Poultry reticuloendotheliosis virus gp90 protein monoclonal antibody

Abstract

The invention discloses a hybridoma cell. The microbial preservation number of the hybridoma cell is CCTCC (China Center For Type Culture Collection) NO: C2024405. The invention also discloses a monoclonal antibody secreted by the hybridoma cell. The invention also discloses an application of the monoclonal antibody in recognition of avian reticuloendotheliosis virus gp90 protein. The monoclonal antibody has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological products and relates to a monoclonal antibody against the gp90 protein of avian reticuloendotheliosis virus. Background Technology

[0002] Avian reticuloendotheliosis virus (REV) is an important retrovirus that infects poultry such as chickens and turkeys. The avian reticuloendotheliosis (RE) it causes is mainly characterized by immunosuppression, tumorigenesis, and growth retardation. REV can significantly reduce flock immunity and production performance through both vertical and horizontal transmission. It is also prone to co-infection or genetic recombination with pathogens such as fowlpox virus and Marek's virus, leading to secondary risks such as enhanced virulence and vaccine contamination. The REV envelope protein gp90 is a key protein mediating viral invasion of host cells. It participates in cell receptor binding and viral assembly and release, and can also induce the production of neutralizing antibodies, making it a core target for diagnostic reagents and vaccine development. Located on the outermost side of the REV particle, gp90 acts as a core mediator of viral invasion, participating in viral adsorption, assembly, and release, and inducing the production of neutralizing antibodies, making it an important target for REV diagnostic reagents and vaccine development. Summary of the Invention

[0003] To address the problems existing in the prior art, the first aspect of the present invention provides a hybridoma cell, wherein the hybridoma cell is a hybridoma cell with the microbial preservation number CCTCC NO:C2024405 or a passaged cell of a hybridoma cell with the microbial preservation number CCTCC NO:C2024405.

[0004] The monoclonal antibody secreted by the passaged cells of the hybridoma cells with the microbial preservation number CCTCC NO:C2024405 maintains specific binding activity to material a.

[0005] The material 'a' is selected from A1, A2, A3, and A4:

[0006] A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2;

[0007] A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2;

[0008] A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0009] A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0010] During the passage of hybridoma cells with the microbial accession number CCTCC NO:C2024405, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the monoclonal antibodies secreted by the passaged cells of the hybridoma cells with the microbial accession number CCTCC NO:C2024405 did not undergo any mutations compared to the monoclonal antibodies secreted by the hybridoma cells with the microbial accession number CCTCC NO:C2024405.

[0011] 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;

[0012] P1: Monoclonal antibody

[0013] The monoclonal antibody is the monoclonal antibody secreted by the hybridoma cells described in the first aspect of this invention;

[0014] P2: Genetically engineered monoclonal antibody

[0015] The genetically engineered monoclonal antibody maintains specific binding activity to material a;

[0016] The material 'a' is selected from A1, A2, A3, and A4:

[0017] A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2;

[0018] A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2;

[0019] A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0020] A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0021] The genetically engineered monoclonal antibody includes a heavy chain and a light chain of the genetically engineered monoclonal antibody.

[0022] The heavy chain of the genetically engineered monoclonal antibody includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the genetically engineered monoclonal antibody.

[0023] The light chain of the genetically engineered monoclonal antibody includes light chain CDR1, light chain CDR2, and light chain CDR3.

[0024] The heavy chain CDR1 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR1 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0025] The heavy chain CDR2 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR2 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0026] The heavy chain CDR3 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR3 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0027] The light chain CDR1 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR1 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0028] The light chain CDR2 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR2 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0029] The light chain CDR3 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR3 of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0030] P3: Antibody derivatives

[0031] The antibody derivative maintains specific binding activity to material a;

[0032] The material 'a' is selected from A1, A2, A3, and A4:

[0033] A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2;

[0034] A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2;

[0035] A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0036] A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0037] The heavy chain CDR1 of the antibody derivative is identical to the heavy chain CDR1 protein sequence of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0038] The heavy chain CDR2 of the antibody derivative is identical to the heavy chain CDR2 protein sequence of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0039] The heavy chain CDR3 of the antibody derivative is identical to the heavy chain CDR3 protein sequence of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0040] The light chain CDR1 of the antibody derivative has the same protein sequence as the light chain CDR1 of the monoclonal antibody secreted by the hybridoma cells described in the first aspect of the present invention.

[0041] The light chain CDR2 of the antibody derivative is identical to the light chain CDR2 protein sequence of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0042] The light chain CDR3 of the antibody derivative is identical to the light chain CDR3 protein sequence of the monoclonal antibody secreted by hybridoma cells as described in the first aspect of the present invention.

[0043] The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, single-chain antibodies, avian-derived antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0044] P4: RNA assembly

[0045] The RNA combination includes genetically engineered monoclonal antibody heavy chain RNA and genetically engineered monoclonal antibody light chain RNA;

[0046] The genetically engineered monoclonal antibody heavy chain RNA can be translated to obtain the heavy chain of the genetically engineered monoclonal antibody described in P2.

[0047] The genetically engineered monoclonal antibody light chain RNA can be translated to obtain the light chain of the genetically engineered monoclonal antibody described in P2.

[0048] P5: Gene Combination

[0049] The gene combination includes a first gene and a second gene;

[0050] The coding sequence of the first gene can encode the genetically engineered monoclonal antibody heavy chain described in P2;

[0051] The coding sequence of the second gene can encode the genetically engineered monoclonal antibody light chain described in P2;

[0052] P6: Gene Expression Catalyst Assembly

[0053] The gene expression cassette assembly includes a first gene expression cassette and a second gene expression cassette.

[0054] The gene expression product in the first gene expression cassette is the monoclonal antibody heavy chain RNA described in P4;

[0055] The gene expression product in the second gene expression cassette is the monoclonal antibody light chain RNA described in P4;

[0056] P7: Genetic Engineering Vector

[0057] 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.

[0058] The first genetic engineering vector encodes the monoclonal antibody heavy chain RNA described in P4 that can be expressed;

[0059] The second genetic engineering vector encodes the monoclonal antibody light chain RNA described in P4 that can be expressed;

[0060] The third genetic engineering vector encodes expressible monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA as described in P4.

[0061] P8: Cells

[0062] The cell is either a first cell or a second cell;

[0063] The first cell contains the first and second genetic engineering vectors described in P7.

[0064] The second cell contains the third gene engineering vector described in P7;

[0065] P9: Composition

[0066] 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

[0067] P10: Reagent Kit

[0068] 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.

[0069] In some embodiments, the heavy chain of the genetically engineered monoclonal antibody further includes a tag peptide and / or a signal peptide for separating and purifying the protein.

[0070] The light chain of the genetically engineered monoclonal antibody also includes a tag peptide and / or a signal peptide for separating and purifying the protein.

[0071] The third aspect of the present invention provides the use of the hybridoma cells described in the first aspect of the present invention, the monoclonal antibody described in the second aspect of the present invention, the genetically engineered monoclonal antibody described in the second aspect of the present invention, the antibody derivative described in the second aspect of the present invention, the genetically engineered vector described in the second aspect of the present invention, or the cells described in the second aspect of the present invention in the preparation of a formulation for recognizing material b.

[0072] Material b is selected from B1, B2, B3, B4, B5, and B6:

[0073] B1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2;

[0074] B2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2;

[0075] B3: A peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0076] B4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2;

[0077] B5: Avian reticuloendotheliosis virus encoding the gp90 protein with the amino acid sequence shown in SEQ ID NO.2;

[0078] A6: Contains GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.1, GQ415643.1, GU012639.1, GU012643.1, GQ415644.1, GU969140.1, GU012644.1, GU012646.1, GQ415645.1, DQ513316.1, D Avian reticuloendotheliosis virus whose gp90 protein amino acid sequence is recorded in any of the GenBank accessions Q513317.1, FJ439120.1, FJ439119.1, DQ387450., GU222420.1 and DQ237901.1.

