Hybridoma cell strain, monoclonal antibody, linear epitope antigen and application thereof

Abstract

The invention discloses a hybridoma cell strain, a monoclonal antibody, a linear epitope antigen and application thereof, and relates to the field of genetic engineering, in particular to a hybridoma cell strain, a monoclonal antibody, a linear epitope antigen and application thereof. The preservation number of the hybridoma cell strain P27-A1 is CGMCC (China General Microbiological Culture Collection Center) No. 46352. The hybridoma cell strain P27-A1 is prepared from feline leukemia virus p27, and a monoclonal antibody secreted by the hybridoma cell strain P27-A1 recognizes a linear epitope antigen of feline leukemia virus p27 protein; the kit for the feline leukemia virus p27 comprises the monoclonal antibody A1 secreted by the hybridoma cell strain P27-A1. The monoclonal antibody secreted by the hybridoma cell strain can greatly improve the sensitivity of the p27 protein for detecting feline leukemia virus. The invention provides the monoclonal antibody secreted by the hybridoma cell strain for identification, and the monoclonal antibody can be used for preparing a diagnostic kit such as a colloidal gold test strip.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a hybridoma cell line, a monoclonal antibody, a linear epitope antigen and applications thereof. Background Art

[0002] Feline leukemia virus (FLV) is a retrovirus containing single-stranded RNA. Its gene sequence, from the 5' end to the 3' end, is LTR-gag-pol-env-LTR. The gag gene encodes internal structural proteins, including p15c, p12, p27, and p10. The p27 protein is abundant in the blood of infected cats and is commonly used in the diagnosis of FLV. The pol gene (polymerase) specifies the virus's reverse transcriptase (RT), which is responsible for synthesizing DNA from an RNA template. The Env gene encodes envelope proteins, including gp70 and p15e. The gp70 protein defines the subtypes of FLV, which are A, B, and C. Of these, only FeLV-A is contagious and horizontally transmitted between cats. Acquiring antibodies to the p27 protein is crucial for diagnosing FLV infection in cats. Summary of the Invention

[0003] The present invention provides a hybridoma cell line, a monoclonal antibody, a linear epitope antigen and applications thereof.

[0004] The hybridoma cell line of the present invention is hybridoma cell P27-A1, with a deposit number of CGMCC No. 46352 and a deposit date of April 24, 2025.

[0005] The hybridoma cell P27-A1 is prepared using feline leukemia virus p27, and the specific preparation method is carried out according to the following steps:

[0006] 1. Synthesize feline leukemia virus p27 prokaryotic expression protein;

[0007] 2. Preparation of splenocytes: Immunize mice with feline leukemia virus p27 prokaryotic expression protein to obtain mouse spleen tissue, then place the mouse spleen tissue in DMEM cell culture medium and repeatedly pipette and aspirate until the spleen tissue turns white. Discard the white spleen tissue to obtain DMEM cell culture medium containing splenocytes;

[0008] 3. Fusion culture: SP2 / 0 cell solution was resuspended with the DMEM cell culture medium containing spleen cells described in step 2, the ratio of SP2 / 0 cells to spleen cells was 5-10:1, and the resuspension was subjected to fusion culture; wherein, the fusion culture was to mix the resuspension evenly and then centrifuge at 1000 r / m for 10 min to remove the supernatant, the precipitate was shaken in a constant temperature water bath at 37°C, and PEG preheated at 37°C with a weight ratio of 10:1 to spleen cells was added dropwise within 1 min, and after standing for 1 min, DMEM culture medium with a weight ratio of 1:2 to spleen cells was added dropwise within 5 min, and then DMEM culture medium with a weight ratio of 1:1 to spleen cells was added dropwise within 5 min, and DMEM culture medium with a weight ratio of 3:2 to spleen cells was added dropwise within 2 min; after the fusion was completed, the cell pellet was centrifuged at 2000 r / m for 10 min, the supernatant was removed, the cell mass was resuspended with complete medium containing HAT and evenly plated into 96-well cell culture plates, with 100 cells per well. µL; 5-10 cell plates were plated for each mouse after fusion. The cells were placed in a 37°C CO2 incubator and half-medium was replaced with HAT medium on the 4th and 7th days of culture. Fusion cells were obtained after 8-12 days of culture.

