Porcine epidemic diarrhea virus subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses a porcine epidemic diarrhea virus PEDV S mutant protein as well as a preparation method and application thereof. The method comprises the following steps: performing site-specific mutagenesis on PEDV S protein, namely mutating the 17th phenylalanine into alanine, mutating the 1097th phenylalanine into cysteine, mutating the 1263rd threonine into cysteine, removing a C-terminal transmembrane region and an intracellular region, adding a 6His tag, and designing to obtain the PEDV S mutant protein. A CHO cell expression system is utilized, codon optimization, vector construction, monoclonal screening and nickel column purification are carried out, the expression quantity of the mutant protein reaches 1.25-1.53 mg / mL, the purity exceeds 95%, and the formation proportion of a trimer is increased to 85%. Animal experiments show that the mutant protein vaccine can induce efficient humoral immunity and cellular immunity, the mouse neutralizing antibody titer reaches 1: 12750, the diarrhea occurrence rate after piglet challenge is 0%, the fecal virus load is significantly lower than that of wild type protein and commercially available vaccines, and a safe and efficient subunit vaccine scheme is provided for PED prevention and control.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and veterinary vaccines, and in particular to a porcine epidemic diarrhea virus subunit vaccine and a preparation method and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease caused by the porcine epidemic diarrhea virus (PEDV). It primarily infects piglets and is characterized by vomiting, watery diarrhea, and dehydration, with a mortality rate exceeding 90%. In recent years, PED outbreaks have occurred frequently worldwide, causing significant economic losses to the swine industry. According to statistics, the 2013-2014 PED outbreak in the United States resulted in the deaths of approximately 8 million piglets and economic losses exceeding US$900 million.

[0003] PEDV belongs to the genus Alphacoronavirus in the family Coronaviridae. Its genome is a single-stranded positive-strand RNA with a total length of approximately 28 kb, encoding four structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). The S protein is a key protein for viral infection of host cells, responsible for binding to host cell receptors and mediating membrane fusion. It is also the main antigen that induces the production of neutralizing antibodies.

[0004] Currently, PEDV vaccines on the market primarily include inactivated and attenuated vaccines. Inactivated vaccines offer superior safety but exhibit weak immunogenicity, requiring multiple immunizations to achieve optimal protection. Attenuated vaccines offer better efficacy but carry the risk of reversion to virulence and have limited protection against different PEDV genotypes. Furthermore, traditional vaccine preparation is complex, requiring large-scale virus cultivation, posing a biosafety risk.

[0005] Subunit vaccines have become a research hotspot due to their safety, ease of production, and storage. Most existing subunit vaccines are based on the full-length or partial fragments of the PEDV S protein, such as the S1 subunit (amino acids 1-789) or the RBD region (amino acids 319-510). However, these vaccines suffer from limited immunogenicity and insufficient protection against variants.

[0006] Furthermore, existing subunit vaccines have shortcomings in their delivery systems and adjuvant formulations. Traditional aluminum adjuvants primarily induce humoral immunity, with weak stimulation of cellular immunity. Newer adjuvants, such as MF59 and AS03, are expensive and have limited application in veterinary vaccines. Therefore, developing an effective, safe, and variant-specific PEDV subunit vaccine is of great practical significance. Summary of the Invention

[0007] In order to solve the existing technical problems, the present invention provides a porcine epidemic diarrhea virus subunit vaccine and a preparation method and application thereof.

[0008] Therefore, the present application discloses a PEDV S mutant protein design: through the structure and function analysis of PEDV S protein, the present application carries out site-directed mutagenesis: the 17th phenylalanine in the N-terminal signal peptide region is mutated to alanine, the signal peptide cleavage efficiency is optimized, and the protein extracellular secretion is promoted; the 1097th phenylalanine and the 1263rd threonine in the heptad repeat region HR1 and HR2 domain are mutated to cysteine respectively, and the trimer interface stability is enhanced by forming disulfide bond; at the same time, the C-terminal transmembrane region and intracellular region are removed, and 6His tag is added for purification. The amino acid sequence of the mutant protein is shown as SEQ ID NO. 2, and the corresponding codon-optimized nucleotide sequence is shown as SEQ ID NO. 3.

