ALV-J envelope protein GP85 FE10 epitope peptide, hybridoma cell strain and monoclonal antibody and application thereof

Abstract

The invention discloses an epitope peptide of avian leukosis virus J subgroup envelope protein GP85, a hybridoma cell strain 3G10E12C4, a monoclonal antibody (mAb) generated by the hybridoma cell strain 3G10E12C4 and application of the monoclonal antibody (mAb). An ALV-J subgroup GP85 protein FE10 epitope peptide is expressed on the surface of an engineering bacterium DH5alpha through a MisL protein passenger-carrying structural domain of a salmonella V-type secretion system in an exhibiting manner and serves as an immunogen to immunize a BALB / c mouse, meanwhile, the FE10 epitope peptide is inserted into a peg pilus operon, an inert carrier bacterium S9H is introduced, the FE10 epitope peptide serves as a detection system, mouse splenocytes and SP2 / 0 cells are fused, and a detection result is obtained. And the monoclonal antibody is obtained through double screening of ELISA (enzyme-linked immuno sorbent assay) and detection targets. The mAb prepared from the hybridoma cell can be specifically combined with the ALV-J strain infected with the HD-11 cell, and has potential application value and prospect in detection, treatment and purification of the ALV.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical technology and immunodiagnostic detection technology, specifically involving the ALV-J envelope protein GP85 FE10 epitope peptide, hybridoma cell line 3G10E12C4 and its monoclonal antibody and applications. Background Technology

[0002] Avian leukosis virus (ALV) can be classified into two main categories based on the antigenicity, structural characteristics, and host infectivity of its viral envelope protein GP85: endogenous and exogenous. Specifically, it includes 11 subgroups: A, B, C, D, E, F, G, H, I, J, and K. Subgroup J (ALV-J) has attracted significant attention due to its unique epidemiological characteristics and pathogenicity. Subgroup J is the most infectious and pathogenic of all ALV subgroups. ALV-J also has strong transmissibility, capable of vertical transmission via hatching eggs, leading to persistent infection of chicken flocks. The ALV-J pathogen adapts to various poultry hosts, such as commercial laying hens and local breeds, and exhibits complex pathogenicity. It can cause myeloid leukosis and multi-organ tumors, induce immunosuppression, and subsequently lead to secondary infections with other viruses and bacteria, posing a serious threat and damage to the poultry farming industry. Currently, ALV-J and ALV-K are the most prevalent subgroups in chicken flocks, with ALV-J being more pathogenic. Furthermore, there are currently no effective drugs or vaccines to prevent ALV-J infection; control is only possible through strict testing and germplasm purification procedures. Therefore, research targeting the mechanisms of action of ALV-J, developing effective antiviral drugs, and precise diagnostic methods are of great significance for the prevention and control of ALV-J.

[0003] Monoclonal antibodies have important applications in the diagnosis, detection, typing, treatment, and vaccine development of ALV. The GP85 protein specifically recognizes viral receptors on the host cell membrane, determining the scope and subgroup specificity of infection. Since ALV-J is currently the main prevalent subgroup, in-depth research into anti-ALV-J GP85-specific monoclonal antibodies will facilitate precise epidemiological investigations. In terms of vaccine development, monoclonal antibodies with neutralizing activity also hold great potential in ALV treatment and vaccine development.

[0004] The preparation of monoclonal antibodies mainly includes two key steps: (1) preparation of immunogens; (2) screening of monoclonal antibodies. (1) Preparation of immunogens: In hybridoma technology, traditional immunogens mainly include three types: naturally extracted protein antigens, recombinant expressed proteins and synthetic short peptides, all of which have certain drawbacks. Although natural proteins can maintain their natural conformation and self-modification, the purification cost is high, and only a few structures such as bacterial flagella and pili can be obtained through this method; heterologous expressed recombinant proteins can be mass-produced, but the antigenic determinant consists of only 4-6 amino acid residues, which accounts for only a very small part of the immunogen protein. The proportion of non-functional regions inside the immunogen is too large, which will lead to the prepared monoclonal antibodies targeting redundant structures with a high probability, wasting the host's limited immune response and making subsequent screening difficult. Moreover, recombinant proteins can only accurately express primary structures, and the conformational epitopes of antigens are difficult to maintain; although synthetic peptides can enrich the dominant epitopes of specific antigens, they are only applicable to linear epitopes and cannot meet the synthesis of spatial conformational epitopes. In addition, the cost of synthetic peptides is high, and subsequent purification is difficult. (2) Screening of monoclonal antibodies: Traditional methods usually use the same intact protein antigen as the immunogen for hybridoma cell screening. However, large molecular weight intact proteins have many redundant regions and unknown antigenic epitope determinants. These redundant components can induce the production of ineffective mAbs or cause non-specific reactions. Moreover, the epitopes targeted by the screened monoclonal antibodies are unknown. In addition, the identification process of antigenic epitopes is complex, time-consuming, costly, and technically demanding.

[0005] The current preparation of ALV monoclonal antibodies mainly revolves around two key proteins, p27 and GP85. GP85, as the viral envelope protein, has promising prospects as a diagnostic and monitoring antigen. However, due to the complexity of its envelope protein structure and function, its expression and purification face significant technical challenges. For example, (1) to overcome its strong hydrophobicity, a fusion tag needs to be added to assist expression. This not only changes the protein's native conformation but also requires laborious tag removal later, and the body may also generate an additional immune response against the tag; (2) the high variability of ALV-J leads to significant differences in gene sequences and antigenic epitopes among different strains, and the monoclonal antibodies obtained targeting a single epitope cannot effectively identify and neutralize the constantly mutating wild-type strains. In view of this, it is necessary to develop broad-spectrum antibodies targeting the conserved epitopes of the ALV-J GP85 protein. It is worth noting that due to the high variability of ALV-J, it is difficult to obtain conserved (conserved within ALV-J subgroups) and specific (specific between different ALV subgroups) epitope peptides. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide an epitope peptide FE10 of the GP85 of the ALV-J envelope protein of avian leukosis virus.

