. Cell fusion technology is an important means of studying cell genetics, gene localization, cell immunity, viruses and tumors.

[0057] In this specification, "limiting dilution method" is a commonly used cloning method, which means that the cell line that needs to be cloned is aspirated from the culture well and the cell count is performed to calculate the number of cells in 1mL. It is often used to screen fused animal cells. Generally, it is diluted with HT culture medium to a cell concentration of 50-60 cells / mL, and 0.1mL (5.5 cells / well) is added to each well of a 96-well culture plate. Inoculate 2 rows, and the remaining cell suspension is diluted in multiples with HT culture medium, and then inoculated in 2 rows, and so on, until each well contains one cell. After 7-10 days of culture, select the positive wells where a single clone grows and clone again. Generally, it is necessary to repeat this 3-5 times until the positive well rate reaches 100% to ensure that the antibody is produced by a single clone.

[0058] In this manual, "IFA" (Indirect Immunological Fluorescence Assay), also known as indirect immunofluorescence detection, combines the specificity and sensitivity of antigen-antibody reaction with the accuracy of microscopic tracing. Fluorescein is used as a marker to bind to known antibodies (or antigens) without affecting their immunological properties. The fluorescein-labeled antibody is then used as a standard reagent to detect and identify unknown antigens. Under a fluorescence microscope, the antigen-antibody complex that exhibits specific fluorescence and its location can be directly observed. In actual work, since the method of using fluorescein-labeled antibodies to examine antigens is more commonly used, it is generally referred to as fluorescent antibody technology.

[0059] In this specification, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0060] In this specification, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can maintain a constant copy number in daughter cells and express the genetic information it carries.

[0061] The present application provides a monoclonal antibody that binds to the APV structural protein VP4, wherein the antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0062] The amino acid sequence of CDR-H1 is shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5;

[0063] The amino acid sequence of CDR-H2 is shown in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8;

[0064] The amino acid sequence of CDR-H3 is shown in SEQ ID NO.9 or SEQ ID NO.10 or SEQ ID NO.11;

[0065] The amino acid sequence of CDR-L1 is shown in SEQ ID NO.12 or SEQ ID NO.13 or SEQ ID NO.14;

[0066] The amino acid sequence of CDR-L2 is shown in SEQ ID NO.15 or SEQ ID NO.16 or SEQ ID NO.17;

[0067] The amino acid sequence of CDR-L3 is shown in SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20.

[0068] In a specific embodiment of the present application, the CDR-H1 has an amino acid sequence as shown in SEQ ID NO.3 (GYTFRSYW), the CDR-H2 has an amino acid sequence as shown in SEQ ID NO.6 (ILPGSGST), the CDR-H3 has an amino acid sequence as shown in SEQ ID NO.9 (ARYRYNGAMDY), the CDR-L1 has an amino acid sequence as shown in SEQ ID NO.12 (KSISKY), the CDR-L2 has an amino acid sequence as shown in SEQ ID NO.15 (SGS), and the CDR-L3 has an amino acid sequence as shown in SEQ ID NO.18 (QQHNEYPWT).

[0069] In a specific embodiment of the present application, the CDR-H1 has an amino acid sequence as shown in SEQ ID NO.4 (GYSITSDYA), the CDR-H2 has an amino acid sequence as shown in SEQ ID NO.7 (ISYSGRN), the CDR-H3 has an amino acid sequence as shown in SEQ ID NO.10 (AGATTIEGGPWFAY), the CDR-L1 has an amino acid sequence as shown in SEQ ID NO.13 (SSVNY), the CDR-L2 has an amino acid sequence as shown in SEQ ID NO.16 (DIS), and the CDR-L3 has an amino acid sequence as shown in SEQ ID NO.19 (QQWNYPLYT).

[0070] In a specific embodiment of the present application, the CDR-H1 has an amino acid sequence as shown in SEQ ID NO.5 (GYTFRSYW), the CDR-H2 has an amino acid sequence as shown in SEQ ID NO.8 (ILPGSATT), the CDR-H3 has an amino acid sequence as shown in SEQ ID NO.11 (ARYRYNGAMDY), the CDR-L1 has an amino acid sequence as shown in SEQ ID NO.14 (KSISRF), the CDR-L2 has an amino acid sequence as shown in SEQ ID NO.17 (SGS), and the CDR-L3 has an amino acid sequence as shown in SEQ ID NO.20 (QQHNEYPWT).

