Monoclonal Antibodies Against VP2 Protein of Infectious Bursal Disease Virus in Chickens and Their Application

Abstract

This invention discloses a hybridoma cell with the microbial preservation number CCTCC NO:C2024404, and also discloses a monoclonal antibody secreted by the aforementioned hybridoma cell. This monoclonal antibody can recognize the VP2 protein of infectious bursal disease virus (IBDV) with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, but cannot recognize the VP2 protein of IBDV with amino acid sequences as shown in SEQ ID NO.2 or SEQ ID NO.3, and has application value for specific identification of IBDV strains.

Description

Technical Field

[0001] This invention belongs to the field of biological products and relates to monoclonal antibodies against VP2 protein of infectious bursal disease virus in chickens and their applications. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, fatal, and immunosuppressive infectious disease primarily affecting chicks, caused by the Infectious Bursal Disease Virus (IBDV). In the late 1980s, an outbreak of a very virulent IBDV strain (vvIBDV) occurred in Europe and rapidly spread to my country. This strain exhibited acute high mortality, posing a serious threat to the healthy development of my country's poultry industry. With the improvement of intensive farming management and the widespread use of vaccines, the vvIBDV outbreak is being effectively controlled. However, in recent years, a novel variant of IBDV (nVarIBDV) has emerged, causing a new atypical IBD outbreak that poses a new threat to the poultry industry. nVarIBDV can evade the immune protection of existing vaccines, directly destroying the central immune organs, the bursa of Fabricius and B lymphocytes, leading to severe immunosuppression and decreased production performance, resulting in significant economic losses.

[0003] Currently, the newly emerging nVarIBDV and the persistently circulating vvIBDV are the two dominant circulating IBDV strains in my country, exhibiting significant antigenic differences. However, their identification and detection rely solely on sequencing analysis, which is time-consuming, labor-intensive, expensive, and requires specialized interpretation. The development of a diagnostic monoclonal antibody for nVarIBDV and the establishment of related immunological detection methods such as ELISA and colloidal gold test strips based on this antibody would greatly improve clinical testing efficiency, but no relevant research reports have yet been published. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention prepares a hybridoma cell line that secretes a monoclonal antibody against the IBDV VP2 protein, which stably secretes the monoclonal antibody IBDV-VP2-2F10. This monoclonal antibody specifically recognizes nVarIBDV and can be used for the identification and detection of the two currently prevalent strains, nVarIBDV and vvIBDV. The aspartic acid (D) at position 318 of VP2 is a key amino acid of the conformational epitope recognized by this monoclonal antibody. This invention provides a new tool and technical means for the specific recognition and detection of nVarIBDV, which is of great significance for the comprehensive prevention and control of IBD.

[0005] The first aspect of the present invention provides a hybridoma cell, wherein the hybridoma cell is hybridoma cell A or hybridoma cell B;

[0006] The hybridoma cell A is a hybridoma cell with the microbial preservation number CCTCC NO: C2024404;

[0007] The hybridoma cell B is a passaged cell of the hybridoma cell with the microbial preservation number CCTCC NO: C2024404;

[0008] In the monoclonal antibody gene contained in the genome of the hybridoma cell B, the heavy chain CDR1 maintains the amino acid sequence encoding positions 50-54 of SEQ ID NO. 6;

[0009] The heavy chain CDR2 retains the amino acid sequence encoding positions 69-85 of SEQ ID NO. 6;

[0010] The heavy chain CDR3 retains the amino acid sequence encoding positions 118-124 of SEQ ID NO. 6;

[0011] The light chain CDR1 retains the amino acid sequence encoding positions 42-53 of SEQ ID NO. 8;

[0012] The light chain CDR2 retains the amino acid sequence encoded as shown in positions 69-75 of SEQ ID NO. 8;

[0013] The light chain CDR3 retains the amino acid sequence encoding positions 108-116 of SEQ ID NO. 8;

[0014] The monoclonal antibody secreted by the hybridoma cell B maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4.

[0015] A second aspect of the present invention provides a biomaterial, said biomaterial being any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0016] P1: Monoclonal antibody

[0017] The monoclonal antibody maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4.

[0018] The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0019] The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0020] The monoclonal antibody light chain includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0021] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6;

[0022] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6;

[0023] The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6;

[0024] The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8;

[0025] The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8;

[0026] The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8;

[0027] P2: Genetically engineered monoclonal antibody

[0028] The genetically engineered monoclonal antibody maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4.

[0029] The genetically engineered monoclonal antibody includes a genetically engineered monoclonal antibody heavy chain and a genetically engineered monoclonal antibody light chain.

[0030] The genetically engineered monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification.

[0031] The genetically engineered monoclonal antibody light chain includes light chain CDR1, light chain CDR2, light chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification.

[0032] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6;

[0033] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6;

[0034] The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6;

[0035] The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8;

[0036] The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8;

[0037] The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8;

[0038] P3: Antibody derivatives

[0039] The antibody derivative maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4;

[0040] The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.

[0041] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6;

[0042] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6;

[0043] The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6;

[0044] The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8;

[0045] The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8;

[0046] The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8;

[0047] The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, avian-derived antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0048] P4: RNA assembly

[0049] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0050] The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2.

[0051] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2.

[0052] P5: Gene Combination

[0053] The gene combination includes monoclonal antibody heavy chain genes and monoclonal antibody light chain genes;

[0054] The coding sequence of the monoclonal antibody heavy chain gene can encode the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2.

[0055] The coding sequence of the monoclonal antibody light chain gene can encode the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2.

[0056] P6: Gene Expression Catalyst Assembly

[0057] The gene expression cassette assembly includes a monoclonal antibody heavy chain gene expression cassette and a monoclonal antibody light chain gene expression cassette.

[0058] The gene expression product in the monoclonal antibody heavy chain gene expression cassette is the monoclonal antibody heavy chain RNA described in P4.

[0059] The gene expression product in the monoclonal antibody light chain gene expression cassette is the monoclonal antibody light chain RNA described in P4.

[0060] The promoters of the monoclonal antibody heavy chain gene expression cassette and the monoclonal antibody light chain gene expression cassette are constitutive expression promoters or artificially inducible promoters;

[0061] P7: Genetic Engineering Vector

[0062] The genetic engineering vector is a combination of a first genetic engineering vector and a second genetic engineering vector or a third genetic engineering vector.

[0063] The first genetic engineering vector encodes the monoclonal antibody heavy chain RNA described in P4 that can be expressed;

[0064] The second genetic engineering vector encodes the monoclonal antibody light chain RNA described in P4 that can be expressed;

[0065] The third genetic engineering vector encodes expressible monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA as described in P4.