[0007] A second objective of this invention is to provide a nucleic acid molecule encoding the GP85 FE10 epitope peptide of the ALV-J envelope protein.

[0008] A third objective of this invention is to provide expression cassettes, recombinant vectors, recombinant cells, recombinant strains, immunogens, or detection systems.

[0009] The fourth objective of this invention is to provide the hybridoma cell line 3G10E12C4 and its monoclonal antibody.

[0010] The fifth objective of this invention is to provide the application of the ALV-J envelope protein GP85 FE10 epitope peptide, the nucleic acid molecule of the GP85 FE10 epitope peptide, expression cassette, recombinant vector, recombinant cell or recombinant strain, immunogen, detection system, hybridoma cell line 3G10E12C4 or monoclonal antibody in the preparation of a kit for detecting ALV-J virus or its antibody.

[0011] The sixth objective of this invention is to provide a kit for detecting ALV-J virus infection or ALV-J virus antibodies.

[0012] To address the bottlenecks in existing monoclonal antibody preparation processes, this invention utilizes the guest-carrying domain of a bacterial type V secretion system to display the epitope peptide, obtained for the first time, on the bacterial surface. This invention also provides a method for preparing recombinant strains or immunogens containing the epitope peptide for immunizing BALB / c mice during monoclonal antibody preparation. Furthermore, this invention provides a method for preparing recombinant strains or detection systems containing the same epitope peptide as the immunogen, which can be used to screen hybridoma cells secreting specific antibody supernatants during monoclonal antibody preparation. The monoclonal antibody (mAb) of this invention facilitates precise epidemiological surveys of ALV-J and its applications in ALV infection treatment and vaccine development.

[0013] Technical solution: In order to solve the above technical problems, the present invention provides an ALV-J envelope protein GP85 FE10 epitope peptide, the amino acid sequence of which is FNGTGGAEAE.

[0014] The present invention also provides a nucleic acid molecule encoding the GP85 FE10 epitope peptide of the ALV-J envelope protein, wherein the sequence of the nucleic acid molecule of the GP85 FE10 epitope peptide is TTTAATGGGACTGGTGGGGCGGAAGCAGAA.

[0015] The present invention also provides expression cassettes, recombinant vectors, recombinant cells or recombinant strains containing the aforementioned nucleic acid molecules.

[0016] The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.1 or SEQ ID NO.3.

[0017] The present invention also provides the recombinant strain or immunogen described above, wherein the recombinant strain or immunogen is obtained by introducing a recombinant vector with the sequence shown in SEQ ID NO.1 into a vector bacterium.

[0018] The recombinant strain or immunogen is obtained by inserting a nucleic acid molecule sequence containing the epitope peptide FE10 into the MisL carrier domain of the Salmonella V-type secretion system and introducing it into a vector bacterium.

[0019] The present invention also provides the recombinant strain or detection system described above, wherein the recombinant strain or detection system is obtained by introducing a recombinant vector with a sequence as shown in SEQ ID NO.3 into a vector bacterium.

[0020] The recombinant strain or detection system is obtained by inserting the nucleic acid molecule sequence encoding the epitope peptide FE10 into the Salmonella peg fimbriae operon gene and introducing it into the inert vector strain S9H.

[0021] This invention also provides a hybridoma cell line 3G10E12C4, which is deposited at the China Center for Type Culture Collection (CCTCC), classified and named Hybridoma cellline 3G10E12C4, with a deposit date of June 17, 2025, accession number CCTCC NO: C2025189, and deposit address in Wuhan, China.

[0022] The present invention also provides a monoclonal antibody, which is secreted by the hybridoma cell line 3G10E12C4.

[0023] The monoclonal antibody 3G10E12C4 is obtained by injecting the hybridoma cell line 3G10E12C4 into the peritoneal cavity of mice, collecting the ascites fluid, centrifuging to remove cell debris and other precipitates, and then purifying the supernatant.

[0024] This invention also provides the application of the ALV-J envelope protein GP85 FE10 epitope peptide, the nucleic acid molecule of the GP85 FE10 epitope peptide, the expression cassette, the recombinant vector, the recombinant cell or recombinant strain, the recombinant strain or immunogen, the recombinant strain or detection system, the hybridoma cell line 3G10E12C4, or the monoclonal antibody in the preparation of a kit for detecting ALV-J virus or its antibody.

[0025] The present invention also provides a kit for detecting ALV-J virus infection or ALV-J virus antibody, the kit comprising the ALV-J envelope protein GP85 FE10 epitope peptide, the nucleic acid molecule of the GP85 FE10 epitope peptide, the expression cassette, recombinant vector, recombinant cells or recombinant strain, the recombinant strain or immunogen, the recombinant strain or detection system, the hybridoma cell line 3G10E12C4 or the monoclonal antibody.

[0026] This invention also provides a specific method for preparing and screening monoclonal antibodies against the ALV-J subset GP85 protein epitope peptide FE10, including the following steps:

[0027] (1) Obtaining the FE10 epitope peptide: Using the B cell epitope prediction online website, the B cell epitope in the ALV-J subset membrane protein GP85 was predicted. Based on the amino acid score and its bioinformatics analysis, the B cell epitope peptide was optimized and selected to obtain the epitope peptide FE10. (2) Preparation of immunogen or recombinant strain: The FE10 epitope peptide was inserted into the MisL carrier domain of the Salmonella V-type secretion system and introduced into DH5α engineered bacteria to obtain recombinant strain DH5α (pBR322-MisL-ALV-J-GP85-FE10) that can display the expression of the FE10 epitope on the cell surface, which was used as an immunogen; (3) Animal immunization: BALB / c mice were subcutaneously injected with the immunogen or recombinant strain expressing the FE10 epitope peptide from (2) at a dose of 1×10⁻⁶. 8 CFU / 0.1mL / animal, immunize once every 12 days, for a total of three immunizations, and give a booster immunization on the third day after the third immunization;

[0028] (4) Preparation of detection system or recombinant strain: The nucleotide sequence encoding the epitope peptide FE10 was inserted into the major subunit pegA of Salmonella peg fimbriae and introduced into the inert vector strain S9H. The FE10 epitope peptide can be functionally presented on the surface of S9H through the peg fimbriae presentation system. S9H (pBR322-peg-ALV-J-GP85-FE10) was used as the detection target and S9H (pBR322-peg) was used as the detection control to qualitatively and quantitatively detect the specific antibodies against the FE10 epitope peptide in the serum of immunized mice.