[0071] In a specific embodiment of the present application, wherein the monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0072] The amino acid sequence of HCVR is as shown in SEQ ID NO.21 or SEQ ID NO.22 or SEQ ID NO.23, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO.21 or SEQ ID NO.22 or SEQ ID NO.23;

[0073] The amino acid sequence of LCVR is shown in SEQ ID NO.24 or SEQ ID NO.25 or SEQ ID NO.26, or is an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO.24 or SEQ ID NO.25 or SEQ ID NO.26.

[0074] In a specific embodiment of the present application, the antibody heavy chain variable region HCVR has an amino acid sequence as shown in SEQ ID NO.21 (QGQLQQSGAELMKPGASEKISCKATGYTFRSYWIEWVKQRPGHGLEWIGEILPGSGSTIYNAKFKGKATLTADTSSNTAYMQLSSLTSEDSGVYYCARYRYNGAMDYWGQGTSVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO.21;

[0075] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.24 (DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK), or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.24.

[0076] In a specific embodiment of the present application, the antibody heavy chain variable region HCVR has SEQ ID NO.22

[0077] The amino acid sequence shown in (DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGFISYSGRNINNPSLKSRISVTRDTSKNQFFLQLNSVTPEDTGTYYCAGATTIEGGPWFAYWGQGTLVTVSA), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO.22;

[0078] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.25, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.25 (EIVLTQSPAITAASLGQKVTITCSASSSVNYMHWYQQKSGTSPKPWIYDISKLASGVPARFSGSGSGTSYSLTISSVEAEDAAIYYCQQWNYPLYTFGGGTKLEIK).

[0079] In a specific embodiment of the present application, the antibody heavy chain variable region HCVR has SEQ ID NO.23

[0080] The amino acid sequence shown in (QVQLQQSGAEVMKPGASEKISCKGTGYTFRSYWIEWVKQRPGHGLEWIGEILPGSATTIYNAKFKGKATFTADTSSNTAYLQLSSLTSEDSGVYYCARYRYNGAMDYWGQGTSVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO.23;

[0081] The antibody light chain variable region LCVR has an amino acid sequence as shown in SEQ ID NO.26 (DVQITQSPSYLAASPGETITINCRASKSISRFLAWYQEKPGKTNTLLIYSGSTLQSGIPSRFSGSGSGTDFTLTIRSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK), or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.25.

[0082] The present application also provides a nucleic acid molecule encoding any of the aforementioned monoclonal antibodies.

[0083] The present application also provides the use of any of the aforementioned monoclonal antibodies or antibodies encoded by the aforementioned nucleic acid molecules in the preparation of reagents for detecting and / or diagnosing animals infected with avian polyomavirus.

[0084] The present application also provides a kit for detecting and / or diagnosing avian polyomavirus infection, which comprises: the monoclonal antibody described in any of the above items or the antibody encoded by the above nucleic acid molecule, which is used to detect APV.

[0085] Preferably, the kit for detecting and / or diagnosing avian polyomavirus infection is a kit based on indirect immunofluorescence assay (IFA).

[0086] The kit may include, but is not limited to: a solid phase carrier (immunosorbent) coated with an antigen or antibody; an enzyme-labeled antigen or antibody; an enzyme substrate; a negative control substance and a positive control substance; a buffer solution; a fixative solution, and the like.

[0087] In a specific embodiment of the present application, the method of producing monoclonal antibodies by hybridoma cell lines is a conventional method in the art.

[0088] The present application also provides the use of any of the aforementioned monoclonal antibodies in the detection and / or diagnosis of avian polyomavirus infection.

[0089] The present application also provides a method for preparing the above-mentioned monoclonal antibody. In a specific embodiment of the present application, the purified VP4 protein is emulsified with an equal volume of Freund's complete adjuvant, and the first immunization is performed by subcutaneous multiple-point injection, with an antigen dose of 60 μg / mouse, and a total of 4 SPF-grade BALB / c female mice are immunized; in a specific embodiment of the present application, the VP4 protein is emulsified with an equal volume of Freund's incomplete adjuvant and used as the antigen for the second and third immunizations, with an antigen dose of 30 μg / mouse, and is injected subcutaneously at multiple points, with an interval of 2 weeks between each immunization; in a specific embodiment of the present application, blood is collected from the eye sockets 10 days after the third immunization to measure the serum titer; in a specific embodiment of the present application, another intraperitoneal shock immunization is performed 3 days before fusion, without adjuvant, and with an antigen dose of 50 μg / mouse.