[0079] A fourth aspect of the present invention provides a method for detecting avian reticuloendotheliosis virus gp90 protein in samples for non-diagnostic purposes, the method comprising the following steps:

[0080] T1: The sample to be tested is fixed onto the solid surface to obtain a fixed sample;

[0081] T2: Incubate the immobilized sample with a monoclonal antibody to obtain the first immobilized sample;

[0082] The monoclonal antibody is the monoclonal antibody described in the second aspect of the present invention or a genetically engineered monoclonal antibody.

[0083] T4: Add the labeled monoclonal antibody-specific conjugate to the immobilized sample after the first incubation and incubate to obtain the immobilized sample after the second incubation.

[0084] T5: The marker that is indirectly associated with the immobilized sample after the second incubation, and the presence or absence of the marker determines whether the avian reticuloendotheliosis virus gp90 protein is present in the sample to be tested.

[0085] In some implementations, any one of the following conditions C1, C2, C3, C4, and C5 is selected:

[0086] C1: In step T1, the sample to be tested is in the form of cell lysate;

[0087] C2: In step T1, the solid surface is the inner surface of the pores of the microporous plate;

[0088] C3: In step T3, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention with fluorescent molecular labeling; In step T4, the immobilized sample after the second incubation is photographed with a fluorescence microscope, and the presence of avian reticuloendotheliosis virus gp90 protein in the sample to be tested is determined based on the photographing results.

[0089] C4: In step T3, the labeled monoclonal antibody-specific conjugate is a secondary antibody labeled with horseradish peroxidase as described in the second aspect of the present invention or a genetically engineered monoclonal antibody; in step T4, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of avian reticuloendotheliosis virus gp90 protein in the sample to be tested is determined based on the chromogenic result.

[0090] C5: Avian reticuloendotheliosis virus gp90 protein is GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.1, GQ415643.1, G The gp90 protein of avian reticuloendotheliosis virus is the amino acid sequence of the gp90 protein recorded in any of the GenBank accessions U012639.1, GU012643.1, GQ415644.1, GU969140.1, GU012644.1, GU012646.1, GQ415645.1, DQ513316.1, DQ513317.1, FJ439120.1, FJ439119.1, DQ387450., GU222420.1, and DQ237901.1.

[0091] The fifth aspect of this invention provides a method for detecting the presence of avian reticuloendotheliosis virus strains in a sample for non-diagnostic purposes;

[0092] The method includes the following steps:

[0093] S1: The sample to be tested is inoculated into avian reticuloendotheliosis virus susceptible cells to obtain inoculated cells;

[0094] S2: The seeded cells are fixed onto a solid surface to obtain immobilized cells;

[0095] S3: Incubate the immobilized cells with a monoclonal antibody to obtain the first incubation of immobilized cells;

[0096] The monoclonal antibody is the monoclonal antibody described in the second aspect of the present invention or a genetically engineered monoclonal antibody.

[0097] 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.

[0098] S5: Characterize the marker that is indirectly bound to the immobilized cells in the second incubation, and determine whether the avian reticuloendotheliosis virus strain is present in the sample to be tested based on the presence or absence of the marker.

[0099] In some implementations, any one of the following conditions D1, D2, D3, and D4 is selected:

[0100] D1: In step S1, the avian reticuloendotheliosis virus susceptible cells are primary chicken embryo fibroblasts;

[0101] D2: In step S2, the solid surface is the inner surface of the pores of the microporous plate;

[0102] D3: 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 avian reticuloendotheliosis virus strain in the sample to be tested is determined based on the photographic results.

[0103] D4: 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 avian reticuloendotheliosis virus strain in the sample to be tested is determined based on the chromogenic result.

[0104] In some embodiments, the avian reticuloendotheliosis virus is selected from GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.1, GQ415643.1, GU012639.1, GU012643.1, GQ415644.1, GU969140.1, GU012644.1, GU012646.1, GQ415645.1, DQ513316.1, DQ513317.1, FJ439120.1, FJ The avian reticuloendotheliosis virus strain corresponding to the gp90 protein amino acid sequence recorded in any of the GenBank accessions 439119.1, DQ387450., GU222420.1, and DQ237901.1. Attached Figure Description

[0105] Figure 1 A statistical graph showing the ELISA titer of gp90 antibody in the serum of immunized mice.

[0106] Figure 2 The identification results of gp90 monoclonal antibody C16 are shown. A. Western blot image of gp90 prokaryotic protein; B. Identification results of monoclonal antibody subclass; C. Western blot image of gp90 eukaryotic protein; D. IFA image of gp90 eukaryotic protein; E. IFA image of REV. Scale bar is 400 μm.

[0107] Figure 3 A schematic diagram of the segmented representation of REV gp90.

[0108] Figure 4The results of antigenic epitope identification for REV gp90 monoclonal antibody C16 are as follows: A: gp90 protein segment identification results, B: P2 segment identification results, C: P2-2 segment identification results, D: R3 segment identification results.

[0109] Figure 5 This is a schematic diagram showing the spatial location of the identified epitopes on the predicted 3D structure of the gp90 protein. Detailed Implementation

[0110] 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.

[0111] Applications of detecting the presence of avian reticuloendotheliosis virus strains in samples for non-diagnostic purposes (not the process of identifying, studying, and determining the etiology or lesion status of living humans or animals) include, but are not limited to, the following: (1) exploring the immune patterns of avian reticuloendotheliosis virus infection; (2) exploring the immune patterns of avian reticuloendotheliosis virus mixed infection with other strains; and (3) providing environmental assessments for developing environmental disinfection strategies for avian reticuloendotheliosis virus strains.

[0112] Example 1: Cloning and expression of gp90 protein of avian reticuloendotheliosis virus

[0113] The circulating strain of avian reticuloendotheliosis virus, HLJR0901 (abbreviated as HLJR0901 strain, REV HLJR0901 strain, or HLJR0901), was isolated, identified, and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The acquisition process is recorded in reference 1, and its genome sequence is recorded in GenBank sequence number GQ415646.2.

[0114] pBlu-HLJR0901 plasmid: The recombinant plasmid pBlu-HLJR0901, which clones the genomic cDNA of strain HLJR0901, was prepared by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The preparation process is described in reference 2.

[0115] Reference 1: Deng Xiaoyun, Qi Xiaole, Gao Yulong, Gao Honglei, Qin Liting, Gao Li, Wang Xiaomei. 2010. Isolation, identification and in vitro replication of avian reticuloendotheliosis virus. Chinese Journal of Animal Infectious Diseases, 18(01):23-27.

[0116] Reference 2: Deng Xiaoyun, Qi Xiaole, Gao Honglei, Gao Yulong, Qin Liting, Gao Li, Wang Yongqiang, Wang Xiaomei. 2010. Cloning and sequence analysis of the whole genome of reticuloendothelial hyperplasia virus strain HLJR0901. Chinese Journal of Veterinary Science, 07:667-672.

[0117] The coding sequence of gp90 protein in REV HLJR0901 strain is as follows (SEQ ID NO.1):