[0029] (5) Cell fusion: Spleen cells from BALB / c mice after booster immunization were fused with myeloma cells SP2 / 0;

[0030] (6) ELISA screening of positive wells: 14 days after fusion, positive wells were screened using indirect ELISA. When the OD of the antigen well was positive, the positive serum was detected. 450nmValue / Negative serum detection antigen well OD 450nm The experiment is valid when the value is ≥2.1; when the detection target in the supernatant of the cells to be tested coats the well OD 450nm Value - OD of the detection control coated well in the supernatant of the cells to be tested 450nm If the value is greater than 0.15, the well is considered a positive well.

[0031] (7) Screening positive wells by glass plate agglutination method: For the preliminary positive wells screened by ELISA, S9H (pBR322-peg-ALV-J-GP85-FE10) expressing the FE10 epitope peptide was used as the detection target, and S9H (pBR322-peg) was used as the detection control. The supernatant of the cells to be tested was subjected to glass plate agglutination detection. If the supernatant to be tested reacts with the detection target and clear white agglutination particles appear (the detection control does not react with the supernatant to be tested), it can be judged as a positive well (this well is a hybridoma cell that can secrete specific antibodies).

[0032] (8) Subcloning of hybridoma cells: The positive wells that were selected and able to secrete antibodies specific to the FE10 epitope were subcloned and expanded three times using the limiting dilution method. The single cell wells in the cell culture plate were detected by ELISA and glass plate agglutination. Both ELISA and glass plate agglutination were positive. At this time, a hybridoma cell line 3G10E12C4 that can stably secrete peptides targeting the FE10 epitope was obtained.

[0033] (9) Preparation and purification of specific monoclonal antibodies: Three 8-week-old BALB / c mice were blocked by intraperitoneal injection of liquid paraffin (0.3 mL / mouse). Hybridoma cell line 3G10E12C4 was injected with 1×10 6 A dose of 1 cell / mouse was injected into the peritoneal cavity of mice. When the abdomen was significantly distended, ascites fluid was collected, centrifuged at 12,000 rpm for 5-10 min, and the precipitate was removed to obtain unpurified specific monoclonal antibody against the FE10 epitope peptide. The ascites antibody was purified using saturated ammonium sulfate to obtain a monoclonal antibody against the ALV-J envelope protein GP85 FE10 epitope peptide.

[0034] (10) Functional verification of 3G10E12C4 monoclonal antibody: Susceptible cells HD-11 were infected with J subgroup avian leukosis virus (JS09GY3 strain). Indirect immunofluorescence detection was performed on the infected cells. The ascites antibody purified in (9), mouse positive serum obtained from immunizing mice, and mouse negative serum were used as primary antibodies, respectively. The results showed that the monoclonal antibody 3G10E12C4 could bind to ALV-J virus and had functionality.

[0035] Beneficial Effects: The monoclonal antibody prepared in this invention targets the antigenic epitope sequence (FE10), which not only avoids the situation of blindly unknown epitopes and functions caused by traditional methods of preparing monoclonal antibodies, but also saves the tedious work and cost of further epitope identification after monoclonal antibody preparation. This invention also uses the guest-carrying domain of the bacterial type V secretion system and bacterial pili to amplify and present the epitope peptide obtained in this invention on the bacterial surface, preparing immunogens and detection targets respectively. Compared with recombinant expressed proteins and synthetic short peptides, the immunogen in this invention can amplify and present the epitope peptide by orders of magnitude. The immunogen does not require expression and purification, reducing the number of immunizations and the total immunization time in the immunization program. No additional adjuvants are needed during immunization, reducing the cost of monoclonal antibody preparation and simplifying the process. Simultaneously, this invention prepared a monoclonal antibody targeting the FE10 epitope peptide of the GP85 protein of ALV-J, successfully verifying the functionality of this monoclonal antibody. This contributes to the accurate epidemiological investigation of ALV-J and its application in ALV infection treatment and vaccine development, providing a powerful tool for the eradication of avian leukosis. Attached Figure Description

[0036] Figure 1 The recombinant plasmid map of pBR322-MisL-ALV-J-GP85-FE10;

[0037] Figure 2 This is an identification diagram of the DH5α (pBR322-MisL-ALV-J-GP85-FE10) recombinant bacteria. Lane M is the Trans2K plus II DNA Marker; Lane 1 is the negative control; Lane 2 is the amplification product of the DH5α (pBR322-MisL) recombinant bacteria; Lane 3 is the amplification product of the DH5α (pBR322-MisL-ALV-J-GP85-FE10) recombinant bacteria.

[0038] Figure 3 This is an identification diagram of the S9H (pBR322-peg-ALV-J-GP85-FE10) recombinant bacteria, where lane M is the Trans 2K DNA Marker; lane 1 is the negative control (template is double-distilled water ddH2O); lane 2 is the amplification product of the S9H (pBR322-peg) recombinant bacteria; and lane 3 is the amplification product of the S9H (pBR322-peg-ALV-J-GP85-FE10) recombinant bacteria.