[0090] In a specific embodiment of the present application, the hybridoma cell line secreting avian polyomavirus (APV) monoclonal antibodies is obtained by fusing mouse spleen cells with sp2 / 0 cells in the logarithmic growth phase; in a specific embodiment of the present application, the cell fusion adopts the PEG cell fusion method; in a specific embodiment of the present application, the fused cells are placed in 10 96-well plates; in a specific embodiment of the present application, the positive hybridoma cells are screened by the indirect ELISA method, the positive wells are subcloned by the limiting dilution method, and the screening is repeated 3 times until the positive rate of each well is 100%, and the cultured cells are expanded.

[0091] In a specific embodiment of the present application, the antigen of the hybridoma cell line secreting APV monoclonal antibodies is the recombinantly expressed APV structural protein VP4.

[0092] In a specific embodiment of the present application, the method for preparing the recombinantly expressed APV structural protein VP4 is to express the APV structural protein VP4 in an Escherichia coli expression system and purify it.

[0093] The Condon plus competent strain used in the examples of the present application is a competent strain obtained by special process treatment of Escherichia coli BL21 (DE3), which can be used for chemical transformation of plasmids.

[0094] Western blot used in the examples of the present application is a method of transferring proteins to a membrane and then using antibodies for detection. For known expressed proteins, corresponding antibodies can be used as primary antibodies for detection, and for the expression products of new genes, antibodies of the fusion part can be used for detection. Western Blot uses polyacrylamide gel electrophoresis, the object to be detected is protein, the probe is antibody, and the secondary antibody labeled is used for color development. The protein sample separated by PAGE is transferred to a solid phase carrier, which adsorbs proteins in the form of non-covalent bonds and can keep the type of polypeptide separated by electrophoresis and its biological activity unchanged. The protein or polypeptide on the solid phase carrier is used as an antigen, and reacts with the corresponding antibody, and then reacts with an enzyme or isotope-labeled second antibody, and the protein component expressed by the specific target gene separated by electrophoresis is detected by substrate color development or radioautography.

[0095] The "recombinantly expressed APV structural protein VP4" and "VP4 protein" mentioned in this application both refer to the protein after expression and are the same protein.

[0096] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0097] Unless otherwise specified, the materials and reagents used in the following examples can be purchased from commercial sources.

[0098] The monoclonal antibody obtained in the present application can be used to detect avian polyomavirus, which specifically binds to CEF cells infected with APV and produces a fluorescent reaction. The antibody has strong specificity, and the antibody titer is equal to or higher than 1:102400, that is, the antibody can still produce an observable standard immune response after being diluted 102400 times, and the affinity index exceeds 5E+0.9, indicating that the antibody has high activity level and affinity, and is tightly bound to the antigen, which also indicates that the drug and treatment method containing the antibody will be more effective.

[0099] The present application obtains a monoclonal antibody against APV, which provides an important theoretical basis for the establishment of APV diagnostic methods and vaccine research. It is also of great significance for the future IFA diagnosis and detection of APV and the development of APV rapid detection kits.

[0100] Example

[0101] Example 1 Preparation of recombinant VP4 protein

[0102] 1. Materials and sources

[0103] 1.1 SPF chicken embryos and animals used in the experiment

[0104] SPF chicken embryos were provided by Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd. 6-8 weeks old SPF-grade BALB / c female mice were purchased from Beijing Liuhe BGI Protein Research and Development Center Co., Ltd.

[0105] 1.2 Viruses and cells

[0106] APV, Egg drop syndrome virus (EDSV), Reticuloendotheliosis virus (REV), Avian leukosis virus (ALV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) are all stored by the China Veterinary Drug Administration. Chicken embryo fibroblasts (CEF) were prepared according to Appendix 3504 of the Chinese Pharmacopoeia of Veterinary Medicine.