[0009] 4. Preparation of hybridoma cells: Select the culture medium of the fused cells with yellow supernatant, use the p27-CRFK cell line to plate as the antigen, the yellow supernatant as the primary antibody, and goat anti-mouse fluorescent antibody as the secondary antibody. Perform IFA test to initially screen out positive hybridoma cells, screen the monoclonal cell wells through subcloning, and repeat the transwell screening to finally obtain the hybridoma cell P27-A1.

[0010] The method for synthesizing the feline leukemia virus p27 prokaryotic expression protein in step 1 is as follows:

[0011] According to the nucleotide sequence of feline leukemia virus p27 protein and the multiple cloning site of the pCold Ⅰ prokaryotic expression vector, EcoRⅠ and SalI were selected as double restriction enzyme sites for vector construction. The upstream primer F-5'-CTCGGTACCCTCGAGGGATCCGAATTCATGcccttgagggaa-3' and the downstream primer R-5'-TTACCTATCTAGACTGCAGGTCGACTTAcagaactttagtca-3' were designed using SnapGene software. The p27 nucleotide sequence was amplified and recombined with the double restriction enzyme linearized vector pcoldI. The cells were transformed into DH5α cells, plated on LB culture dishes containing ampicillin resistance, and single colonies were picked. The recombinant plasmid pcold I-p27 containing 802 bp of the p27 gene product was screened by bacterial liquid PCR. The recombinant plasmid pcoldI-p27 was cloned. I-p27 was transformed into BL21, and a single colony was picked and inoculated into 5 ml of ampicillin-resistant LB culture medium. The culture was shaken on a shaker at 37°C and 180 r / min until the bacterial solution became turbid. 200 ul was pipetted into 200 ml of ampicillin-resistant LB culture medium for expansion culture. The culture was shaken on a shaker at 37°C and 180 r / min. After 4-5 hours, IPTG inducer was added, and expression was induced on a shaker at 16°C and 150 r / min. After 24 hours, the bacterial precipitate was collected by centrifugation, resuspended in 10 ml of PBS, ultrasonically disrupted, and centrifuged to obtain the supernatant. The supernatant was bound to 2 ml of his-tagged protein agarose purification resin overnight and then passed through a nickel column. The impurities were washed with 40 mmol / L imidazole, and the protein was eluted with 250 mmol / L imidazole to obtain the p27 prokaryotic expressed protein.

[0012] The mouse immunization method in step 2 is as follows: the prepared feline leukemia virus p27 prokaryotic expression protein is mixed with Biolong 3w adjuvant at a ratio of 1:1 per mouse at a ratio of 50ug, and the mice are immunized by intramuscular injection. The immunization is boosted every 14 days, and blood is collected 7 days after the third immunization. The p27-CRFK cell line is used as an antigen, and the serum of the mice is determined by the IFA method until the titer of the prepared monoclonal antibody reaches 1024; the method for obtaining mouse spleen tissue in step 2 is as follows: blood is collected from the eyeball of the mouse with a titer of 1024 as positive serum; the mouse is then killed by cervical dislocation and soaked in 75% alcohol for 5 min; the mouse is fixed on a sterilized plate with the abdomen facing up in a biological safety cabinet, the abdominal skin is clamped with tweezers, a small hole is cut in the skin near the fat layer below the abdomen of the mouse, the skin is gradually torn open at the opening with scissors and tweezers, the skin is fixed, and the abdominal cavity is exposed; the peritoneum is cut open, and the spleen is found on the left side of the mouse abdominal cavity. New scissors and tweezers are used to separate the spleen and remove the surrounding connective tissue to obtain mouse spleen tissue.

[0013] As described above, the SP2 / 0 cell recovery method in step 3: SP2 / 0 cells are cultured in 20% FBS culture medium until they are uniform in size and morphology and have neat edges.

[0014] As described above, in the cell fusion in step three, under the action of HAT, the cells that successfully fuse will grow, and the unsuccessful cells will die. Under a microscope, the fused cells are round and translucent.

[0015] In the p27-CRFK cell line in step 4, the main plasmid pLvx-IRES-mCherry-p27 is inserted into the p27 gene of the feline leukemia virus by homologous recombination, and the auxiliary plasmid psPAX2 and the auxiliary plasmid pMD2.G are respectively in the amounts of 9ug, 6ug, and 3ug, mixed for a total of 18ug, and transfected with 54ul of transfection reagent PEI, i.e., a ratio of 1:3 to transfect 293T cells. After 48 hours, the supernatant is collected, and the supernatant after filtering through a 0.45um filter is infected with CRFK cells. After 48 hours, the supernatant is discarded, washed twice with PBS, resuspended in basic DMEM medium, and cells containing the mCherry tag are sorted by flow cytometry and expanded for culture.

