Celine leukemia virus p27 protein monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The invention discloses a feline leukemia virus p27 protein monoclonal antibody, a hybridoma cell strain and application thereof, and relates to the technical field of biology, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.1 in a sequence table, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.2 in the sequence table. According to the invention, a cell fusion technology is utilized to establish a hybridoma cell strain p27-3A5 capable of secreting a p27 protein monoclonal antibody, and a specific monoclonal antibody 3A5 is obtained. And the minimum linear epitope identified by the antibody is identified, so that the establishment of subsequent preparation of a diagnostic kit such as a colloidal gold test strip is facilitated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against the p27 protein of feline leukemia virus, a hybridoma cell line, and their applications. Background Technology

[0002] Feline leukemia virus (FeLV) belongs to the family Retroviridae, subfamily Orthoretrovirinae, and genus Gamma-retrovirus. It is an enveloped, single-stranded, positive-sense RNA virus that appears as spherical particles about 100 nm in size under an electron microscope. In 1964, Jarrett's team first reported on the transmission of feline lymphomavirus in *Nature*, observing typical type C retrovirus particles under an electron microscope, which were later named FeLV.

[0003] The FeLV gene sequence is LTR-gag-pol-env-LTR. LTRs (long terminal repeats) play a crucial role in viral tissue orientation and pathogenicity. The gag (group-associated antigen) gene encodes internal structural proteins, including p15c, p12, p27, and p10. p27 is abundant in the cytoplasm of infected cells and is also present in large quantities in the blood of infected cats. Furthermore, free p27 protein is shed in tears and saliva and can be detected in these bodily fluids; therefore, most existing immunochromatographic assays (such as enzyme-linked immunosorbent assays, colloidal gold test strips, and immunofluorescence assays) are designed to detect p27 protein. The pol (polymerase) gene encodes the viral reverse transcriptase (RT), which is responsible for synthesizing DNA using RNA as a template. The env (env) gene encodes the envelope components gp70 and p15e. gp70 determines the viral subtype, while the transmembrane protein p15e is thought to interfere with the host cell's immune response. Obtaining specific antibodies against the p27 protein is crucial for diagnosing whether a cat is infected with leukemia virus. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody against feline leukemia virus p27 protein, a hybridoma cell line, and their applications.

[0005] The monoclonal antibody against feline leukemia virus p27 protein of the present invention has the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.1 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO.2.

[0006] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-39 in SEQ ID NO.1.

[0007] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 52-66 in SEQ ID NO.1.

[0008] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-114 in SEQ ID NO.1.

[0009] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-35 in SEQ ID NO.2;

[0010] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 51-57 in SEQ ID NO.2;

[0011] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 90-98 in SEQ ID NO.2.

[0012] An expression vector containing a monoclonal antibody against the feline leukemia virus p27 protein.

[0013] A hybridoma cell line p27-3A5 that secretes a monoclonal antibody against the feline leukemia virus p27 protein, and a hybridoma cell line mAb3A5 that secretes a monoclonal antibody, are deposited at the China General Microbiological Culture Collection Center on October 17, 2025, with accession number CGMCC No. 46706.

[0014] The application of the above-mentioned feline leukemia virus p27 protein monoclonal antibody in the preparation of feline leukemia virus detection kits and / or test strips.

[0015] A feline leukemia virus p27 detection kit containing a monoclonal antibody against the feline leukemia virus p27 protein.

[0016] The beneficial effects of this invention are:

[0017] This invention provides a monoclonal antibody against the p27 protein of feline leukemia virus. A hybridoma cell line, p27-3A5, capable of stably secreting anti-p27 protein specificity, was successfully prepared. The recognition site of antibody 3A5 differs from the recognition site of antibody (A1) in patent CN120738129A; the two complement each other in use. Using antibody A1 as a capture antibody and 3A5 as a detection antibody forms a paired antibody for immunological detection purposes.

