A method for preparing a monoclonal cell strain stably expressing VP1 fusion EGFP

Abstract

A method for preparing a stable monoclonal cell line expressing VP1 fusion with EGFP belongs to the field of vaccine technology. The technical solution of this invention includes the following steps: culturing PK15 cells; the appropriate concentration of G418 should be 500 μg / mL; transfecting PK15 cells with Lipofectamine™ 2000 and pEGFP-N1-VP1 plasmid at a 1:1 ratio; G418 selection; and elution with weak acid to obtain SLA-I class presenting peptides.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a method for preparing a monoclonal cell line that stably expresses VP1 fused with EGFP. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious and devastating infectious disease of livestock. Previously widespread in Africa, South America, and Asia, FMD led to a significant decline in livestock production and severely impacted the livestock industry. Currently, FMD continues to spread annually in the pig farming sector, causing serious disruption to pig farming. Serological studies have identified seven serotypes of FMDV: A, O, and C (European types); Asia 1; and SAT1, SAT2, and SAT3 (South African types). FMDV consists of 60 particles, each primarily composed of four capsid proteins: VP1, VP2, VP3, and VP4. The FMDV structural protein VP1 is the main antigenic fragment and plays a crucial role in inducing animal immunity. Therefore, research on the FMDV structural protein VP1 is of great significance for vaccine development.

[0003] Currently, the method used to control foot-and-mouth disease (FMD) in endemic areas is the injection of traditional inactivated FMD vaccines. These inactivated vaccines are produced by chemically inactivating live viruses, which may result in compound residues and incomplete inactivation. Traditional live attenuated vaccines were previously used, but they were found to pose serious biosafety risks, with FMD sometimes exhibiting virulence enhancement during vaccination. Furthermore, serological testing makes it difficult to distinguish between viral infection and vaccination in animals. Therefore, in some Western European countries, both inactivated and live attenuated FMD vaccines are banned. Researchers are currently exploring a new vaccine to replace traditional vaccines. This new vaccine consists of many short peptides located in the antigenic immunodeterminant region and does not carry any viral genes, making it a safer multi-epitope vaccine. Viral epitopes consist of B-cell epitopes, helper T-cell (Th) epitopes, and cytotoxic T-lymphocyte (CTL) epitopes. Previous research on FMDV antigens has mainly focused on B-cell and Th epitopes, with less research on CTL epitopes. Recent studies have shown that cytotoxic T lymphocyte (CTL) epitopes bind to the polypeptide binding groove of the major compatibility complex (MHC) class I heavy chain and simultaneously bind non-covalently to the light chain β2 microglobulin (β2m). Research indicates that once these epitopes are presented to the cell surface, they induce cellular immunity in vivo, playing a crucial role in combating foot-and-mouth disease virus.

[0004] To screen for CTL epitopes of the foot-and-mouth disease virus (FMDV) VP1 protein, various methods have been attempted, including constructing covalent complexes of the heavy and light chains of porcine major histocompatibility complex (MHC) class I molecules (i.e., swine leukocyte antigen (SLA) class I molecules) to screen for FMDV antigenic peptides; and refolding separately expressed heavy chain, light chain, and FMDV peptides. However, these methods all involve screening for peptides bound to SLA-I molecules in vitro, and the screened peptides are not naturally presented in vivo by SLA-I molecules. In other words, these methods have low efficiency in screening for CTL epitopes. Previously, researchers attempted to extract peptides from the surface of human or mouse MHC class I positive expression cell lines using a mild acid elution method and demonstrated that the isolated antigenic peptides were naturally expressed by MHC class I molecules, belonging to functional peptides that could be used for vaccine development. To date, no porcine cell lines have been established for screening and isolating FMDV-derived antigenic peptides. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a method for preparing a monoclonal cell line fused with VP1 and EGFP, which can effectively isolate naturally presented antigen peptides.