[0118] ATGGACTGTCTCACCAACCTCCGATCCGCTGAGGGTAAAGTTGACCAGGCGGGCAAAACCCTAATTCTTCTTGTGGTTTGGTGGGGGTTTGGGACCACTGCCGAGGGTTACCCCTTGCAGCAACTTTGGGGACTGCCTTGTGACTGCTCCGGGGGATATGTCTTCTCCATACCTACCTATTACACCAACTCCCTCGATTGCGGTAGCTCCACCGCCTACCTGACTTACGGGTCCGGTACAGGGAGTTGGGGCTGGGGAGGGGGATTTAGACAACAGTGGGAGTGTGTATTTAAACCTAAGATCATACCCTCTGTGCAGGGGCAGCCAGGGCCCTGCCCATCCGAATGCCTCACGATAGCTACCCAAATGCATTCCACCTGTTATGAAAAGGCTCAGGAATGCACCCTCCTGGGAAAAACTTATTTTACTGCCATCCTACAGAAAACAAAGCTAGGTTCGTATGAAGACGGGCCTAATAAACTGCTTCAAGCCTCTTGCACGGGAACCATAGGGAAACCAGTATGTTGGGACCCCGTAGCCCCTGTGTATGTCTCTGATGGGGGCGGTCCCACTGACATGATTCGGGAAGAATCTGTGCGTGAAAGACTAGAGGAAATCATCAGGCACAGCTACCCCTCCGTACAGTATCACCCTTTAGCCCTGCCCCGACCAAGAGGAGTAGATCTGGATCCCCAGACGTCTGACATACTGGAAGCTACTCACCAGGTCCTTAACGCCACTAATCCCCAGCTAGCAGAGAACTGCTGGCTTTGTATGACTCTTGGAACTCCAATCCCCGCAGCCATCCCGGCGAATGGCAATGTCACTCTCGATGGAAATTGCAGCCTTAGCCTCCCCTTTCGGGTGCAACCCACCGGGTCGATAGATGTCAACTGCTATGCAGGGGAAGCAGACAATAGGACTGGTATACCCATAGGGTATGTTCATTTCACTAACTGCACTAGCATCCAGGAGGTCTCTAACGAGACAAGTCATATAAGAAATCTTACGAGGCTATGTCCTCCACCGGGTCATGTATTTGTGTGTGGGAACAACATGGCCTACACGGCGCTCCCTAATAAATGGATAGGGCTGTGCATACTGGCATCAATCGTACCCGACATGAGCATAATATCCGGGGAAGAGCCTATCCCACTCCCATCCATCGAGTACACCGCTGGGCGTCATAAG

[0119] The amino acid sequence of gp90 protein from strain REV HLJR0901 is as follows (SEQ ID NO.2):

[0120] MDCLTNLRSAEGKVDQAGKTLILLVVWWGFGTTAEGYPLQQLWGLPCDCSGGYVFSIPTYYTNSLDCGSSTAYLTYGSGTGSWGWGGGFRQQWECVFKP KIIPSVQGQPGPCPSECLTIATQMHSTCYEKAQECTLLGKTYFTAILQKTKLGSYEDGPNKLLQASCTGTIGKPVCWDPVAPVYVSDGGGPTDMIREES VRERLEEIIRHSYPSVQYHPLALPRPRGVDLDPQTSDILEATHQVLNATNPQLAENCWLCMTLGTPIPAAIPANGNVTLDGNCSLSLPFRVQPTGSIDVNCYAGEADNRTGIPIGYVHFTNCTSIQEVSNETSHIRNLTRLCPPPGHVFVCGNNMAYTALPNKWIGLCILASIVPDMSIISGEEPIPLPSIEYTAGRHK

[0121] Based on the gene sequence of REV HLJR0901 strain, upstream primer gp90-pCold-F (SEQ ID NO.3) and downstream primer gp90-pCold-R (SEQ ID NO.4) were designed to clone the gp90 gene of REV into the prokaryotic vector (pColdⅠ), and upstream primer gp90-pCAGGS-F (SEQ ID NO.5) and downstream primer gp90-pCAGGS-R (SEQ ID NO.6) were designed to clone the gp90 gene into the eukaryotic vector (pCAGGS).

[0122] gp90-pCold-F:TCGAAGGTAGGCATATGGAGCTCGGTACCCTCGAGCATCATCATCATCATGACTGTCTCACCAACCTCCGATCCGCTGAGGGTAA

[0123] gp90-pCold-R: ACCTATCTAGACTGCAGGTCGACAAGCTTGAATTCTTACTTATGACGCCCAGCGGTGTACTCGATGGATG

[0124] gp90-pCAGGS-F: TGTCTCATCATTTTGGCAAAGAATTCATGGACTGTCTCACCAACCTCCGATCCGCTGAG

[0125] gp90-pCAGGS-R:GAGGGAAAAAGATCTGCTAGCTCGAGTCACTTATCGTCGTCATCCTTGTAATCGCTACCCCCGCCCTTATGACGCCCAGC

[0126] Using recombinant plasmid pBlu-HLJR0901 as a template, PCR amplification was performed using gp90-pCold-F and gp90-pCold-R as primers. The amplified fragment 1 was purified and recovered via gel electrophoresis. The prokaryotic expression vector pColdⅠ (TaKaRa) was double-digested with EcoRI and Xho I restriction endonucleases, respectively. Homologous recombination of the recovered pColdⅠ vector with the purified amplified fragment 1 was performed using a homologous recombination kit (Novozan) according to the manufacturer's instructions. The ligation product was transformed into DH5α competent cells. The plasmid extracted and confirmed by Sanger sequencing was named pCold-gp90. The recombinant plasmid pCold-gp90 was transformed into BL21(DE3) competent cells. Single colonies were screened and inoculated into 4 mL of ampicillin-containing LB medium. After 12 h of shaking culture at 37℃ and 200 rpm, the colonies were transferred to 20 mL of ampicillin-containing LB medium at a 1% inoculation rate and cultured at 37℃ and 200 rpm for an extended period. When the bacterial culture OD... 450nm When the growth rate reaches 0.6–0.8 (logarithmic growth phase), add a final concentration of 1.0 mmol / L IPTG and induce induction at 20°C and 180 rpm for 22 h. After induction, collect the bacterial cells by centrifugation at 4500 g for 10 min, resuspend in 10 mL of pre-cooled PBS and centrifuge repeatedly, repeating the washing process three times. Finally, resuspend the bacterial cell pellet in 1.5 mL of PBS. Place the bacterial suspension in an ice-water mixture and sonicate to disrupt the cell structure. Centrifuge the disrupted solution at 13000 g at 4°C for 30 min to separate the supernatant and precipitate.

[0127] Supernatant and precipitate samples were prepared by adding 5× loading buffer in specific proportions. The expression form and efficiency of gp90 protein were analyzed by 12.5% ​​SDS-PAGE electrophoresis and Coomassie brilliant blue staining. SDS-PAGE results showed that the recombinant REV envelope protein gp90 was mainly present in the lysed cell pellet, indicating that the expressed recombinant REV envelope protein gp90 was primarily expressed in inclusion body form, with a molecular weight of approximately 46 kDa. Using electroelution, 4 mL of recombinant gp90 protein was obtained from 800 mL of bacterial cells, at a concentration of 1.6 mg / mL. Western blot analysis of the purified gp90 protein showed that it could be specifically recognized by the His-tagged antibody.

[0128] Using recombinant plasmid pBlu-HLJR0901 as a template, PCR amplification was performed using gp90-pCAGGS-F and gp90-pCAGGS-R as primers, and the amplified fragment 2 was recovered by gel electrophoresis. The eukaryotic expression vector pCAGGS was double-digested with EcoRI and Xho I restriction endonucleases, respectively. Homologous recombination of the recovered pCAGGS vector with the previously purified amplified fragment 2 was performed using a homologous recombination kit (Novozan) according to the manufacturer's instructions. The ligation product was transformed into DH5α competent cells. The gp90 eukaryotic expression recombinant plasmid, confirmed by Sanger sequencing, was named pCAGGS-gp90.

[0129] Example 2: Preparation and identification of monoclonal antibodies against avian reticuloendotheliosis virus gp90 protein

[0130] I. Mouse Immunization

[0131] The purified gp90 protein prepared in Example 1 was diluted to a concentration of 1 mg / ml with sterile PBS (0.01 mol / L, pH 7.4). The gp90 protein solution was then mixed with Freund's complete adjuvant at a 1:1 volume ratio and emulsified using a tissue homogenizer until a stable, water-resistant emulsion was formed, yielding immunogen composition 1. The same gp90 protein solution was then mixed with Freund's incomplete adjuvant at a 1:1 volume ratio and emulsified using the same method to obtain immunogen composition 2.

[0132] Five 6-week-old BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were immunized via subcutaneous injection at multiple sites on their backs. Immunogen composition 1 was administered to the mice via subcutaneous injection at multiple sites on their backs, with an immunization dose of 0.1 mg Gp90 protein per mouse. A second immunization was administered 14 days after the initial immunization using immunogen composition 2, and a third immunization was administered 14 days after the second immunization using immunogen composition 2. The method and dosage of the second and third immunizations were the same as the initial immunization.