[0039] Figure 4The image shows the antibody titer results for the mouse ascites monoclonal antibody 3G10E12C4. The detection control is S9H (pBR322-peg) (left), and the detection target is S9H (pBR322-peg-ALV-J-GP85-FE10) (right). If the agglutination reaction is negative, there are no agglutinated particles in the reaction solution. If the agglutination reaction is positive, white agglutinated particles are produced in the reaction solution, and the particles aggregate to both sides. The detection control shows no agglutination reaction. Positive agglutinated particles are indicated by red arrows in the image.

[0040] Figure 5 This image shows the indirect immunofluorescence results of the monoclonal antibody 3G10E12C4 against HD-11 cells infected with the ALV-J subset JS09GY3 strain. The primary antibody used in the 3G10E12C4 group was obtained by diluting the ascites fluid from mice prepared with 3G10E12C4 at a 1:200 ratio. The primary antibody used in the positive serum group was positive serum collected after immunization with fusion mice. The primary antibody used in the negative serum group was negative serum collected from feeder mice before treatment. The ALV strain used to infect HD-11 cells was JS09GY3. Specific green fluorescence was observed in the infected HD-11 cells, as well as in the 3G10E12C4 group and the positive serum group. No specific green fluorescence was observed in the negative serum from feeder mice. Detailed Implementation

[0041] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the following specific embodiments. The descriptions in the embodiments are merely examples of the present invention and are not intended to limit the scope of protection of the present invention. In addition to the specific methods, devices, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of the present invention, any prior art methods, devices, and materials similar to or the same as those described in the embodiments of the present invention can be used to implement the present invention.

[0042] Example 1: Construction of DH5α (pBR322-MisL-ALV-J-GP85-FE10) recombinant bacteria

[0043] 1. Obtaining the ALV-J GP85 protein epitope peptide FE10

[0044] The amino acid sequence of the envelope protein GP85 of avian leukosis virus subgroup J (ALV-J) was used to predict B-cell epitopes using the B-cell epitope prediction website BepiPred (https: / / services.healthtech.dtu.dk / services / BepiPred-3.0 / ). This website scores each amino acid residue in the predicted sequence, with a default threshold of 0.1512. If multiple consecutive amino acid residues have a score >0.1512, it is considered a B-cell epitope. Based on the amino acid residue scoring results, a B-cell epitope peptide of the GP85 protein was obtained and named FE10. The average amino acid residue score of this epitope peptide was 0.1817, and it was present in all ALV-J strains, but not in other ALV subgroup strains. Its amino acid sequence is FNGTGGAEAE, and the nucleic acid molecule sequence encoding the amino acid sequence of FE10 is: TTTAATGGGACTGGTGGGGCGGAAGCAGAA.

[0045] 2. Selection of FE10 epitope peptide substitution sites in MisL protein of bacterial type V secretion system

[0046] MisL is a type V secretion system in bacteria, containing a guest-carrying domain that can be displayed on the bacterial surface. It has been developed as a bacterial surface display tool for presenting exogenous antigens. Using a B-cell epitope prediction website, B-cell epitopes in the MisL protein (GenBank accession number: CAR39455.1) of *Salmonella typhimurium* strain 287 / 91 were analyzed. The immunogenic amino acids 310-319 of the MisL protein (replaced with the sequence ASKAASYSKA) were replaced with the FE10 epitope peptide. The nucleotide sequence of the replaced misl gene is GCCAGTAAGGCTGCATCCTATAGTAAAGCC. The recombinant expression vectors pBR322-MisL-ALV-J-GP85-FE10 (SEQ ID NO. 1) and pBR322-MisL (SEQ ID NO. 2) containing the replaced epitope peptide were artificially synthesized (Nanjing Qingke Biotechnology Co., Ltd.). The recombinant plasmid pBR322-MisL-ALV-J-GP85-FE10 is shown in the image. Figure 1 .

[0047] 3. Preparation of immunogens

[0048] The synthetically produced pBR322-MisL-ALV-J-GP85-FE10 and pBR322-MisL plasmids were diluted to 100 ng / μL and electroporated into DH5α competent cells (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: D1031S) using a Bio-Rad electroporator. Resistance selection was performed on LB solid medium containing 100 μg / mL ampicillin (Amp; Beijing Solarbio Science & Technology Co., Ltd., catalog number: A8180). After incubation at 37℃ for 16 h, single colonies were picked and inoculated into Amp containing 100 μg / mL. + After overnight incubation in LB liquid medium at 37°C and 200 rpm, bacterial colony PCR was performed for identification. The upstream primer sequences were F1: 5'-ATGCCAACTCCCCAAAATTACT-3' and R1: 5'-TCAGAAACTGTATTTCATCCCCAA-3'. The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; amplification for 35 cycles, including denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; and further amplification at 72°C for 5 min. The PCR products of positive colonies showed a 2868 bp target band after 1% agarose gel electrophoresis, confirming the successful construction of DH5α (pBR322-MisL-ALV-J-GP85-FE10) and DH5α (pBR322-MisL). Results are shown in the figure below. Figure 2 The constructed positive clones were stored at -70°C for later use.

[0049] Example 2: Preparation and screening of monoclonal antibodies targeting the FE10 epitope peptide of ALV-J GP85 protein

[0050] 1. Preparation of the detection system

[0051] To monitor the specific antibodies against the FE10 epitope peptide produced in BALB / c mice after immunization with DH5α (pBR322-MisL-ALV-J-GP85-FE10) immunogen, an antibody detection system targeting the FE10 epitope peptide was constructed. First, bioinformatics analysis was performed on the major subunit pegA (amino acids 1-177 of the PEG fimbriae) of the peg operon in Salmonella pullorum CVCC 526 (laboratory preserved, Yang Weifeng. Development and Preliminary Clinical Application of Anti-Salmonella PEG Filiform Antibody [D]. Yangzhou University, 2016.). A highly immunogenic position in pegA was selected and replaced with the FE10 epitope peptide. The replacement sites were amino acids 59-68 from the N-terminus of the pegA protein, with the replacement sequence: DRLTDLNPGD; and the replacement sites were 175-204 bp in the pegA gene, with the replacement sequence: GATAGATTGACTGACTTAAACCCTGGCGAT. The recombinant expression vector pBR322-peg-ALV-J-GP85-FE10 (SEQ ID NO.3), containing the ALV-J-GP85-FE10 epitope peptide nucleic acid sequence, and identification primers targeting pegA were artificially synthesized by Nanjing Qingke Biotechnology Co., Ltd. The primer sequences are as follows: upstream primer pegA-F: 5'-ATGAAACGTTCACTTATTGCTGCT-3', downstream primer pegA-R: 5'-TTAATCAGTTAATACCGTCATCGTCA-3'. The identification primers were used to confirm the successful construction of the subsequent recombinant strain (i.e., the detection system). The identification results are shown in […]. Figure 3 .