[0107] 1.3 Vectors and strains

[0108] The vector pcDNA3.1-Flag was preserved by the China Veterinary Drug Administration; the Condon plus strain and protein expression and purification reagents were provided by Beijing Liuhe BGI Protein Research and Development Center Co., Ltd.

[0109] 1.4 Main Reagents

[0110] Fetal bovine serum, FITC-labeled goat anti-mouse IgG, Invitrogen; M199 cell culture medium, Gibco; Ni Sepharose 6Fast Flow, GE Healthcare; Q5 Hot Start High-Fidelity 2X MasterMix, EcoRI and XhoI restriction endonucleases, T4 DNA ligase, NEB; viral DNA / RNA extraction kit, Axygen, USA; complete Freund's adjuvant and incomplete Freund's adjuvant, Sigma; protein molecular weight standard, TaKaRa; transfection reagent, Transfection Reagent was a product of Mirus; HRP-labeled goat anti-mouse IgG was from Beijing Solebao Technology Co., Ltd.; ECL color development solution was from Shanghai Tianneng Life Science Co., Ltd.

[0111] 1.5 Main instruments

[0112] Electrophoresis apparatus, Beijing Liuyi Biotechnology Co., Ltd.; SDS-PAGE electrophoresis apparatus, membrane transfer apparatus, protein gel imager, Bio-Rad; fluorescence microscope, OLYMPUS; microplate reader, Molecular Devices.

[0113] 2. Primer design and synthesis, PCR amplification of VP4 gene fragment

[0114] According to the VP4 gene of APV sequence published on GenBank, the appropriate region was selected through bioinformatics analysis, and a pair of specific primers VP4-F / VP4-R were designed using Primer Express 5.0 to amplify the VP4 gene of APV strain, VP4-F: 5'-ACCCTGCGCCAGGAATTAAA-3' (SEQ ID No. 1), containing EcoRI restriction site. VP4-R: 5'-GGGTGGTAGCAGTAGGGGTA-3' (SEQ ID No. 2), containing XhoI restriction site. The above primers were synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.

[0115] The nucleic acid was extracted from the APV virus liquid using a viral DNA / RNA extraction kit and used as a template for PCR amplification. The reaction system was: Q5 Hot Start High-Fidelity 2X MasterMix 25 μL, upstream and downstream primers 2.5 μL each, template 2 μL, RNase H-free 2 O 18μL. The reaction conditions were: 94℃2min; 94℃30s, 56℃30s, 72℃1 min, 30 cycles, 72℃7min. The amplified products were analyzed by 1% agarose gel electrophoresis, and the remaining PCR products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing.

[0116] The total gene extracted from the infected CEF was detected by PCR using the specific primers VP4-F and VP4-R of the structural protein VP4 of APV, and an electrophoresis band of about 500 bp was amplified ( Figure 1 . Among them, M: DNA standard DL 2000; 1: APVVP4 gene PCR product; 2: negative control), which is consistent with the expected size. The sequencing results showed that the fragment length was 531bp, and the BLAST sequence comparison showed that the sequence was consistent with the GenBank database (accession number: AYV96736.1).

[0117] 3. Expression and purification of VP4 protein

[0118] The VP4 gene was verified by sequencing, and the purified PCR product and plasmid pET30a were digested with restriction endonucleases EcoRI and XhoI, respectively. The digested products were connected by T4 DNA ligase and transferred into Escherichia coli DH5α. After PCR screening, positive single colonies were selected and stored in LB medium and sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing to confirm whether the sequence of the recombinantly expressed vp4 gene was correct. The positive clone obtained by screening was named pET-VP4. The positive clone pET-VP4 obtained by screening was transformed into the prepared Condonplus competent cells, and cultured in LB liquid medium containing corresponding resistance at 37°C, 200r / min until OD 600 reached 0.6-0.8, and IPTG (0.5mM) was induced at 37°C, 200rpm for 2h. The induced bacterial solution was taken for SDS-PAGE gel electrophoresis to detect the expression of the target protein.

[0119] After a small amount of expression by the recombinant expression bacteria, SDS-PAGE electrophoresis was performed, indicating that the VP4 protein was successfully expressed ( Figure 2A Among them, M: pre-stained protein standard; 1: uninduced control (Codon plus); 2: IPTG induced (Codon plus).