[0133] II. Detection of antibodies in mouse serum

[0134] Seven days after the third immunization, mouse tail blood was collected. Purified gp90 protein (prepared in Example 1) was used as the antigen to coat ELISA plates, and the antibody titer of the immunized mouse serum was detected by indirect ELISA. The specific operation is as follows: (1) Coating antigen: The purified gp90 protein was diluted to 10 μg / mL with carbonate coating solution. 100 μL of protein dilution solution was added to each well of the ELISA plate and incubated at 4℃ for 12-16 h. (2) Washing coating solution: The coated plate was removed, the protein solution was discarded, 300 μL of PBST was added to each well, and the plate was washed 4 times and then patted dry. (3) Blocking: 100 μL of 5% skim milk (solvent is PBS) was added to each well and the plate was incubated at 37℃ for 1 h. The liquid in the well was discarded and then patted dry. (4) Washing blocking solution: 300 μL of PBST was added to each well and the plate was washed 4 times and then patted dry. (5) Diluting the serum samples to be tested: Prepare serum diluent (PBS containing 5% fetal bovine serum). Serially dilute the mouse serum samples to be tested with the serum diluent at a ratio of 1:1000 (volume ratio), establishing a total of 7 dilution gradients. Specific dilution method: a. Add 120 μL of serum diluent per well in each row of the serum dilution plate, corresponding to the number of samples to be tested, leaving the first row empty. b. Thoroughly mix the serum samples to be tested. Dilute the original serum solution to be tested to 1000-fold using the serum diluent in a two-step method. After thorough mixing, add 240 μL to the first row for later use. c. Transfer 120 μL of diluent from the first row to the second row, aspirate 30 times at a uniform speed, then transfer 120 μL of diluent to the third row. Repeat this process until all gradients are completed. The negative control is serum from unimmunized mice diluted with serum diluent at a ratio of 1:1000 (volume ratio). (6) Incubation with primary antibody: Add 100 μL of the serum sample to be tested (immune serum or control serum) to each well, then incubate at 37°C for 1 h, discard the primary antibody diluent, and pat dry. (7) Washing primary antibody: Add 300 μL of PBST to each well, wash 5 times, and pat dry. (8) Dilute HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Sigma) in PBS buffer at a dilution ratio of 1:3000 (volume ratio), add 100 μL of secondary antibody diluent (PBS) to each well, incubate at 37°C for 1 h, discard the solution, and pat dry. Washing secondary antibody: Add 300 μL of PBST to each well, wash 5 times, and pat dry. (9) Color development: Add 100 μL of TMB substrate color development solution to each well, then incubate at 37°C for 15 min. (10) Termination of color development and result determination: Add 100 μL of stop solution (2M H2SO4) to each well, and immediately place the microplate into the microplate reader to measure the absorbance OD value. 450 nmA sample well absorbance greater than 0.2 indicates a positive result for REV gp90 antibody. For the ELISA titer statistics of REV gp90 antibody in the serum of the immunized mouse with the highest antibody titer (mouse a), please refer to [link to ELISA data]. Figure 1 Therefore, it can be seen that the OD of mouse serum immunized with gp90 protein diluted 32,000 times is... 450nm The value is still greater than 0.2, and the ELISA titer of gp90 serum antibody can reach 1:32000.

[0135] III. Hybridoma Cell Preparation

[0136] 1. Resuscitation of SP2 / 0 myeloma cells

[0137] (1) Remove the frozen SP2 / 0 cells from liquid nitrogen and thaw them quickly in a 37°C water bath. (2) In a biosafety cabinet, transfer the thawed cell culture to a 15mL centrifuge tube containing 10mL of pre-warmed SP2 / 0 cell culture medium (DMEM medium), gently mix, and centrifuge at 300g for 5 minutes. (3) After centrifugation, discard the supernatant, gently resuspend the cells enriched at the bottom of the tube in 6mL of SP2 / 0 cell culture medium, transfer to a cell culture flask, and incubate statically in a 37°C, 5% CO2 incubator. After 12 hours, replenish the medium or passage the cells according to their condition to avoid excessive cell density that could reduce cell quality or cause resuscitation failure.

[0138] 2. Passage of SP2 / 0 myeloma cells

[0139] (1) When the cell density reaches 90% and the cells are uniform in shape (transparent, round, and with clear boundaries), discard the old culture medium and gently wash the cells twice with 4 mL of pre-warmed sterile PBS along the flask wall to remove metabolites and dead cells. (2) After discarding the PBS, add an appropriate amount of pre-warmed SP2 / 0 cell culture medium, and use a tilting pipette to remove the semi-adherent SP2 / 0 cells. Add the cell suspension to a new cell culture flask, add an appropriate amount of culture medium, mix well, and place it in a 37℃, 5% CO2 incubator for static culture. (3) Preparation before fusion: Passage the cells 2-3 times until they are in the logarithmic growth phase. Replace the SP2 / 0 cell culture medium with fresh medium 24 hours before fusion to ensure that the cells are in the optimal metabolic state. After obtaining mouse spleen cells, gently resuspend the SP2 / 0 cells cultured to the optimal metabolic state with 25 mL of culture medium and transfer them to a 50 mL centrifuge tube for later use.

[0140] 3. Preparation of mouse spleen cells

[0141] (1) Boost the immunization of mice with gp90 protein without adjuvant by intraperitoneal injection at a dose of 0.1 mg / mouse. Three to four days after the booster immunization, the mice were euthanized by enucleation and blood samples from the orbital venous plexus were collected simultaneously. (2) The mice carcasses were then immersed in 75% ethanol aqueous solution for 15 minutes. (3) After the mice were transferred to a biosafety cabinet, their limbs were fixed in an extended position using sterile foam boards and sterile steel nails, allowing the residual ethanol on the body surface to evaporate naturally. (4) Using sterile surgical instruments, the abdominal cavity was exposed by opening the mouse's abdominal cavity along the midline to fully expose the spleen. (5) The spleen was removed using blunt dissection. The mouse's spleen was located on the right posterior side and was surrounded by abundant fat. During the removal process, excess fat and tissue were removed as much as possible. (6) Spleen cell isolation: Transfer mouse spleen to sterile culture dish, add 20-30 mL of DMEM culture medium, make holes evenly on the side of spleen with 1 mL needle, use a 20 mL syringe combined with a 1 mL syringe needle to draw an appropriate amount of DMEM and flush the spleen from both ends and the middle, repeat several times until the spleen turns white (most spleen cells are blown out); after fully suspending the extracted spleen cells, transfer them into a 50 mL centrifuge tube to prepare for subsequent fusion.

[0142] 4. Preparation before cell fusion

[0143] Place DMEM culture medium, an 800mL beaker (containing an appropriate amount of ultrapure water), PEG (P7306), HybridomaFeeder additive, and HAT culture medium in a 37℃ water bath for preheating.