[0006] The technical solution adopted by this invention to solve the technical problem includes the following steps:

[0007] (1) Culture PK15 cells;

[0008] (2) Prepare a G418 solution with a concentration of 500 μg / mL;

[0009] (3) Determine Lipofectamine TM PK15 cells were transfected with 2000 and pEGFP-N1-VP1 plasmids at a 1:1 ratio.

[0010] (4) G418 screening;

[0011] (5) SLA-I type presenting peptides were obtained by weak acid elution.

[0012] Beneficial effects:

[0013] The advantage of this technology is that it provides a method for directly isolating and identifying naturally presented cellular epitopes of foot-and-mouth disease virus (FMDV) at the cellular level. Compared to in vitro binding methods for screening cellular epitopes, the viral epitopes obtained by this method have undergone binding with SLA-I class molecules and natural presentation within cells, maintaining their natural activity. Therefore, they can serve as candidate components for FMD epitope vaccines and antigen presentation research. Furthermore, compared to in vitro binding methods for epitope screening, this invention is more convenient and faster to operate at the cellular level, yielding a greater number of epitopes with higher efficiency. Attached Figure Description

[0014] Figure 1 This is a graph showing the lethal concentration of G418 in step 2 of Example 1.

[0015] Figure 2 This is a graph showing the transfection efficiency of PK15 cells in step 3 of Example 1.

[0016] Figure 3 The images show transfection at different time points after pEGFP-N1-VP1 transfection in step 4 of Example 1.

[0017] Figure 4 This is a diagram showing the expression of green fluorescent protein in stably transfected cells during step 4 of Example 1.

[0018] Figure 5 This is an electrophoresis diagram used in step 5 of Example 1 to verify VP1 gene expression. Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments.

[0020] Raw material sources: Plasmid pEGFP-N1-VP1 (transient expression vector) was kindly provided by China Agricultural University. PK15 cell line was purchased from the China Veterinary Microbial Culture Collection Center (CVCC). DMEM and fetal bovine serum were purchased from Gibco. Horseradish peroxidase-labeled goat anti-mouse IgG monoclonal antibody, plasmid extraction kit, and gel recovery kit were all purchased from Sangon Biotech (Shanghai) Co., Ltd. Reverse transcription kit, T4 DNA Ligase, and Taq polymerase were purchased from Takara Bio (Dalian) Co., Ltd. Lipofectamine TM2000 was purchased from Invitrogen Corporation, USA. G418 was purchased from Sigma-Aldrich, USA. Mouse anti-GFP monoclonal antibody was purchased from Beyotime, USA. Acetonitrile (HPLC grade) was purchased from Kermel, USA. Ultrasensitive luminescent solution was purchased from Solarbio, Beijing, China. A pair of primers was designed to detect VP1 gene expression, including upstream primer VP1-F, 5'-ACCACCTCCACAGGTGAGTCG-3' and downstream primer VP1-R, 5'-CAAAAGCTGTTTCACAGGCG-3'. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0021] Example 1

[0022] (1) Cell Culture. Cells were removed from liquid nitrogen and cultured at 37°C and 5% CO2 in DMEM medium containing 10% FBS for 12-18 hours until the cells recovered. The medium was then changed. After the cells had grown and covered the entire bottom of the culture flask, they were digested with 0.25% trypsin at 37°C for approximately 5 minutes. The digested cells were then cultured at 7-8 × 10⁶ cells per well. 4 Seed cells into 24-well plates and incubate at 37°C with 5% CO2 for 24 hours until the cell density reaches 70%-80%.