[0120] The results of SDS-PAGE gel electrophoresis showed that a large amount of target protein was detected in the supernatant, and further verification of the specific band at 37kD indicated that its expression form was soluble protein expression ( Figure 2B Among them, M: pre-stained protein standard; 1: whole bacteria after ultrasound; 2: supernatant after ultrasound; 3: precipitate after ultrasound).

[0121] The VP4 protein was purified by nickel column (Ni Sepharose 6 Fast Flow, GE Healthcare) and eluted with 10 mM Tris-HCl (pH 8.0) (containing 0.5 M sodium chloride) solution containing 15 mM imidazole, 60 mM imidazole, and 300 mM imidazole, respectively. Finally, the protein peaks were collected and the protein purification effect was detected by SDS-PAGE gel electrophoresis.

[0122] The results of SDS-PAG gel electrophoresis showed that VP4 protein could be purified and the purification effect was good ( Figure 3 Among them, M: pre-stained protein standard; 1: original protein; 2: flow-through; 3: 15mM imidazole elution; 4: 60mM imidazole elution; 5-7: 300mM imidazole elution).

[0123] Example 2 Screening of hybridoma cell lines with monoclonal antibodies against APV structural protein VP4

[0124] 1. Animal immunization

[0125] The purified VP4 protein was emulsified with an equal volume of Freund's complete adjuvant, and the first immunization was injected subcutaneously at multiple points, with an antigen dose of 60 μg / mouse, and a total of 4 SPF-grade BALB / c female mice were immunized; the VP4 protein was then emulsified with an equal volume of Freund's incomplete adjuvant as the antigen for the second, third, and fourth immunizations, with an antigen dose of 30 μg / mouse, and injected subcutaneously at multiple points, with an interval of 2 weeks between each immunization. Blood was collected from the eye sockets 10 days after the fourth immunization, and the serum titer was measured. Three days before fusion, another intraperitoneal impact immunization was performed, without adjuvant, and the antigen dose was 50 μg / mouse.

[0126] The serum antibody titers of immunized mice were tested by indirect ELISA. The results showed that the titers of immune sera from mice No. 1 to No. 4 were all 1:12800. Figure 4 ,in Figure 4 The horizontal axis shows the dilution multiple of mouse serum antibody, and the vertical axis is the OD value measured at a wavelength of 450nm). The spleen cells of mouse No. 3 were randomly selected and fused with SP2 / 0 cells using the PEG method.

[0127] 2. Establishment of animal immune-positive hybridoma cell lines

[0128] Under sterile conditions, spleen cells of immunized mice were taken and fused with sp2 / 0 cells in the logarithmic growth phase by PEG method, and the fused cells were placed in 10 96-well plates. VP4 protein and His protein were used as coating antigens, respectively, and positive hybridoma cells were screened by indirect ELISA method. Positive wells were subcloned by limiting dilution method, and screened for 3 consecutive times until the positive rate of each well was 100%, and the cells were expanded and cultured.

[0129] The purified recombinant VP4 protein was used to coat the ELISA plate, and finally 15 positive hybridoma cell lines that can stably secrete anti-recombinant VP4 protein were obtained, which were named Anti-A-VP4:1, Anti-A-VP4:2, Anti-A-VP4:3, Anti-A-VP4:4, Anti-A-VP4:5, Anti-A-VP4:6, Anti-A-VP4:7, Anti-A-VP4:8, Anti-A-VP4:9, Anti-A-VP4:10, Anti-A-VP4:11, Anti-A-VP4:12, Anti-A-VP4:13, Anti-A-VP4:14, and Anti-A-VP4:15 respectively.

[0130] Example 3 Biological Specificity Detection

[0131] 1. Identification of monoclonal cell subtypes

[0132] The supernatants of the 15 positive hybridoma cells producing VP4 monoclonal antibodies obtained in Example 2 were taken as primary antibodies and identified by the ELISA method in the mouse antibody subclass identification kit.

[0133] The results showed that the subtype of monoclonal antibodies produced by VP4:9 and VP4:15 hybridoma cell lines was IgG2b, and the others were IgG1, and the light chain type was κ. Anti-A-VP4:9, Anti-A-VP4:10, and Anti-A-VP4:15 cell lines with relatively high OD values ​​when identifying subtypes were selected for subsequent testing.