[0018] This invention also identifies the smallest linear antigenic epitope recognized by the antibody, which is beneficial for the subsequent development of diagnostic kits such as colloidal gold test strips. The antibody of this invention can be used for scientific research on viruses. Attached Figure Description

[0019] Figure 1 Results of SDS-PAGE and Western Blot analysis of the p27 truncated protein;

[0020] Figure 2 To construct the p27-CRFK cell line and determine the serum IFA titer in mice;

[0021] Figure 3 Results of serum IFA in mice;

[0022] Figure 4 The results of IFA on monoclonal antibody 3A5 in p27-CRFK cell line;

[0023] Figure 5 Western blot results for recognition of p27 protein and epitope identification by monoclonal antibody 3A5;

[0024] Figure 6 This is a schematic diagram of the recognition site of monoclonal antibody 3A5;

[0025] Figure 7 This is a graph showing the antigen-P / N ratio relationship in Example 3;

[0026] Figure 8 This is a graph showing the sample-P / N value relationship in Example 3. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0028] Example 1: Prokaryotic expression of p27 truncated protein and construction of p27-CRFK cells

[0029] 1. Prokaryotic expression of p27 truncated protein:

[0030] Based on the nucleotide sequence of the truncated p27 protein and the multiple cloning site of the pET32a prokaryotic expression vector, EcoRI and HindIII were selected as the double restriction enzyme sites for vector construction. Upstream and downstream primers were designed using SnapGene software: primer F: 5'-CTGATATCGGATCCGAATTCAGTGTTATCCTGTCCTTTAT-3' and primer R: 5'-TCGAGTGCGGCCGCAAGCTTCAGAACTTTAGTCATCTCCT-3', respectively. These primers were synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd. The truncated p27 protein nucleotide sequence was amplified, recombined with the double-restricted linearized vector pET32a, and transformed into DH5 cells. Single colonies were picked from LB agar plates containing ampicillin and screened using colony PCR to identify the p27 gene product (256 bp). Samples were sent to the company for sequencing, and the sequencing results were correct, confirming the recombinant plasmid pET32a-p27. Recombinant plasmid pET32a-p27 was transformed into Escherichia coli BL21(DE3). Single colonies were picked and inoculated into 5 mL of ampicillin-resistant LB broth, and placed on a shaker at 37°C and 180 rpm. When the bacterial culture became turbid, 200 μL was transferred to 200 mL of ampicillin-resistant LB broth for expansion culture. After shaking on a shaker at 37°C and 180 rpm for 4-5 hours, when the bacterial culture became turbid, IPTG inducer was added, and expression was induced on a shaker at 16°C and 150 rpm. After 24 hours, the bacterial pellet was collected by centrifugation, resuspended in 10 mL of PBS, sonicated, and centrifuged again. The supernatant and pellet were sampled separately for SDS-PAGE analysis and Coomassie brilliant blue staining to analyze the solubility of the truncated p27 protein. The p27 truncated protein expressed in the precipitate was continuously dissolved in 1 mol / L urea and 2 mol / L urea. The 1 mol / L and 2 mol / L urea solutions after dissolving the p27 protein were mixed and subjected to SDS-PAGE Coomassie staining to analyze the elution of the precipitated p27 truncated protein. The protein was then detected by Western blotting using anti-His antibody as the primary antibody. After overnight dialyzing with 2 L of PBS, the protein concentration was determined by the BCA method.

[0031] 2. Construction of p27-CRFK cells

[0032] The pLvx-IRES-mCherry plasmid vector was preserved in our laboratory. EcoRI and BamHI were selected as the double restriction enzyme sites for the plasmid vector. The reference strain for the inserted p27 gene was FeLV-FAIDS (GenBank: M18247.10). The upstream primer pLVX-p27-F and the downstream primer pLVX-p27-R were designed. The p27 fragment synthesized using the pcDNA3.1(+)-FeLV-FAIDS whole genome as a template was recombined with the double-digested pLvx-IRES-mCherry linearized vector. The recombinant was transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and single colonies were picked and cultured. After the bacterial culture with correct sequencing was expanded, the recombinant plasmid was extracted and named pLvx-IRES-mCherry-p27.