[0009] In one aspect, the present application also discloses a preparation method of PEDV S mutant protein: the optimized mutant protein gene is cloned into pCDNA3.1(+) expression vector, and after transformation of CHO-K1 cells, stable high expression monoclonal cell strain is obtained by G418 screening. After serum-free fermentation culture, when the cell density reaches 7-8x10 6 mg / mL, the protein is purified by nickel column affinity chromatography, and the PEDV S mutant protein with purity of more than 95% and concentration of 6.89 mg / mL is obtained. The preparation method realizes high expression of mutant protein in CHO cells, which can reach 1.25-1.5 mg / mL, and the expression amount fluctuates by not more than 10% after continuous passage of the cell strain for 20 generations.

[0010] In one aspect, the present application also discloses a porcine epidemic diarrhea vaccine composition: the vaccine composition comprises the above-mentioned PEDV S mutant protein, M903 adjuvant or M108L adjuvant. The piglets are immunized by muscle injection, and the immunization is strengthened 2 weeks after the first immunization, which can induce high-efficiency immune response.

[0011] Based on this, the beneficial effects of the present application are:

[0012] 1. The protein expression and purification efficiency are significantly improved: the secretion expression efficiency of recombinant protein is significantly improved by mutation of key sites in signal peptide (F17A), and the expression amount of mutant in CHO cells reaches 1.25-1.53 mg / mL, which is 8-10 times higher than that of wild type. The mutation effectively enhances the extracellular secretion capacity of protein, and more than 95% of high-purity product can be obtained by one-step purification of nickel column, which lays an important foundation for industrial large-scale production.

[0013] 2. The stability of trimer structure is enhanced: the disulfide bond is formed by F1097C and T1263C mutations in HR1 and HR2 regions, so that the trimer proportion is increased from 50% of wild type to 85%, which is closer to the natural virus conformation, and provides a more effective spatial structure basis for antigen presentation.

[0014] 3. Comprehensive enhancement of immune response: The results of mouse immunization experiments showed that the neutralizing antibody titer induced by the mutant protein was 1:12750, which was twice that of the wild-type protein; the levels of IFN-γ and IL-4 secreted by spleen cells were 256±32pg / mL and 187±25pg / mL, respectively, which were 1.64 times and 1.49 times higher than those of the wild type, indicating that it can synergistically activate Th1 / Th2 immune responses.

[0015] 4. The animal protection effect is significant: In the piglet challenge experiment, the diarrhea incidence rate in the mutant protein vaccine group was 0%, and the fecal viral load CT value reached 37.8±1.3 on the 7th day after challenge, which was significantly higher than that of the wild type group (33.1±2.3) and the commercially available inactivated vaccine group (31.0±2.0) (the higher the CT value, the lower the viral load), and there was no vomiting symptom. The protection effect was significantly better than that of the control vaccine.

[0016] 5. Optimization of vaccine preparation process: The use of a CHO cell expression system combined with a codon optimization strategy avoids the biosafety risks of traditional virus culture; the serum-free fermentation and affinity chromatography purification process simplifies the production process, reduces costs, and is suitable for industrial scale-up.

[0017] Therefore, the present invention solves the problem of insufficient immunogenicity of traditional subunit vaccines through reasonable mutation design and efficient expression system. The mutant protein vaccine shows significant advantages in immunogenicity, animal protection effect and production process, providing an innovative technical solution for the prevention and control of porcine epidemic diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of double enzyme digestion of recombinant plasmid were shown, where M: DL10000 marker, 1: double enzyme digestion of recombinant plasmid, band size is about 5.4kb / 4.1kb.

[0019] Figure 2 Western blot identification of the mutant S protein of porcine epidemic diarrhea virus, where M is a marker and 1 is the supernatant collected 48 hours after transfection.

[0020] Figure 3 SDS-PAGE results of porcine epidemic diarrhea virus S mutant protein; M is marker; 1 is S protein.

[0021] Figure 4 SDS-PAGE identification results of wild-type PEDV S protein; M is a marker; 1 is the supernatant of the collected cells. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Preparation and testing of PEDV S mutant protein

[0025] 1. Sequence Analysis and Mutation

[0026] 1. Signal peptide optimization

[0027] Analysis of the amino acid sequence of the PEDV S protein (shown in SEQ ID NO. 1) revealed that the first 18 amino acids at the N-terminus are a signal peptide (positions 1-18), the cleavage efficiency and hydrophobicity of which affect protein secretion.

[0028] Analysis of the sequence revealed that the 17th F in the signal peptide is located in the hydrophobic core region. Replacing it with a less hydrophobic A can optimize the exposure of the signal peptidase cleavage site, promote efficient removal of the signal peptide, reduce the accumulation of immature proteins, and improve extracellular secretion efficiency. Based on this, a mutation was set in the signal peptide to improve the extracellular secretion efficiency of the protein, as shown below:

[0029] Mutation site: phenylalanine (F) at position 17 → alanine (A) (F17A).

