Construction method and application of recombinant cell strain for resisting porcine diarrhea virus

Abstract

The invention relates to the technical field of biological medicine, in particular to a construction method and application of an anti-porcine diarrhea virus recombinant cell strain. Aiming at the problems of low PEDV S protein expression efficiency and insufficient cell strain stability in the prior art, the invention aims to provide a construction method and application of a recombinant cell strain for resisting porcine diarrhea virus, the recombinant CHO cell strain capable of efficiently secreting PEDV S protein is obtained by optimizing expression vector design, a screening strategy and a suspension domestication process, and the recombinant CHO cell strain can be used for efficiently secreting PEDV S protein. And a foundation is laid for preparation of subunit vaccines and detection reagents.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing and applying a recombinant cell line against swine diarrhea virus. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting, dehydration, and decreased appetite. This virus belongs to the Coronaviridae family and primarily attacks the epithelial cells of the small intestine in pigs, causing damage to the intestinal barrier and severe electrolyte imbalance. Pigs of all ages are susceptible, but it is particularly deadly to suckling piglets, with an infection mortality rate as high as 90%-100%.

[0003] Virological studies have shown that the spike protein (S protein) of PEDV is a key antigen mediating host cell infection and a major target of neutralizing antibodies. Currently, PEDV control mainly relies on inactivated vaccines and live attenuated vaccines, but both have significant drawbacks: Firstly, inactivated vaccines, prepared by inactivating the virus with formaldehyde or β-propiolactone, while highly safe, have weak immunogenicity, requiring multiple booster immunizations. Studies have shown that neutralizing antibodies are produced slowly after immunization with inactivated vaccines; in the low-dose group (4 mL / head), antibodies are only detectable after 14 days, and the peak titer is low, with a short duration of immunity (approximately 49 days). Live attenuated vaccines, obtained through viral attenuation, have the advantage of inducing mucosal immunity, but carry the risk of virulence reversion.

[0004] To address the shortcomings of traditional vaccines, there is an urgent need to develop a method for constructing recombinant cell lines that can directly utilize the natural S gene, possess high-efficiency secretion, long-term stable expression, and high-purity protein production, thereby overcoming the bottleneck in PED vaccine development. Currently, such technologies mainly suffer from the following drawbacks: 1. Low secretion efficiency: It relies on the single transmembrane protease serine 9 (sp9) signal peptide to mediate secretion, resulting in a large amount of S protein remaining in the cell. The expression level in the culture supernatant is only 0.63 mg / mL, and complex modifications to the S gene (such as inserting a secretion enhancer) are required to achieve basic expression. 2. Insufficient expression stability: Exogenous genes are easily silenced due to chromatin position effects during continuous passage. The expression level of S protein in the 15th generation (P15) cells decreases by more than 10%, and single antibody selection (such as G418 only) leads to a false positive rate of more than 20%, resulting in low efficiency in constructing stable cell lines. Therefore, developing a method for constructing recombinant CHO cell lines that does not require additional modification of the S gene and can simultaneously improve secretion efficiency and expression stability is of great significance for the research and industrialization of PEDV vaccines. Summary of the Invention

[0005] To address the problems of low PEDVS protein expression efficiency and insufficient cell line stability in existing technologies, the present invention aims to provide a method for constructing and applying a recombinant cell line against porcine diarrhea virus. By optimizing expression vector design, screening strategies, and suspension acclimation processes, a recombinant CHO cell line capable of efficiently secreting PEDVS protein is obtained, laying the foundation for the preparation of subunit vaccines and diagnostic reagents.

[0006] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention aims to provide a method for constructing a recombinant cell line against porcine diarrhea virus, comprising the following steps: (1) Construction of recombinant plasmids: A chimeric signal peptide was designed and fused with the PEDVS gene and tag sequence to form a fusion gene fragment; this fragment was then cloned into an expression vector, and a human β-interferon MAR element and a dual-resistance marker were simultaneously inserted to obtain a recombinant plasmid. (2) Cell transfection and screening: The recombinant plasmid was transfected into CHO cells, and after screening with a two-drug gradient, monoclonal cell lines were obtained by limiting dilution. (3) Stability verification and suspension acclimatization: The monoclonal cell line was subjected to passage stability verification, and a three-step suspension acclimatization method was used to obtain a recombinant CHO cell line.

[0007] Preferably, in step (1), the chimeric signal peptide is composed of amino acids 1-15 at the N-terminus of the human IgG2a signal peptide, the glycine-serine linker GGGS, and amino acids 16-24 at the C-terminus of the transmembrane protease serine 9 sp9 signal peptide, and its amino acid sequence is shown in SEQ ID NO:4.

[0008] Preferably, the PEDV S gene is a gene with a nucleotide sequence as shown in SEQ ID NO:1, which encodes the S protein (amino acid sites 19-1383) of the PEDV variant strain AH2012 / 12; the tag sequence is a 6×His tag.

[0009] Preferably, the fusion gene fragment is a nucleotide sequence as shown in SEQ ID NO:2, with BamHI / EcoRI restriction sites introduced at both ends, and cloned into the corresponding restriction sites of the pcDNA3.1 (+) vector; the human β-interferon MAR element is inserted through the XhoI site, and the dual resistance marker is the original G418 resistance gene of the vector and the puromycin resistance gene inserted through the EcoRV site.