[0016] As described above, in step 4, the positive hybridoma cells are expanded and cultured, the supernatant is collected to identify the antigen epitope recognized by the monoclonal antibody, the cells are transferred to cell freezing tubes, placed at -80°C overnight, and stored in a liquid nitrogen tank for long-term storage.

[0017] The monoclonal antibody secreted by the hybridoma cell line of the present invention is monoclonal antibody A1.

[0018] The monoclonal antibody A1 of the present invention can recognize the feline leukemia virus p27 epitope antigen with a sequence as shown in SEQ ID NO: 1.

[0019] The linear epitope antigen of the feline leukemia virus p27 protein of the present invention has a sequence as shown in SEQ ID NO: 1.

[0020] The preparation of the linear epitope antigen of the feline leukemia virus p27 protein is carried out according to the following steps:

[0021] 1. Expanding and culturing the hybridoma cell P27-A1 of the present invention, and collecting the supernatant to obtain the monoclonal antibody A1;

[0022] Second, the p27 protein was continuously truncated and expressed, i.e., the truncated gene fragments were connected to the pCMV-HA-DsRed plasmid. The recombinant plasmid and the empty plasmid were transfected into 293T cells using a eukaryotic expression method to obtain the corresponding protein. The epitope was identified by Western blot assay, using monoclonal antibody A1 as the primary antibody. The shortest protein recognized by A1 was the linear epitope antigen.

[0023] The feline leukemia virus p27 test kit of the present invention comprises the monoclonal antibody A1.

[0024] The p27 protein, a capsid protein of the feline leukemia virus, is highly specific and stable, and is virtually resistant to mutation. Furthermore, it is abundant in the blood of sick cats, making it an optimal protein for feline leukemia virus diagnosis. The antibodies secreted by the cell lines provided herein recognize the minimal linear epitope of the p27 protein, significantly improving the sensitivity of feline leukemia virus detection. The antibodies secreted by the cell lines provided herein identify and recognize the minimal linear epitope of the feline leukemia virus, facilitating the subsequent development of diagnostic kits, such as colloidal gold test strips.

[0025] The hybridoma cell line described in the present invention is hybridoma cell P27-A1, which is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 46352 and a deposit date of April 24, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of protein purification of feline leukemia virus p27 protein expressed in E. coli;

[0027] Figure 2 The results of the IFA experiment on the serum of mice after immunization in Example 1;

[0028] Figure 3 The results of the IFA experiment of monoclonal antibody A1 in Example 1 on the p27-CRFK cell line are shown;

[0029] Figure 4 Western Blot results of monoclonal antibody A1 recognizing FL74 cell freeze-thaw supernatant (strain KT-FeLV-UCD-1);

[0030] Figure 5 It is a truncated sequence of feline leukemia virus p27 protein;

[0031] Figure 6 These are the Western Blot results of various truncated sequences of feline leukemia virus p27 protein. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0034] Example 1 Preparation of hybridoma cell line P27-A1, feline leukemia virus p27 protein monoclonal antibody and antigenic epitope

[0035] 1. Materials and Methods

[0036] 1.1 Main experimental materials

[0037] (1) The cDNA sequence of the FeLV-A virus genome was synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd., and the reference sequence was the complete nucleotide sequence of subgroup A (GenBank accession number: M18247.1).

[0038] (2) SP2 / 0 cells, p27-CRFK cell line, 293T cells, FL74 cells (ATCC number CRL-8012, this cell line can continuously produce feline leukemia virus, strain KT-FeLV-UCD-1, GenBank: MT129531.1), plasmid pcold I, plasmid pCMV-HA-DsRed were all preserved by the Pet Disease Innovation Team of Harbin Veterinary Research Institute; DH5α competent cells and BL21 (DE3) competent cells were purchased from Tiangen Biochemical Technology Co., Ltd.; 6-8 week old Balb / c SPF female mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0039] (3) DNA gel recovery kit was purchased from Omega Biotechnology Co., Ltd.; endotoxin-free plasmid miniprep kit was purchased from Tiangen Biochemical Technology Co., Ltd.; Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L) was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.; EcoRI and SalI restriction endonucleases, 2 × Taq Master Mix (Dye Plus), DNA Marker, and protein marker were purchased from TaKaRa; KOD-Plus-Neo high-fidelity enzyme was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; ClonExpress Ultra One Step Cloning Kit V2 was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; FITC-labeled goat anti-mouse IgG (H+L) and His-tag protein agarose purification resin were purchased from Yisheng Biotechnology; Quick Antibody-Mouse3W immune adjuvant was purchased from Biolong; polyethylene glycol (PEG) and HAT / HT were purchased from Sigma-Aldrich.