[0033] Upstream primer pLVX-p27-F:

[0034] 5'-TAGAGGATCTATTTCCGGTGAATTCGCCACCATGCCCCTCCGG GA-3',

[0035] Downstream primer pLVX-p27-R:

[0036] 5'-GGGGGGAGGGAGAGGGGCGGGATCCTTACAGCACCTTAGTCATCT-3',

[0037] The master plasmid pLvx-IRES-mCherry-p27 (inserted into the feline leukemia virus p27 gene via homologous recombination) was mixed with helper plasmids psPAX2 and pMD2.G at a ratio of 9 μg, 6 μg, and 3 μg, respectively, to obtain a plasmid mixture with a total mass of 18 μg. This plasmid mixture was then mixed with 54 μL of PEI transfection reagent (plasmid to PEI ratio of 1:3) and transfected into 293T cells. Forty-eight hours after transfection, the cell culture supernatant was collected, filtered through a 0.45 μm filter, and used to infect CRFK cells. Forty-eight hours after infection, the culture supernatant was discarded, the cells were washed twice with PBS, and then resuspended in basal DMEM medium. Cells expressing the mCherry fluorescent tag were obtained by flow cytometry sorting and then expanded into p27-CRFK cell lines by sequentially culturing them in 96-well, 48-well, 24-well, 12-well, 6-well, and 10cm culture dishes.

[0038] 3. Results

[0039] (1) Expression of p27 truncated protein

[0040] The recombinant plasmid pET32a-p27, truncated and expressed in E. coli BL21(DE3), was analyzed by SDS-PAGE and Western Blot of the supernatant after elution with 1M and 2M urea, as shown below. Figure 1 As shown, M is the protein marker, 1 is the mixed supernatant after dissolving the p27 truncated protein in 1M and 2M urea, and 2 is the mixed supernatant after dissolving the p27 truncated protein in 1M and 2M urea. The expected molecular weight of the p27 truncated protein is approximately 28.3 kDa. The target protein was successfully obtained by Western blotting with anti-his antibody as the primary antibody.

[0041] (2) Construction of p27-CRFK cell line

[0042] Construction of p27-CRFK cell line and determination of mouse serum IFA titer, as follows Figure 2 As shown in the figure, A: LV-pLVX-p27-IRES-mCherry-CRFK cells, B: LV-pLVX-IRES-mCherry-Control-CRFK cells, C: IFA results of mouse serum diluted 1:1024 in CRFK cells containing p27 lentivirus, D: IFA results of mouse serum diluted 1:1024 in CRFK cells without p27 lentivirus, E: IFA results of blank mouse serum in CRFK cells containing p27 lentivirus, and F: IFA results of blank mouse serum in CRFK cells without p27 lentivirus. The results indicate that lentivirus containing the p27 gene was successfully expressed in CRFK cells, and IFA verification was performed using mouse serum immunized with p27 truncated protein.

[0043] Example 2: Preparation of Monoclonal Antibodies

[0044] 1. Immunity in mice

[0045] The prepared p27 truncated prokaryotic protein was mixed with 50 μg of Bio-Drone 3w adjuvant at a 1:1 ratio per mouse and injected intramuscularly into the mice. A booster immunization was performed 14 days later. Blood was collected 7 days after the third immunization, and the serum of the mice was tested using the p27-CRFK cell line as the antigen to determine whether the titer of the mouse serum reached the required level for the preparation of monoclonal antibodies. After confirming that the titer was reached, 50 μg of p27 truncated prokaryotic protein was injected intraperitoneally three days before cell fusion to boost the immunization.

[0046] 2. Cell fusion

[0047] (1) SP2 / 0 cell resuscitation and culture

[0048] SP2 / 0 cells were revived 10 days before fusion. SP2 / 0 cells were cultured in 20% FBS medium until they were uniform in size and morphology and had neat edges.

[0049] (2) Preparation of spleen cells

[0050] Blood was collected from the eyeballs of mice that met the titer in the experimental group as positive serum. The mice were then euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. In a biosafety cabinet, the mice were fixed abdomen-up onto a sterile plate. The abdominal skin was grasped with forceps, and a small incision was made in the skin near the fat layer on the lower abdomen. The skin was then gradually torn open with scissors and forceps, and the skin was secured to expose the abdominal cavity. The peritoneum was cut open, and the spleen was located on the left side of the abdominal cavity. Using new scissors and forceps, the spleen was separated, and the surrounding connective tissue was removed. The spleen tissue was placed in a petri dish containing 10 mL of DMEM cell culture medium. The culture medium was injected into the spleen using a syringe, and repeated aspiration and respiration continued until the spleen tissue turned white. The whitened spleen tissue was discarded, and the DMEM cell culture medium containing spleen cells was transferred to a 50 mL centrifuge tube.