[0010] Preferably, the transfection is performed using Lipofectamine 3000 reagent.

[0011] Preferably, the dual-drug gradient pressurization screening is performed as follows: screening is conducted at 24-72 h using 1-3 μg / mL G418 and 0.4-0.6 μg / mL (e.g., 0.5 μg / mL) puromycin, and screening is conducted at 3-14 days using 4-6 μg / mL (e.g., 5 μg / mL) G418 and 1-3 μg / mL (e.g., 2 μg / mL) puromycin.

[0012] Preferably, in step (3), the three-step suspension acclimatization includes: (a) Adhesion-floating transition: Culture in CD FortiCHO™ medium containing 9-11% fetal bovine serum with shaking for 2-3 days until the viable cell density is ≥ Once cells / mL and viability ≥95% are achieved, proceed to the next stage; (b) Microcarrier-assisted: Add 0.08-0.12 g / L Cytodex-3 microcarriers to the culture system, replace with CDFortiCHO™ medium containing 4-6% FBS, and continue culturing for 3-7 days. During this period, ensure cell viability ≥93% and cell density ≥ using trypan blue staining. cells / mL; (c) Serum-free amplification: Replace the medium completely with serum-free CD FortiCHO™ medium and culture in batches, adding 4%-6% volume of concentrated nutrient solution every 22-26 hours. This concentrated nutrient solution contains 7-9 mM glutamine, 0.5-2% insulin-transferrin-selenium supplement, and 0.4-1% Pluronic F-68. Continue culturing until the viable cell density is ≥ A stable suspension culture cell line was obtained by achieving a cell density of 90% / mL and a viability of ≥90%.

[0013] Most preferably, in step (3), the three-step suspension acclimatization includes: (a) Adhesion-floating transition: CD FortiCHO™ medium containing 10% fetal bovine serum was used and incubated at 37°C with 5% [unclear - possibly referring to a specific concentration or concentration]. In an incubator, culture with shaking at 100-140 rpm for 2-3 days until the viable cell density is ≥ Once cells / mL and viability ≥ 95% are achieved, proceed to the next stage; (b) Microcarrier-assisted: Add 0.1 g / L Cytodex-3 microcarriers to the culture system, replace with CD FortiCHO™ medium containing 5% FBS, and maintain at 37°C and 5% FBS. Under the following conditions, adjust the rotation speed to 120-160 rpm and culture for 3-7 days. During this period, ensure a viability of ≥93% and a viable cell density of ≥ [missing information - likely a specific value]. cells / mL; (c) Serum-free amplification: Replace the medium with serum-free CD FortiCHO™ medium, increase the rotation speed to 160-180 rpm for fed-batch culture, and add 5% volume of concentrated nutrient solution every 24 hours. This concentrated nutrient solution contains 7-9 mM glutamine, 1% insulin-transferrin-selenium additive, and 0.5% Pluronic F-68. Continue culturing until the viable cell density is ≥ A stable suspension culture cell line was obtained by achieving a cell density of 90% / mL and a viability of ≥90%.

[0014] Secondly, the present invention aims to provide a recombinant CHO cell line obtained by the construction method provided in the first aspect above.

[0015] Furthermore, the present invention also provides the use of the recombinant CHO cell line provided in the second aspect above in the preparation of a subunit vaccine against porcine epidemic diarrhea virus.

[0016] Alternatively, the aforementioned recombinant CHO cell line may be used in the preparation of a diagnostic reagent for porcine epidemic diarrhea virus.

[0017] Thirdly, the present invention also provides a PEDV S protein expressed by the recombinant CHO cell line provided in the second aspect above, and its amino acid sequence is shown in SEQ ID NO:3.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves highly efficient preparation of PEDV vaccines through multiple technological innovations and process optimizations. At the technical level, the innovatively designed chimeric signal peptide (sp9 / IgG2a) fundamentally overcomes the protein secretion bottleneck, significantly reducing intracellular retention and improving secretion efficiency. The integrated human β-interferon MAR element effectively solves the gene silencing problem, significantly improving gene retention and long-term expression stability. The synergistic effect of these two components results in a significantly improved overall performance compared to a single optimization scheme, achieving the dual goals of "highly efficient secretion + stable passage." In terms of production process, step-by-step suspension acclimatization combined with precise rotation speed control enables high-density cell culture, significantly improving culture efficiency and cell viability, laying a solid foundation for industrial production.

[0019] At the vaccine product level, subunit vaccines based on high-purity S protein outperform commercially available products in terms of immunogenicity, challenge protection, and intestinal tissue protection: they induce higher levels of neutralizing antibodies, achieve 100% challenge protection, significantly enhance virus clearance capabilities, minimize intestinal pathological damage, and have the safety advantage of no virus residue.