[0052] The synthesized recombinant expression vector plasmid pBR322-peg-ALV-J-GP85-FE10 was diluted to a final concentration of 100 ng / μL with enzyme-free water (Biosharp, catalog number: BL510B). It was then electroporated into competent cells of the inert vector bacterium S9H (S9H inert vector bacterium was isolated in our laboratory and has been granted a national patent, patent number: CN111500504A) (prepared and preserved in the laboratory). The cells were plated on LB solid medium containing 100 μg / mL ampicillin for resistance selection. The cells were incubated upside down at 37°C for 16 h. Single colonies from the solid medium were picked and inoculated into Ampicillin-containing medium. + LB liquid medium (Amp) + After obtaining bacterial culture by overnight incubation at a concentration of 100 μg / mL, the bacterial culture was identified by PCR using pegA identification primers. The verification results are shown in [link to results]. Figure 3The correctly identified recombinant bacteria were named S9H (pBR322-peg-ALV-J-GP85-FE10). After overnight incubation, the bacteria were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacteria were washed twice with PBS (1×PBS granules from Novizan dissolved in 1 L of ultrapure water, product number: G101). After the final centrifugation, the supernatant was discarded, the bacterial pellet was resuspended in PBS, and the bacterial concentration was adjusted to 5×10⁻⁶. 9 CFU / mL, stored at 4℃ for use as a detection system; the control system used for detection was S9H (pBR322-peg) (previously constructed and preserved in the laboratory, and has been granted a national invention patent, patent number: CN118496323A).

[0053] 2. Animal immunization and determination of antibody titers in immune serum

[0054] Three eight-week-old BALB / c mice were immunized three times by subcutaneous injection at multiple sites on the nape of their necks using the immunogen DH5α (pBR322-MisL-ALV-J-GP85-FE10) prepared in Example 1. Each immunization was performed 12 days apart, with a booster immunization given on the third day after the third immunization. The immunization dose was 0.1 mL / mouse for each mouse, and the bacterial concentration of the immunogen (recombinant strain DH5α (pBR322-MisL-ALV-J-GP85-FE10)) was 1×10⁻⁶. 9 CFU / mL. Blood was collected from the infraorbital venous plexus after booster immunization, and mouse serum was separated. The collected serum was subjected to plate agglutination reactions with S9H (pBR322-peg-ALV-J-GP85-FE10) and S9H (pBR322-peg) respectively to detect the antibody level against the FE10 epitope peptide in the immunized mice. The results are shown in Table 1 below. Mouse No. 1, which had the highest antibody titer against the FE10 epitope peptide among the three mice, was selected for subsequent cell fusion experiments.

[0055] Table 1. Monitoring of antibody titers in mouse serum after immunization Mouse No. 1 Mouse No. 2 Mouse No. 3 Post-exempt efficacy 1:64 1:64 1:16 Post-secondary immunization efficacy 1:521 1:256 1:32 Post-treatment efficacy 1:1024 1:512 1:64

[0056] 3. Cell fusion and positive well screening

[0057] (1) Preparation of SP2 / 0 cells

[0058] About two weeks before fusion, SP2 / 0 cells (preserved in our laboratory, Xia Pengpeng. Study on the specific protein receptor of enterotoxigenic Escherichia coli (ETEC) F4 fimbriae—porcine aminopeptidase N [D]. Yangzhou University, 2016.) were revived, their condition was adjusted and cultured to ensure that the SP2 / 0 cells were in the logarithmic growth phase at the time of fusion, and that the cells were plump, with clear outlines, in good condition, and with a viable cell count of more than 95%. On the day of fusion, SP2 / 0 cells were gently blown off with DMEM basal medium (Gibco, catalog number: C11995500BT), collected in centrifuge tubes, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the cells were resuspended with an appropriate amount of culture medium, washed once by centrifugation with DMEM (1000 rpm, 10 min), and finally the cell pellet was resuspended in 10 mL of DMEM to prepare a cell suspension, which was then mixed and used for later use.

[0059] (2) Preparation of feeder cells

[0060] In addition, one unimmunized BALB / c mouse was purchased, its eyeball was removed, and blood was collected. The serum was then separated to serve as a negative serum control for antibody testing. After euthanizing the BALB / c mouse by cervical dislocation, it was immersed in 75% alcohol for 5 minutes and fixed on a dissection board. Using sterile scissors and forceps, the abdominal skin was lifted from the posterior abdomen to expose the peritoneum, which was then disinfected by wiping with alcohol swabs. 8 mL of DMEM basal culture medium was injected into the peritoneal cavity in divided doses using a syringe, taking care to avoid puncturing the intestines and causing contamination. The syringe was then fixed so that the needle remained in the peritoneal cavity. Simultaneously, the abdomen was gently massaged with alcohol swabs, and the injected culture medium was slowly aspirated. Subsequently, the cells were centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in HAT medium (DMEM basal medium + 10% fetal bovine serum + 1% penicillin-streptomycin mixed antibiotic + 1% 50×HAT) (fetal bovine serum: Biochannel, catalog number BC-SE-FBS08; HAT: Gibco, catalog number 21060017; penicillin-streptomycin mixed antibiotic: Biochannel, catalog number BC-CE-007) to achieve a cell concentration of 2×10⁶ cells / min. 5 The feeder cell suspension was prepared by increasing the cell count to mL. 100 μL of the cell suspension was added to each well of a 96-well plate and incubated at 37°C in a 6% CO2 incubator.