[0134] 2. VP4 monoclonal antibody purity and concentration detection

[0135] The above three monoclonal antibodies were selected, and the three positive hybridoma cells were injected into mice by the ascites injection method. The ascites was collected, purified by ammonium sulfate precipitation, and the concentration was detected by BCA protein quantification method. The purity was analyzed by SDS-PAGE gel electrophoresis (12% separation gel).

[0136] The purity of the three monoclonal antibodies screened out was tested by SDS-PAGE gel electrophoresis. The results showed that the three monoclonal antibodies all had obvious bands at around 25 and 55 kDa, which were the light chain and heavy chain of the monoclonal antibodies, respectively, and no other mixed bands ( Figure 5 . 1: Anti-A-VP4:9; 2: Anti-A-VP4:10; 3: Anti-A-VP4:15; M: prestained protein standard). The concentrations of Anti-A-VP4:9, Anti-A-VP4:10 and Anti-A-VP4:15 were detected by BCA protein mass concentration assay kit and were 2.9 mg / mL, 2.6 mg / mL and 3.2 mg / mL, respectively. In summary, the three monoclonal antibodies obtained in this experiment have good purity and high concentration.

[0137] 3. Determination of VP4 monoclonal antibody titer and affinity constant

[0138] The selected monoclonal antibody was diluted 2 times from 200 times. The antibody titer and affinity constant were determined by indirect ELISA method, with VP4 recombinant protein as the coating antigen, the concentration was 2μg / mL, and the antibody diluted in multiples was tested as the primary antibody. The absorbance value at a wavelength of 450nm was measured by an enzyme marker, and a curve of OD value corresponding to antibody dilution multiple was drawn to find the dilution multiple A corresponding to ≥1 / 2 "platform OD value", affinity constant ≈ (150000×A) / antibody concentration, and dilution multiple A is the antibody titer.

[0139] The results showed that the affinity constants of Anti-A-VP4:9, Anti-A-VP4:10 and Anti-A-VP4:15 were 1.06E+10; 5.91E+09; 9.60E+09, respectively. The titers of Anti-A-VP4:9 and Anti-A-VP4:15 were both 1∶204800, and the titer of Anti-A-VP4:10 was 1∶102400, indicating that the titers of the three antibodies were all high. The test results are shown in Tables 1 and Figure 6 (in, Figure 6 -A is a schematic diagram of the relationship between the dilution multiple and OD value of Anti-A-VP4:9; Figure 6 -B is a schematic diagram of the relationship between the dilution multiple and OD value of Anti-A-VP4:10; Figure 6 - Figure C is a schematic diagram of the relationship between the dilution multiple and OD value of Anti-A-VP4:15).

[0140] Table 1 Monoclonal antibody affinity constant and titer test results

[0141]

[0142]

[0143] A represents the antibody dilution factor corresponding to the 1 / 2 OD value of the platform

[0144] 4. Western blot identification

[0145] Western blot analysis was performed on APV-infected CEF cells and uninfected CEF cells (control group) using Anti-A-VP4:9, Anti-A-VP4:10 and Anti-A-VP4:15. CEF cells were inoculated with APV at a dose of 0.1 MOI, and uninfected CEF cells were set as the control group. After 72 hours of infection, the protein was lysed for SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane using the wet transfer method (100v, 1h). The three monoclonal antibodies collected were used as primary antibodies, and HRP-labeled goat anti-mouse was used as the secondary antibody. The ultrasensitive ECL luminescent liquid was used for color development and the results were judged. .

[0146] The results showed that the cell samples infected with APV virus could specifically bind to VP4, while the cell samples not infected with the virus did not show any bands ( Figure 7 .in, Figure 7 -A: Anti-A-VP4:9; Figure 7 -B: Anti-A-VP4:10; Figure 7-C: Anti-A-VP4:15; M: protein molecular weight standard; 1: CEF cells not infected with APV; 2: CEF cells infected with APV).