[0020] In summary, this invention, through its core design of "chimeric signal peptide directed secretion + MAR element anchoring stabilization" and step-by-step domestication process, has for the first time achieved high-yield, stable-yield, and high-purity preparation of PEDV S protein in CHO cells. This provides a safe, efficient, and economical solution for the prevention and control of porcine epidemic diarrhea virus, and has significant industrialization value. Detailed Implementation

[0021] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention.

[0022] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0023] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass.

[0024] Example 1 This embodiment provides a method for constructing a recombinant CHO cell line (CHO-PEDV / S-MAR), as detailed below: 1. Material Preparation Cells: CHO-K1 cells (ATCC CCL-61), cultured in DMEM / F12 medium containing 10% FBS (37℃, 5%). ). Vectors and genes: pcDNA3.1 (+) vector (Invitrogen), human β-interferon MAR element (GenBank M28622.1), PEDVS gene (SEQ ID NO:1, encoding amino acid SEQ ID NO:3), chimeric signal peptide (SEQ ID NO:4), 6×His tag sequence. Reagents: restriction enzymes (BamHI / EcoRI / XhoI / EcoRV), Lipofectamine 3000, G418, puromycin, CD FortiCHO™ medium, Cytodex-3 microcarriers, concentrated nutrient solution (containing 8 mM glutamine, 1% insulin-transferrin-selenium additive and 0.5% Pluronic F-68), etc. 2. Construction of recombinant CHO cell line (CHO-PEDV / S-MAR): (1) Construction of recombinant plasmids A chimeric signal peptide-PEDV S-6×His fusion gene (SEQ ID NO:2) was synthesized, and BamHI / EcoRI sites were introduced at both ends to obtain the fusion gene fragment, the sequence of which is shown in SEQ ID NO:3. The fragment was then cloned into the pUC57 vector. Vector modification: The pcDNA3.1 (+) vector was digested with BamHI / EcoRI and then ligated to the fusion gene; the MAR element was inserted at the XhoI site and the puromycin resistance gene was inserted at the EcoRV site to construct the recombinant plasmid pCDNA3.1-sp9 / IgG2a-PEDVS-MAR (sequencing verification). (2) Cell transfection and screening: Transfection procedure: CHO-K1 cells were transfected according to... Cells / well were seeded into 6-well plates, and transfected with liposomes (Lipofectamine 3000, 2 μg plasmid / well) 24 h later. The medium was replaced with complete medium (containing 10% FBS) 6 h after transfection. Two-drug gradient selection was initiated 24 h later. Dual-drug screening: In the first stage, 2 μg / mLG418 + 0.5 μg / mL puromycin was used (24-72 h), and in the second stage, the drug was changed to 5 μg / mLG418 + 2 μg / mL puromycin (3-14 days) until all cells in the control group died. Monoclonal screening: Monoclonal antibodies were obtained using the limiting dilution method for initial screening against His-ELISA to determine OD. 450 Positive clones with a value >1.0 were selected, and then Western blot was used to verify the expression of 180 kDa S protein. The highest expression strain was named CHO-PEDV / S-MAR-1. (3) Stability verification and suspension acclimatization: The cells were continuously passaged to P25, and S protein expression levels (ELISA) and gene copy number (qPCR) were measured every 5 generations to ensure that the target protein expression level would not drop sharply due to gene silencing during passage. P25 represents a fluctuation of <3% in expression level. Then, suspension acclimatization was performed using the following method: Transition phase: Monoclonal cells are inoculated into CD FortiCHO™ medium (Cytiva) containing 10% FBS in 125 mL shake flasks (30 mL working volume) and cultured with shaking at 120 rpm for 3 days. Daily samples are collected for trypan blue staining to measure viable cell density (VCD) and viability. Target value: VCD ≥ cells / mL, viability ≥95%. Microcarrier assistance: Add 0.1 g / L Cytodex-3 microcarriers (Cytiva), replace with 5% FBS medium, and incubate at 120 rpm for 5 days. Monitor VCD and viability daily (target value: VCD ≥ (cells / mL, viability ≥93%).

[0025] Serum-free amplification: Replace with serum-free CD FortiCHO™ medium, incubate at 170 rpm in batches, adding 5% concentrated nutrient solution (containing 8 mM glutamine, 1% ITS-X (Gibco), and 0.5% Pluronic F-68) every 24 h. Incubate until VCD ≥ cells / mL (viability ≥90%).

[0026] Comparative Example 1 1. Experimental Methods To systematically evaluate the contribution of the chimeric signal peptide to the MAR element, this comparative study included two parallel control groups: Control group 1 (single signal peptide group): Based on the recombinant plasmid of Example 1, the hydrophobic core sequence of IgG2a in the chimeric signal peptide was deleted by site-directed mutagenesis (the sp9 signal peptide was retained, and SEQ ID NO:4 was truncated to 23 amino acids containing only sp9). At the same time, the human β-interferon MAR element (GenBank M28622.1) was removed by XhoI enzyme digestion. The G418+ puromycin double resistance gene was retained as the resistance marker (consistent with Example 1).

[0027] The recombinant plasmid was confirmed by Sanger sequencing to have the signal peptide truncated and the MAR element deleted correctly, ensuring that no frameshift mutations occurred in the PEDV S gene (SEQ ID NO:1) and the 6×His tag sequence.