[0030] 2. Trimer Optimization

[0031] The heptad repeat regions (HR1 and HR2) of the PEDV S protein are key to trimer formation. The hydrophobic amino acid residues in the HR1 region are crucial for trimer stability. To address this, the F residue at position 1097 in the C-terminus of the HR1 domain was mutated to a C, while the T residue at position 1263 in the N-terminus of the HR2 domain was mutated to a C. This allows these two cysteine ​​residues to form a disulfide bond, thereby enhancing the HR1-HR2 interaction, stabilizing the trimer interface, and increasing the trimer ratio. The details are as follows:

[0032] Mutation site: phenylalanine (F) at position 1097 → cysteine ​​(C) (F1097C);

[0033] Mutation site: Threonine (T) at position 1263 → Cysteine ​​(C) (T1263C);

[0034] The PEDV S protein after the above mutation treatment (the transmembrane region and intracellular region at the C terminal are removed, and 6His is added at the C terminal to facilitate subsequent purification) is marked as PEDV S mutant protein, and the amino acid sequence is as shown in SEQ ID NO. 2.

[0035] II. Preparation of PEDV S mutant protein

[0036] (I) Experimental materials

[0037] 1. Cell line: CHO-K1 cells.

[0038] 2. Vector: pCDNA3.1 expression vector.

[0039] 3. Strain: E. coli DH5a competent cells.

[0040] 4. Tool enzyme: restriction enzymes EcoRI, XhoI, T4 DNA ligase.

[0041] 5. Medium: DMEM / F12 medium (Gibco) containing 10% FBS (Gibco); CD-CHO medium (Gibco), serum-free; screening medium: CD-CHO medium + 800 μg / mL G418 (Sigma)

[0042] 6. Other reagents: Lipofectamine 3000 transfection reagent (Thermo Fisher); PureLink TM HiPlasmid Maxiprep Kit (Thermo Fisher); Trypsin-EDTA solution (Gibco); cell freezing solution: 90% FBS + 10% DMSO (Sigma).

[0043] (II) Experimental steps and results

[0044] 1. Plasmid construction

[0045] According to the PEDV S mutant protein amino acid sequence (SEQ ID NO. 2), the codon optimization (the optimized nucleotide sequence is shown in SEQ ID NO. 3) is carried out, and the primer containing the enzyme cutting site (EcoRI and XhoI enzyme cutting site) is designed.

[0046] The PEDV S mutant protein gene sequence (SEQ ID NO. 3) was used as a template for PCR amplification, and the reaction system was 50 μL, containing 1 μL of template DNA (100 ng / μL), 2 μL of each upstream and downstream primers (10 μM), 25 μL of 2×Phusion Master Mix and 20 μL of ddH2O. The reaction conditions were as follows: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 5 minutes, a total of 35 cycles; 72℃ final extension for 10 minutes. The amplification product was detected by 1% agarose gel electrophoresis, which was consistent with the expected size.

[0047] After the PCR product was purified using the GeneJET PCR Purification Kit, the vector and the target gene were double-digested, respectively. The enzyme digestion system was 50 μL, and the vector enzyme digestion system contained 5 μg of vector, 5 μL of 10×CutSmart Buffer, 2 μL of EcoRI and XhoI; the target gene enzyme digestion system contained 3 μg of PCR product, and the other components were the same. After 37℃ enzyme digestion for 2 hours, 1% agarose gel electrophoresis was used for separation, and the GeneJET Gel Extraction Kit was used to recover about 5.4 kb of the vector fragment and about 4.1 kb of the target gene fragment.

[0048] The enzyme-digested vector and target gene were ligated at a molar ratio of 1:3, and the reaction system was 20 μL, containing 100 ng of vector, 300 ng of target gene, 2 μL of 10×T4 DNA Ligase Buffer and 1 μL of T4 DNA Ligase, 16℃ ligation overnight. 10 μL of the ligation product was used to transform E. coli DH5α competent cells, which were plated on LB plates containing 100 μg / mL of ampicillin and incubated at 37℃ overnight.