[0040] 1.2 Main experimental methods

[0041] 1.2.1 Prokaryotic expression and purification of feline leukemia virus p27 protein

[0042] Based on the p27 protein nucleotide sequence and the multiple cloning site of the pCold I prokaryotic expression vector, EcoRI and SalI were selected as double restriction enzyme sites for vector construction. SnapGene software was used to design the upstream and downstream primers: F-5'-CTCGGTACCCTCGAGGGATCCGAATTCATGcccttgagggaa-3' and R-5'-TTACCTATCTAGACTGCAGGTCGACTTAcagaactttagtca-3', respectively. These primers were synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd. The p27 nucleotide sequence was amplified, recombined with the double-restricted linearized vector pcold I, and transformed into DH5α cells. The cells were plated on LB culture plates containing ampicillin resistance, and single colonies were picked. Samples expressing the p27 gene product (802 bp) were screened by PCR and sent to the company for sequencing. Samples with positive sequencing results were returned to the plasmid. The recombinant plasmid pcold I-p27 was transformed into BL21 (DE3) cells. A single colony was picked and inoculated into 5 ml of ampicillin-resistant LB medium, which was shaken at 37°C and 180 rpm. When the culture became turbid, 200 μL of the culture was transferred to 200 ml of ampicillin-resistant LB medium and cultured at 37°C and 180 rpm. After 4-5 hours, IPTG was added, and expression was induced at 16°C and 150 rpm. After 24 hours, the pellet was collected by centrifugation, resuspended in 10 ml of PBS, sonicated, and centrifuged. The supernatant and pellet were sampled separately for SDS-PAGE analysis and Coomassie blue staining to analyze the solubility of p27 protein. The supernatant was bound to 2 ml of His-tagged protein agarose purification resin overnight and then passed through a nickel column. The protein was eluted with a low concentration (40 mmol / L) of imidazole and then a high concentration gradient (60-250 mol / L). The protein elution was analyzed by SDS-PAGE, and the protein expression was analyzed by Western Blot. The target protein was dialyzed with PBS overnight, and the protein concentration was measured by BCA method.

[0043] Figure 1 The results of prokaryotic expression and purification of feline leukemia virus p27 protein were identified by SDS-PAGE (M: protein marker, 1: flow-through buffer, 2: wash buffer 40mM imidazole, 3: 60mM imidazole, 4: 80mM imidazole, 5: 100mM imidazole, 6: 120mM imidazole, 7: 140mM imidazole, 8: 160mM imidazole, 9: 250mM imidazole). As shown in Figure 1, the constructed recombinant plasmid pCold I-p27 was successfully expressed in the supernatant of Escherichia coli BL21 (DE3). The protein was purified by nickel column affinity chromatography and identified by SDS-PAGE. The expected molecular weight of the p27 protein was approximately 32.3KDa.

[0044] 1.2.2 Preparation of splenocytes

[0045] First, mice were immunized, that is, the prepared p27 prokaryotic expression protein was mixed with Biolong 3w adjuvant at a ratio of 1:1 at a rate of 50 μg per mouse, and the mice were immunized by intramuscular injection. After 14-day booster immunization, blood was collected 7 days after the third immunization. The p27-CRFK cell line (the main plasmid pLvx-IRES-mCherry-p27 was inserted into the p27 gene of the feline leukemia virus by homologous recombination, and the auxiliary plasmid psPAX2 and the auxiliary plasmid pMD2.G were respectively 9 μg, 6 μg, and 3 μg, and the total amount was 18 μg) was mixed with the transfection reagent PEI. 54ul, or a 1:3 ratio, was used to transfect 293T cells. After 48 hours, the supernatant was collected and filtered through a 0.45um filter to infect CRFK cells. After 48 hours, the supernatant was discarded, washed twice with PBS, and resuspended in basal DMEM medium. Cells containing the mCherry tag were isolated by flow cytometry and expanded for culture. ) was used as an antigen. The mouse serum was tested by IFA to determine whether it had reached the titer for preparing monoclonal antibodies. Once the titer was confirmed, 50ug of protein was injected intraperitoneally three days before cell fusion. Immunization was boosted. Blood was collected from the eyeballs of mice with a titer of 1024 as positive serum. The mice were then sacrificed by cervical dislocation and immersed in 75% alcohol for 5 minutes. In a biosafety cabinet, the mice were mounted on a sterile plate with their abdomen facing upward. Forceps were used to grasp the abdominal skin and a small incision was made in the skin below the abdomen, near the fat layer. Scissors and forceps were used to gradually tear the skin apart at the opening and secure it to expose the abdominal cavity. The peritoneum was cut open, and the spleen was located on the left side of the abdominal cavity. Using fresh scissors and forceps, the spleen was isolated and the surrounding connective tissue removed. The mouse spleen tissue was placed in a dish containing 10 mL of DMEM cell culture medium. Cell culture medium was aspirated into the spleen using a syringe and repeatedly pipetted and pumped until the spleen tissue turned whitish. The whitish spleen tissue was discarded, and the DMEM cell culture medium containing the spleen cells was transferred to a 50 mL centrifuge tube.