[0023] (2) Measurement of G418 lethal concentration curve. 4×10 5 PK15 cells were seeded into 24-well plates and cultured at 37°C and 5% CO2. When the cell density reached 70%-80%, G418 was diluted with DMEM medium at concentration gradients of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg / mL and added to the 24-well plates. The plates were then incubated at 37°C and 5% CO2, with each treatment repeated three times. Every 48 hours, the medium was changed with fresh DMEM medium containing the gradient concentrations of G418. The lowest G418 concentration that killed all cells within 7 days was determined as the optimal concentration for screening for stable transfection of exogenous plasmids. Curves were plotted to assess the relationship between viable cell count and G418 concentration after 7 days of lethal assays using a series of G418 gradient concentrations, as shown in the figure. Figure 1 As shown in the figure. The results showed that when the G418 concentration reached 500 μg / mL, all cells were killed. Therefore, the appropriate concentration of G418 for screening stable transfectants should be 500 μg / mL.

[0024] (3) PK15 cell transfection efficiency determination. 4×10⁶ cells were transfected... 5PK15 cells were seeded into 24-well plates and cultured at 37°C with 5% CO2. When the PK15 cell density reached 70%-80% per well, transfection was performed using Lipofectamine. TM PK15 cells were transfected with pEGFP-N1-VP1 plasmid at a ratio of 1:1 to 1:3, with each ratio repeated three times. Transfection efficiency was then assessed by flow cytometry. PK15 cells without any liposomes or plasmids were used as a control. Figure 2 Flow cytometry was used to determine the transfection efficiency of PK15 cells. (A) A representative result for PK15 control cells without any transfectants. 0 on the X-axis represents no transfectants, and 0.5 represents the background value. (B–D) Representative transfection efficiencies were obtained when the ratio of liposomes to recombinant plasmids was 1:1, 1:2, and 1:3, respectively, which were 13.5%, 9.83%, and 7.45%. (E) Statistical analysis of the results after transfecting PK15 cells with different ratios of liposomes and recombinant plasmids. **, p<0.01.

[0025] (4) PK15 cell transfection and G418 selection. 4×10⁶ cells were transfected with PK15 cells and selected for G418. 5 PK15 cells were seeded into 24-well plates and cultured at 37°C with 5% CO2. When the density of transfected PK15 cells reached 70%-80% per well, liposomes and plasmid Lipofectamine were added. TM2000 / pEGFP-N1-VP1 was transfected into cells at a 1:1 ratio. Twenty-four hours after transfection, the transfected cells were washed twice with PBS, and then DMEM medium containing 10% FBS and 500 μg / mL G418 was added to kill the cells. The medium was changed every 48 hours thereafter. After 7 days, cell clusters emitting clear green fluorescence were observed in the wells. The locations of these clusters were marked, and 2 μL of 0.25% trypsin was gently aspirated from each well using a micropipette to digest the marked cells. The cells were then transferred to new 96-well plates for culture. Cells in the 96-well plates were observed using a fluorescence inverted microscope until their number was clearly visible and they covered the bottom of the culture dish. The cells from the 96-well plates were then transferred to 24-well plates, and cell growth was maintained in medium containing 10% FBS and 500 μg / mL G418. After cells grew to completely cover the wells, they were transferred to 6-well plates and cultured in medium containing 10% FBS and 500 μg / mL G418, then transferred to 6 cm diameter culture dishes. Finally, the cells were transferred to 10 cm culture dishes for further culture, and a portion of the cells were stored at -80°C. At 3, 6, 12, 24, 48, 72, 168, 336, and 480 hours post-transfection, a portion of the transfected cells was analyzed to evaluate the transfection and selection efficiency of PK15 cells. In summary, transfected cells were fixed with 4% paraformaldehyde and incubated at room temperature for 30 minutes, followed by three washes with PBS. Cells were permeabilized with 0.2% Triton X-100 for 10 minutes, followed by three washes with PBS. The nuclei were stained with 1 μg / mL 4,6-diamidinyl-2-phenylindole (DAPI) for 10 minutes, followed by three washes with PBS. Cells were observed under a 20x objective lens using the ImageXpress MicroXLS wide-field high-content analysis system (Megu Molecular, USA), and images were taken using DAPI and FITC channels. The images were then transmitted via Thermo Scientific. TM HCS Studio TM 2.0 Analyze the data.