[0049] The plasmid was extracted after single colony inoculation and culture, and enzyme digestion and PCR identification were performed. The enzyme digestion identification result showed that the recombinant plasmid was digested by EcoRI / XhoI, and about 5.4 kb of the vector fragment and about 4.1 kb of the target gene fragment were visible on the agarose gel, which was consistent with the expected size. Figure 1 ), and the recombinant plasmid was digested by EcoRI / XhoI, and about 5.4 kb of the vector fragment and about 4.1 kb of the target gene fragment were visible on the agarose gel, which was consistent with the expected size. The positive clone was sent for sequencing verification, and the sequencing result showed that the inserted target gene sequence was completely consistent with the designed PEDV S mutant protein sequence, without base mutation or frame shift.

[0050] The single clone with correct sequencing was inoculated in 500 mL of LB medium containing ampicillin, and after 37℃ shaking culture for 16 hours, the PureLink HiSpeed PCR Purification Kit was used to extract the plasmid. TMHigh-purity plasmid was extracted using the HiPure Plasmid Maxiprep Kit. The concentration was 1.2 μg / μL, and the A260 / A280 ratio was 1.85, indicating good plasmid purity. The plasmid was stored at -20°C until use.

[0051] 2. CHO cell transfection and monoclonal screening

[0052] Remove the cryovial of CHO-K1 cells from liquid nitrogen and quickly thaw in a 37°C water bath. Transfer the cells to a 15 mL centrifuge tube containing 10 mL of DMEM / F12 medium and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in 10 mL of fresh medium, plate them in a T25 culture flask, and culture in a 37°C, 5% CO2 incubator. Change the medium every 2-3 days and passage the cells when they reach 80-90% confluence.

[0053] One day before transfection, CHO-K1 cells were plated at 1×10 5 The density of cells / well was inoculated into 24-well plates, and transfection was performed when the cell confluence reached 70-80%. Each well of the transfection system included: Tube A contained 50μL Opti-MEM medium and 1μg plasmid DNA, and Tube B contained 50μL Opti-MEM medium and 2.5μL Lipofectamine3000. Tube B was added to Tube A, incubated at room temperature for 15 minutes, and then added to the cell culture well and gently mixed. After culturing at 37℃ for 4-6 hours, the medium was replaced with fresh medium. 48 hours after transfection, the expression of the target protein was detected by Western blot, and the results showed ( Figure 2 ) A specific band was observed at approximately 180 kDa, which was consistent with the expected molecular weight of the PEDV S mutant protein (which contained glycosylation modification and had a molecular weight of approximately 150 kDa before glycosylation modification).

[0054] One day before transfection, CHO-K1 cells were plated at 5 × 10 5 Cells were seeded into 6-well plates at a density of 10 cells / well and transfected using optimized transfection conditions. 48 hours after transfection, cells were passaged at a 1:10 ratio into selection medium containing 800 μg / mL G418. Selection medium was changed every 3-4 days for 2-3 weeks until all untransfected cells had died. When resistant clones formed visible colonies, single clones were picked using a cloning ring and transferred to 24-well plates for further culture.

[0055] When the monoclonal cells are cultured to 80-90% confluence, the supernatant is collected for ELISA detection of the expression level of the target protein, and the cells are digested and stored for backup. The ELISA detection results show that among the 48 obtained resistant clones, 12 clones express PEDV S mutant protein, and the expression amount ranges from 50 to 820 μg / mL. The three clones with the highest expression (S8, S14, and S22) are selected for limited dilution purification.

[0056] The high-expression monoclonal cells are digested and diluted to 1 cell / mL with the screening medium, 100 μL (i.e., 0.1 cell / well) is inoculated into each well of a 96-well plate, and it is ensured that there is only a single cell in most wells. After 7-10 days of culture, the wells with single-cell growth are observed and marked under a microscope, the single-cell clones are expanded, and the expression level is detected again. The limited dilution method is repeated 2-3 times, and finally three stable high-expression monoclonal cell strains (S8-3, S14-2, and S22-5) are obtained, with expression amounts of 1250 μg / mL, 1530 μg / mL, and 1470 μg / mL, respectively.

[0057] The monoclonal cell strains are continuously passaged for 20 generations, and the protein expression level is detected every 5 generations. The results show that the expression amounts of the three cell strains remain stable during the passage process, with a fluctuation of no more than 10%. The freeze-thaw recovery experiment shows that the cell strains still maintain good growth state and protein expression level after freeze-thaw recovery.