[0066] P8: Cells

[0067] The cell is either a first cell or a second cell;

[0068] The first cell contains the first and second genetic engineering vectors described in P7.

[0069] The second cell contains the third gene engineering vector described in P7;

[0070] P9: Composition

[0071] The composition comprises the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA assembly described in P4, the genetically engineered vector described in P7, or the cell described in P8; and

[0072] P10: Reagent Kit

[0073] The kit contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7, or the cells described in P8.

[0074] In some embodiments, the monoclonal antibody is a monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0075] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or the heavy chain variable region of the genetically engineered monoclonal antibody is shown as positions 20-135 of SEQ ID NO. 6;

[0076] The amino acid sequence of the light chain variable region of the monoclonal antibody or the light chain variable region of the genetically engineered monoclonal antibody is shown in positions 20-126 of SEQ ID NO. 8.

[0077] The third aspect of this invention provides the use of the hybridoma cells described in the first aspect of this invention, the monoclonal antibody described in the second aspect of this invention, the genetically engineered monoclonal antibody described in the second aspect of this invention, the antibody derivative described in the second aspect of this invention, the genetically engineered vector described in the second aspect of this invention, or the cells described in the second aspect of this invention in the preparation of formulations for recognizing proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4.

[0078] The fourth aspect of the present invention provides a method for detecting the presence of a strain of infectious bursal disease virus in a sample for non-diagnostic purposes, wherein the amino acid sequence of the VP2 protein of the infectious bursal disease virus strain is shown in SEQ ID NO.1 or SEQ ID NO.4.

[0079] The method includes the following steps:

[0080] S1: Inoculate the sample to be tested into susceptible cells for infectious bursal disease virus in chickens to obtain the inoculated cells;

[0081] S2: The seeded cells are fixed onto a solid surface to obtain immobilized cells;

[0082] S3: Incubate the immobilized cells with a monoclonal antibody to obtain the first incubation of immobilized cells;

[0083] The monoclonal antibody is the monoclonal antibody described in the second aspect of the present invention or a genetically engineered monoclonal antibody.

[0084] S4: Add the labeled monoclonal antibody-specific conjugate to the immobilized cells after the first incubation and incubate to obtain immobilized cells after the second incubation.

[0085] S5: Characterize the marker that is indirectly bound to the immobilized cells in the second incubation, and determine whether the chicken infectious bursal disease virus strain is present in the sample to be tested based on the presence or absence of the marker.

[0086] In some embodiments, in step S1, the susceptible cells for infectious bursal disease virus in chickens are DF1 cells;

[0087] In some embodiments, in step S2, the solid surface is the inner surface of the pores of a microporous plate;

[0088] In some embodiments, in step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step S5, the immobilized cells after the second incubation are photographed using a fluorescence microscope, and the presence of the infectious bursal disease virus strain in the sample to be tested is determined based on the photographic results.

[0089] In some embodiments, in step S4, the labeled monoclonal antibody-specific conjugate is a horseradish peroxidase-labeled secondary antibody of the monoclonal antibody; in step S5, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of the infectious bursal disease virus strain in the sample to be tested is determined based on the chromogenic result. Attached Figure Description

[0090] Figure 1 The curve shows the ELISA titer results of monoclonal antibodies.

[0091] Figure 2 The results of monoclonal antibody subtype identification are shown.

[0092] Figure 3 Results of antibody stability assay for hybridoma cells; A. SHG19-VP2 coated indirect ELISA; B. HLJ0504-VP2 coated indirect ELISA.

[0093] Figure 4 The results of the detection of nVarIBDV by the monoclonal antibody IBDV-VP2-2F10 are shown. A. IBDV infection (SHG19 and HLJ0504); B. IBDV VP2 protein expression (SHG19-VP2 and HLJ0504-VP2).

[0094] Figure 5 The results show the recognition and detection of different VP2 strains and their truncated variants by monoclonal antibodies; A. Detection of different VP2 strains by monoclonal antibodies based on Western blot (WB); B. Truncated expression scheme of SHG19-VP2; C. Detection of SHG19-VP2 and its truncated variants by monoclonal antibodies based on infusion factor (IFA).

[0095] Figure 6 The amino acid sequence alignment diagrams for VP2 of nVarIBDV (SHG19) and vvIBDV (HLJ0504) are shown below. A. Amino acid sequence alignment of VP2; B. Positions of the differentially expressed amino acids in the three-dimensional structure of VP2.

[0096] Figure 7 This is a photograph of the IFA detection results of monoclonal antibody against SHG19-VP2 and its 19 mutants.

[0097] The scale bar in each fluorescence photograph is 200 μm. Detailed Implementation

[0098] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0099] Materials and instruments not described in this invention are conventional materials and instruments in the art. Operational details not described in this invention are conventional operations in the art. The software used in this invention is operated by conventional methods in accordance with the software provider's instructions. The reagent kits used in this invention are operated by conventional methods in accordance with the reagent kit's instruction manual.

[0100] Applications of detecting the presence of infectious bursal disease virus strains in samples for non-diagnostic purposes (not the process of identifying, studying, and determining the cause or lesion status in living humans or animals) include, but are not limited to, the following:

[0101] (1) Explore the immune patterns of infection with chicken infectious bursal disease virus strains.

[0102] (2) Explore the immune patterns of mixed infection of chicken infectious bursal disease virus strains with other strains.

[0103] (3) Provide environmental assessment for developing environmental disinfection methods for infectious bursal disease virus strains in chickens.

[0104] Material

[0105] (1) IBDV SHG19 strain: The isolation and identification of the novel IBDV variant (nVarIBDV) representative strain SHG19 is described in Patent 1 and Journal 1 below. The gene sequence can be found in GenBank accession numbers MN393076.1 and MN393077.1.

[0106] (2) IBDV HLJ0504 strain (also called HLJ-0504 strain): IBDV super-virulent strain vvIBDV HLJ0504 strain, its isolation and identification are recorded in the following journal 2, and the gene sequence can be found in GenBank accession numbers GQ451330.1 and GQ451331.1.

[0107] (3) IBDV recombinant virus rGtHLJVP2 strain: a recombinant virus expressing vvIBDV HLJ0504 strain VP2 using IBDV attenuated strain Gt as parental backbone. The preparation steps of rGtHLJVP2 strain are recorded in the following patent 2. The microbial preservation number of rGtHLJVP2 strain is: CGMCC No.3749.

[0108] (4) IBDV rGtVarVP2 strain: a recombinant virus expressing nVarIBDV SHG19 strain VP2 using IBDV Gt strain as parental backbone. The preparation steps of rGtVarVP2 strain are described in the following patent 1. The microbial preservation number of rGtVarVP2 strain is: CGMCC No.19383.