[0026] (5) Identification of stable VP1 gene expression in pEGFP-N1-VP1 / PK15 cells. To confirm successful transfection of the target plasmid into PK15 cells and to identify pEGFP-N1-VP1 gene expression in cells, total RNA was extracted from transfected cells using the TRizol Reagents kit (Invitrogen Inc., USA) according to the methods described in the literature. The samples were stored at -80°C for later use. Following the manufacturer's recommendations, the extracted total RNA was reverse transcribed into cDNA using avian fibroblast virus (AMV) reverse transcriptase (TaKaRa, Japan) and oligo(dT) primers. Using 1 μg of cDNA as a template, the VP1 gene was amplified using the VP1-F / VP1-R primer pair. The PCR reaction was as follows: 94°C, 5 min pre-denaturation; 94°C, 30 s; 56°C, 45 s; 72°C, 1 min, 30 cycles; finally, 72°C, 10 min to terminate the reaction. The PCR product was stored at -20°C for gene cloning. The PCR products were recovered using a gel extraction kit and ligated into the pMD 18-T vector, followed by sequencing.

[0027] To identify VP1 protein expression in pEGFP-N1-VP1 / PK15 cells, 2 × 10⁻⁶ cells were collected. 6 Stable transfected cells were used to extract intracellular proteins using RIPA lysis buffer. Protein concentration was then determined using the BCA method. A 20 μg sample of denatured protein was analyzed by SDS-PAGE and transferred to a 0.45 μm PVDF membrane. The membrane was blocked for 1 hour at room temperature with phosphate-buffered saline (PBS) containing 5% BSA. It was then incubated at 4°C with VP1 antibody (1:1000, mouse-derived, kindly provided by China Agricultural University) and GAPDH mouse monoclonal antibody (Proteintech, Chicago, USA) as an internal control. After incubation, the membrane was washed three times with TBS containing 0.3% Tween 20 for 10 minutes each time. The membrane was then incubated for 1 hour at room temperature with horseradish peroxidase (HRP) labeled with goat anti-mouse IgG monoclonal antibody (1:5000). Finally, the PVDF membrane was treated with ultrasensitive chromogenic solution (Solarbia, Beijing).

[0028] (6) Detection of SLA-I class presenting peptides eluted with weak acid by LC-MS / MS. pEGFP-N1-VP1 / PK15 cells were cultured in DMEM medium containing 10% FBS, 300 μg / mL G418, 37℃, and 5% CO2 to the logarithmic growth phase, and then 1×10⁻⁶ cells were collected. 8Cells were centrifuged at 1500 rpm for 5 minutes at room temperature, washed twice with PBS, and centrifuged again. Then, following the method reported by Storkus et al., cells were eluted for 30 seconds at room temperature with phosphate buffer (0.131 mol / L citrate, 0.066 mol / L Na2HPO4, pH 3.3). The elution mixture was centrifuged at 1500 rpm for 5 minutes to remove cells and debris. The eluted sample was centrifuged at 4500 × g for 5 minutes through an Amicon Ultra-15 protein centrifuge filter (3000) to remove large fragments of proteins or other macromolecules. Based on literature reports and with slight modifications, Stage-tip C18 (Empore) was used. TM The filtrate was desalted using an Octadecyl C18, 47mm Extraction Disk (PA, USA). First, 100 μL of acetonitrile was added to the stage-tip C18 membrane, and centrifuged at 500 × g until the acetonitrile had completely passed through the membrane. The C18 column was equilibrated with 100 μL of 0.1% TFA / H2O, and then centrifuged at 500 × g until the solution had completely passed through the membrane, repeated three times. The column was transferred to a new 2 mL EP tube, and the peptide sample was loaded onto the membrane. The sample was incubated at room temperature for 5 minutes, and then centrifuged at 500 × g until the sample had completely passed through. The filtrate was reloaded onto the membrane and the filtration was repeated. As described above, the column was washed three times with 100 μL of 0.1% TFA / H2O, and centrifuged three times at 500 × g. The column was then transferred to another new 2 mL EP tube. The peptide sample adsorbed onto the membrane was eluted with 100 μL of elution buffer containing 0.1% TFA and 50% acetonitrile, while centrifuging at 400 × g until the buffer was completely passed through. The elution was repeated twice. The two eluates were combined and collected in a 500 μL EP tube and lyophilized in a Speedvac concentrator (ZLS-2 Origin: Hunan, China).