[0058] 3. Purification of PEDV S mutant protein

[0059] The above-mentioned screened cell strains are inoculated in CD-CHO medium containing 10% FBS and cultured at 37°C, 5% CO2, and a shaking speed of 120 r / min. The FBS concentration is gradually reduced until the cells are completely adapted to the serum-free culture environment. When the cell density is about 7-8 × 105 / mL, the medium is replaced with serum-free CD-CHO medium, and the cells are cultured for 7-10 days. The culture supernatant is collected and centrifuged at 4°C and 3000 r / min for 10 min. The supernatant is collected and stored at -20°C for further use. 6The cell density, cell viability and glucose content were recorded daily, and the glucose content was controlled at 6-8 g / L, and 2% Feed WM2 feed medium was added daily to provide sufficient nutrients for cell growth and protein expression. Fermentation was carried out to the 10th day, and the culture was ended, and the fermentation broth was harvested. The fermentation material was centrifuged at 4°C and 5000 rpm for 20 minutes to remove cells and cell debris, and the supernatant was collected. Nickel column was used for purification, first the nickel column was equilibrated with equilibration buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.9), then the supernatant was loaded, and the impurity proteins were removed by washing with washing buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 7.9), and finally the target protein was eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 7.9). The elution peak was collected, and the protein solution was replaced with PBS buffer using a membrane bag, and finally sterilized through a 220 nm filter membrane, and stored at -80°C for standby.

[0060] The purified PEDV S mutant protein was detected by SDS-PAGE, and the results showed that Figure 3 ), a single bright band appeared at about 180 kDa on the Coomassie blue-stained gel, and the purity of the PEDV S mutant protein was more than 95% by gray scale analysis; the protein content was determined by BCA concentration detection kit, and the result was 6.89 mg / ml.

[0061] 4. Preparation of PEDV S protein

[0062] According to the above method for preparing PEDV S mutant protein, after removing the transmembrane region and intracellular region, PEDV S protein (without mutation treatment, i.e. wild type PEDV S protein) was prepared. Under the same culture conditions and purification process, it was found that the expression amount in CHO cells was generally lower than that of PEDV S mutant protein, and the protein expression amount of the highest expression clone was only 150 μg / mL by ELISA detection, and the purity of the purified protein was about 92%. Figure 4 At the same time, in the trimer formation ratio detection, the same HPLC detection method as the mutant protein was used, and the results showed that the trimer formation ratio was about 50%, which was significantly lower than that of PEDV S mutant protein of 85%. This shows that by optimizing the signal peptide and trimer interface of PEDV S protein, the expression amount of the protein in CHO cells and the trimer formation ratio are significantly improved.

[0063] Example 2: Analysis of immunogenicity of PEDV S mutant protein

[0064] 1. Experimental Materials

[0065] 1. Experimental animals: 6-8 week old SPF-grade BALB / c female mice, weighing 18-22 g.

[0066] 2. Reagents: M903 adjuvant was produced by Hangzhou Yisikang Pharmaceutical Technology Co., Ltd.; anti-PEDV S protein monoclonal antibody was produced by Zhejiang Hongsheng Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG secondary antibody and TMB colorimetric solution were produced by Beijing Solebold Technology Co., Ltd.; 2 M H2SO4 stop solution was produced in-house; mouse IFN-γ ELISA kit was produced by Beijing Solebold Technology Co., Ltd.; mouse IL-4 ELISA kit was produced by Beijing Solebold Technology Co., Ltd.

[0067] 2. Experimental steps and results

[0068] 1. Vaccine Preparation: The PEDV S mutant protein was diluted to 1 mg / mL in PBS and emulsified with an equal volume of M903 adjuvant to prepare the vaccine. After emulsification, the vaccine was dropped into water and diffused in a cloud-like manner. The wild-type PEDV S protein vaccine was prepared using the same method. The adjuvant control vaccine was an emulsification of equal volumes of M903 adjuvant and PBS; the control group was a PBS solution.

[0069] 2. Forty BALB / c mice were randomly divided into four groups, with 10 mice in each group. Each mouse was injected subcutaneously at multiple points on the back with 100 μL of vaccine containing 50 μg protein. The first vaccination was on day 0, and the second vaccination was on day 14.

[0070] 3. Serum collection: Before immunization (day 0), 2 weeks after the first immunization (day 14), and 2 weeks after the second immunization (day 28); blood was collected from the tip of the tail, with approximately 100 μL of blood collected each time. The serum was separated and stored at -20°C.