[0144] 5. Fusion of SP2 / 0 cells with spleen cells

[0145] (1) Count the SP2 / 0 cells obtained in section 2 and the spleen cells obtained in section 3 separately, and adjust the cell density to a ratio of SP2 / 0 cells to spleen cells of 1:9 (cell number ratio). Then centrifuge at 1000g at room temperature for 10 min. (2) Discard the supernatant, take 20 mL of preheated DMEM and gently resuspend the SP2 / 0 cells and mix well. Then gently resuspend the spleen cells with the cell suspension to mix the two types of cells thoroughly. Centrifuge at 1000g at room temperature for 10 min. (3) Discard as much supernatant as possible, tap the bottom of the centrifuge tube, and mix the SP2 / 0 cell layer (white) and the spleen cell layer (red) thoroughly until they form a pink paste. (4) Place the centrifuge tube at 37℃ and add 1 mL of PEG (P7306) dropwise over 1 min. The specific procedure is as follows: Wipe the liquid outside of the preheated 800mL beaker dry and disinfect it with alcohol, transfer it to the ultra-clean workbench, place the centrifuge tube along the notch of the beaker, aspirate 1mL of PEG (P7306), insert the pipette tip into the bottom of the tube, and slowly and evenly add it over 1 minute, trying to drop it onto the pink cells as much as possible. This process requires two people to work together, one person is responsible for timing 1 minute, and the other person strictly controls the dropping speed throughout the process according to the timing time. A preliminary experiment can be conducted in advance to check the number of drops per 1mL. After the addition is completed, let it stand at room temperature for 3 minutes. (5) Add 20mL of DMEM along the wall of the centrifuge tube to stop the effect of PEG (P7306) (the order of addition is 1mL in the first 30s, 2mL in the next 30s, and 13mL in the last 2 minutes). (6) After the fusion tube is placed in a 37℃, 5% CO2 cell culture incubator for 10 minutes, centrifuge at 1000g at room temperature for 10 minutes. (7) Discard the supernatant, gently resuspend the cells in an appropriate amount of HAT culture medium, mix until there are no visible cell clumps, add 8 mL of Hybridoma Feeder, and then bring the total volume to 80 mL, gently mix. (8) Distribute the cell suspension evenly into 3-4 96-well cell culture plates, 200 μL / well (cells around the edges of the plate will dry more easily due to the edge effect, so 300 μL / well can be added to the edge wells). (9) Fix the cell plates to the four corners with sterile tape and incubate them in a 37°C, 5% CO2 cell culture incubator. Observe whether the culture medium turns yellow (bacterial contamination) within 3 days, and observe the cell growth status after 5 days. After 7 days of fusion, when replenishing the culture medium with HAT culture medium, hybridoma cells that have initially formed monoclonal cell clumps can be seen in the 96-well plate. After 10 days of fusion culture, the hybridoma cell clumps are observed to increase in volume, the cells are round in shape, and the growth status is good.

[0146] 6. Screening of positive hybridoma cells

[0147] Five days after cell fusion, the cells were observed under a microscope for positive wells (wells containing cell clones), with particular emphasis on wells containing single cell clones. After 7–10 days of culture, the gp90 antibody titer in the hybridoma cell supernatant was detected using an indirect ELISA method (specific procedure as described in Section 2 of Example 2). The detected OD values ​​were combined with the previous single-clone cell recordings for comprehensive analysis. Twenty wells with high OD values ​​and containing single-cell clones were selected. The culture medium was changed from HAT to HT, and the cells in each well were dispersed. Once the cells reached confluence, they were transferred to 48-well plates for further culture until expansion to 12-well plates, at which point cell subcloning was performed. During the expansion culture process, the supernatant in each well was periodically analyzed using ELISA to determine the titer, and the optimal clones were selected for subcloning based on a comprehensive assessment.

[0148] 7. Preparation before hybridoma cell subcloning

[0149] Prepare a 96-well cell culture plate, label it with the corresponding clone name, and add 200 μL of HT culture medium per well, and 300 μL per well in the edge wells around the plate. Aspirate the supernatant of the cells to be subcloned, gently wash the cells with HT culture medium, and discard the wash solution; mix the cells with an appropriate amount of HT culture medium by blowing, and add 400 μL of cell suspension through the filter membrane at the end of a flow cytometry tube, labeling the tube with the corresponding cell line name. Passage or cryopreserve the remaining cell suspension for later use.

[0150] 8. Subclonal sorting

[0151] Hybridoma cell lines were sorted using a flow cytometry system (SONY-MA900 Flow Cell Sorter). Cloned cells were grown in a 37°C, 5% CO2 cell incubator for 7–10 days. Each well was observed and labeled for the presence of cells, cell size, whether it was a monoclonal cell cluster, and whether it was contaminated. Once the monoclonal cell clusters reached a suitable size, the titer was measured. Cell lines with positive results, high OD values, and monoclonal cell clusters underwent a second subcloning. After the second subcloning, cell lines with positive results, high OD values, and monoclonal cell clusters were identified as hybridoma cell lines capable of secreting REVgp90 monoclonal antibody. After two subclonings using flow cytometry, one hybridoma cell line capable of secreting gp90 monoclonal antibody was obtained, named REV-gp90-C16 (abbreviated as C16). The monoclonal antibody secreted by this line shares the same name as the hybridoma cell line (abbreviated as MAb C16).

[0152] 9. Culture and cryopreservation of hybridoma cells

[0153] Positive hybridoma cell clones selected after two subcloning processes were expanded and cultured to 6-well plates for titer testing. If the results were consistent with previous positive results, the culture was expanded further. A portion was used for subsequent ascites fluid preparation, and the other portion was cryopreserved at -80°C for future use.

[0154] 10. Preparation of monoclonal antibody ascites

[0155] Cultured monoclonal hybridoma cells were injected intraperitoneally into 6-week-old BALB / c female mice to prepare gp90 monoclonal antibody ascites. The ascites was purified using Protein G affinity chromatography medium.

[0156] IV. Identification of Monoclonal Antibodies

[0157] 1. ELISA titer determination of monoclonal antibodies

[0158] The monoclonal antibody ascites fluid was serially diluted 2-fold starting at 1:2000 using PBS, and its ELISA titer was measured (the specific procedure is the same as in Section 2 of Example 2). The indirect ELISA results showed that the gp90 antibody titer in the C16 monoclonal antibody ascites fluid could reach 1:64000.

[0159] 2. Subclass identification of monoclonal antibodies

[0160] The subclass of gp90 monoclonal antibody C16 was analyzed using a mouse monoclonal antibody Ig subclass identification ELISA kit (supplier: Beijing Bio-Long Immunotherapy Co., Ltd.). The kit results showed that gp90 monoclonal antibody C16 is an IgG1 type, Kappa class (…). Figure 2 B).

[0161] IV. Preliminary Applications of Monoclonal Antibodies

[0162] 1. Detection of gp90 prokaryotic expressed protein by monoclonal antibody

[0163] The purified gp90 protein prepared in Example 1 was subjected to SDS-PAGE and then transferred to an NC membrane. Using diluted gp90 monoclonal antibody C16 (1:1000, diluted with PBS) as the primary antibody and IRDye800CW-labeled goat anti-mouse IgG as the secondary antibody (Sigma), Western blot was used to detect the reactivity of the gp90 monoclonal antibody to the gp90 prokaryotic expressed protein. Western blot results showed that the target band appeared at approximately 46 kDa, indicating that the prepared gp90 monoclonal antibody C16 exhibited a good immunoreactivity with the gp90 prokaryotic expressed protein (see...). Figure 2 A). This indicates that the prepared gp90 monoclonal antibody can be used to detect gp90 prokaryotic expression protein using Western blot technology.

[0164] 2. Detection of gp90 eukaryotic expressed protein by monoclonal antibody

[0165] When DF-1 cells were in good condition and their density was 70%-80%, the recombinant eukaryotic expression plasmid pCAGGS-gp90 and the empty plasmid pCAGGS obtained in Example 1 were transfected into DF-1 cells for 30 hours. Western blot analysis was performed using gp90 monoclonal antibody C16 (1:1000) as the primary antibody, anti-Flag antibody (Sigma) as the primary antibody positive control, anti-β-actin antibody (Sigma) as the primary antibody internal control, and IRDye800CW-labeled goat anti-mouse IgG (Sigma) as the secondary antibody. Results are shown below. Figure 2 C. Western blot analysis showed that in the group transfected with the recombinant eukaryotic expression vector pCAGGS-gp90, the gp90 monoclonal antibody could recognize a specific band of approximately 60 kDa, and the Flag tag antibody could also detect this band; however, in the control group transfected with the empty vector pCAGGS, neither the gp90 monoclonal antibody nor the Flag tag antibody detected a specific band. Cellular β-actin (C-actin) was detectable in both groups. Figure 2 C). This indicates that the prepared gp90 monoclonal antibody C16 can be used to detect gp90 eukaryotic expression protein by Western blot technology.

[0166] Indirect immunofluorescence (IFA) was performed using gp90 monoclonal antibody C16 (1:1000, diluted in PBS) as the primary antibody and FITC-labeled goat anti-mouse IgG (Sigma) as the secondary antibody. Results are shown below. Figure 2 D. Therefore, specific green fluorescence was observed in DF-1 cells transfected with pCAGGS-gp90, while no fluorescence was observed in the pCAGGS transfected group, indicating that the prepared monoclonal antibody C16 specifically reacts with the gp90 protein expressed by the eukaryotic expression plasmid. The prepared monoclonal antibody C16 can be used to detect eukaryotically expressed gp90 protein using IFA technology.