[0046] Determination of serum titer after mouse immunization: The serum titer of mice immunized three times was determined by IFA test, as shown in FIG. Figure 2 It was shown that when the mouse polyclonal antiserum was diluted 1:1024, clear green fluorescence was still visible.

[0047] 1.2.3 Cell fusion

[0048] First, resuscitate and culture SP2 / 0 cells. 10 days before cell fusion, resuscitate SP2 / 0 cells and culture them in 20% FBS until they are uniform in size, morphology, and have neat edges. Then, discard the SP2 / 0 cell culture medium, wash the cells with 5 mL of PBS, and discard the PBS. Add 2 mL of trypsin to evenly cover the cells, then discard the trypsin. Incubate the cells at 37°C for 2 min to digest the SP2 / 0 cells, then resuspend the cells in 10 mL of DMEM. Add the SP2 / 0 cell suspension to a 50 mL centrifuge tube containing 10 mL of spleen cells in DMEM. Mix the cells by repeated pipetting and agitation. Centrifuge at 1000 rpm for 10 min, and discard the supernatant. Gently tap the centrifuge tube to loosen the cells. Place the centrifuge tube in a 37°C water bath pre-placed in a biosafety cabinet to maintain a constant temperature of 37°C throughout the fusion process. Shake the tube frequently to ensure even heating. Slowly add 1 mL of 37°C preheated PEG dropwise within 1 minute. After standing for 1 minute, add 5 mL of DMEM medium dropwise within 5 minutes, then add 10 mL of DMEM medium dropwise within 5 minutes, and then add 15 mL of DMEM medium dropwise within 2 minutes to obtain cell pellets. Centrifuge the cell pellets at 2000 rpm for 10 minutes, remove the supernatant, resuspend the cell pellets in complete medium containing HAT and evenly plate them into 96-well cell culture plates, 100 µL per well (5-10 cell plates per mouse cell pellets are controlled). Culture in a 37°C carbon dioxide incubator for 10 days to obtain post-fusion cells. Perform half-medium replacement with HAT medium after 4 and 7 days, depending on the growth status.

[0049] 1.2.4 Hybridoma Cell Preparation

[0050] During cell fusion, under the action of HAT, cells that successfully fuse grow, while unsuccessful cells die. Microscopically, round, translucent cells are considered preliminary hybridomas. After 10 days, culture medium from wells where the supernatant has turned yellow is selected. The p27-CRFK cell line is plated as an antigen, the yellowed supernatant is used as the primary antibody, and a goat anti-mouse fluorescent antibody is used as the secondary antibody. Initially, positive hybridoma cells are screened for IFA. Subcloning is then performed to identify single clones in the wells. Repeated transwell screening ultimately yields hybridomas.

[0051] Hybridoma cells P27-A1 were cultured in 96-well plates, 48-well plates, 24-well plates, 12-well plates, 6-well plates, and 10 cm culture dishes in sequence. The supernatant was collected to identify the antigen epitope recognized by the monoclonal antibody. The cells were transferred to cell cryopreservation tubes, placed at -80°C overnight, and stored in liquid nitrogen tanks for long-term storage.