[0071] 4.ELISA antibody test and results

[0072] Coating: PEDV S protein (wild type) was diluted to 1 μg / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), 100 μL per well, and incubated at 4°C overnight;

[0073] Blocking: 5% BSA-PBS, 200 μL / well, 37°C for 1 hour;

[0074] Sample addition: Serum samples were diluted at a ratio of 1:100, with 3 replicate wells for each dilution, and incubated at 37°C for 1 hour;

[0075] Add secondary antibody: HRP-labeled goat anti-mouse IgG (1:5000), 100 μL / well, 37°C for 1 hour;

[0076] Color development: TMB substrate, 100 μL / well, room temperature in the dark for 15 minutes;

[0077] Stop: 2M H2SO4, 50 μL / well;

[0078] Detection: Read OD with microplate reader 450 nm value.

[0079] Antibody titer determination standard: The highest dilution with an OD value ≥ 2.1 times that of the negative control is the antibody titer.

[0080] The results of ELISA antibody testing are shown in Table 1. Before immunization, the antibody titers of all four groups of mice were below 1:100, indicating that they were in a non-immunized state. Two weeks after the first immunization, the average antibody titer of the PEDV S mutant protein immunization group reached 1:820±65, significantly higher than the 1:380±40 of the wild-type PEDV S protein immunization group (P<0.001). However, the antibody titers of the adjuvant control and PBS control groups remained below 1:100, indicating that the mutant protein can quickly stimulate a strong and relatively stable antibody response after the first immunization. Two weeks after the second immunization, the average antibody titer of the PEDV S mutant protein immunization group increased to 1:12750±850, approximately twice the 1:6300±700 of the wild-type group, demonstrating a typical secondary immune response enhancement effect, and the inter-group differences were extremely significant (P<0.001). The adjuvant control and PBS control groups did not produce an effective antibody response throughout the experiment, further confirming that they were non-immunogenic. Overall, the immunogenicity of the PEDV S mutant protein is significantly stronger than that of the wild-type protein. It can not only efficiently induce mice to produce high-titer antibodies, but also the differences in antibody titers between individuals are relatively small, and the immune effect is more stable.

[0081] Table 1 Antibody detection results

[0082]

[0083] 5. Cytokine detection and results

[0084] Sample preparation: Two weeks after the second immunization, five mice were randomly selected from each group and killed by cervical dislocation, and their spleens were removed aseptically;

[0085] Preparation of single cell suspension: Spleen was ground and passed through a 200-mesh cell sieve. After erythrocyte lysis, the cell concentration was adjusted to 5 × 10 cells / mL using RPMI 1640 complete medium. 6 / mL;

[0086] Stimulation culture: 1 mL of cell suspension was added to a 24-well plate, and PEDV S protein antigen was added at a final concentration of 5 μg / mL. An unstimulated control group was also set up and cultured at 37°C, 5% CO2 for 72 hours.

[0087] Collect the supernatant: Centrifuge at 1200 rpm for 10 minutes, collect the supernatant, and store at -80°C until testing;

[0088] ELISA test: Strictly follow the kit instructions and set up 3 replicate wells for each sample;

[0089] The test results are shown in Table 2. The levels of IFN-γ and IL-4 secreted by mouse spleen cells in the PEDV S mutant protein group were 256±32 pg / mL and 187±25 pg / mL, respectively, compared to 156±24 pg / mL and 125±16 pg / mL, respectively, in the wild-type PEDV S protein group. The IFN-γ and IL-4 levels in the mutant protein group were significantly higher than those in the wild-type group (P<0.01), reaching 1.64-fold and 1.49-fold higher, respectively. The IFN-γ and IL-4 levels in the adjuvant and PBS control groups were significantly lower, at 32±8 pg / mL and 28±6 pg / mL, and 25±7 pg / mL and 22±5 pg / mL, respectively, which were significantly different from those in the mutant protein and wild-type groups (P<0.01). This indicates that the PEDV S mutant protein can more effectively induce Th1 (IFN-γ) and Th2 (IL-4) immune responses, promote the synergistic effect of cellular immunity and humoral immunity, and its immunogenicity is better than that of the wild-type PEDV S protein, while adjuvants and PBS can hardly induce the production of related cytokines.

[0090] Table 2 Cytokine detection results

[0091]

[0092]

[0093] Example 3: Evaluation of the protective effect of PEDV S mutant protein vaccine on animals

[0094] 1. Experimental Materials

[0095] 1. Experimental animals: 2-5 day-old SPF Duroc×Landrace×Large White hybrid piglets.

[0096] 2. Virus strain: Porcine epidemic diarrhea virus JH strain (the latest isolated genotype is a virulent strain of G2c), isolated, identified and preserved by Hangzhou Yisikang Pharmaceutical Technology Co., Ltd., with a virus titer of 10 6.5 TCID 50 / mL.