[0029] The lyophilized peptides were dissolved in 20 μL of 0.1% formic acid / water solution, and 4 μL of the sample was injected into a C18 (3 μm) container. In 75 μm × 15 cm), and in a high-performance liquid chromatograph (HPLC, Thermo Scientific) TMSeparation was performed using an Easy nLC 1200 (USA) with a chromatographic gradient for 90 min, followed by equilibrium shift phase A (0.1% formic acid aqueous solution) and phase B (0.1% formic acid), 80% acetonitrile / H₂O, at a flow rate of 350 nL / min. Mass spectrometry data were obtained using an Orbitrap Fusion Lumos ultra-high resolution mass spectrometer (ThermoScientific Orbitrap Fusion Lumos, USA) with the following parameters: spray voltage, 2.0 kV; capillary temperature, 320 °C; RF lens, 40°C. For the primary MS data, the resolution was set to 120,000 m / z 200, while for MS / MS data, the resolution was set to 30,000 m / z 200. The precursor ion m / z scan range was 350 to 1550, while the daughter ion m / z scan started at 110. The ion screening window was set to 1.6 m / z. The fragmentation mode was set to HCD. The energy was NCE 32. The data search first prioritized the top 20 relevant MS / MS values, followed by manual analysis. A dynamic exclusion timeout was set to 60 seconds. Data was analyzed using Proteome Discoverer 2.1 (Thermo Fisher, MA, USA).

[0030] The test results for each step are as follows:

[0031] Since the pEGFP-N1 plasmid fuses a foreign gene with EGFP for expression, the expression status of VP1 can be indirectly determined by detecting the fluorescence of EGFP expression. Using the ImageXpress Micro XLS Widefield High-Content Analysis System, the results showed that pEGFP-N1-VP1, with a liposome to recombinant plasmid transfection ratio of 1:1, was successfully transfected into PK15 cells, exhibiting different levels of fluorescence at different time points, such as... Figure 3 As shown. Screening at a G418 concentration of 500 μg / mL revealed detectable green fluorescence in PK15 cells 3 hours post-transfection, but cell proliferation ceased within 6 hours, indicating that many transiently expressing cells were killed during this period. From 12 hours onwards, the number of cells exhibiting green fluorescence gradually increased, and the fluorescence became increasingly intense (3A-F and af). On day 7 (168 hours), aggregated, green fluorescent cell clusters appeared (…). Figure 3G and g). The labeled cell clusters were then transferred to 96-well plates and further screened for killing using DMEM medium containing 500 μg / mL G418. After 10 days, wells still showing stable green fluorescence were selected as positive cells, and the positive cells were expanded from 96-well to 24-well plates, then to 6-well plates, 6 cm culture dishes, and finally 10 cm culture dishes under the same conditions as above. Results showed that on day 14 (336 hours), over 90% of the cells emitted fluorescence (G and g). Figure 3 H and h). Further screening and culture were conducted. On day 20 (480 hours) post-transfection, nearly 100% of transfected cells emitted green fluorescence, indicating that all PK15-transfected cells expressed the VP1 fusion EGFP protein. This also demonstrates the successful construction of EGFP-N1-VP1 / PK15 cells stably expressing VP1. Figure 4 ).