[0061] (3) Preparation of spleen B lymphocytes from immunized mice

[0062] Based on agglutination titers, mice with the highest agglutination antibody titers after triple immunization (mouse #1) were selected for booster immunization. On day 3 of the booster immunization, blood was collected from the immunized mice after enucleation, and the resulting serum was used as a positive control serum for antibody detection. After cervical dislocation and euthanasia, the mice were immersed in 75% alcohol for 5 minutes, fixed on a dissection board, and the abdominal cavity was opened to expose the spleen. The spleen was aseptically removed in a laminar flow hood and placed in a petri dish containing 10 mL of HAT medium. The spleen was washed and the connective tissue was removed. After rinsing, the spleen was transferred to a new petri dish, and the spleen cells were squeezed to enter the HAT medium. The cells were then pipetted to prepare a single-cell suspension. The single-cell suspension was centrifuged at 1000 rpm for 5 minutes, washed twice with HAT medium, resuspended in HAT medium, and the cells were counted for later use.

[0063] (4) Cell fusion

[0064] The spleen cells prepared in (3) and SP2 / 0 cells were added to a fusion tube at a ratio of 5:1 and mixed thoroughly. The mixture was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The bottom of the fusion tube was gently tapped to loosen and evenly distribute the cell pellet. 1 mL of preheated polyethylene glycol PEG1500 (Roche, catalog number: 10783641001) at 37°C was slowly added to the fusion tube as a fusion agent over 45 s, while gently tapping the bottom of the fusion tube. Then, preheated DMEM basal medium at 37°C was added to 30 mL at a rate that was slow at first and then fast. The fusion tube was then sealed and incubated in a constant temperature water bath at 37°C for 30 min. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded. The cell pellet was gently resuspended in 5 mL of HAT medium, and HAT medium was added to 40 mL. The cell suspension was added to a 96-well plate containing feeder cells at a volume of 100 μL / well and then incubated in a 37°C, 6% CO2 incubator. Ten days after fusion, the culture medium in the 96-well plate was aspirated, and 200 μL of HT complete culture medium (DMEM basal medium + 10% fetal bovine serum + 1% 100× HT; HT: Gibco, catalog number 11067030) was added to each well. When the cell culture supernatant turned yellow, the cell culture supernatant was tested by ELISA and glass plate agglutination assay.

[0065] (5) Screening of hybridoma cells in positive wells

[0066] (5.1) ELISA screening

[0067] ① Antigen Coating: S9H (pBR322-peg-ALV-J-GP85-FE10) was used as the detection antigen, and S9H (pBR322-peg) was used as the control antigen. Both were mixed with carbonate buffer (0.73g sodium bicarbonate, 0.4g sodium carbonate, and 250mL ultrapure water; sodium bicarbonate and sodium carbonate were purchased from Sinopharm Chemical Reagent Co., Ltd., catalog numbers 10018960 and 10019260, respectively) at a ratio of 1:10. 50μL of S9H (pBR322-peg-ALV-J-GP85-FE10) (bacterial concentration 1×10⁻⁶) was added to each of two 96-well plates. 10 The antigen bacteria and S9H (pBR322-peg) control antigen bacteria were detected by CFU / mL, and dried at 37℃ to obtain 96-well plates coated with specific antigens and controls;

[0068] ② Add 100 μL of pre-cooled methanol (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10014118) at -20℃ to each well, fix at room temperature for 15 min, wash 3 times with PBS for 5 min each time, and pat dry;

[0069] ③ Add 150 μL of 5% BSA blocking buffer (Beijing Solarbio Technology Co., Ltd., catalog number: A8020) to each well, block overnight at 4℃, wash 3 times with PBST (PBS with 0.5% Tween-20 added, Tween-20 purchased from Solarbio, catalog number: T8220), 5 min each time, and pat dry;

[0070] ④ Add 100 μL of hybridoma cell supernatant to each well of the two 96-well plates coated with the antigen bacteria for detection and the control antigen bacteria. At the same time, set up positive serum control and negative serum control (obtained in "(2) Preparation of feeder cells" and "(3) Preparation of spleen B lymphocytes of immunized mice" in "3. Cell fusion and positive well screening"). The serum was diluted 1:200 with PBS, and 100 μL was added to each well. The plate was incubated at 37°C for 2 h, washed 3 times with PBST for 5 min each time, and patted dry.

[0071] ⑤ Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (diluted 1:20000 with PBS; HRP-labeled goat anti-mouse IgG secondary antibody was purchased from Beijing Bio-Sen Biotechnology Co., Ltd., catalog number: bs-0296G-HRP) to each well, incubate at 37℃ for 1 h, wash 3 times with PBST for 5 min each time, and pat dry.

[0072] ⑥ Finally, add 100 μL of TMB chromogenic solution (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0209-500mL) to each well of the 96-well plate, and incubate at 37°C in the dark for 20-30 min. Then, add 50 μL of ELISA stop solution (Absin (Shanghai) Biotechnology Co., Ltd., catalog number: abs9472) to each well to terminate the reaction. Use a microplate reader (BioTek) to read the OD values ​​of each well in the 96-well plate. 450nm Numerical value.