[0109] (5) Monoclonal antibody 7D4: The applicant's laboratory-made monoclonal antibody 7D4 against IBDV VP2 protein (also called IBDV-VP2-7D4) is a broad-spectrum monoclonal antibody that can recognize multiple types of IBDV VP2 protein. It has been tested and can recognize VP2 protein of strains HLJ0504 (GenBank No. GQ451330.1 for segment A of genome), Gx strain (GenBank No. AY444873.3 for segment A of genome), SHG19 strain (GenBank No. MN393076.1 for segment A of genome), SHG352 strain (GenBank No. MT179720.1 for segment A of genome), and Gt strain (GenBank No. DQ403248.1 for segment A of genome).

[0110] (6) Other materials: SP2 / 0 myeloma cells, DF-1 cells, and DT40 cells were preserved by the applicant’s laboratory.

[0111] Patent 1: Chinese patent application No. CN202010312138.0

[0112] Patent 2: Chinese patent application No. CN201010215328.7

[0113] Journal 1: Fan L, Wang Y, Jiang N, Gao L, Li K, Gao Y, Cui H, Pan Q, LiuC, Zhang Y, Wang X, Qi X. A reassortment vaccine candidate of the novelvariant infectious bursal disease virus. Vet Microbiol. 2020 Dec;251:108905.doi: 10.1016 / j.vetmic.2020.108905. Epub 2020 Nov 7. PMID: 33186757.

[0114] Journal 2: Qi X, Gao L, Qin L, Deng X, Wu G, Zhang L, Yu F, Ren X, Gao Y, Gao H, Wang Y, Wang DOI:10.16175 / j.cnki.1009-4229.2011.12.004.

[0115] Example 1: Preparation and identification of monoclonal antibodies against VP2 protein of a novel variant of infectious bursal disease virus in chickens.

[0116] I. Construction of plasmid pCASHGVP2

[0117] The VP2 coding sequence of nVarIBDV SHG19 strain (refer to GenBank ID: MN393076.1) was cloned into the eukaryotic vector pCAGGS. After the sequence of the transformed gene was verified to be correct by recombinant vector sequencing, it was used for eukaryotic expression of the VP2 protein of nVarIBDV SHG19 strain (named pCASHGVP2).

[0118] The VP2 protein sequence of SHG19 strain is as follows (SEQ ID NO.1):

[0119] MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIVGAHYILQSDGSYKFDQMLLTAQNLPASYNYCRLVSRSLTVRS STLPGGVYALNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAVGLDPKMVATCDSSDRPRVYTITAADNYQFSSQY KTGGVTITLFSANIDAITSLSVGGELVFKTSIQNLVLGATIYLIGFDGTAVITRAVAANNGLTAGIDNLMPFNLVIPTSEITQPITSIKLEIVTSKSDGQAGEQMSWSAS GSLAVTIHGGNYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGA

[0120] The plasmid pCASHGVP2 was transformed into DF1 cells to express the VP2 protein of the nVarIBDV SHG19 strain in eukaryotes, which was then used for IFA detection of the prepared monoclonal antibody.

[0121] II. Construction of plasmid pCAHLJVP2

[0122] The VP2 coding sequence of vvIBDV HLJ0504 strain (refer to GenBank number: GQ451330.1) was cloned into the eukaryotic vector pCAGGS. After the sequence of the transformed gene was verified to be correct by recombinant vector sequencing, it was used for eukaryotic expression of the VP2 protein of vvIBDV HLJ0504 strain (named pCAHLJVP2).

[0123] The VP2 protein sequence of strain vvIBDV HLJ0504 is as follows (SEQ ID NO.2):

[0124] MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIVGAHYTLQSNGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRS STLPGGVYALNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAANDYQFSSQY QAGGVTITLFSANIDAITSLSIGGELVFQTSVQGLILGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNIVIPTSEITQPITSIKLEIVTSKSGGQAGDQMSWSAS GSLAVTIHGGNYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKNLITEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGA

[0125] The plasmid pCAHLJVP2 was transformed into DF1 cells to express the VP2 protein of the nVarIBDV SHG19 strain in eukaryotes, which was then used for IFA detection of the prepared monoclonal antibody.

[0126] III. Prokaryotic Expression of VP2 Protein

[0127] The VP2 coding sequence of the aforementioned nVarIBDV SHG19 strain was cloned into the prokaryotic vector pCold I. After the sequence of the transformed gene was verified to be correct by recombinant vector sequencing, it was put into use (named pCo-HHT28-SHG19VP2-466) for prokaryotic expression of the VP2 protein of the nVarIBDVSHG19 strain.

[0128] The plasmid pCo-HHT28-SHG19VP2-466 ​​was transformed into E. coli Transetta (DE3) expression engineered bacteria for expression and purification to obtain the nVarIBDV SHG19 strain VP2 protein, named SHG19-VP2, for use in immunizing mice and coating ELISA plates.

[0129] The VP2 coding sequence of the vvIBDV HLJ0504 strain was cloned into the prokaryotic vector pCold I. After the sequence of the transformed gene was verified to be correct by recombinant vector sequencing, it was used for prokaryotic expression of the VP2 protein of the vvIBDVHLJ0504 strain (named pCo-HHT28-HLJ0504VP2-466).

[0130] The plasmid pCo-HHT28-HLJ0504VP2-466 ​​was transformed into E. coli Transetta (DE3) expression engineered bacteria, expressed and purified to obtain the vvIBDV HLJ0504 strain VP2 protein, named HLJ0504-VP2, for use in coating ELISA plates.

[0131] IV. Animal Immunization

[0132] The aforementioned SHG19-VP2 protein solution (solvent: sterile PBS (0.01 mol / L, pH 7.4)) was mixed with Freund's complete adjuvant at a 1:1 volume ratio and emulsified thoroughly on a tissue homogenizer to obtain immunogen composition 1. The same SHG19-VP2 protein solution was mixed with Freund's incomplete adjuvant at a 1:1 volume ratio and emulsified using the same method to obtain immunogen composition 2.

[0133] Six-week-old BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were used for immunization, with a total of 5 mice immunized. Immunogen composition 1 was administered to the mice via multiple subcutaneous injections on the back, with an immunization dose of 70 μg SHG19-VP2 protein per mouse. A second immunization was administered 14 days after the first immunization with immunogen composition 2, and a third immunization was administered 14 days after the second immunization with immunogen composition 2, using the same immunization method and dosage as the first immunization.