[0032] Figure 3 To detect the transfection results at different time points after pEGFP-N1-VP1 transfection. (A–G) The expression of VP1 fusion EGFP protein in transfected PK15 cells was detected using the ImageXpress Micro XLS Widefield High-Content Analysis System. Positive cells emitted bright green fluorescence. The detection time points were 3, 6, 12, 24, 48, 72, and 168 hours after transfection. (a–g) Corresponding to Figure AH, the green fluorescence of cells expressed was detected simultaneously after localization with 4',6-diamidinyl-2-phenylindole (DAPI) nuclear staining. The nuclei of negative cells stained blue, while the cytoplasm of positive cells expressing VP1 fusion EGFP protein stained dark green, and the nuclei stained cyan. (H) Similar detection as (A–G) was performed after expanding culture from a cell cluster emitting green fluorescence to 336 hours. (h) Corresponding to Figure (H), similar detection as (a–g) was performed.

[0033] Figure 4 To detect the expression of VP1 fusion EGFP protein in stably transfected pEGFP-N1-VP1 / PK15 cells 480 hours after transfection. (B) Corresponding to Figure A, the condition of cells after DAPI nuclear staining was detected. (C) Corresponding to Figures (A) and (B), the expression of cytoplasmic green fluorescent protein and the staining of the cell nucleus were detected simultaneously. In negative cells, only the nucleus was stained blue, while in positive cells expressing EGFP-VP1, the cytoplasm was stained dark green, and the cell body was stained cyan.

[0034] To determine whether VP1 is stably expressed at the mRNA level in pEGFP-N1-VP1 / PK15 cells, RT-PCR was used to detect the VP1 transcription product. The results showed that the specifically amplified fragment was approximately 600 bp, consistent with the theoretical value of 639 bp for stably transcribed VP1 in pEGFP-N1-VP1 / PK15 cells. Figure 5 A) The PCR product was recovered using a gel extraction kit, ligated into the pMD 18-T vector, and then transferred to TOP10 competent cells. Positive clones were selected for sequencing, and the results showed that the VP1 clone sequence was completely identical to the VP1 sequence in the pEGFP-N1-VP1 plasmid. Western blotting of the VP1 fusion protein with an anti-VP1 monoclonal antibody showed that the VP1 fusion protein was stably expressed in the transfected PK15 cell line, with a size of approximately 50 kDa. Based on the EGFP protein size of 28.0 kDa and the VP1 protein size of 23.1 kDa, the fusion protein size was calculated to be 51.1 kDa, consistent with the theoretical value. However, control PK15 cells and PK15 cells transfected with the empty vector pEGFP-N1 did not express the VP1 fusion protein. Figure 5 As shown in B.

[0035] Eluted peptide samples were analyzed using high-performance liquid chromatography (HPLC) and an Orbitrap Fusion Lumos ultra-high resolution mass spectrometer. The master data were analyzed using Proteome Discoverer 2.1. Peptides were searched in the self-constructed O-VP1.fasta database and screened using a PSM validator with a medium screening criterion. Analysis revealed at least 37 peptides of 8–11 amino acids from O-VP1.fasta, which precisely fit the size of the 8–11 amino acid binding groove in SLA-I class molecules (see Table 1). Most peptides were modified post-presentation, such as by oxidation or deamidation. Twelve peptides had an XCorrSequest HT value (peptide abundance value) greater than 1.

[0036] Table 1. Peptides eluted from pEGFP-N1-VP1 / PK15 cell line as determined by LS–MS / MS

[0037]

[0038]

[0039]

[0040] Note: * indicates that the polypeptide has undergone modifications such as deamination or oxidation; a indicates the abundance value of the polypeptide; b indicates that two polypeptides with the same sequence have undergone different modifications and have different molecular weights.