[0073] ⑦ ELISA result interpretation: If the serum test antigen well OD is positive... 450nm Value / Negative serum detection antigen well OD 450nm The test is valid if the OD value of the recombinant bacterial wells, the detection target, is ≥2.1 in the supernatant of the tested cells. 450nm Value - OD of recombinant bacterial wells as a control for the detection of cell supernatant. 450nm A value > 0.15 indicates a positive well, which is designated as well 3G10. The indirect ELISA results of the positive hybridoma cell supernatant are shown in Table 2 below:

[0074] Table 2. Indirect ELISA results of 3G10-well positive hybridoma cell supernatant. <![CDATA[Sample OD 450nm value]]> Positive serum negative serum 3G10 Detection of target recombinant bacteria 1.463 0.179 0.338 Detection control recombinant bacteria 1.269 0.172 0.18 <![CDATA[Target OD 450nm - Control OD 450nm > 0.194 0.007 0.158

[0075] (5.2) Glass plate agglomeration screening

[0076] Cell supernatant from ELISA positive wells was used for plate agglutination detection. Two 6µL drops of hybridoma cell culture supernatant from the wells to be tested were placed on a glass slide. 6µL of the S9H (pBR322-peg-ALV-J-GP85-FE10) detection system and the S9H (pBR322-peg) control were added to each drop of hybridoma cell supernatant. The mixture was gently shaken on the slide to ensure adequate antigen-antibody contact. The presence of obvious white agglutination particles was observed. If white agglutination particles appeared on the target (but not in the control), the hybridoma cell positive well was considered positive.

[0077] Finally, a primitive hybridoma cell line was selected and named 3G10.

[0078] 4. Cell subcloning

[0079] Three subclonings were performed on the positive cell well 3G10, which produced antibodies specifically against the GP85-FE10 epitope peptide, using a limiting dilution method. The specific steps were as follows: The hybridoma cells to be cloned were diluted with HT complete medium to adjust the cell concentration to 10 cells / mL; the diluted hybridoma cells were aliquoted into 96-well plates, 100 μL per well, with one hybridoma cell per well; the cells were incubated at 37°C and 6% CO2 for 7 days. When cell clusters appeared in the wells, the cell supernatant was collected for identification by indirect ELISA and glass plate agglutination assay. This process was repeated until all single cell wells in the culture plate tested positive for ELISA and glass plate agglutination, at which point a hybridoma cell line 3G10E12C4 that stably secretes antibodies against the GP85-FE10 epitope peptide was obtained.

[0080] The hybridoma cell line 3G10E12C4 is deposited at the China Center for Type Culture Collection (CCTCC), and its classification name is Hybridoma cell line 3G10E12C4. The deposit date is June 17, 2025, the accession number is CCTCC NO: C2025189, and the deposit address is Wuhan, China.

[0081] 5. Preparation of monoclonal antibodies against ascites fluid from hybridoma cell line 3G10E12C4

[0082] Eight-week-old BALB / c mice were intraperitoneally injected with 0.3 mL of liquid paraffin per mouse. Seven days later, they were intraperitoneally injected with 1 × 10⁻⁶ 3G10E12C4 hybridoma cell line. 6 The injection volume was 0.1 mL per mouse. The condition of the mice was observed daily. When the mice's abdomen swelled and they felt tense to the touch, ascites fluid was collected using a syringe. The collected ascites fluid was centrifuged at 4000 rpm for 10 min. The supernatant was then collected to obtain the ascites monoclonal antibody of hybridoma cell line 3G10E12C4.

[0083] Example 3: Characterization of monoclonal antibody targeting ALV-J-GP85-FE10 epitope peptide

[0084] 1. Glass plate agglutination assay to detect the titer of mAb targeting the GP85-FE10 epitope peptide.

[0085] The ascites antibody prepared in Example 2, "5. Preparation of Monoclonal Antibody in Ascites Fluid of Hybridoma Cell Line 3G10E12C4", was first diluted 1:200 using sterile physiological saline, followed by serial dilutions: 30 µL of sterile physiological saline was added to each well of a 96-well plate, and 30 µL of the 1:200 diluted ascites antibody was added to the first well of each row, mixed thoroughly with sterile physiological saline, and then 30 µL of diluted ascites was added to the next well. This process was repeated 12 times for each dilution step. 6 µL of S9H (pBR322-peg-ALV-J-GP85-FE10) detection system and S9H (pBR322-peg) detection control (both with a bacterial concentration of 1×10⁻⁶) were added to each dilution of the ascites antibody. 10 (CFU / mL) Gently shake the mixture on a glass slide to ensure adequate contact between antigen and antibody, and observe for the appearance of obvious white agglutination particles. If white agglutination particles appear on the target (but not in the control), continue testing at the next dilution until no white agglutination particles appear on the target. The results show that the titer of the prepared ascites monoclonal antibody targeting the ALV-J-GP85-FE10 epitope peptide is 1:25600. (See attached table for test results.) Figure 4 .

[0086] 2. Indirect immunofluorescence assay to verify the specificity of mAb targeting the GP85-FE10 epitope peptide.

[0087] The prepared mouse ascites antibody was used as the primary antibody for indirect immunofluorescence detection to verify the reactivity of the prepared ascites fluid with the ALV-J subgroup JS09GY3 strain. Simultaneously, positive serum prepared during mouse immunization was used as a positive control, and negative serum from fed mice was used as a negative control for IFA detection. The specific IFA steps are as follows:

[0088] (1) One day in advance, seed the well-condition HD-11 cells in a 24-well plate (Luo Huan. Exploring the Glucose Metabolism Pathway Regulated by c-Myc Protein in Chickens to Counteract ALV-J Inhibitors [D]. Yangzhou University, 2023.). When the cell density reaches 60%-70%, discard the DMEM medium and add 200 μL of diluted JS09GY3 strain virus solution (MOI value of 5) to each well (the ALV-J strain used is JS09GY3 strain, Luo Huan. Exploring the Glucose Metabolism Pathway Regulated by c-Myc Protein in Chickens to Counteract ALV-J Inhibitors [D]. Yangzhou University, 2023.). After incubating the cell culture plate in the incubator for 2 hours, discard the virus solution and wash once with PBS. Add 200 μL of DMEM containing 1% FBS and continue culturing for 48 hours. Then discard the supernatant culture medium and wash three times with PBS for 5 minutes each time.