[0134] V. Determination of Antibody Titer in Mouse Serum

[0135] Seven days after the third immunization, blood was collected from the tail tip of the mice, and serum was separated to determine the antibody ELISA titer. The mouse with the highest titer was selected for hybridoma preparation. This mouse was then boosted with the same immunogen, immunization method, and dosage as the third immunization. Three days later, the serum was diluted 1:16000 (using PBS as the diluent) and OD was measured. 450nm Mice with >0.7 were used for subsequent cell fusion.

[0136] The specific steps for detecting antibody titers in mice using the indirect ELISA method are as follows:

[0137] ① Coating: Using SHG19-VP2 as the coating antigen, dilute it to 100 ng / 100 μl with carbonate buffer (pH=9.6), add 100 μl to each well of a 96-well microplate, and coat overnight at 4°C;

[0138] ② Blocking: Discard the liquid in the wells, wash the wells 3 times with PBST, add 100 μl of 5% skim milk to each well, and incubate at 37°C for 1 h;

[0139] ③ Dilution of the serum to be tested: Dilute the mouse serum 1000 times with serum diluent (PBS containing 5 v / v% fetal bovine serum), and then perform a 2-fold serial dilution to 1:64000 for later use;

[0140] ④ After blocking, discard the skim milk, wash once with PBST, add 100 μl of diluted serum to each well, one well for each gradient, and incubate at 37°C for 1 h.

[0141] ⑤ Discard the supernatant, wash 5 times with PBST, add 100 μl of enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG antibody) (1:3000 volume dilution) to each well, and incubate at 37°C for 1 h;

[0142] ⑥ Discard the supernatant, wash 5 times with PBST, add 100 μl of TMB colorimetric solution to each well, and react at room temperature for 15 min;

[0143] ⑦ Add 100 μl of 2M H2SO4 stop solution to each well and read the OD using a microplate reader. 450nm .

[0144] VI. Cell Fusion

[0145] SP2 / 0 myeloma cells were resuscitated one week before cell fusion. After the cells recovered, they were cultured in a larger volume. The medium was changed the night before fusion to maintain cell growth activity.

[0146] The specific steps of cell fusion are as follows:

[0147] (1) Spleen isolation in mice: Mice were anesthetized and euthanized by cervical dislocation, and their bodies were disinfected by immersion in 75% alcohol for 15 minutes. The mice were then placed supine on a dissection board in a laminar flow hood and fixed to fully expose the abdomen. All subsequent steps were performed under aseptic conditions. The skin below the midline of the abdomen was lifted with forceps, and the abdominal cavity was cut open to the muscle layer with scissors. Starting from the incision, the cuts were made upward to the thoracic cavity, and 1.5 cm were cut to the left and right. The skin was then spread out to expose the inside of the abdominal cavity. Throughout the process, the forceps and scissors should not touch the organs inside the abdominal cavity. The spleen (dark reddish-brown) was located on the left side of the abdominal cavity using unused autoclaved forceps. After locating the spleen, the connective tissue and fat around the spleen were carefully separated with forceps. After cleaning, the spleen was transferred to preheated DMEM medium without serum at 37°C.

[0148] (2) Spleen cell isolation: The spleen surface fat was cleaned in serum-free DMEM medium and transferred to a cell culture dish. Using a 10 ml sterile syringe with a 1 ml syringe needle, 10 ml of serum-free DMEM medium was drawn up. Holes were made evenly on the surface of the spleen, and the medium was injected from both ends of the spleen to wash the spleen cells. Initially, the flushed medium was turbid, but the process was stopped when the medium became clear and the spleen turned white. The turbid medium in the cell culture dish was then transferred to a 50 ml centrifuge tube, centrifuged at 1000 g for 10 min, the supernatant was discarded, and 10 ml of serum-free DMEM medium was added to resuspend the cells.

[0149] (3) Preparation of SP2 / 0 cells: Take 3 bottles of SP2 / 0 cells in good growth condition, discard the culture medium, and wash away dead cells with serum-free DMEM medium. Then blow off the cells with 25 ml of serum-free DMEM medium, transfer them to a 50 ml centrifuge tube, centrifuge at 1000 g for 10 min, discard the supernatant, and resuspend them in 10 ml of serum-free DMEM medium.

[0150] (4) Cell fusion: After uniformly mixing the suspensions of immune mouse spleen cells and SP2 / 0 myeloma cells, centrifuge at 1000 g for 10 min and discard the supernatant. Gently tap the bottom of the tube to loosen the cell pellet, place it in a beaker in a 37℃ water bath, and evenly drop 1 ml of fusion reagent PEG onto the mixed cells, adding it slowly for 1 min, not too quickly. Then allow it to stand for 4 min, add 20 ml of blank DMEM to terminate the fusion, adding it slowly at first and then quickly for 4 min.

[0151] (5) Centrifuge the fused cell suspension at 1000 g for 10 min, discard the supernatant, resuspend the cells in 100 ml of DMEM complete medium containing HAT and 10% Hybridoma Feeder, and seed 200 μl per well into a 96-well cell culture plate. Incubate at 37°C and 5% CO2 for 7 days.

[0152] VII. Screening of positive cell lines with significance for the differential detection of nVarIBDV

[0153] Seven days after fusion, all 96-well plates were examined microscopically, and all wells showing cell growth were marked. The number of clones and cell status in each well were recorded. Eight days later, the supernatant from each hybridoma cell was aspirated, and the antibody titer was detected using an IBDV SHG19-VP2-coated ELISA plate. Specific procedures are detailed in Section 5. OD was selected. 450nm The 15 wells with the highest values ​​and largest P / N ratios were designated as positive wells and cultured further. These 15 positive wells were then cultured in 6-well plates, and positive cell lines that specifically recognized nVarBDV but not vvIBDV were selected using indirect immunofluorescence (IFA) and ELISA for subsequent processing.

[0154] (1) Specific steps of IFA

[0155] ① Recombinant IBDV rGtHLJVP2 (expressing vvIBDV HLJ0504 VP2) and rGtVarVP2 (expressing nVarIBDV SHG19 VP2) were inoculated into 24-well plates containing DF1 cells at a seeding volume of 0.1 MOI. After 36 h, the culture medium was discarded, the cells were washed 3 times with PBS, and fixed with 5 v / v% paraformaldehyde aqueous solution for 20 min.

[0156] ② Discard the 5 v / v % paraformaldehyde aqueous solution, wash 3 times with PBS, add 5% skim milk, and incubate at 37°C for 30 min;

[0157] ③ Discard the skim milk, wash once with PBS, add the supernatant of hybridoma cells from the 6-well plate as the primary antibody, and incubate at 37°C for 1 hour;

[0158] ④ Discard the supernatant, wash 5 times with PBS, add FITC-labeled goat anti-mouse IgG (secondary antibody, purchased from Sigma), and incubate at 37°C for 1 h;

[0159] ⑤ Discard the secondary antibody, wash three times with PBS, add 500 μl of PBS to each well to cover, and finally observe the fluorescence using an inverted fluorescence microscope. Select hybridoma cell wells infected with rGtVarVP2 that show fluorescence, while those infected with rGtHLJVP2 do not, for subsequent operations.