[0097] 3. Vaccines

[0098] PEDV S mutant protein vaccine: Dilute the PEDV S mutant protein with PBS to 55.5 μg / ml, then mix it with M108L adjuvant (produced by Hangzhou Yisikang Pharmaceutical Technology Co., Ltd.) at a mass ratio of 9:1. After mixing, package it and store it at 2-8°C until use. The target protein content is 50 μg / ml.

[0099] Wild-type PEDV S protein vaccine: Preparation method is the same as that of PEDV S mutant protein vaccine, and the content is the same;

[0100] Commercial inactivated PEDV vaccine: a well-known brand of commercially available vaccine, 2 ml / dose;

[0101] PBS control group: sterile PBS solution.

[0102] 4. Reagents: Viral RNA extraction kit (Beijing Solebold Technology Co., Ltd.) and PEDV fluorescence quantitative PCR detection kit (Guangzhou Weiboxin Biotechnology Co., Ltd.).

[0103] 2. Experimental steps and results

[0104] 1. Grouping and immunization: 25 piglets were randomly divided into 5 groups, with 5 pigs in each group. The grouping is as follows:

[0105] Group 1: PEDV S mutant protein vaccine group;

[0106] Group 2: wild-type PEDV S protein vaccine group;

[0107] Group 3: commercial inactivated vaccine group;

[0108] Group 4: adjuvant control group (PBS + M108L adjuvant);

[0109] Group 5: PBS control group

[0110] 2. Immunization method: intramuscular injection, 1 mL per piglet, second vaccination 2 weeks after the first vaccination.

[0111] 3. Serological testing and results

[0112] Blood collection time points: before immunization (day 0), 2 weeks after the first immunization (before the second immunization), and 1 week after the second immunization (day 21);

[0113] Detection method: Detection is carried out according to the neutralizing antibody detection method;

[0114] The test results are shown in Table 3. Before immunization, the neutralizing antibody levels of pigs in each group were only negative (≤1:4), and there were no significant differences between the groups. Two weeks after the first immunization, the neutralizing antibody titer of the PEDV S mutant protein group increased significantly, higher than the wild-type PEDV S protein group and the commercial inactivated vaccine group, while the adjuvant control group and the PBS control group remained negative. One week after the second immunization, the neutralizing antibody titer of the PEDV S mutant protein group further increased significantly and remained at a high level. Although the wild-type PEDV S protein group increased, the increase was small, the commercial inactivated vaccine group did not change much, and the adjuvant control group and the PBS control group did not have an effective immune response. Overall, the PEDV S mutant protein group had the strongest ability to induce pigs to produce neutralizing antibodies after immunization, which was significantly better than the other experimental groups. The adjuvant control group and the PBS control group had no immune effect.

[0115] Table 3 Results of porcine epidemic diarrhea virus neutralizing antibody test

[0116]

[0117] 4. Virus challenge experiment

[0118] Time of challenge: 1 week after the second vaccination (21 days);

[0119] Challenge dose: Oral inoculation of 10 6.0 TCID 50 / mL of porcine epidemic diarrhea virus JH strain, inoculation volume 10mL;

[0120] Observation period: Observe continuously for 7 days after the challenge, and record the following indicators every day:

[0121] Clinical symptoms: diarrhea score (0-4 points), vomiting, and mental state (1-3 points);

[0122] Fecal shedding: Fecal samples are collected daily to test for viral RNA levels.

[0123] The results of clinical symptom observation are shown in Table 4: In this experimental result, the PEDV S mutant protein group performed extremely well, with the incidence of diarrhea and vomiting both being 0% (0 / 5), the average number of diarrhea days and the highest diarrhea score both being 0, which means that the pigs in this group did not show any diarrhea or vomiting symptoms; the wild-type PEDV The S protein group had a diarrhea incidence of 20% (1 / 5), an average diarrhea duration of 0.6 days, a maximum diarrhea score of 3.0, and a vomiting incidence of 20% (1 / 5). The commercial inactivated vaccine group had a diarrhea incidence of 40% (2 / 5), an average diarrhea duration of 1.4 days, a maximum diarrhea score of 3.0, and a vomiting incidence of 20% (1 / 5). The situation in the adjuvant control group and the PBS control group was not optimistic, with both having a diarrhea incidence of 100% (5 / 5). The adjuvant control group had an average diarrhea duration of 4.6 days, a maximum diarrhea score of 4.0, and a vomiting incidence of 60% (3 / 5). The PBS control group had an average diarrhea duration of 5.2 days, a maximum diarrhea score of 4.0, and a vomiting incidence of 80% (4 / 5). It is clear that the PEDV S mutant protein group has a significantly stronger protective effect on pigs than the other groups, can effectively resist the occurrence of related symptoms, and shows a significant advantage in preventing diarrhea and vomiting.