[0147] 5. Indirect Immunofluorescence Assay (IFA)

[0148] APV was inoculated into CEF cells at 0.1 MOI, washed twice with PBS after 72 hours, fixed with ice-cold anhydrous methanol; washed three times with PBS, added the culture supernatants of three hybridoma cells to the corresponding wells, respectively, with Anti-A-VP4:9, Anti-A-VP4:10 and Anti-A-VP4:15 as primary antibodies, incubated at 37°C for 1 hour; washed three times with PBS, used FITC-goat anti-mouse IgG as secondary antibody, incubated at 37°C in the dark for 1 hour; washed three times with PBS. Normally grown CEF cells were set up as negative controls.

[0149] The results were observed under a fluorescence microscope. Figure 8 ( Figure 8 -A: Anti-A-VP4:9; Figure 8 -B: Anti-A-VP4:10; Figure 8 -C: Anti-A-VP4:15; 1: CEF cells infected with APV; 2: CEF normal cell control) It can be seen that the three prepared VP4 monoclonal antibodies can all specifically bind to CEF cells infected with APV, and bright green fluorescence can be observed under a fluorescence microscope, while no green fluorescence is observed in CEF cells that are not infected with the virus, indicating that the three monoclonal antibodies prepared in this study can specifically bind to the VP4 protein of APV.

[0150] Example 4 Establishment of indirect immunofluorescence detection method for APV in cells

[0151] Establishment of IFA test method

[0152] CEF cells were cultured in a monolayer in a 24-well plate. APV was inoculated into the CEF cells at an MOI of 0.1 and placed in a 37°C, CO 2 The cells were cultured in a 5% volume fraction incubator for 72 h, the culture medium was discarded, the cells were washed twice with PBS, ice-cold anhydrous methanol was added and placed in a 4°C refrigerator for 20 min to fix the cells; washed three times with PBS, and Anti-A-VP4:15 diluted appropriately with PBS was used as the primary antibody, and incubated at 37°C for 1 h; washed three times with PBS, and FITC-sheep anti-mouse IgG (1:200) diluted with PBS was used as the secondary antibody, and incubated at 37°C in the dark for 1 h; washed three times with PBS, and finally an appropriate amount of PBS was added to each well and the cells were observed under an inverted fluorescence microscope.

[0153] Example 5 Optimization and specificity test of APV indirect immunofluorescence detection method in cells

[0154] 1. Optimization of IFA test method

[0155] The experiment mainly optimized the primary antibody working concentration and incubation time, and the secondary antibody working concentration.

[0156] Anti-A-VP4:15 was diluted with PBS at 1:500, 1:1000, 1:2000, 1:5000, and 1:10000. The working concentration of FITC-labeled goat anti-mouse IgG was diluted at 1:200. After determining the optimal dilution multiple of the primary antibody, the primary antibody was diluted at this dilution concentration.

[0157] Working concentrations of FITC-labeled goat anti-mouse IgG: diluted by volume ratio of 1:200, 1:500, 1:1000, and 1:2000, respectively;

[0158] Primary antibody incubation time: 1 hour at 37°C, overnight at 4°C. Observe the results under a microscope and select the best condition with clear green fluorescence.

[0159] (1) Determination of the optimal dilution factor of Anti-A-VP4:15

[0160] To the CEF cells inoculated with APV, VP4 protein monoclonal antibody diluted in multiple ratios with PBS and FITC-goat anti-mouse IgG secondary antibody diluted 1:200 were added, and a blank control was set up.

[0161] Depend on Fig. 9 (in, Fig. 9 -A: dilution ratio 1:500; Fig. 9 -B: dilution ratio 1:1000; Fig. 9 -C: dilution ratio 1:2000; Fig. 9 -D: dilution ratio 1:5000; Fig. 9 -E: dilution ratio 1:10000; Fig. 9 -F: blank control) It can be seen that when the APV VP4 protein monoclonal antibody is diluted 1000 times, the fluorescent spot is clearest, so the optimal dilution multiple of Anti-A-VP4:15 is 1:1000.

[0162] (2) Determination of the optimal dilution factor of FITC-labeled goat anti-mouse IgG

[0163] In CEF cells inoculated with APV, the primary antibody was Anti-A-VP4:15 diluted 1:1000, the secondary antibody was FITC-goat anti-mouse IgG diluted in PBS, and a blank control was set up.