[0028] Control group 2 (no MAR group): The chimeric signal peptide (sp9 / IgG2a, SEQ ID NO:4) and the dual resistance gene of Example 1 were retained, and the MAR element was deleted only through the XhoI site. The rest of the backbone (pcDNA3.1 (+)) and gene sequence were completely consistent with Example 1.

[0029] Both control groups used the same two-drug screening strategy as in Example 1, with the same specific parameters and other operations as in Example 1, until monoclonal cells were obtained.

[0030] 2. Experimental Results and Analysis 2.1 Suspension Culture and Detection Methods Culture system: Single-clonal cells were seeded in 125 mL shake flasks (Corning), initial density... cells / mL, working volume 30mL, CD FortiCHO™ medium (containing 8mM glutamine + 1% ITS-X), 37℃, 5% Cultured by shaking at 120 rpm.

[0031] Sampling time point: Daily sampling with trypan blue staining and counting, when the viable cell density (VCD) reaches... When the cell count reaches 100 cells / mL (approximately 5-6 days of culture), simultaneously collect the supernatant and cell pellet: Supernatant S protein: Detected using the BCA method (Thermo 23225), with a standard curve range of 0.1-10 μg / mL, and three replicates per sample; Intracellular retention analysis: Cell pellets were washed three times with pre-cooled PBS, lysed on ice for 30 min with RIPA lysis buffer (containing 1% PMSF), centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected for Western blot analysis (primary antibody: mouse anti-His-tagged antibody, Abcam ab18184, 1:5000 dilution; secondary antibody: HRP-labeled goat anti-mouse IgG, CST #7076, 1:10000 dilution). The gray values ​​of the bands were analyzed using ImageJv1.53e software. The intracellular retention percentage = (intracellular S protein gray value) / (intracellular + supernatant S protein gray value) × 100%.

[0032] 4. Long-term stability testing parameters RNA extraction and qPCR: Total RNA was extracted from each passage (P5 / P15 / P25) of cells using TRIzol reagent (Invitrogen 15596026), quantified using Nanodrop 2000 (OD260 / 280=1.8-2.0), and reverse transcribed into cDNA using PrimeScript RT reagent Kit (TaKaRa RR047A). The qPCR system (20 μL) contained SYBR Green Master Mix (Roche4887352001), forward and reverse primers (10 μM, see Table 1), and cDNA template (100 ng). Reaction conditions: 95℃ pre-denaturation for 30 s, 40 cycles (95℃ 5 s, 60℃ 30 s). Melting curves were used to verify primer specificity.

[0033] Apoptosis rate detection: Annexin V-FITC / PI double staining kit (BD 556547), flow cytometry (BDFACSCanto II) detection. The early apoptosis rate of cells was defined as Annexin V. / PI Cell percentage.

[0034] The experimental results are shown in Tables 1 and 2: Table 1 Comparison of experimental results between Control Group 1 and Example 1

[0035] The comparative experiment between Example 1 (chimeric signal peptide + MAR element) and Control Group 1 (single sp9 signal peptide + no MAR element) showed that the chimeric signal peptide (sp9 / IgG2a) is the core factor in improving the secretion efficiency of PEDV S protein. In the early passages (P0-P3) of suspension culture after monoclonal selection, the supernatant S protein concentration of Example 1 reached 0.82±0.04 mg / mL, which was significantly higher than that of Control Group 1 (0.59±0.03 mg / mL, a decrease of 28%, p<0.01). This difference is due to the optimization of intracellular protein transport by the chimeric signal peptide: the intracellular retention rate of Example 1 was only 18.7±1.9%, while that of Control Group 1 was as high as 42.3±3.1% (an increase of 2.3 times, p<0.001), and the secretion efficiency (supernatant / intracellular) increased from 1.4±0.2 to 4.4±0.3 (an increase of 3.1 times, p<0.001).

[0036] Table 2 Comparison of experimental results between Control Group 2 and Example 1

[0037] Note: p<0.05, p<0.01 vs. same generation Example 1 (two-tailed t-test); data are the mean ± SD of 3 independent passage experiments.

[0038] The above experimental results indicate that the regulatory effect of MAR elements on gene stability - short passage (P5): there were no significant differences between the two groups in S gene retention rate (99.2% vs 98.1%), protein expression level (0.82 vs 0.80 mg / mL), and apoptosis rate (4.8% vs 5.2%), suggesting that the absence of MAR elements has little impact on gene expression in the initial integration stage.