[0124] Table 4 Statistics of clinical observation results after challenge

[0125] Group Incidence of diarrhea Average number of days with diarrhea Maximum diarrhea score Incidence of vomiting PEDV S mutant protein group 0％(0 / 5) 0 0 0％ Wild-type PEDV S protein group 20％(1 / 5) 0.6 3.0 20％(1 / 5) Commercial inactivated vaccine group 40％(2 / 5) 1.4 3.0 20％(1 / 5) Adjuvant control group 100％(5 / 5) 4.6 4.0 60％(3 / 5) PBS control group 100％(5 / 5) 5.2 4.0 80％(4 / 5)

[0126] The results of fecal viral load detection are shown in Table 5: After infection, the changes in CT values ​​corresponding to fecal viral loads in each group showed significant differences. The CT value of the PEDV S mutant protein group was 32.6±1.7 on the first day after infection, which was at a relatively high level, indicating that the viral load was low. As time went on, the CT value gradually increased, reaching 37.8±1.3 on the 7th day after infection, indicating that the viral load continued to decline; the wild-type PEDV The CT value of the S protein group was 31.8±2.3 on day 1 after challenge, dropping to 26.0±2.0 on day 3, and fluctuating thereafter, reaching 33.1±2.3 on day 7. The CT value of the commercial inactivated vaccine group was 31.2±2.6 on day 1 after challenge, significantly decreasing to 24.0±1.7 on day 3, with a slight rebound later. The CT values ​​of the adjuvant control and PBS control groups were generally lower, at 30.6±2.0 and 31.9±1.7, respectively, on day 1 after challenge, and remained low thereafter. The CT values ​​of the adjuvant control group and the PBS control group were 24.6±2.6 and 23.5±2.2 on day 7 after challenge, indicating that the viral load in both groups remained high. Overall, the PEDV S mutant protein group was significantly more effective than the other groups in resisting viral proliferation and reducing viral load, demonstrating stronger immune protection.

[0127] Table 5 Fecal viral load test results after challenge (CT value)

[0128]

[0129] It should be noted that the present invention also used the original PEDV ZJ / 15 strain (a strain with genotype G2a isolated in 2015 and maintained by Zhejiang Hongsheng Biotechnology Co., Ltd.) for challenge. The challenge results showed that the protection rate of the PEDV S mutant protein group was still 100%, consistent with the results in Table 5. This shows that the PEDV S mutant protein prepared in the present invention has good protection against both the currently prevalent G2a and G2c genotypes.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A porcine epidemic diarrhea virus (PEDV) S mutant protein, characterized in that: The mutant protein comprises the following amino acid mutations: phenylalanine at position 17 is mutated to alanine, phenylalanine at position 1097 is mutated to cysteine, threonine at position 1263 is mutated to cysteine, and the transmembrane region and intracellular region are removed from the C-terminus and a 6His tag is added.

2. The PEDV S mutant protein according to claim 1, characterized in that The amino acid sequence of the PEDV S mutant protein is shown in SEQ ID NO.

2.

3. The PEDV S mutant protein according to claim 1, characterized in that The nucleotide sequence of the codon-optimized PEDV S mutant protein is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that: The vector comprises the nucleotide sequence according to claim 3, and the vector is a pCDNA3.1 expression vector.

5. A recombinant cell line, characterized in that The cell line contains the recombinant expression vector according to claim 4, and the cell line is a CHO-K1 cell.

6. A method for preparing a PEDV S mutant protein, characterized in that: The method comprises the following steps: (1) cloning the nucleotide sequence of claim 3 into an expression vector and transforming the host cell; (2) Screening positive clones and inducing expression; (3) PEDV S mutant protein was obtained by nickel column affinity chromatography purification.

7. The preparation method according to claim 6, characterized in that The host cell is a CHO-K1 cell.

8. A porcine epidemic diarrhea vaccine composition, characterized in that: The composition comprises the PEDV S mutant protein according to claim 1 or 2 and a pharmaceutically acceptable adjuvant.

9. The vaccine composition according to claim 8, characterized in that The adjuvant is M903 adjuvant or M108L adjuvant.

10. Use of the PEDV S mutant protein according to claim 1 or 2 in the preparation of porcine epidemic diarrhea virus vaccine.

Images