[0052] 1.2.5 Identification of hybridoma cells and preparation of monoclonal antibodies

[0053] Hybridoma cells with unlimited in vitro proliferation ability were obtained by cell fusion technology. The yellow supernatant of the hybridoma cells was used as the primary antibody, and the p27-CRFK cell line was used as the antigen. Initial screening was carried out by the IFA method, and after two subclones, a hybridoma cell line that can stably secrete anti-p27 protein-specific antibodies was obtained, named hybridoma cell P27-A1, and the monoclonal antibody produced by this hybridoma cell was named A1 (the above hybridoma cell P27-A1 was expanded and the supernatant was collected to obtain monoclonal antibody A1). Figure 3 The results of IFA of monoclonal antibody A1 on p27-CRFK cell line are shown in Figure 2. Figure 3 It can be seen that monoclonal antibody A1 specifically recognizes P27 protein expressed in cells and does not react with intrinsic cell components.

[0054] From the hybridoma cells producing MAb P27-A1 (mouse hybridoma cell P27-A1), RNA was extracted using the Trizol method and reverse transcribed to obtain cDNA. The full-length heavy and light chains of the mouse antibody were amplified and cloned into the pMD18-T vector, and monoclonal antibody A1 was sequenced. Sequencing results were compared with the IMGT / V-QUEST database for further analysis. Among them, the amino acid sequence of the variable region of the heavy chain (full-length sequence, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) of MAbP27-A1 is as follows: QIQLVQSGPELKKPGETVKISCKSSGYSFTDPGNVMDPVRQAPGKGLKWMAWLDTERGQTFEFLARFAFSLEASASTAYLQINNLKNEDTATYFCVRNIVYGQLKVYAMNKWGQGTSVTVSS; the amino acid sequence of the variable region of the light chain (full-length sequence, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) is as follows: DIVMTQSHKFMSTSVGDRVTITCETGKDVSGVTTAWYQQKPGQSPKLLIYYTASTVSGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCEQSTYHFPSFGSGTKLEIK.

[0055] 1.2.6 Recognition of Feline Leukemia Virus by Monoclonal Antibody A1

[0056] The frozen FL74 suspension cells were revived and serially passaged after the cells recovered. The supernatants of the second, fourth, and sixth generation cells after freezing and thawing were collected and Western Blot validation was performed using the supernatant of P27-A1 as the primary antibody. Figure 4 Shown: 1: FL74 second generation supernatant, 2: FL74 fourth generation supernatant, 3: FL74 sixth generation supernatant, 4: pcold-I-p27 prokaryotic expression protein. The results show that monoclonal antibody A1 can recognize feline leukemia virus.

[0057] 1.2.7 The linear epitope antigen sequence of the feline leukemia virus p27 protein is shown in SEQ ID NO: 1. The linear epitope antigen of the feline leukemia virus p27 protein is prepared as follows: the hybridoma cell P27-A1 is cultured and the supernatant is collected to obtain the monoclonal antibody A1; the p27 protein is 248 amino acids in length, and the p27 protein is continuously truncated for expression, i.e., the truncated gene fragments are ligated to the pCMV-HA-DsRed plasmid, and the recombinant plasmid and the empty plasmid are transfected into 293T cells for expression using a eukaryotic expression method to obtain the corresponding protein. The epitope is identified by Western blot, wherein the monoclonal antibody A1 is used as the primary antibody, and the shortest protein recognized by A1 is the linear epitope antigen.

[0058] Specifically, the p27 protein was expressed by truncating the truncated gene fragments into the pCMV-HA-DsRed plasmid. Using a eukaryotic expression approach, the recombinant plasmids and the empty vector were transfected into 293T cells for expression. The DsRed-tagged fusion protein was visualized under a fluorescence microscope. Proteins expressed by the transfected plasmids were harvested using cell lysate and validated by Western blot. The primary antibody used was the supernatant from the positive hybridoma cell line P27-A1 (monoclonal antibody A1 was used as the primary antibody), and the secondary antibody used was FITC-conjugated goat anti-mouse IgG (H+L).

[0059] In order to determine the shortest linear epitope antigen prepared by the monoclonal antibody produced by hybridoma cell P27-A1, the p27 gene (1-248aa) was gradually truncated to express the protein. Figure 5 For truncated sequences, Figure 5 The 8 groups of truncated base sequences were recombined with pCMV-HA-Dsred plasmid and expressed in 293T cells to obtain the corresponding proteins. Epitope identification was performed by Western blot, using monoclonal antibody A1 as the primary antibody. Figure 6 Western Blot results, Figure 6Middle M: ​​Protein Marker, 1: 1-70aa, 2: 51-117aa, 3: 102-184aa, 4: 177-248aa, 5: 124-184aa, 6: negative control, 7: 139-169aa, 8: 132-169aa, 9: negative control, 10: 137-169aa, 11: 135-169aa, 12: 133-169aa, 13: negative control, 14: 133-164aa, 15: 133-166aa, 16: 133-168aa, 17: negative control, 18: 133-167aa, 19: 133-168aa, 20: negative control. Figure 6 It can be seen that the minimum linear antigen epitope recognized by monoclonal antibody A1 is group 8, and the sequence is " 133 PTNLAQVKQVVQGKEETPASFLERLKEAYRMYTPY 168 ”.