[0160] (2) Indirect ELISA

[0161] Hybridoma cell supernatant from 6-well plates was used to detect antibody titers in ELISA plates coated with SHG19-VP2 (see Section 5 for details) and HLJ0504-VP2 (see Section 5 for details, the difference being that HLJ0504-VP2 was used instead of SHG19-VP2 for coating; all other materials, parameters, and procedures were the same). Hybridoma cell wells that reacted with SHG19-VP2 but not with HLJ0504-VP2 were selected for subsequent operations.

[0162] The hybridoma cell line that meets the requirements for both IFA and ELISA in 15 positive wells and has the best effect is called hybridoma cell a.

[0163] VIII. Subcloning of Positive Hybrid Cell Lines

[0164] Two subclonings of the selected positive hybridoma cells (a) were performed using a flow cytometry sorting system. The supernatant of the hybridoma cells to be subcloned in the six-well plates was discarded, and the cells were resuspended in 1 ml of HT medium. Four 96-well cell culture plates were prepared, with 200 μl of HT medium added to each well. Using an ultra-high-speed flow cytometry sorting system, the hybridoma cells to be cloned were transferred into the 96-well plates, one cell per well. The plates were incubated at 37°C for 5-8 days. Wells showing single-clone growth were strictly selected, and the supernatant was subjected to antibody detection and differential screening using IFA and ELISA, following the same methods as in Section 7. Subsequently, positive clones were expanded through transfer from the 96-well plates to 24-well plates and gradually expanded. Finally, the cells were frozen in multiple passages to obtain the selected hybridoma cells capable of secreting the nVarBDV differential monoclonal antibody.

[0165] A hybridoma cell line that secretes a monoclonal antibody against IBDVVP2 protein was obtained by screening using cell fusion technology, flow cytometry, IFA, and indirect ELISA. It was named IBDV-VP2-2F10 (abbreviated as 2F10), and the monoclonal antibody it secretes has the same name.

[0166] IX. Preparation and Purification of Monoclonal Antibody Ascites Fluid

[0167] Ten healthy six-week-old female BALB / c mice were intraperitoneally injected with Freund's complete adjuvant, 300 μl per mouse, one week prior to the treatment. Positive hybridoma cells (2F10) in the logarithmic growth phase were dispersed into a suspension using serum-free DMEM medium, and the cell number was adjusted to 6.6 × 10⁻⁶. 7 0.3 mL (approximately 2 × 10⁶ cells / mL) was administered intraperitoneally to each mouse. 6 (Number of cells). Four days later, the abdominal swelling of the mice was observed daily. Ascites fluid was collected when the abdomen became rounded, and slowly aspirated using a syringe. The ascites fluid was centrifuged at 12000 g for 30 min, and the middle layer of pale yellow, clear ascites fluid was collected and stored at -80℃ for later use. After injecting positive hybridoma cells into mice via intraperitoneal injection, 22 ml of ascites fluid containing IBDV-VP2-2F10 monoclonal antibody was successfully prepared and stored at -80℃.

[0168] 10. Monoclonal antibody ELISA titer determination

[0169] The monoclonal antibody ascites fluid was serially diluted 2-fold, and 2 samples were selected. 1 -2 19 For the monoclonal antibody ascites of the dilution range, the antibody titer was detected using ELISA plates coated with SHG19-VP2 protein and HLJ0504-VP2 protein, respectively. The specific steps are described in Section 7 (2).

[0170] Data showed that the indirect ELISA result coated with HLJ0504-VP2 was negative; the indirect ELISA result coated with SHG19-VP2 was positive, and the IBDV-VP2-2F10 monoclonal antibody titer was 2. 17 ( Figure 1 Therefore, it can be seen that the IBDV-VP2-2F10 monoclonal antibody can identify SHG19-VP2 by indirect ELISA, but not HLJ0504-VP2.

[0171] XI. Monoclonal Antibody Subtype Determination

[0172] The supernatant of the selected monoclonal antibody hybridoma cell line 2F10 was identified using the ELISA kit for mouse monoclonal antibody Ig class / subtype identification (Biodragon). For specific steps, please refer to the kit's instructions.

[0173] The results of the ELISA kit for identifying mouse monoclonal antibody Ig class / subtypes showed that the IBDV-VP2-2F10 monoclonal antibody heavy chain type was IgG1, and the light chain type was Kappa (IgG1). Figure 2 ).

[0174] XII. Determination of the stability of antibodies secreted by hybridoma cells

[0175] Hybridoma cell line 2F10 was passaged 20 times, and the cell supernatant from each generation was collected for ELISA antibody detection. The specific method is described in Section 5.

[0176] Hybridoma cells IBDV-VP2-2F10 were passaged to the 20th generation. The OD (oxidative stress) in the cell supernatant was detected using both SHG19-VP2 protein-coated indirect ELISA and HLJ0504-VP2 protein-coated indirect ELISA. 450nm For specific steps, see Section 7(2). The test results show ( Figure 3 For ELISA coated with HLJ0504-VP2 protein, the antibody in the cell supernatant was negative; for ELISA coated with SHG19-VLP protein, the OD in the cell supernatant was negative. 450nm Fluctuations between 0.6 and 0.8 indicate a positive antibody result; hybridoma cells IBDV-VP2-2F10 can stably secrete monoclonal antibodies against SHG19-VP2.

[0177] XIII. Preliminary Applications of Monoclonal Antibodies

[0178] The monoclonal antibody IBDV-VP2-2F10 was used as the primary antibody, and IFA was used to detect the representative strain of nVarIBDV (SHG19), the representative strain of vvIBDV HLJ0504, and their encoded VP2 protein. The ability of this monoclonal antibody to identify and detect nVarIBDV was analyzed from both viral and protein perspectives.

[0179] (a) Identification and detection of IBDV

[0180] DT40 cells cultured in 96-well plates were inoculated with nVarIBDV (SHG19) and vvIBDV (HLJ0504) as representative strains, respectively, at a dose of 1×10⁶. 3 TCID 50 / well. After 48 hours, the recognition of the virus by the monoclonal antibody IBDV-VP2-2F10 was detected by IFA. Simultaneously, a positive control group was set up using the universal IBDV VP2 monoclonal antibody 7D4 as the primary antibody; a negative control group (Mock) was set up using PBS instead of the primary antibody. See Section 7 for the specific IFA procedure.