[0039] Long-term passaging experiments (P5 / P15 / P25) of Example 1 and Control Group 2 (chimeric signal peptide + no MAR element) confirmed that the MAR element is crucial for maintaining the genetic stability of recombinant cell lines. In short-term passaging (P5), there were no significant differences between the two groups in S gene retention rate (99.2% vs 98.1%), protein expression level (0.82 vs 0.80 mg / mL), and apoptosis rate (4.8% vs 5.2%), indicating that MAR deficiency has a relatively small impact on the initial integration stage. With increasing passage number, the role of MAR elements gradually became apparent: at passage P15, the S gene retention rate of control group 2 decreased to 82.4±2.7% (a decrease of 15.5% compared to Example 1, p<0.05), the S protein concentration decreased by 13.6% (0.70 vs 0.81 mg / mL, p<0.05), and the apoptosis rate increased by 129% (12.6% vs 5.5%, p<0.05); by passage P25, the gene retention rate of control group 2 was only 71.3±3.5% (a decrease of 26.3%, p<0.01), the protein expression level decreased by 27.8% (0.57 vs 0.79 mg / mL, p<0.01), and the apoptosis rate reached 24.3±2.1% (an increase of 298%, p<0.01). This result is closely related to the chromatin anchoring function of MAR elements: MAR maintains the open state of gene loci by binding to nuclear matrix proteins (such as SATB1), reducing epigenetic silencing and chromosome breakage, thereby inhibiting gene loss and activation of apoptosis pathways during passage (ROS levels decreased by 2.1-fold and caspase-3 activity decreased by 1.8-fold).

[0040] The comparative experiments of the two groups show that the chimeric signal peptide and the MAR element have an irreplaceable synergistic effect in the production of recombinant proteins. The chimeric signal peptide is responsible for "short-term high-efficiency secretion," solving the "transport bottleneck" of proteins from the intracellular to the extracellular space. Its absence (control group 1) directly leads to a 3.1-fold decrease in secretion efficiency. The MAR element is responsible for "long-term stable expression," solving the "gene drift bottleneck" during passage. Its absence (control group 2) leads to a 27.8% decrease in the amount expressed at P25. After the synergy of the two, Example 1 still maintains a high expression level of 0.79 mg / mL at the P25 generation. The overall performance (secretion efficiency × gene retention rate) is 3.3 times higher than that of control group 1 (1.00) and 39% higher than that of control group 2 (3.07). This combination of technologies, through the dual guarantee of "improved secretion efficiency + maintenance of long-term stability," meets the core requirements of "high yield and low attenuation" for the industrial production of recombinant proteins, laying a key technological foundation for the large-scale preparation of PEDV subunit vaccines.

[0041] Test case 1. Vaccine preparation based on the recombinant cell line of Example 1. S1: Cell Culture and Bioreactor Scale-up CHO-PEDV / S-MAR-1 cells were cultured in suspension using a 5L Sartorius BIOSTAT® B-DCU stirred bioreactor. The seeding density was [not specified]. The cells / mL culture was maintained at CD FortiCHO™ (Gibco) as the basal medium, with a feeding strategy of adding 4% Feed C (Thermo) daily starting from day 3. Throughout the culture, the culture parameters were maintained at pH 7.2 ± 0.1 (via...). The dissolved oxygen level was adjusted with NaOH (30±5% dissolved oxygen / nitrogen mixture), the temperature was 37℃, and the stirring speed was 120 rpm. The culture period was 7 days, and the endpoint viable cell density reached 8.3±0.4× The cell count was 90.5 ± 1.2%, and 12 L of culture supernatant was harvested (titer 0.82 ± 0.05 mg / mL).

[0042] S2: Step-by-step purification process Clarification and Capture: After clarification using a 0.22 μm filter, the cell culture harvest medium was loaded onto a HisTrap HP 5 mL nickel affinity chromatography column pre-equilibrated with equilibration buffer (20 mM Tris-HCl, 500 mM NaCl, pH 8.0). After loading, the column was washed with 5–10 column volumes of equilibration buffer until the baseline stabilized to remove unbound contaminating proteins. Subsequently, the column was washed with wash buffer containing 50 mM imidazole to remove weakly bound contaminating proteins. Finally, the column was eluted specifically with elution buffer containing 100 mM imidazole, and the protein absorption peak was collected.

[0043] Purification and Inactivation Preservation: The eluent obtained from the affinity capture was concentrated to approximately 1 mg / mL using a 30 kDa ultrafiltration membrane, and then loaded onto a Superdex 200 Increase 10 / 300 GL gel filter column equilibrated with PBS (pH 7.4) for purification. Based on the chromatogram, the main peak corresponding to the PEDV S protein monomer (molecular weight approximately 180 kDa) was collected (typically, retention time 12.2–14.8 min) to obtain the purified S protein; then, the potential virus was inactivated by incubating in a 60°C water bath for 30 min, sterilized by 0.22 μm filter membrane, aliquoted, and stored at 2–8°C.

[0044] S3: Quality Control Results The monomer purity results showed that SDS-PAGE with Coomassie Brilliant Blue staining showed a single band, indicating a purity of ≥95%. The polymer residue results showed that the polymer content detected by HPLC-SEC (Agilent 1260) was ≤1.5% (which meets the requirements of the 2020 edition of the Chinese Veterinary Pharmacopoeia for recombinant protein vaccines).

[0045] Endotoxin results showed that the endotoxin level detected by the LAL gel method (Lonza) was <0.1 EU / μg, which meets the standards for veterinary vaccines.

[0046] 2. Mechanism study of purified S protein inducing intestinal epithelial barrier dysfunction in vitro. Using the purified S protein obtained in the aforementioned steps, the key pathogenic mechanism of the S protein was verified in an in vitro porcine small intestinal epithelial cell (IPEC-J2) model, as follows: 2.1 S protein preparation: The PEDV S protein was derived from the purified S protein obtained in the aforementioned steps, with an irrelevant protein control group (bovine serum albumin, BSA).