[0051] (3) Cell fusion

[0052] Discard the SP2 / 0 cell culture medium, add 5 mL of PBS to wash the cells, then discard the PBS. Add 2 mL of trypsin to evenly cover the cells, then discard the trypsin. Incubate at 37°C for 2 min to digest the SP2 / 0 cells, then resuspend the cells in 10 mL of DMEM cell culture medium. Add the SP2 / 0 cell suspension to a 50 mL centrifuge tube containing 10 mL of spleen cells in DMEM cell culture medium, and repeatedly pipette to mix the cells thoroughly. Centrifuge at 1000 r / m for 10 min, then discard the supernatant. Gently tap the centrifuge tube to loosen the cells. Place the centrifuge tube in a 37°C constant temperature water bath pre-placed in a biosafety cabinet to maintain a constant temperature of 37°C throughout the fusion process. During the fusion process, continuously shake the centrifuge tube to ensure even heating. Slowly add 1 mL of PEG preheated to 37°C over 1 min, let stand for 1 min, then add 5 mL of DMEM culture medium over 5 min, followed by 10 mL of DMEM culture medium over 5 min, and finally 15 mL of DMEM culture medium over 2 min. After fusion, the cells were centrifuged at 2000 rpm for 10 min, the supernatant was removed, and the cell clusters were resuspended in complete medium containing HAT and evenly seeded into 96-well cell culture plates, 100 µL per well. The number of cells seeded from each mouse was controlled to be 5-10 plates. The plates were incubated at 37°C in a CO2 incubator. Depending on the growth status, the medium was partially replaced with HAT medium after 4 and 7 days.

[0053] 3. Screening of positive hybridoma cells and subcloning of cells

[0054] During cell fusion, under the influence of HAT (Hybridoma Acid), successfully fused hybridoma cells grow while non-hybridoma cells die. Under a microscope, round, translucent cells are identified as hybridoma cells. After 10 days, culture medium from wells with yellowed supernatant was selected. p27-CRFK cell line was plated as the antigen, the yellowed supernatant served as the primary antibody, and goat anti-mouse fluorescent antibody was used as the secondary antibody. An IFA (Inductively Coupled Acid) assay was performed to initially screen for positive hybridoma cells. Subclonal screening of monoclonal cell wells was then repeated until positive monoclonal hybridoma cells (p27-3A5) were obtained. Positive hybridoma cells were sequentially cultured in 96-well, 48-well, 24-well, 12-well, and 6-well plates, and finally in 10cm culture dishes. Supernatants were collected to identify the antigenic epitopes recognized by the monoclonal antibodies. Cells were then transferred to cryopreservation tubes, incubated overnight at -80°C, and then stored long-term in liquid nitrogen.

[0055] 4. Recognition of p27 protein by monoclonal antibodies and identification of antigenic epitopes.

[0056] To verify whether monoclonal antibodies obtained from truncated p27 protein recognize the full-length p27 protein expressed in prokaryotes and eukaryotes, as well as the native feline leukemia virus p27 protein, Western blotting was performed using the supernatant of positive hybridoma cells as the primary antibody. The full-length p27 truncated protein is 72 amino acids long. To determine the minimal linear epitope recognized by the monoclonal antibody, the p27 truncated protein was continuously truncated and expressed by ligating the truncated gene fragment into the pCMV-HA-DsRed plasmid. Using eukaryotic expression, the recombinant plasmid and the empty vector plasmid were transfected into 293T cells. The expression effect was observed under a fluorescence microscope, showing the fusion protein with the DsRed tag. The protein expressed by the transfected plasmid was obtained using cell lysis buffer and Western blotting was performed. The primary antibody was the supernatant produced by positive hybridoma cells, and the secondary antibody was FITC-labeled goat anti-mouse IgG (H+L).

[0057] 5. Variable region sequence analysis of monoclonal antibody mAb 3A5

[0058] RNA was extracted from hybridoma cells (mouse hybridoma cells p27-3A5) producing mAb 3A5 using the Trizol method, and cDNA was obtained after reverse transcription. The full-length heavy and light chains of the mouse antibody were amplified and obtained. The cDNA was cloned into the pMD18-T vector and sequenced. The sequencing results were compared with those from the IMGT / V-QUEST database for further analysis.