[0060] The p27 protein, a capsid protein of the feline leukemia virus, is highly specific, stable, and resistant to mutation. Furthermore, it is abundant in the blood of sick cats, making it an optimal protein for feline leukemia virus diagnosis. This example generated a hybridoma cell line capable of stably secreting anti-p27 protein-specific antigens. The shortest linear epitope antigens produced are also useful for the subsequent development of diagnostic kits, such as colloidal gold test strips.

Claims

1. A hybridoma cell line, characterized in that The hybridoma cell line is hybridoma cell P27-A1, the preservation number is CGMCC No 46352, and the preservation time is April 24, 2025.

2. The hybridoma cell line according to claim 1, characterized in that: The hybridoma cell line is prepared using feline leukemia virus p27, and the specific preparation method is carried out according to the following steps:

1. Synthesize feline leukemia virus p27 prokaryotic expression protein; 2. Preparation of splenocytes: Immunize mice with feline leukemia virus p27 prokaryotic expression protein to obtain mouse spleen tissue, then place the mouse spleen tissue in DMEM cell culture medium and repeatedly pipette and aspirate until the spleen tissue turns white. Discard the white spleen tissue to obtain DMEM cell culture medium containing splenocytes; 3. Fusion culture: SP2 / 0 cell solution was resuspended with the DMEM cell culture medium containing spleen cells described in step 2, the ratio of SP2 / 0 cells to spleen cells was 5-10:1, and the resuspension was subjected to fusion culture; wherein, the fusion culture was to mix the resuspension evenly and then centrifuge at 1000 r / m for 10 min to remove the supernatant, the precipitate was shaken in a constant temperature water bath at 37°C, and PEG preheated at 37°C with a weight ratio of 10:1 to spleen cells was added dropwise within 1 min, and after standing for 1 min, DMEM culture medium with a weight ratio of 1:2 to spleen cells was added dropwise within 5 min, and then DMEM culture medium with a weight ratio of 1:1 to spleen cells was added dropwise within 5 min, and DMEM culture medium with a weight ratio of 3:2 to spleen cells was added dropwise within 2 min; after the fusion was completed, the cell pellet was centrifuged at 2000 r / m for 10 min, the supernatant was removed, the cell mass was resuspended with complete medium containing HAT and evenly plated into 96-well cell culture plates, with 100 cells per well. µL; 5-10 cell plates were plated for each mouse after fusion. The cells were placed in a 37°C CO2 incubator and half-medium was replaced with HAT medium on the 4th and 7th days of culture. Fusion cells were obtained after 8-12 days of culture.

4. Preparation of hybridoma cells: Select the culture medium of the fused cells with yellow supernatant, use the p27-CRFK cell line to plate as the antigen, the yellow supernatant as the primary antibody, and goat anti-mouse fluorescent antibody as the secondary antibody. Perform IFA test to initially screen out positive hybridoma cells, screen the monoclonal cell wells through subcloning, and repeat the transwell screening to finally obtain the hybridoma cell P27-A1.

3. The method for preparing a hybridoma cell line according to claim 2, characterized in that: Method for synthesizing feline leukemia virus p27 prokaryotic expression protein in step 1: According to the nucleotide sequence of feline leukemia virus p27 protein and the multiple cloning site of pCold Ⅰ prokaryotic expression vector, EcoRⅠ and SalI were selected as double restriction enzyme sites for vector construction. The upstream primer F-5'-CTCGGTACCCTCGAGGGATCCGAATTCATGcccttgagggaa-3' and the downstream primer R-5'-TTACCTATCTAGACTGCAGGTCGACTTAcagaactttagtca-3' were designed using SnapGene software. The p27 nucleotide sequence was amplified and recombined with the double restriction enzyme linearized vector pcold I. The cells were transformed into DH5α cells, plated on LB culture dishes containing ampicillin resistance, and single colonies were picked. The recombinant plasmid pcold I-p27 containing 802 bp of the p27 gene product was screened by bacterial liquid PCR. The recombinant plasmid pcold I-p27 was cloned. I-p27 was transformed into BL21, and a single colony was picked and inoculated into 5 ml of ampicillin-resistant LB culture medium. The culture was shaken on a shaker at 37°C and 180 r / min until the bacterial solution became turbid. 200 ul was pipetted into 200 ml of ampicillin-resistant LB culture medium for expansion culture. The culture was shaken on a shaker at 37°C and 180 r / min. After 4-5 hours, IPTG inducer was added, and expression was induced on a shaker at 16°C and 150 r / min. After 24 hours, the bacterial precipitate was collected by centrifugation, resuspended with 10 ml of PBS, ultrasonically disrupted, and centrifuged to obtain the supernatant. 2 ml of his-tagged protein agarose purification resin was used to bind to the supernatant overnight, then passed through a nickel column, washed with 40 mmol / L imidazole, and the protein was eluted with 250 mmol / L imidazole to obtain the p27 prokaryotic expressed protein.