[0041] Currently, multiepitope vaccines, due to their safety and efficacy against different serotypes of the virus, may become an alternative to traditional inactivated or live attenuated foot-and-mouth disease (FMD) vaccines. A key task in designing FMD multiepitope vaccines is identifying novel epitopes, especially CTL epitopes. Previously, it was believed that FMDV infection did not elicit significant cellular immunity, particularly CD8+ T lymphocyte immunity. In fact, many researchers have begun to study CTL epitopes, which can induce CD8+ T lymphocyte immunity. + Cellular immunity is primarily driven by T lymphocytes. While CTL epitopes are typically designed using computer prediction tools, these predictions cannot accurately mimic in vivo antigen processing mechanisms.

[0042] This study successfully constructed a PK15 cell line transfected with the VP1 gene, stably expressing the VP1 gene of type O foot-and-mouth disease virus. Further RT-PCR and Western blotting confirmed the stable expression of the EGFP-fused VP1 gene in PK15 cells. Because of the VP1-EGFP fusion expression, cells stably expressing the VP1 gene also express EGFP, and positive cells emitted green fluorescence as detected by the ImageXpress Micro XLS Widefield high-content analysis system. According to the antigen presentation mechanism, the VP1 protein, expressed as an endogenous antigen, is processed into peptides after digestion by the proteasome. Some peptides are transported to the endoplasmic reticulum and bind to the heavy chain of MHC class I molecules. The heavy chain simultaneously binds non-covalently to the light chain (β2m), forming a complex. This complex refolds in the Golgi apparatus and is then presented to the cell membrane surface to bind CD8. + T lymphocytes induce cellular immunity. By eluting and extracting peptides bound to MHC class I molecules, naturally presented CTL epitopes can be identified. In this study, a key objective was to screen PK15-positive cell lines that stably express VP1. After extensive screening, it was found that a G418 concentration of 500 μg / mL and a liposome-to-plasmid ratio of 1:1 were sufficient to screen and isolate PK15 cell lines stably expressing the EGFP-fused VP1 gene, which were then gradually expanded. However, once a cell line stably expressing green fluorescence was established, the G418 concentration in subsequent cell cultures was reduced to 300 μg / mL to ensure stable expression of the EGFP-fused VP1 gene in PK15 cells.

[0043] To identify whether VP1 fused with the EGFP gene was stably expressed, we successfully demonstrated, through RT-PCR and Western blotting, that VP1 fused with the EGFP gene was expressed at both the nucleic acid and protein levels. These results indicate that we successfully constructed a cell line stably expressing the type O foot-and-mouth disease virus VP1 gene, which can be used for peptide elution as previously reported. However, to date, all reports related to acid-eluted antigenic peptides have been conducted in human or mouse cell lines, without corresponding porcine cell lines, such as PK15, for studying antigenic peptide presentation and elution associated with porcine viruses. In this study, using a mild acid elution method, we determined 37 eluted peptides from the surface of the constructed, stably VP1-expressing pEGFP-N1-VP1 / PK15 cell line by LC-MS / MS. All eluted peptides were in the range of 8-11 amino acids, and 12 of them, with an XCorr Sequest HT (abundance value) greater than 1, were likely dominant antigenic peptides bound to and presented by SLA-I class molecules, as shown in Table 1. Because the peptide-binding grooves of SLA-I class molecules can mostly accommodate 8-11 amino acids, this is consistent with the length of the eluted peptides in this experiment. Furthermore, these results indicate that eluted peptides derived from the VP1 protein can be processed, assembled, and presented to the surface of pEGFP-N1-VP1 / PK15 cells by SLA-I molecules. Therefore, according to literature reports, the peptides eluted in this experiment should be SLA-I class molecule-restricted CTL epitopes. This experiment also demonstrates that the constructed pEGFP-N1-VP1 / PK15 cell line can be used to screen and study naturally presented SLA-I class molecule-restricted CTL epitopes in vivo.

[0044] In summary, we have constructed a cell line that stably expresses the VP1 recombinant plasmid in PK15 cells. This cell line can be used to study the presentation and elution of antigenic peptides, and to further identify functional CTL epitopes that match SLA-I class molecules. This research will lay the foundation for screening CTL epitopes and developing multi-epitope vaccines for foot-and-mouth disease.