[0181] The reactivity of the monoclonal antibody to two IBDV strains (SHG19 or HLJ0504) showed ( Figure 4 a) In DT40 cells infected with the SHG19 strain, the 2F10 group showed obvious specific green fluorescence signal; in DT40 cells infected with the HLJ0504 strain, the 2F10 group did not show green fluorescence signal; the positive control 7D4 group showed specific green fluorescence signal in cells infected with both viruses; the Mock group did not show specific green fluorescence signal in cells infected with either virus.

[0182] (II) Identification and detection of IBDV VP2 protein

[0183] The recombinant eukaryotic expression plasmid pCASHGVP2 (expressing SHG19-VP2 protein) was used to express the protein. P CAHLJVP2 (expressing HLJ0504-VP2 protein) was transfected into DF1 cells cultured in 24-well plates at a transfection dose of 1 μg / well. After 48 h, the recognition of the viral VP2 protein by the monoclonal antibody IBDV-VP2-2F10 was detected using in vitro anabolism (IFA). A positive control group was established using the universal IBDV VP2 monoclonal antibody 7D4 as the primary antibody; a negative control group (Mock) was established using PBS instead of the primary antibody. The specific IFA procedure is detailed in Section 7.

[0184] The reactivity of the monoclonal antibody to the VP2 protein (SHG19 or HLJ0504) of two IBDV strains showed ( Figure 4(b) In DF1 cells expressing SHG19-VP2, the 2F10 group showed a distinct green fluorescence signal; in DF1 cells expressing HLJ0504-VP2, the 2F10 group did not show a green fluorescence signal; the positive control 7D4 group showed a distinct green fluorescence signal in cells expressing both types of VP2; the Mock group showed no distinct green fluorescence signal in cells expressing both types of VP2.

[0185] This invention also detected vvIBDV Gx strain (GenBank accession numbers: AY444873.3 and AY705393.2, VP2 protein sequence shown in GenBank accession number AAR14320.1, positions 1-441) and nVarIBDV SHG352 strain (GenBank accession numbers: MT179720.1 and MT179722.1, VP2 protein sequence shown in GenBank accession number QKI85215.1, positions 1-441) and obtained results consistent with... Figure 4 The same pattern yielded results (vvIBDV Gx strain did not react with IBDV-VP2-2F10, while SHG352 strain reacted with IBDV-VP2-2F10; both strains reacted with 7D4). This demonstrates that the monoclonal antibody IBDV-VP2-2F10 specifically recognizes and detects nVarIBDV.

[0186] The VP2 protein sequence of strain Gx is as follows (SEQ ID NO.3):

[0187] MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIVGAHYTLQSNGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYALNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAADDYQFSSQYQAGGVTITLFSANIDAITSLSIGGELVFQTSVQGLILGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNIVIPTSEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGA

[0188] The VP2 protein sequence of the SHG352 strain is as follows (SEQ ID NO.4):

[0189] MTNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIVGAHYTLQSDGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYALNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAADNYQFSSQYKTGGVTITLFSANIDAITSLSVGGELVFKTSIQNLVLGATIYLIGFDGTAVITRAVAANNGLTAGIDNLMPFNLVIPTSEITQPITSIKLEIVTSKSDGQAGEQMSWSASGSLAVTIHGGNYPGALRPVTLVAYERVAKGSVVTVAGVSNFELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGA

[0190] XIV. Depositing of Hybridoma Cells

[0191] The hybridoma cell line 2F10 prepared by the present invention was submitted to a patent procedure-approved preservation institution for preservation. The preservation unit is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial preservation number is CCTCC NO: C2024404; the name of the culture is: Hybridoma cell line IBDV-VP2-2F10 Hybridoma cell line IBDV-VP2-2F10; the Chinese classification name is: Hybridoma cell line; the English classification name is: Hybridoma Cell Line; the preservation time is December 18, 2024; the identified survival time is December 26, 2024.

[0192] Example 2 Identification of the antigenic epitope of monoclonal antibody IBDV-VP2-2F10

[0193] I. Identification of the epitope recognized by monoclonal antibody based on peptide scanning technology

[0194] (1) Western blotting

[0195] The recombinant plasmids pCASHGVP2 and pCAHLJVP2 were transfected into DF1 cells cultured in a six-well plate, and the transfection dose was 2 μg / well. After 48 h, the cells were collected and protein samples were prepared. The primary antibody was monoclonal antibody IBDV-VP2-2F10, and the secondary antibody was IRDye800CW-conjugated goat anti-mouse IgG antibody. Western blotting was used to detect the reaction of monoclonal antibody IBDV-VP2-2F10 to SHG19-VP2 and HLJ504-VP2, respectively.

[0196] The results of Western blotting showed that in the transfected DF1 cells, monoclonal antibody IBDV-VP2-2F10 could neither recognize SHG19-VP2 nor HLJ050 Figure 5 4-VP2 (

[0197] (2) IFA

[0198] To identify the antigenic epitopes recognized by the monoclonal antibody IBDV-VP2-2F10 using peptide scanning technology, three overlapping truncated variants covering the full length of SHG19-VP2—SHG19VP2-TP1 (aa 1-161), SHG19VP2-TP2 (aa 141-305), and SHG19VP2-TP3 (aa 261-441)—were constructed using SHG19-VP2 (i.e., the corresponding recombinant eukaryotic expression plasmid pCAGGS) as the parental backbone. These were then cloned into the eukaryotic expression vector pCAGGS to construct the corresponding recombinant eukaryotic expression plasmids. pCASHGVP2 and the three truncated recombinant eukaryotic expression plasmids were transfected into DF-1 cells. Simultaneously, a pCAHLJVP2 transfection control and a blank cell control (Mock) without plasmid transfection were set up. Twenty-four hours after transfection, the reaction of the monoclonal antibody IBDV-VP2-2F10 with the plasmid was detected using IFA. For the specific operating steps of IFA, please refer to Example 1. The difference is that FITC-labeled goat anti-mouse IgG is replaced with TRITC-labeled rabbit anti-mouse IgG as the secondary antibody (the secondary antibody was purchased from Sigma).

[0199] IFA results showed that the monoclonal antibody IBDV-VP2-2F10 reacted only with the full-length SHG19-VP2, and did not react with any of the three truncated variants of SHG19-VP2 or HLJ0504-VP2. Figure 5 B and Figure 5 C). Because IFA detects proteins with a stereoconformation, this data indicates that the epitope recognized by the monoclonal antibody IBDV-VP2-2F10 is a stereoconformation epitope, and its specific recognition of SHG19-VP2 depends on the full-length stereoconformation of the VP2 protein.