[0047] 2.2 Experimental Methods Cell Culture and Treatment: IPEC-J2 cells (porcine jejunal epithelial cell line) were seeded in Transwell chambers (polycarbonate membrane, 0.4 μm pore size) and cultured until a dense monolayer formed (transmembrane resistance TEER ≥ 500 Ω·cm²). The experiment was then divided into three groups: Control group: Only fresh culture medium was added.

[0048] BSA control group: Culture medium containing 50 μg / mL BSA was added.

[0049] S protein treatment group: The medium containing 50 μg / mL PEDV S protein was added.

[0050] 2.3 Detection Indicators and Methods: Transmembrane resistance (TEER): TEER values ​​were measured using a Millicell ERS-2 voltmeter at 0, 12, 24, and 48 h after treatment, and the percentage change relative to 0 h was calculated.

[0051] Bypass permeability: After 24 h of treatment, 4 kDa FITC-glucan (1 mg / mL) was added to the upper chamber, and after incubation for 2 h, the fluorescence intensity in the lower chamber was measured to calculate the glucan flux.

[0052] Apoptosis detection: After 24 h of treatment, flow cytometry analysis was performed using the Annexin V-FITC / PI apoptosis detection kit to calculate the total apoptosis rate (early apoptosis + late apoptosis / necrosis).

[0053] Tight junction protein expression: The expression levels of key tight junction proteins ZO-1 and Occludin were detected by Western blotting, and GAPDH was used as an internal control for semi-quantitative analysis by grayscale values.

[0054] 2.3. Experimental Results Table 3 shows the results of various indicators after treating IPEC-J2 cells with S protein for 24 hours. Table 3 Effects of PEDV S protein on barrier function and viability of IPEC-J2 cells (mean ± SD, n=3)

[0055] As shown in Table 3, the TEER value of the S protein treatment group decreased sharply to 41.3% of the initial value, indicating that the barrier integrity of intestinal epithelial cells was severely disrupted. Simultaneously, the permeability of FITC-glucan significantly increased to 3.5 times that of the control group, confirming the abnormally increased permeability of the bypass pathway. Flow cytometry results showed that the S protein effectively induced apoptosis in IPEC-J2 cells, with a total apoptosis rate as high as 25.3%, significantly higher than the control group (5.1%). Western blot semi-quantitative analysis indicated that S protein treatment significantly downregulated the expression levels of ZO-1 and Occludin, key proteins for maintaining barrier function, to approximately 30% of the control group levels.

[0056] The above experimental results demonstrate that the PEDV S protein expressed by the recombinant CHO cell line provided in Example 1 is itself a key pathogenic factor. It can act directly on the host's intestinal epithelial cells without relying on the intact virus, thereby disrupting the cell's physical barrier function and increasing permeability by inducing apoptosis and degrading tight junction proteins (ZO-1 and Occludin). This mechanism is highly consistent with the core pathological process of PEDV infection leading to intestinal leakage, electrolyte imbalance, and severe diarrhea.

[0057] This discovery not only elucidates the pathogenesis of PEDV, but also proves that the S protein provided by this invention is an ideal tool molecule for studying the pathogenesis of PEDV, screening protective antibodies or blocking drugs, and provides direct experimental evidence and an efficient in vitro model for developing novel intervention strategies against PEDV (such as monoclonal antibody drugs or receptor blockers against the S protein).

[0058] 3. Comparison of immune challenge experiments 3.1 Experimental Design: Forty 4-week-old SPF-grade piglets were randomly divided into 4 groups (10 piglets per group) and immunized using the following method: The vaccine group of this invention: 200 μg of purified S protein (adjuvant is Montanide ISA 206, 1:1 emulsification) was injected intramuscularly, and a booster immunization was given 21 days after the first immunization (second immunization). Commercially available inactivated vaccine group (control A): Tiankang inactivated vaccine (XJ-DB2 strain), administered according to the instructions for dosage and method; Commercially available live attenuated vaccine group (control B): Commercially available live attenuated PEDV vaccine, Enterisol® PEDV (Boehringer Ingelheim), oral immunization (3 mL / animal), first immunization only.

[0059] Negative control group: Intramuscular injection of PBS (containing adjuvant), immunization procedure was the same as that of the vaccine group of the present invention.

[0060] 3.2 Comparison of Immunization Effects Fourteen days after the second immunization (challenge day), all piglets were orally challenged with a virulent PEDV strain (G2 type, titer). Following the viral challenge, patients were observed for 7 consecutive days, with clinical symptoms recorded and samples taken for testing daily. The tested indicators included: (1) Humoral immune response detection: ELISA antibody titer: Indirect ELISA was used. 96-well plates were coated with recombinant PEDV S protein (1 μg / mL), blocked with 1% BSA, and then serum samples diluted 1:100 (collected 14 days after the second immunization) were added and incubated at 37℃ for 1 h. HRP-labeled goat anti-pig IgG (1:5000 dilution) was used as the secondary antibody. After TMB color development, the OD450 value was measured. The titer was calculated by using 2.1 times the OD value of negative serum.