[0045] The above embodiments are merely illustrative and explanatory of the present invention and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many more variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention.

Claims

1. A method for isolating and identifying a SLA class I molecule-restricted naturally presented CTL epitope of the VP1 protein of foot-and-mouth disease virus, characterized in that, The method includes the following steps: S1. Cultured cells PK15; S2. Adjust the concentration of G418 to a suitable concentration of 500 μg / mL; S3. Transfect PK15 cells with Lipofectamine™ 2000 and pEGFP-N1-VP1 plasmid at a 1:1 ratio; S4.G418 screening; S5. SLA-I class presenting peptides were obtained by weak acid elution: pEGFP-N1-VP1 / PK15 cells were cultured in DMEM medium containing 10% FBS, 300 μg / mL G418, 37℃, and 5% CO2 to the logarithmic growth phase, and then 1×10⁻⁶ cells were collected. 8 Cells were centrifuged at 1500 rpm for 5 minutes at room temperature. Cells were washed twice with PBS, centrifuged again, and eluted for 30 seconds at room temperature with a phosphate buffer (pH 3.3) containing 0.131 mol / L citrate and 0.066 mol / L Na₂HPO₄. The elution mixture was centrifuged at 1500 rpm for 5 minutes, and the eluted sample was centrifuged at 4500 × g for 5 minutes through an Amicon Ultra-15 protein centrifuge filter. The filtrate was desalted using a Stage-tip C18 column. First, 100 μL of acetonitrile was added to the Stage-tip C18 membrane, and the column was centrifuged at 500 × g until the acetonitrile completely flowed through the membrane. The C18 column was equilibrated with 100 μL of 0.1% TFA / H₂O, and then centrifuged at 500 × g until the solution completely flowed through the membrane. This process was repeated three times. The column was then transferred to a fresh 2 mL aliquot. In an EP tube, the peptide sample was loaded onto the membrane and incubated at room temperature for 5 minutes. Then, the membrane was centrifuged at 500×g until all the sample had passed through. The filtrate was reloaded onto the membrane and the filtration was repeated. The column was washed three times with 100 μL of 0.1% TFA / H2O and centrifuged three times at 500×g. The column was then transferred to another new 2 mL EP tube and the peptide sample adsorbed on the membrane was eluted with 100 μL of elution buffer containing 0.1% TFA and 50% acetonitrile and centrifuged at 400×g until the buffer had completely passed through. The elution was repeated twice. The two eluates were combined and collected in a 500 μL EP tube and lyophilized in a Speedvac concentrator. Elution yielded 37 presenting peptides, with the following amino acid sequences: VGALLRTA, KDQINVLDLM*, QINVLDLMQ, QINVLDLMQ*, EVAVKHEGNL*, VLTQKAARTLP, QINVLDLMQTP*b, LRTATYYFA, NVLDLMQTPA*, LTQK AARTL*, QINVLDLMQTP*b, PNGAPEAAL, KVTPKDQIN*, VAVKHEGNLT*, INVLDLMQTP, KDQINVLDLM*, QINVLDLMQT*, VTNPRGDL*, RGDLQVLT Q*, PNGAPEAAL*, LTWVPNGAPE, QKAARTLP*, NLTWVPNGA, TPKDQINVLD*, GDLQVLTQ, ENYGGETQVQ, QINVLDLMQ*, PRGDLQVLTQ, KAARTLPTSFN, INVLDLMQTP, INVLDLMQTP*, LQVLTQKA*, KAARTLPTSF, IKATRVTE, INVLDLMQ*, TQVQRRQH*, RGDLQVLTQ*; * indicates that the peptide has undergone modifications such as deamination and oxidation; S6, identifying CTL epitopes for restricted natural presentation of SLA-I class molecules.

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