[0200] II. Identification of recognition epitopes for monoclonal antibodies based on point mutations

[0201] (1) Sequence comparison

[0202] To further identify the key amino acids that the monoclonal antibody IBDV-VP2-2F10 specifically recognizes the conformational epitope of nVarIBDV, the amino acid sequences of VP2 from the representative nVarIBDV strain SHG19 (GenBank ID: MN393076.1) and the representative vvIBDV strain HLJ0504 (GenBank ID: GQ451330.1) were compared.

[0203] The comparison results showed that SHG19-VP2 had 19 amino acid differences compared to HLJ0504-VP2. Figure 6 ).

[0204] (2) IFA detection of SHG19-VP2 mutant

[0205] To target 19 differentially expressed amino acids in SHG19-VP2 and HLJ0504-VP2, 19 single-point mutations of VP2 were constructed using SHG19-VP2 as the parental backbone, targeting 19 differentially expressed amino acids from SHG19-VP2 to HLJ0504-VP2. (That is, the 19 differentially expressed amino acids from SHG19-VP2 were mutated to amino acids from HLJ0504-VP2, while the other 18 sites retained the amino acids from SHG19-VP2.) These mutants were cloned into the eukaryotic expression vector pCAGGS to construct the corresponding recombinant eukaryotic expression plasmids. pCAGGS and the recombinant eukaryotic expression plasmids of the 19 mutants were transfected into DF1 cells cultured in six-well plates at a transfection dose of 2 μg / well. A pCAGGS empty plasmid transfection control and a blank cell control (Mock) without plasmid transfection were also included. 24 hours after transfection, the reaction of monoclonal antibody IBDV-VP2-2F10 with IBDV broad-spectrum monoclonal antibody 7D4 was detected by IFA using monoclonal antibody IBDV-VP2-2F10 and IBDV broad-spectrum monoclonal antibody 7D4, respectively. The specific IFA operation procedure was the same as in Example 1.

[0206] IFA testing results for these 19 amino acid mutants showed that, among all 19 VP2 mutants, the broad-spectrum IBDV monoclonal antibody 7D4 could recognize all VP2 mutants; while the monoclonal antibody IBDV-VP2-2F10 could recognize 18 of the VP2 mutants, but could not recognize SHGVP2-D318G (where amino acid 318 of SHGVP2 is mutated from D to G). Figure 7 Therefore, the aspartic acid (D) at position 318 of VP2 is a key amino acid in the conformational epitope recognized by the monoclonal antibody IBDV-VP2-2F10; mutations in the other 18 amino acids do not affect the recognition and binding of the monoclonal antibody IBDV-VP2-2F10 to nVarIBDV. Sequence analysis shows that D318G is a conserved difference between nVarIBDV and vvIBDV.

[0207] In summary, the monoclonal antibody IBDV-VP2-2F10 can specifically recognize nVarIBDV and can be used for the identification and detection of the two current epidemic strains, nVarIBDV and vvIBDV. The aspartic acid (D) at position 318 of VP2 is the key amino acid of the conformational epitope recognized by the monoclonal antibody IBDV-VP2-2F10.

[0208] Example 3: Sequencing of the variable region of monoclonal antibody IBDV-VP2-2F10

[0209] The hybridoma cell line IBDV-VP2-2F10 was revived and cultured in DMEM medium containing 10% fetal bovine serum. After the cells reached confluence, the cell density was measured and the cells were aliquoted, with each tube containing at least 1 × 10⁶ cells. 6 Cells were aliquoted and centrifuged at 300g for 5 min at room temperature. The culture medium was discarded, and the cells in each aliquot were resuspended in 1 ml of Trizol. Genomic RNA was extracted from hybridoma cells and reverse transcribed into cDNA using SMARTScribe Reverse Transcriptase. The antibody heavy chain variable region nucleotide sequence and antibody light chain variable region nucleotide sequence of the monoclonal antibody IBDV-VP2-2F10 cDNA were amplified using a rapid amplification of cDNA ends (RACE) kit (GenScript). The obtained PCR products were cloned into a T vector and sequenced using the Sanger method. Each cDNA fragment was sequenced 5 times, and the results consistently confirmed the accuracy of the sequence.

[0210] The nucleotide sequences corresponding to the heavy chain variable region and light chain variable region of the monoclonal antibody IBDV-VP2-2F10 were obtained by sequencing and analysis using the IMGT method. The sequence information is as follows.

[0211] (1) The nucleotide sequence (405 bp) of the heavy chain variable region is as follows (SEQ ID NO.5):

[0212] ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTATCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACTTTCACAGACTATTCAATGCACTGGGTGAAGCAGACTCCAGGAAAGGGTTTAAAGTGGATGG GCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAGACCTCTGACAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGATCTGCGGGGGGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0213] Among them, the signal peptide is at positions 1-57, FR1 is at positions 58-147, CDR1 is at positions 148-162, FR2 is at positions 163-204, CDR2 is at positions 205-255, FR3 is at positions 256-351, CDR3 is at positions 352-372, and FR4 is at positions 373-405.

[0214] (2) The amino acid sequence (135 aa) of the heavy chain variable region is as follows (SEQ ID NO.6):

[0215] MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQTPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSDSTAYLQINNLKNEDTATYFCARSAGGFTYWGQGTLVTVSA

[0216] Among them, the signal peptide is at positions 1-19, FR1 is at positions 20-49, CDR1 is at positions 50-54, FR2 is at positions 55-68, CDR2 is at positions 69-85, FR3 is at positions 86-117, CDR3 is at positions 118-124, and FR4 is at positions 125-135.

[0217] (3) The nucleotide sequence of the light chain variable region (378 bp) is as follows (SEQ ID NO.7):

[0218] ATGCAGATTATCAGCTTGCTGCTAATCAGTGTCACAGTCATAGTGTCTGATGGAGAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCGGTGTCAGTTCAAGTATAAGTTCCAAGTACTTGTATTGGTTTCAGCAGAAGCCAGGATTCTCCCCT AAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0219] Among them, the signal peptide is at positions 1-54, FR1 is at positions 55-123, CDR1 is at positions 124-159, FR2 is at positions 160-204, CDR2 is at positions 205-225, FR3 is at positions 226-321, CDR3 is at positions 322-348, and FR4 is at positions 349-378.

[0220] (4) The amino acid sequence (126 aa) of the light chain variable region is as follows (SEQ ID NO.8):

[0221] MQIISLLLISVTVIVSDGEIVLTQSPTTMAASPGEKITITCGVSSSISSKYLYWFQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPLTFGAGTKLELK

[0222] Among them, the signal peptide is at positions 1-18, FR1 is at positions 19-41, CDR1 is at positions 42-53, FR2 is at positions 54-68, CDR2 is at positions 69-75, FR3 is at positions 76-107, CDR3 is at positions 108-116, and FR4 is at positions 117-126.