[0167] 3. Detection of REV with monoclonal antibodies

[0168] HLJR0901 REV strain was seeded at a dose of 1 MOI into CEF cells (primary chicken embryo fibroblasts) cultured in 96-well plates, with an uninoculated cell control. After 7 days of culture at 37℃ and 5% CO2, in vitro anabolism (IFA) was performed using gp90 monoclonal antibody C16. gp90 monoclonal antibody C16 (1:200 dilution in PBS) was used as the primary antibody, and FITC-labeled goat anti-mouse IgG (Sigma) was used as the secondary antibody. Results are shown below. Figure 2Specific green fluorescence was visible in CEF cells in the E, REV infection group, while no fluorescence was seen in the non-infected cell control group. The results showed that the gp90 monoclonal antibody C16 could specifically recognize REV infecting CEF, showing green fluorescence( Figure 2 E). This indicates that the prepared gp90 monoclonal antibody C16 can be used to detect REV.

[0169] V. Preservation of Hybridomas

[0170] The hybridoma cell line C16 prepared in the present invention was submitted to a preservation institution recognized by the patent procedure 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: C2024405; the name of the culture is: Hybridoma cell line REV-gp90-C16 Hybridoma cellline REV-gp90-C16; the Chinese classification name is: Hybridoma cell line; the English classification name is: Hybridoma Cell Line; the preservation time is December 18, 2024; the identification survival time is December 26, 2024.

[0171] Example 3: Identification of the Antigenic Epitope Recognized by Monoclonal Antibody

[0172] I. Construction of Recombinant Plasmids for Truncated Expression of REV gp90

[0173] In order to screen and identify the smallest antigenic epitope recognized by REV gp90 monoclonal antibody, the gp90 protein of REV HLJR0901 strain was truncated and eukaryotically expressed in the present invention.

[0174] II. Primer Design

[0175] Primers were designed according to the truncated expression scheme of gp90. The corresponding primer sequences used in this example are shown below. Among them, F in the primer name represents the upstream primer, R represents the downstream primer, and the code before F and R is the name of the protein fragment amplified by the primer.

[0176] P1-F (SEQ ID NO.7): ACTCTCGGCATGGACGAGCTGTACAAGATGGACTGTCTCACCAACCTCCGATCCGCTGAGGGTAAA

[0177] P1-R (SEQ ID NO.8): GAAAAAGATCTGCTAGCTCGAGTCACAGGAGGGTGCATTCCTGAGCCTTTTCATAACA

[0178] P2-F(SEQ ID NO.9):ACTCTCGGCATGGACGAGCTGTACAAGGAATGCACCCTCCTGGGAAAAACTTATTTTACTGCCATC

[0179] P2-R(SEQ ID NO.10):GAAAAAGATCTGCTAGCTCGAGTCAGATGGCTGCGGGGATTGGAGTTCCAAGAGTCAT

[0180] P3-F(SEQ ID NO.11):ACTCTCGGCATGGACGAGCGTACAAGATCCCCGCAGCCATCCCGGCGAATGGCAATGTCACTCTC

[0181] P3-R(SEQ ID NO.12):AAAAAGATCTGCTAGCTCGAGTCACTTATGACGCCCAGCGGTGTACTCGATGGATGG

[0182] P2-1-F(SEQ ID NO.13):ACTCTCGGCATGGACGAGCTGTACAAGGAATGCACCCTCCTGGGAAAAACTTATTTTACT

[0183] P2-1-R(SEQ ID NO.14):GAAAAAGATCTGCTAGCTCGAGTCAGGTTCCCGTGCAAGAGGCTTGAAGCAGTTTATT

[0184] P2-2-F(SEQ ID NO.15):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTCTTGCACGGGAACCATAGGGAA

[0185] P2-2-R(SEQ ID NO.16):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCACGCACAGATTCTTC

[0186] P2-3-F(SEQ ID NO.17):GGGATCACTCTCGGCATGGACGAGCTGTACAAGCGTGAAAGACTAGAGGAAATCATCAGGCAC

[0187] P2-3-R(SEQ ID NO.18):GAAAAAGATCTGCTAGCTCGAGTCATATGTCAGACGTCTGGGGATCCAGATCTACTCC

[0188] P2-4-F(SEQ ID NO.19):GGGATCACTCTCGGCATGGACGAGCTGTACAAGCAGACGTCTGACATACTGGAAGCTACTCACCAG

[0189] P2-4-R(SEQ ID NO.20):GAAAAAGATCTGCTAGCTCGAGTCAGATGGCTGCGGGGATTGGAGTTCCAAGAGTCAT

[0190] L1-F(SEQ ID NO.21):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTCTTGCACGGGAACCATAGGGAA

[0191] L1-R(SEQ ID NO.22):GAAAAAGATCTGCTAGCTCGAGTCAAATCATGTCAGTGGGACCGCC

[0192] L2-F(SEQ ID NO.23):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTCTTGCACGGGAACCATAGGGAA

[0193] L2-R(SEQ ID NO.24):GAAAAAGATCTGCTAGCTCGAGTCAGACATACACAGGGGCTACGGG

[0194] L3-F(SEQ ID NO.25):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTCTTGCACGGGAACCATAGGGAA

[0195] L3-R(SEQ ID NO.26):GAAAAAGATCTGCTAGCTCGAGTCATACTGGTTTCCCTAT

[0196] R1-F(SEQ ID NO.27):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTGTTGGGACCCCGTAGCCCCTGTGTAT

[0197] R1-R(SEQ ID NO.28):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCACGCACAGATTCTTC

[0198] R2-F(SEQ ID NO.29):GGGATCACTCTCGGCATGGACGAGCTGTACAAGTCTGATGGGGGCGGTCCCACTGAC

[0199] R2-R(SEQ ID NO.30):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCACGCACAGATTCTTC

[0200] R3-F(SEQ ID NO.31):ATCACTCTCGGCATGGACGAGCTGTACAAGCGGGAAGAATCTGTGCGTGAA

[0201] R3-R(SEQ ID NO.32):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCACGCACAGATTCTTC

[0202] L9-F(SEQ ID NO.33):ATCACTCTCGGCATGGACGAGCTGTACAAGCGGGAAGAATCTGTGCGTGAA

[0203] L9-R(SEQ ID NO.34):GAAAAAGATCTGCTAGCTCGAGTCATAGTCTTTCACGC ACAGATTCTTC

[0204] L8-F(SEQ ID NO.35):ATCACTCTCGGCATGGACGAGCTGTACAAGCGGGAAGAATCTGTGCGTGAA

[0205] L8-R(SEQ ID NO.36):GAAAAAGATCTGCTAGCTCGAGTCATCTTTCACGCACA GATTCTTC

[0206] L7-F(SEQ ID NO.37):ATCACTCTCGGCATGGACGAGCTGTACAAGCGGGAAGAATCTGTGCGTGAA

[0207] L7-R(SEQ ID NO.38):GAAAAAGATCTGCTAGCTCGAGTCATTCACGCACAGAT TCTTC

[0208] R9-F(SEQ ID NO.39):CTCGGCATGGACGAGCTGTACAAGGAAGAATCTGTGC GTGAAAGACTA

[0209] R9-R(SEQ ID NO.40):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCA CGCACAGA

[0210] R8-F(SEQ ID NO.41):ACTCTCGGCATGGACGAGCTGTACAAGGAATCTGTGCG TGAAAGACTA

[0211] R8-R(SEQ ID NO.42):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCA CGCACAGA

[0212] R7-F(SEQ ID NO.43):ACTCTCGGCATGGACGAGCTGTACAAGTCTGTGCGTGA AAGACTA

[0213] R7-R(SEQ ID NO.44):GAAAAAGATCTGCTAGCTCGAGTCACTCTAGTCTTTCA CGCACAGA

[0214] III. Construction of recombinant expression plasmids expressing truncated gp90 fragments

[0215] Using recombinant plasmid pBlu-HLJR0901 as a template, gp90 truncated fragments were amplified by PCR using appropriate primers, and then cloned into the pCAGGS vector to construct recombinant eukaryotic expression plasmids expressing the gp90 truncated fragments. All gp90 truncated fragments had an EGFP tag fused to their N-terminus. These plasmids were then sequenced for identification.