[0164] The results showed that when the dilution ratio of FITC-labeled goat anti-mouse IgG was 1:200, the fluorescence was clearest and most stable ( Fig.10 .in, Fig.10 -A: dilution ratio 1:200; Fig.10 -B: dilution ratio 1:500; Fig.10 -C: dilution ratio 1:1000; Fig.10 -D: dilution ratio 1:2000; Fig.10 -E: blank control).

[0165] (3) Anti-A-VP4:15 incubation time

[0166] The fluorescence of VP4 protein monoclonal antibody is clearer and more stable when incubated overnight at 4°C, so the best incubation time for Anti-A-VP4:15 is 4°C overnight ( Fig.11 .in, Fig.11 -A: incubate at 37°C for 1 h; Fig.11 -B: overnight incubation at 4°C; Fig.11 -C: negative control).

[0167] 2. Specificity test

[0168] After the CEF cell growth density reached 80%, the cells were inoculated with egg drop syndrome virus (EDSV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and APV, and normal cells without virus were set up as controls. After 72 hours of inoculation, the cells were tested using the IFA test method established in Example 4.

[0169] In the CEF cell culture plates inoculated with EDSV, REV, ALV, and APV, 1:1000 diluted Anti-A-VP4:15, 1:200 diluted FITC-goat anti-mouse IgG, and normal CEF cells were added as negative controls, and then observed under a fluorescence microscope. The results showed that the cells inoculated with EDSV, REV, ALV, NDV, and IBV had no specific fluorescence, while the cells inoculated with APV showed obvious specific fluorescence color development, indicating that the established IFA method has good specificity ( Fig.12 .in, Fig.12 -A: Chicken egg drop syndrome virus; Fig.12 -B: avian reticuloendotheliosis virus; Fig.12 -C: avian leukosis virus; Fig.12 -D: Newcastle disease virus; Fig.12 -E: infectious bronchitis virus; Fig.12 -F: avian polyomavirus; Fig.12 -G: negative control).

[0170] The monoclonal antibody in the present application can undergo a specific fluorescent reaction with CEF cells infected with APV, indicating that the monoclonal antibody obtained in the present application can undergo a specific reaction with APV, while CEF cells not infected with the virus showed no green fluorescence, indicating that the monoclonal antibody in the present application has good specificity.

[0171] The present application provides a monoclonal antibody that binds to the APV structural protein VP4 and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only the preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the scope of protection of the present application. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A monoclonal antibody that binds to the APV structural protein VP4, wherein: It comprises three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, of which: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.3, The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6, The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.9, The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.12, The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.15, The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

18.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: The amino acid sequence of the HCVR is shown in SEQ ID NO.21, The amino acid sequence of the LCVR is shown in SEQ ID NO.

24.

3. A monoclonal antibody that binds to the APV structural protein VP4, wherein: It comprises three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, of which: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.4, The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.7, The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.10, The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.13, The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.16, The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

19.

4. The monoclonal antibody according to claim 3, characterized in that The monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: The amino acid sequence of the HCVR is shown in SEQ ID NO.22, The amino acid sequence of the LCVR is shown in SEQ ID NO.

25.

5. A monoclonal antibody that binds to APV structural protein VP4, wherein: It comprises three heavy chain complementary determining regions CDR-H1, CDR-H2, CDR-H3 and three light chain complementary determining regions CDR-L1, CDR-L2, CDR-L3, of which: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.5, The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.8, The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.11, The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.14, The amino acid sequence of the CDR-L2 is shown in SEQ ID NO.17, The amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

20.

6. The monoclonal antibody according to claim 5, characterized in that The monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: The amino acid sequence of the HCVR is shown in SEQ ID NO.23, The amino acid sequence of the LCVR is shown in SEQ ID NO.

26.

7. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 6.

8. Use of the monoclonal antibody according to any one of claims 1 to 6 or the antibody encoded by the nucleic acid molecule according to claim 7 in the preparation of a reagent for detecting and / or diagnosing animals infected with avian polyomavirus.

9. A kit for detecting and / or diagnosing avian polyomavirus infection, comprising: The monoclonal antibody according to any one of claims 1 to 6 or the antibody encoded by the nucleic acid molecule according to claim 7.

10. The kit according to claim 9, wherein The kit is a kit based on indirect immunofluorescence detection method.

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