[0059] 6. Results

[0060] (1) Determination of serum titer after mouse immunization

[0061] The titer of serum from mice immunized three times was determined using the IFA assay, and the results are as follows: Figure 3 As shown, 1: IFA results of 1:1024% immunized mouse serum, 2: IFA results of 1:8192% immunized mouse serum, 3: IFA results of 1:16384% immunized mouse serum, and 4: IFA results of blank mouse serum. Clear green fluorescence was still visible even when the polyclonal antibody serum from immunized mice was diluted to 1:16384%.

[0062] (2) Identification of positive hybridoma cells and preparation of monoclonal antibodies

[0063] Hybridoma cells with unlimited in vitro proliferation capacity were obtained using cell fusion technology. Using the yellowing supernatant of hybridoma cells as the primary antibody and the p27-CRFK cell line as the antigen, preliminary screening was performed using the IFA method. After two subcloning processes, a hybridoma cell line p27-3A5 stably secreting anti-p27 protein-specific antibody was obtained, and the monoclonal antibody was named 3A5. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2025, with accession number CGMCC No. 46706. The IFA results are as follows: Figure 4 As shown, 1: IFA results with 3A5 supernatant as the primary antibody, and 2: IFA results with blank mouse serum as the primary antibody. Preliminary verification indicates that hybridoma cell p27-3A5 can recognize the P27 protein expressed in the p27-CRFK cell line.

[0064] The recognition site of monoclonal antibody 3A5 differs from that of existing antibodies (A1). A schematic diagram of the antibody recognition site is shown below. Figure 6 As shown, the two complement each other in use. When antibody A1 is used as the capture antibody and 3A5 as the detection antibody, they form a pair of antibodies that can be used for immunological detection.

[0065] (3) Recognition of p27 protein by monoclonal antibodies and identification of antigenic epitopes

[0066] To determine the antigenic epitope recognized by the prepared monoclonal antibody, the p27 truncated gene (177-248aa) was stepwise truncated to express the protein, in the following order: Figure 5 As shown in the figure. The truncated base sequences of Groups 2-Group 6 were recombined with the pCMV-HA-Dsred plasmid, and the corresponding proteins were obtained by eukaryotic expression in 293T cells. Epitope identification was performed using a Western blot assay, with monoclonal antibody 3A5 as the primary antibody. The results are shown in the figure. Figure 5 As shown, the smallest linear antigenic epitope recognized by 3A5 is Group 6, with the sequence “225REERLW230”. Figure 5M: Protein Marker, 1: p27 protein expressed in prokaryotic cells using the pcold I vector, 2: pcold 1. Vector empty vector expressing protein; 3. p27-CRFK cell line protein; 4. Lentiviral CRFK cell line protein without p27 gene; 5. FL74 supernatant; 6. FL74 medium; 7. 177-222 aa; 8. 203-248 aa; 9. Negative control; 10. 203-230 aa; 11. 221-248 aa; 12. Negative control; 13. 221-230 aa; 14. Negative control; 15. 223-230 aa; 16. 221-228 aa; 17. Negative control; 18. 221-229 aa; 19. 224-230 aa; 20. 225-230 aa; 21. 226-230 aa; 22. Negative control.

[0067] like Figure 5 As shown, monoclonal antibody 3A5 can recognize prokaryotic and eukaryotic p27 protein expression, as well as feline leukemia virus p27 protein. The minimal linear epitope recognized was also identified.

[0068] The obtained p27 protein monoclonal antibody was sequenced, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows:

[0069] QIQLVQSGPELKKPGETVKISCKSSGYSFTQCGDPNVMVRQAPGKGLKWMARGQTFESGEFLLDTARFAFSLEASASTAYLQINNLKNEDTATYFCVRYPEGLNIAMVKEDTKYWGQGTSVTVSS.

[0070] The amino acid sequence of CDR1 in the heavy chain variable region is: QCGDPNVMV

[0071] The amino acid sequence of CDR2 in the heavy chain variable region is: RGQTFESGEFLLDTA

[0072] The amino acid sequence of CDR3 in the heavy chain variable region is: YPEGLNIAMVKEDTKY

[0073] The amino acid sequence of the light chain variable region of this monoclonal antibody is as follows:

[0074] DIVMTQSHKFMSTSVGDRVTITCSGVDRVTGKDTAWYQQKPGQSPKLLIYQVVSPSTGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCFPEHQSTYSFGSGTKLEIK.