[0223] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A hybridoma cell, wherein the hybridoma cell is hybridoma cell A or hybridoma cell B; The hybridoma cell A is a hybridoma cell with the microbial preservation number CCTCC NO: C2024404; The hybridoma cell B is a passaged cell of the hybridoma cell with the microbial preservation number CCTCC NO: C2024404; In the monoclonal antibody gene contained in the genome of the hybridoma cell B, the heavy chain CDR1 maintains the amino acid sequence encoding positions 50-54 of SEQ ID NO. 6; The heavy chain CDR2 retains the amino acid sequence encoding positions 69-85 of SEQ ID NO. 6; The heavy chain CDR3 retains the amino acid sequence encoding positions 118-124 of SEQ ID NO. 6; The light chain CDR1 retains the amino acid sequence encoding positions 42-53 of SEQ ID NO. 8; The light chain CDR2 retains the amino acid sequence encoded as shown in positions 69-75 of SEQ ID NO. 8; The light chain CDR3 retains the amino acid sequence encoding positions 108-116 of SEQ ID NO. 8; The monoclonal antibody secreted by the hybridoma cell B maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.

4.

2. A biomaterial, said biomaterial being any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.

4. The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The monoclonal antibody light chain includes light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6; The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6; The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8; The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8; The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8; P2: Genetically engineered monoclonal antibody The genetically engineered monoclonal antibody maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.

4. The genetically engineered monoclonal antibody includes a genetically engineered monoclonal antibody heavy chain and a genetically engineered monoclonal antibody light chain. The genetically engineered monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification. The genetically engineered monoclonal antibody light chain includes light chain CDR1, light chain CDR2, light chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification. The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6; The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6; The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8; The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8; The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8; P3: Antibody derivatives The antibody derivative maintains specific binding activity to proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4, or to fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.4; The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3. The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO. 6; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO. 6; The heavy chain CDR3 protein sequence is shown in positions 118-124 of SEQ ID NO. 6; The light chain CDR1 protein sequence is shown in positions 42-53 of SEQ ID NO. 8; The light chain CDR2 protein sequence is shown in positions 69-75 of SEQ ID NO. 8; The light chain CDR3 protein sequence is shown in positions 108-116 of SEQ ID NO. 8; The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, avian-derived antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA assembly The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2. The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2. P5: Gene Combination The gene combination includes monoclonal antibody heavy chain genes and monoclonal antibody light chain genes; The coding sequence of the monoclonal antibody heavy chain gene can encode the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2. The coding sequence of the monoclonal antibody light chain gene can encode the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2. P6: Gene Expression Catalyst Assembly The gene expression cassette assembly includes a monoclonal antibody heavy chain gene expression cassette and a monoclonal antibody light chain gene expression cassette. The gene expression product in the monoclonal antibody heavy chain gene expression cassette is the monoclonal antibody heavy chain RNA described in P4. The gene expression product in the monoclonal antibody light chain gene expression cassette is the monoclonal antibody light chain RNA described in P4. The promoters of the monoclonal antibody heavy chain gene expression cassette and the monoclonal antibody light chain gene expression cassette are constitutive expression promoters or artificially inducible promoters; P7: Genetic Engineering Vector The genetic engineering vector is a combination of a first genetic engineering vector and a second genetic engineering vector or a third genetic engineering vector. The first genetic engineering vector encodes the monoclonal antibody heavy chain RNA described in P4 that can be expressed; The second genetic engineering vector encodes the monoclonal antibody light chain RNA described in P4 that can be expressed; The third genetic engineering vector encodes expressible monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA as described in P4. P8: Cells The cell is either a first cell or a second cell; The first cell contains the first and second genetic engineering vectors described in P7. The second cell contains the third gene engineering vector described in P7; P9: Composition The composition comprises the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA assembly described in P4, the genetically engineered vector described in P7, or the cell described in P8; and P10: Reagent Kit The kit contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7, or the cells described in P8.

3. The biomaterial as described in claim 2, characterized in that, In P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cells as described in claim 1.

4. The biomaterial as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody or the heavy chain variable region of the genetically engineered monoclonal antibody is shown in positions 20-135 of SEQ ID NO. 6; The amino acid sequence of the light chain variable region of the monoclonal antibody or the light chain variable region of the genetically engineered monoclonal antibody is shown in positions 20-126 of SEQ ID NO.

8.

5. Use of the hybridoma cells of claim 1, the monoclonal antibody of claim 2 or 3, the genetically engineered monoclonal antibody of claim 2 or 4, the antibody derivative of claim 2, the genetically engineered vector of claim 2 or 4, or the cells of any one of claims 2-4 in the preparation of formulations for recognizing proteins with amino acid sequences as shown in SEQ ID NO. 1 or SEQ ID NO. 4, or fusion proteins containing peptides with amino acid sequences as shown in SEQ ID NO. 1 or SEQ ID NO.

4.

6. A method for detecting the presence of an infectious bursal disease virus strain in a sample for non-diagnostic purposes, wherein the amino acid sequence of the VP2 protein of the infectious bursal disease virus strain is shown in SEQ ID NO.1 or SEQ ID NO.4; The method includes the following steps: S1: Inoculate the sample to be tested into susceptible cells for infectious bursal disease virus in chickens to obtain the inoculated cells; S2: The seeded cells are fixed onto a solid surface to obtain immobilized cells; S3: Incubate the immobilized cells with a monoclonal antibody to obtain the first incubation of immobilized cells; The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody as described in any one of claims 2-4; S4: Add the labeled monoclonal antibody-specific conjugate to the immobilized cells after the first incubation and incubate to obtain immobilized cells after the second incubation. S5: Characterize the marker that is indirectly bound to the immobilized cells in the second incubation, and determine whether the chicken infectious bursal disease virus strain is present in the sample to be tested based on the presence or absence of the marker.

7. The method as described in claim 6, characterized in that, In step S1, the susceptible cells for infectious bursal disease virus in chickens are DF1 cells.

8. The method as described in claim 6 or 7, characterized in that, In step S2, the solid surface is the inner surface of the pores of the microporous plate.

9. The method according to any one of claims 6-8, characterized in that, In step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step S5, the immobilized cells after the second incubation are photographed using a fluorescence microscope, and the presence of the infectious bursal disease virus strain in the sample to be tested is determined based on the photographic results.

10. The method according to any one of claims 6-8, characterized in that, In step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with horseradish peroxidase; in step S5, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of the chicken infectious bursal disease virus strain in the sample to be tested is determined based on the chromogenic result.

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