[0089] Preparation of viral solution: One day in advance, seed DF-1 cells in cell culture flasks. When the cell density reaches 60%~70%, discard the supernatant, wash 2-3 times with PBS, and inoculate with serum-free DMEM containing ALV-J isolate JS09GY3. Incubate at 37℃ for 1 hour, discard the supernatant, add DMEM medium containing 1% FBS, and incubate at 37℃ for 7 days. Repeat freeze-thaw cycles 2-3 times, centrifuge at 12000rpm for 5-10 minutes at 4℃, collect the supernatant, aliquot, and store at -80℃.

[0090] (2) Add 200 μL of 4% paraformaldehyde (Biosharp, catalog number: BL539A) to each well, fix at room temperature for 10 min, and wash with PBS 3 times for 3 min each time;

[0091] (3) Add 200 μL of 5% BSA blocking solution to each well, block at 37°C for 1 h, and wash with PBS 3 times for 5 min each time;

[0092] (4) Add 200 μL of 3G10E12C4 ascites antibody (diluted 1:200 with PBS) to each well, incubate overnight at 4°C, and wash 3 times with PBS for 5 min each time;

[0093] (5) Add the secondary antibody working solution (FITC-goat anti-mouse IgG, green fluorescence, diluted 1:200 with PBS, purchased from Boster Biological Engineering Co., Ltd., catalog number: BA1101) in the dark, 200 μL per well, incubate at 37℃ in the dark for 1 h, wash 3 times with PBS for 5 min each time;

[0094] (6) Add 100 μL of DAPI dye (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0131-25mL), incubate at 37°C in the dark for 10 min, wash 3 times with PBS for 5 min each time (all in the dark).

[0095] (7) Observe the results using an inverted fluorescence microscope (OLYMPUS) in the dark.

[0096] The results are as follows Figure 5 As shown, the blank control group without viral infection showed no green fluorescence, while 1:200 diluted 3G10E12C4 ascites fluid specifically bound to HD-11 infected with ALV-J (JS09GY3), and green fluorescence was observed. Obvious green fluorescence was also observed in the polyclonal antibody serum (positive serum) of immunized mice; however, no specific green fluorescence was observed in the negative serum of fed mice. These results indicate that the prepared ascites fluid monoclonal antibody can specifically bind to the ALV-J subset strain.

[0097] In summary, this invention successfully prepared the FE10 epitope peptide of the GP85 protein of avian leukosis virus (ALV) subgroup J, the hybridoma cell line 3G10E12C4, and a monoclonal antibody, verifying that the FE10 epitope peptide of the ALV subgroup J GP85 protein can produce specific antibodies. The monoclonal antibody prepared in this invention was demonstrated by indirect immunofluorescence assay that the 3G10E12C4 monoclonal antibody can specifically bind to ALV subgroup J infected with HD-11 cells, and the agglutination titer of the prepared ascites fluid reached 1:25600. This invention provides a powerful tool for developing effective antiviral drugs and accurate diagnostic detection methods against ALV-J, and also provides new ideas for the eradication of avian leukosis. Further research can provide a foundation for the development of fluorescent antibodies for ALV-J detection.

[0098] SEQ ID NO.1: Recombinant expression vector pBR322-MisL-ALV-J-GP85-FE10 nucleotide sequence

[0099] SEQ ID NO.2: Nucleotide sequence of recombinant expression vector pBR322-MisL

[0100] SEQ ID NO.3: Nucleotide sequence of recombinant expression vector pBR322-peg-ALV-J-GP85-FE10

Claims

1. The ALV-J envelope protein GP85 FE10 epitope peptide, characterized in that, Its amino acid sequence is FNGTGGAEAE.

2. A nucleic acid molecule encoding the GP85 FE10 epitope peptide of the ALV-J envelope protein according to claim 1, characterized in that, The nucleic acid sequence of the GP85 FE10 epitope peptide is TTTAATGGGACTGGTGGGGCGGAAGCAGAA.

3. An expression cassette, recombinant vector, recombinant cell or recombinant strain containing the nucleic acid molecule of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The nucleotide sequence of the recombinant vector is shown in SEQ ID NO.1 or SEQ ID NO.

3.

5. The recombinant strain or immunogen according to claim 3, characterized in that, The recombinant strain or immunogen is obtained by introducing the recombinant vector of claim 4, as shown in SEQ ID NO.1, into the vector bacteria.

6. The recombinant strain or detection system according to claim 3, characterized in that, The recombinant strain or detection system is obtained by introducing the recombinant vector of claim 4, as shown in SEQ ID NO.3, into the vector bacteria.

7. A hybridoma cell line 3G10E12C4, characterized in that, The hybridoma cell line 3G10E12C4 is deposited at the China Center for Type Culture Collection (CCTCC), and its classification name is Hybridoma cell line 3G10E12C4. The deposit date is June 17, 2025, and the accession number is CCTCC NO: C2025189.

8. A monoclonal antibody, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line 3G10E12C4 described in claim 1.

9. The use of the ALV-J envelope protein GP85 FE10 epitope peptide of claim 1, the nucleic acid molecule of the GP85 FE10 epitope peptide of claim 2, the expression cassette, recombinant vector, recombinant cell or recombinant strain of claim 3, the recombinant strain or immunogen of claim 5, the recombinant strain or detection system of claim 6, the hybridoma cell line 3G10E12C4 of claim 7, or the monoclonal antibody of claim 8 in the preparation of a kit for detecting ALV-J virus or its antibody.

10. A kit for detecting ALV-J virus infection or ALV-J virus antibodies, characterized in that, The kit comprises the ALV-J envelope protein GP85 FE10 epitope peptide of claim 1, the nucleic acid molecule of the GP85 FE10 epitope peptide of claim 2, the expression cassette of claim 3, the recombinant vector, the recombinant cell or recombinant strain of claim 3, the recombinant strain or immunogen of claim 5, the recombinant strain or detection system of claim 6, the hybridoma cell line 3G10E12C4 of claim 7, or the monoclonal antibody of claim 8.

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