[0216] IV. Preparation of REV gp90 truncated expression protein

[0217] The obtained sequencing verification of the correctly recombinant eukaryotic expression plasmid was then... After transfection into DF-1 cells with the transfection reagent, cell lysis products were collected 30 hours later, and protein samples of the lysis products were prepared and stored at 4°C for later use.

[0218] V. Identification of REV gp90 antigenic epitopes

[0219] Using Western blot and peptide scanning techniques, the REV gp90 monoclonal antibody C16 prepared in this invention was used to screen and identify the antigenic epitopes of the REV gp90 protein. A series of screenings were performed on truncated REV gp90 proteins using the monoclonal antibody. Short peptides that reacted with the REV gp90 monoclonal antibody C16 strain and whose band size was consistent with the incubation results of the EGFP-tagged antibody were identified as positive short peptides, containing the gp90 antigenic epitope. These short peptides were further truncated and detected until the smallest short peptide recognizable by the monoclonal antibody was identified, which was the REV gp90 antigenic epitope.

[0220] Two samples of a fusion protein were prepared. One sample used REV gp90 monoclonal antibody C16 as the primary antibody and IRDye800CW-labeled goat anti-mouse IgG as the secondary antibody. Western blot analysis was used to identify the regions recognized by the monoclonal antibody to determine its recognition characteristics. The other sample used an antibody (Sigma) containing the fusion tag (EGFP) of each peptide as the primary antibody and IRDye800CW-labeled goat anti-mouse IgG as the secondary antibody to detect the expression of each peptide. Western blot analysis was performed using anti-β-actin antibody (Sigma) as the primary antibody (internal control) and IRDye800CW-labeled goat anti-mouse IgG (Sigma) as the secondary antibody.

[0221] VI. GP90 Truncation Scheme

[0222] (1) First round of truncation: The complete gp90 protein was divided into three overlapping peptide segments: P1 (aa1-137), P2 (aa 133-269), and P3 (aa 265-397). (2) Second round of truncation: P2 was further divided into four overlapping peptide segments: P2-1 (aa 133-169), P2-2 (aa 165-204), P2-3 (aa 200-236), and P2-4 (aa 232-269). (3) Third round of truncation: P2-2 is truncated from both ends into six peptide segments: L1 (aa 165-194), L2 (aa 165-184), L3 (aa 165-174), R1 (aa 175-204), R2 (aa 185-204), and R3 (aa 195-204). (4) Fourth round of truncation: R3 is further truncated from both ends into six overlapping peptide segments: L9 (aa 195-203), L8 (aa 195-202), L7 (aa 195-201), R9 (aa 196-204), R8 (aa 197-204), and R7 (aa 198-204).

[0223] All of the above gp90 truncated variants have an EGFP tag fused to their N-terminus. The position relative to gp90 is indicated in parentheses after each peptide name. Primers used for amplifying the template when expressing each peptide are listed below.

[0224] This invention performed four rounds of truncation on the gp90 protein. Figure 3 These short peptides were detected using Western blot-based peptide scanning with the REV gp90 monoclonal antibody C16. In the first round of detection, MAb C16 specifically recognized the short peptide P2(aa 133-269) (…). Figure 4 A). In the subsequent second round of testing, MAb C16 further identified the short peptide P2-2 (aa 165-204) ( Figure 4 B). The results of the third round of Western blot analysis showed that MAb C16 and R3 (aa 195-204) had a specific reactivity. Figure 4 C) indicates the presence of an antigenic epitope between aa195-204 of gp90. To precisely pinpoint the shortest antigenic epitope recognized by C16, a fourth round of truncated expression was performed on R3. The results showed that reducing the C-terminus of R3 by one amino acid (L9) resulted in a reaction with MAbC16; further reducing the C-terminus of R3 by two amino acids (L8) resulted in no further reaction with MAbC16; and reducing the N-terminus of R3 by one amino acid (R9) resulted in no further reaction with MAbC16. Figure 4 D). These results indicate that gp90's 195 REESVRERL 203 It is the smallest unit of the MAb C16 identification table (called the MAb C16 identification field).

[0225] Example 4: Spatial Structure Analysis of Antigenic Epitopes

[0226] The tertiary structure of the gp90 protein of the REVHLJR0901 strain was predicted and analyzed using the Alfha-Fold3 online prediction software (https: / / alphafoldserver.com / ). The prediction results with the highest confidence were selected for amino acid site labeling, marking the N-terminus and C-terminus of the protein, as well as the spatial location of the antigenic epitope recognized by the REV gp90 monoclonal antibody C16 strain.

[0227] The three-dimensional structure of the gp90 protein shows the antigenic epitopes identified in this study. 195 REESVRERL 203 Located on the outer side of the middle segment of the gp90 protein; this epitope forms a ring-like structure. Figure 5 ).

[0228] Example 5: Conservation Analysis of Antigenic Epitopes

[0229] The gp90 sequences of representative REV strains from China and abroad were selected, and the amino acid sequences of the identified gp90 antigenic epitopes were compared to analyze the conservation of these epitopes. The reference strains used and their corresponding GenBank accession numbers are as follows: HLJR0901 (GQ415646.2), HA9901 (AY842951.1), ZD0708 (FJ496333.1), HLJ07I (GQ375848.1), HLJR0801 (GU012640.1), HLJR0902 (GU012638.1), HLJR0903 (GQ415643.1), HLJR0904 (GU012639.1), HLJR0905 (GU012643.1), JLR0801 (GQ4156 44.1), JLR0802 (GU969140.1), JLR0803 (GU012644.1), JLR0901 (GU012646.1), JLR0902 (GQ415645.1), 3122 / 03 (DQ513316.1), 3295 / 04(DQ513317.1), 3337 / 05(FJ439120.1), 3410 / 06(FJ439119.1), APC-566(DQ387450.1), 170A(GU222420.1), PC-R92(DQ237901.1).

[0230] Sequence alignment showed that the gp90 antigenic epitope identified in this study... 195 REESVRERL 203 The (MAb C16 recognition domain) is sequence conserved across different REV strains.

[0231] according to Figure 4 It is evident that truncated peptides of gp90 protein containing the C16 recognition domain can bind to monoclonal antibody C16, reflecting that the portion of these truncated peptides outside the C16 recognition domain does not spatially hinder the binding of the C16 recognition domain to monoclonal antibody C16. According to Figure 5 Looking at the three-dimensional structure shown, 195 REESVRERL 203 Located on the outer side of the middle segment of the gp90 protein, this domain is exposed to the outside of the protein, and there are no obvious spatial barriers from amino acid residues upstream and downstream. Furthermore, the gp90 antigenic epitope... 195 REESVRERL 203 The (MAbC16 recognition domain) is sequence conserved in different REV strains, from which it can be reasonably inferred that the monoclonal antibody C16 of the present invention has a broad-spectrum recognition ability against different REV strains.

[0232] 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 a hybridoma cell with the microbial preservation number CCTCC NO:C2024405 or a passaged cell of a hybridoma cell with the microbial preservation number CCTCC NO:C2024405; The monoclonal antibody secreted by the passaged cells of the hybridoma cells with the microbial preservation number CCTCC NO:C2024405 maintains specific binding activity to material a. The material 'a' is selected from A1, A2, A3, and A4: A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2; A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2; A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2; A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2; During the passage of hybridoma cells with the microbial accession number CCTCC NO:C2024405, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the monoclonal antibodies secreted by the passaged cells of the hybridoma cells with the microbial accession number CCTCC NO:C2024405 did not undergo any mutations compared to the monoclonal antibodies secreted by the hybridoma cells with the microbial accession number CCTCC NO:C2024405.