[0075] The amino acid sequence of CDR1 in the light chain variable region is: SGVDRVTGKDTA

[0076] The amino acid sequence of CDR2 in the light chain variable region is: QVVSPST

[0077] The amino acid sequence of CDR3 in the light chain variable region is: FPEHQSTYS

[0078] The p27 protein is the capsid protein of feline leukemia virus (FLV), exhibiting high specificity and stability, and is not prone to mutation. Furthermore, it is abundant in the blood of infected cats, making it an optimal protein for FLV diagnosis. This invention successfully prepared a hybridoma cell line capable of stably secreting anti-p27 protein specificity and identified the minimal linear antigenic epitope, facilitating the subsequent development of diagnostic kits such as colloidal gold test strips. This antibody can be used for scientific research on viruses.

[0079] Example 3: Establishment of a sandwich ELISA method for feline leukemia virus antigen

[0080] 1. Determination of capture antibody and enzyme-labeled antibody

[0081] The antibodies p27-A1 and p27-3A5 were labeled with peroxidase, and these two antibodies were used as capture antibodies and enzyme-labeled antibodies respectively for testing. The p27-A1 and p27 proteins are derived from patent CN120738129A.

[0082] (1) Capture antibody (p27-A1 / p27-3A5) coating: 2 μg / mL, 100 μL per well, 37℃, 2 h. Wash with PBST for 5 min each time, 3 times.

[0083] (2) Block with 5% skim milk, 200 μL / well, 37℃, 2h. Wash with PBST for 5 min / time, 3 times.

[0084] (3) Incubate p27 protein antigen at 2 μg / mL, 100 μL per well, at 37°C for 1 h. Wash with PBST for 5 min each time, 3 times.

[0085] (4) HRP-labeled antibodies (p27-3A5 / p27-A1) were diluted 1:3200 and incubated in 100 μL per well, 3 times, at 37°C for 1 h. Wash with PBST for 5 min each time, 3 times.

[0086] (5) TMB color development (100 μL), 37℃, 15 min, 2M sulfuric acid (50 μL) to terminate the reaction, reading OD 490 nm.

[0087] (6) Results: The antigen-P / N ratio relationship graph is shown below. Figure 7As shown in the test, using P27-A1 as the capture antibody and P27-3A5 as the enzyme-labeled antibody, a higher value can be obtained for the P27 protein.

[0088] 2. Sensitivity and specificity tests

[0089] After experimental optimization, the concentration of the capture antibody p27-A1 was 2 μg / mL, and the concentration of the enzyme-labeled antibody p27-3A5 was 0.475 μg / mL. Tests were conducted against A-feline coronavirus, B-feline herpesvirus, C-feline calicivirus, D-feline chlamydia, E-feline parvovirus, F-feline leukemia virus, G-cell culture medium used for propagating feline leukemia virus, H-pcold-1 empty vector prokaryotic expression of P27 protein, I-pcold-l-p27 vector prokaryotic expression of P27 protein, J-CRFK cell lysis buffer, and K-lentiviral p27-CRFK cell lysis buffer. The sample-P / N value relationship is shown in the figure below. Figure 8 As shown in the figure. The results indicate that this ELISA method can effectively detect samples containing the feline leukemia pathogen P27.

Claims

1. A monoclonal antibody against feline leukemia virus p27 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2. The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 31-39 in SEQ ID NO.

1. The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 52-66 in SEQ ID NO.

1. The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 99-114 in SEQ ID NO.

1. The amino acid sequence of CDR1 in the light chain variable region is shown as positions 24-35 in SEQ ID NO.2; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 51-57 in SEQ ID NO.2; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 90-98 in SEQ ID NO.

2.

2. An expression carrier, characterized in that, It contains a monoclonal antibody against the feline leukemia virus p27 protein as described in claim 1.

3. A hybridoma cell line p27-3A5 that secretes a monoclonal antibody against the feline leukemia virus p27 protein as described in claim 1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2025, with accession number CGMCC No. 46706.

4. The use of the feline leukemia virus p27 protein monoclonal antibody as described in claim 1 in the preparation of feline leukemia virus detection kits and / or test strips.

5. A feline leukemia virus p27 detection kit, characterized in that, Including the feline leukemia virus p27 protein monoclonal antibody as described in claim 1.

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