4. The method for preparing a hybridoma cell line according to claim 2, characterized in that: The mouse immunization method in step 2 is as follows: the prepared feline leukemia virus p27 prokaryotic expression protein is mixed with Biolong 3w adjuvant at a ratio of 1:1 at a ratio of 50ug per mouse, and the mice are immunized by intramuscular injection. The immunization is strengthened every 14 days, and blood is collected 7 days after the third immunization. The p27-CRFK cell line is used as the antigen, and the mouse serum is determined by the IFA method until the titer of the prepared monoclonal antibody reaches 1024; the method for obtaining mouse spleen tissue in step 2 is as follows: blood is collected from the eyeball of the mouse with a titer of 1024 as the positive serum; then the mouse is killed by cervical dislocation and soaked in 75% alcohol for 5 min; the mouse is fixed on a sterilized plate with the abdomen facing up in a biosafety cabinet, the abdominal skin is clamped with tweezers, a small hole is cut in the skin near the fat layer under the mouse abdomen, the skin is gradually torn open at the opening with scissors and tweezers, the skin is fixed, and the abdominal cavity is exposed; the peritoneum is cut open, and the spleen is found on the left side of the mouse abdominal cavity. New scissors and tweezers are used to separate the spleen and remove the surrounding connective tissue to obtain the mouse spleen tissue.

5. The method for preparing a hybridoma cell line according to claim 2, characterized in that: In step 4, the p27-CRFK cell line: the main plasmid pLvx-IRES-mCherry-p27 is inserted into the p27 gene of the feline leukemia virus by homologous recombination. The auxiliary plasmid psPAX2 and the auxiliary plasmid pMD2.G are respectively in the amounts of 9ug, 6ug, and 3ug, mixed together for a total of 18ug, and transfected with 54ul of transfection reagent PEI, i.e., a ratio of 1:3, into 293T cells. After 48 hours, the supernatant is collected and filtered through a 0.45um filter to infect CRFK cells. After 48 hours, the supernatant is discarded, washed twice with PBS, resuspended in basic DMEM medium, and cells containing the mCherry tag are sorted by flow cytometry and expanded for culture.

6. The monoclonal antibody secreted by the hybridoma cell line according to claim 1, characterized in that The monoclonal antibody is monoclonal antibody A1.

7. The monoclonal antibody according to claim 6, characterized in that Monoclonal antibody A1 can recognize the feline leukemia virus p27 epitope antigen whose sequence is shown in SEQ ID NO:

1.

8. A linear epitope antigen of the feline leukemia virus p27 protein, characterized in that The sequence of the linear epitope antigen is shown in SEQ ID NO:

1.

9. The linear epitope antigen of the feline leukemia virus p27 protein according to claim 8, characterized in that The preparation of linear epitope antigen of feline leukemia virus p27 protein is carried out according to the following steps:

1. Expanding and culturing the hybridoma cell P27-A1 according to claim 2, and collecting the supernatant to obtain the monoclonal antibody A1; Second, the p27 protein was continuously truncated and expressed, i.e., the truncated gene fragments were connected to the pCMV-HA-DsRed plasmid. The recombinant plasmid and the empty plasmid were transfected into 293T cells using a eukaryotic expression method to obtain the corresponding protein. The epitope was identified by Western blot assay, using monoclonal antibody A1 as the primary antibody. The shortest protein recognized by A1 was the linear epitope antigen.

10. A feline leukemia virus p27 kit, characterized in that: Comprising the monoclonal antibody A1 according to claim 3.

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