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 is the monoclonal antibody secreted by the hybridoma cells as described in claim 1; P2: Genetically engineered monoclonal antibody The genetically engineered monoclonal antibody maintains specific binding activity to material a; The material 'a' is selected from A1, A2, A3, and A4: A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2; A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2; A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2; A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2; The genetically engineered monoclonal antibody includes a heavy chain and a light chain of the genetically engineered monoclonal antibody. The heavy chain of the genetically engineered monoclonal antibody includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the genetically engineered monoclonal antibody. The light chain of the genetically engineered monoclonal antibody includes light chain CDR1, light chain CDR2, and light chain CDR3. The heavy chain CDR1 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR1 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The heavy chain CDR2 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR2 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The heavy chain CDR3 of the genetically engineered monoclonal antibody has the same protein sequence as the heavy chain CDR3 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR1 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR1 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR2 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR2 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR3 of the genetically engineered monoclonal antibody has the same protein sequence as the light chain CDR3 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. P3: Antibody derivatives The antibody derivative maintains specific binding activity to material a; The material 'a' is selected from A1, A2, A3, and A4: A1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2; A2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2; A3: A peptide with an amino acid sequence as shown in SEQ ID NO.2; A4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2; The heavy chain CDR1 of the antibody derivative has the same protein sequence as the heavy chain CDR1 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The heavy chain CDR2 of the antibody derivative has the same protein sequence as the heavy chain CDR2 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The heavy chain CDR3 of the antibody derivative has the same protein sequence as the heavy chain CDR3 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR1 of the antibody derivative has the same protein sequence as the light chain CDR1 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR2 of the antibody derivative has the same protein sequence as the light chain CDR2 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The light chain CDR3 of the antibody derivative has the same protein sequence as the light chain CDR3 of the monoclonal antibody secreted by the hybridoma cells according to claim 1. The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, single-chain antibodies, avian-derived antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA assembly The RNA combination includes genetically engineered monoclonal antibody heavy chain RNA and genetically engineered monoclonal antibody light chain RNA; The genetically engineered monoclonal antibody heavy chain RNA can be translated to obtain the heavy chain of the genetically engineered monoclonal antibody described in P2. The genetically engineered monoclonal antibody light chain RNA can be translated to obtain the light chain of the genetically engineered monoclonal antibody described in P2. P5: Gene Combination The gene combination includes a first gene and a second gene; The coding sequence of the first gene can encode the genetically engineered monoclonal antibody heavy chain described in P2; The coding sequence of the second gene can encode the genetically engineered monoclonal antibody light chain described in P2; P6: Gene Expression Catalyst Assembly The gene expression cassette assembly includes a first gene expression cassette and a second gene expression cassette. The gene expression product in the first gene expression cassette is the monoclonal antibody heavy chain RNA described in P4; The gene expression product in the second gene expression cassette is the monoclonal antibody light chain RNA described in P4; 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 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 cell described in P8. as well as 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, The heavy chain of the genetically engineered monoclonal antibody also includes a tag peptide and / or a signal peptide for separating and purifying the protein. The light chain of the genetically engineered monoclonal antibody also includes a tag peptide and / or a signal peptide for separating and purifying the protein.

4. The use of the hybridoma cells of claim 1, the monoclonal antibody of claim 2, the genetically engineered monoclonal antibody of claim 2 or 3, the antibody derivative of claim 2, the genetically engineered vector of claim 2, or the cells of claim 2 in the preparation of a formulation for recognizing material b; Material b is selected from B1, B2, B3, B4, B5, and B6: B1: The amino acid sequence of the peptide segment shown in positions 195 to 203 of SEQ ID NO.2; B2: A fusion protein containing a peptide with an amino acid sequence as shown in positions 195 to 203 of SEQ ID NO.2; B3: A peptide with an amino acid sequence as shown in SEQ ID NO.2; B4: A fusion protein containing a peptide with an amino acid sequence as shown in SEQ ID NO.2; B5: Avian reticuloendotheliosis virus encoding the gp90 protein with the amino acid sequence shown in SEQ ID NO.2; A6: Contains GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.

1. GQ415643.1, GU012639.1, GU012643.1, GQ415644.1, GU969140.1, GU012644.1, GU Avian reticuloendotheliosis virus with the gp90 protein amino acid sequence recorded in any of the GenBank accessions 012646.1, GQ415645.1, DQ513316.1, DQ513317.1, FJ439120.1, FJ439119.1, DQ387450., GU222420.1, and DQ237901.

1.

5. A method for detecting avian reticuloendotheliosis virus gp90 protein in a sample for non-diagnostic purposes, the method comprising the following steps: T1: The sample to be tested is fixed onto the solid surface to obtain a fixed sample; T2: Incubate the immobilized sample with a monoclonal antibody to obtain the first immobilized sample; The monoclonal antibody is the monoclonal antibody described in claim 2 or 3 or a genetically engineered monoclonal antibody. T4: Add the labeled monoclonal antibody-specific conjugate to the immobilized sample after the first incubation and incubate to obtain the immobilized sample after the second incubation. T5: The marker that is indirectly associated with the immobilized sample after the second incubation, and the presence or absence of the marker determines whether the avian reticuloendotheliosis virus gp90 protein is present in the sample to be tested.

6. The method as described in claim 5, characterized in that, Choose from any one of the following: C1, C2, C3, C4, and C5. C1: In step T1, the sample to be tested is in the form of cell lysate; C2: In step T1, the solid surface is the inner surface of the pores of the microporous plate; C3: In step T3, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in claim 2 or 3 with fluorescent molecular labeling; In step T4, the immobilized sample after the second incubation is photographed using a fluorescence microscope, and the presence of avian reticuloendotheliosis virus gp90 protein in the sample to be tested is determined based on the photographing results. C4: In step T3, the labeled monoclonal antibody-specific conjugate is a secondary antibody labeled with horseradish peroxidase as described in claim 2 or 3, or a genetically engineered monoclonal antibody; in step T4, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of avian reticuloendotheliosis virus gp90 protein in the sample to be tested is determined based on the chromogenic result. C5: Avian reticuloendotheliosis virus gp90 protein is GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.1, GQ415643.1, GU012639.1, GU012643.1, GQ415644.1, GU969140.1, GU012644. The avian reticuloendotheliosis virus gp90 protein whose amino acid sequence is recorded in any of the GenBank accessions: .1, GU012646.1, GQ415645.1, DQ513316.1, DQ513317.1, FJ439120.1, FJ439119.1, DQ387450., GU222420.1, and DQ237901.

1.

7. A method for detecting the presence of avian reticuloendotheliosis virus strains in a sample for non-diagnostic purposes; The method includes the following steps: S1: The sample to be tested is inoculated into avian reticuloendotheliosis virus susceptible cells to obtain 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 described in claim 2 or 3 or a genetically engineered monoclonal antibody. 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 avian reticuloendotheliosis virus strain is present in the sample to be tested based on the presence or absence of the marker.

8. The method as described in claim 7, characterized in that, Choose from any one of the following options: D1, D2, D3, and D4. D1: In step S1, the avian reticuloendotheliosis virus susceptible cells are primary chicken embryo fibroblasts; D2: In step S2, the solid surface is the inner surface of the pores of the microporous plate; D3: 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 avian reticuloendotheliosis virus strain in the sample to be tested is determined based on the photographic results. D4: 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 avian reticuloendotheliosis virus strain in the sample to be tested is determined based on the chromogenic result.

9. The method as described in claim 8, characterized in that, The avian reticuloendotheliosis virus was selected from GQ415646.2, AY842951.1, FJ496333.1, GQ375848.1, GU012640.1, GU012638.1, GQ415643.1, GU012639.1, GU012643.1, GQ415644.1, GU969140.1, and GU012644.

1. The avian reticuloendotheliosis virus strain corresponding to the gp90 protein amino acid sequence recorded in any of the GenBank accessions: GU012646.1, GQ415645.1, DQ513316.1, DQ513317.1, FJ439120.1, FJ439119.1, DQ387450, GU222420.1, and DQ237901.1.

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