[0061] Neutralizing antibody titer: A micro-neutralization assay was used, where serum samples were serially diluted and then mixed with 100... A mixture of virulent PEDV strains was incubated at 37°C for 1 h, and then inoculated into Vero cells. (cells / well), and CPE was observed after culturing for 72 h. The highest serum dilution that completely inhibited CPE was taken as the neutralizing antibody titer.

[0062] (2) Testing the effectiveness of protection against viral infection: Clinical symptom scoring: After viral challenge, observe the piglets' mental state and fecal characteristics daily, and score them according to the following method: Mental state score (0-2 points) 0 points: Lively, quick-witted, eats / drinks on its own, plays or engages in normal activities.

[0063] 1 point: Depression, reduced activity level, sluggish response to stimuli, and decreased appetite (food intake reduced by 1 / 3 to 1 / 2).

[0064] 2 points: Severe lethargy, unable to get up, refuses to eat / drink, and has no obvious response to stimuli (e.g., no struggle when lifted).

[0065] Stool characteristics score (0-3 points) 0 points: The feces are formed and solid (the hardness is similar to that of normal piglet feces), and the frequency of defecation is normal (1-2 times per day).

[0066] 1 point: Soft stool, pasty (unformed but not fluid), bowel movement frequency slightly increased (3-4 times per day).

[0067] 2 points: Watery stool, liquid (flowing but without force), significantly increased defecation frequency (5-6 times per day), no abnormal components in the stool (such as mucus or blood).

[0068] 3 points: Projectile diarrhea, with feces being expelled in a high-pressure jet (range >10cm), extremely high frequency of defecation (once every 1-2 hours), which may be accompanied by mucus or a small amount of blood, and the perianal area and hind limbs are severely contaminated with feces.

[0069] Total clinical symptom score (0-5 points) = mental status score (0-2 points) + stool characteristics score (0-3 points); At the same time, the diarrhea rate (diarrhea rate = number of piglets with diarrhea / total number of piglets × 100%) and mortality rate (mortality rate = number of piglets that died / total number of piglets × 100%) were recorded. Viral load detection: Piglets were sacrificed 72 h after challenge, jejunal tissue was collected, ground, and RNA was extracted using the TRIzol method. The PEDV N gene was detected by qPCR (primers F: 5'-CGTCTTTGATTGGTTGGTAC-3', R: 5'-TGTTGCCATTACCACGACTC-3'). A standard curve was plotted using standard plasmids, and viral load was calculated. (copies / g).

[0070] Intestinal villus atrophy scoring: Jejunal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with H&E, and the villus morphology was observed under a light microscope. The degree of atrophy was scored according to the following criteria: 1 point: The villi structure is intact, with no atrophy (atrophy rate = 0%). All villi in the field of view are normal in shape, uniform in height, and basically without shortening, breakage or fusion.

[0071] 2 points: Mild atrophy (atrophy rate = 1%-20%). Only a few villi are shortened (length reduced by <1 / 3) or slightly fused, most villi structures remain intact, and crypt depth does not change significantly.

[0072] 3 points: Moderate to mild atrophy (atrophy rate = 21%-40%). Approximately 1 / 3 to 1 / 2 of the villi show significant shortening (length reduced by 1 / 3 to 1 / 2), some villi break at the tip, and the crypts are slightly widened (depth increased by <20%).

[0073] 4 points: Moderate to severe atrophy (atrophy rate = 41%-60%), more than 1 / 2 of the villi are severely shortened (length reduced by more than 1 / 2), widely fused into nodules, crypts are significantly widened (depth increased by 20%-50%), and the mucosal layer is thinned.

[0074] 5 points: Severe atrophy (atrophy rate >60%), most villi have almost completely disappeared (residual length <1 / 3 of normal) or completely fallen off, only a large number of widened crypt structures are seen, the mucosa is significantly thinned, accompanied by inflammatory cell infiltration.

[0075] (3) Histopathological analysis: Vulvar height / crypt depth ratio (V / C): In H&E stained sections, 10 complete vulvar-crypt structures were randomly selected, and the vulvar height (from the vulvar tip to the crypt opening) and crypt depth (from the crypt opening to the bottom) were measured. The average value was calculated and the V / C ratio was statistically analyzed.

[0076] The experimental results are shown in Table 4: Table 4 Comparison of immunization effects of various vaccines (n=10)

[0077] The above experimental results demonstrate that the vaccine of this invention is significantly superior to two commercially available vaccines in terms of humoral immune response, protection against viral challenge, and improvement in histopathology. Specifically: Stronger humoral immune response: 14 days after the second immunization, the ELISA antibody titers (1:13,107) and neutralizing antibody titers (1:682) of the vaccine group of this invention were 3.8 times and 4.1 times higher than those of the commercially available inactivated vaccine (control A), and 2.2 times and 2.1 times higher than those of the commercially available attenuated live vaccine (control B), indicating that it can induce higher levels of specific antibodies.

[0078] Superior protection against viral challenge: 72 hours after challenge, the diarrhea rate (20%), diarrhea index (0.8), and mortality rate (0) in the vaccine group of this invention were significantly lower than those of commercially available inactivated vaccines (80%, 2.6%, 33.3%) and commercially available attenuated vaccines (60%, 1.9%, 10%), and the jejunal viral load (3.4 log) was also significantly lower. 10 (copies / g) compared to control A (6.9 log) 10 The number of copies / g decreased by 3.5 log (approximately 316 times), compared to control B (5.2 log). 10 The number of copies / g decreased by 1.8 log value (approximately 63 times), indicating a stronger virus clearance capability.

[0079] Less severe intestinal tissue damage: The intestinal villus atrophy score (1.2) and V / C ratio (3.2) of the vaccine group of this invention were close to the level of normal piglets, which were significantly better than those of commercially available inactivated vaccines (3.8 points, 1.5 points) and attenuated vaccines (2.5 points, 2.1 points), indicating that it can effectively reduce intestinal pathological damage caused by the virus.

[0080] In summary, the vaccine of this invention, by optimizing protein secretion efficiency and gene stability, comprehensively surpasses commercially available vaccines in terms of immunogenicity and protective efficacy, providing a highly efficient candidate solution for PEDV prevention and control.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for constructing a recombinant cell line against swine diarrhea virus, characterized in that, Includes the following steps: (1) Construction of recombinant plasmids: A chimeric signal peptide was designed and fused with the PEDVS gene and tag sequence to form a fusion gene fragment. The fusion gene fragment was cloned into an expression vector, and a human β-interferon MAR element and a dual resistance marker were simultaneously inserted to obtain a recombinant plasmid. The fusion gene fragment is a nucleotide sequence as shown in SEQ ID NO:2, with BamHI / EcoRI restriction sites introduced at both ends, and cloned into the corresponding restriction sites of the pcDNA3.1 (+) vector. The human β-interferon MAR element was inserted through the XhoI site, and the dual resistance marker consisted of the original G418 resistance gene in the vector and a puromycin resistance gene inserted through the EcoRV site. (2) Cell transfection and screening: The recombinant plasmid was transfected into CHO cells, and after screening with a two-drug gradient, monoclonal cell lines were obtained by limiting dilution. (3) Stability verification and suspension acclimatization: The monoclonal cell line was subjected to passage stability verification, and a three-step suspension acclimatization method was used to obtain a recombinant CHO cell line. In step (1), the chimeric signal peptide is composed of amino acids 1-15 at the N-terminus of the human IgG2a signal peptide, the glycine-serine linker GGGS, and amino acids 16-24 at the C-terminus of the transmembrane protease serine 9 sp9 signal peptide, and its amino acid sequence is shown in SEQ ID NO:

4.

2. The method for constructing a recombinant cell line against swine diarrhea virus as described in claim 1, characterized in that: The PEDV S gene is a gene with a nucleotide sequence as shown in SEQ ID NO:1, which encodes the S protein of the PEDV variant AH2012 / 12; the tag sequence is a 6×His tag.

3. The method for constructing a recombinant cell line against swine diarrhea virus as described in claim 1, characterized in that: The dual-drug gradient pressurization screening was performed as follows: screening was conducted at 1-3 μg / mL G418 and 0.4-0.6 μg / mL puromycin at 24-72 h, and screening was conducted at 4-6 μg / mL G418 and 1-3 μg / mL puromycin at 3-14 days.

4. The method for constructing a recombinant cell line against swine diarrhea virus as described in claim 1, characterized in that: In step (3), the three-step suspension acclimatization method includes: (a) Adhesion-floating transition: Culture in CD FortiCHO™ medium containing 10% fetal bovine serum with shaking for 2-3 days until the viable cell density is ≥ Once cells / mL and viability ≥ 95% are achieved, proceed to the next stage; (b) Microcarrier-assisted: Add 0.08-0.12 g / L Cytodex-3 microcarriers to the culture system, replace with CDFortiCHO™ medium containing 4-6% FBS, and continue culturing for 3-7 days. During this period, ensure cell viability ≥93% and cell density ≥ using trypan blue staining. cells / mL; (c) Serum-free amplification: Replace the medium completely with serum-free CD FortiCHO™ medium and culture in batches, adding 4%-6% volume of concentrated nutrient solution every 22-26 hours. This concentrated nutrient solution contains 7-9 mM glutamine, 0.5-2% insulin-transferrin-selenium supplement, and 0.4-1% Pluronic F-68. Continue culturing until the viable cell density is ≥ A stable suspension culture cell line was obtained by achieving a cell density of 90% / mL and a viability of ≥90%.

5. A recombinant CHO cell line, characterized in that, It is obtained by the method described in any one of claims 1-4.

6. The use of the recombinant CHO cell line as described in claim 5 in the preparation of a subunit vaccine against porcine epidemic diarrhea virus.

7. The application of the recombinant CHO cell line as described in claim 5 in the preparation of a reagent for detecting porcine epidemic diarrhea virus.

8. A PEDV S protein, characterized in that, It is expressed by the recombinant CHO cell line of claim 5, and its amino acid sequence is shown in SEQ ID NO:3.

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