Porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses a porcine epidemic diarrhea virus PEDV S mutant protein, a porcine rotavirus PoRV VP4 optimized protein and a preparation method of a bivalent subunit vaccine of the porcine epidemic diarrhea virus PEDV S mutant protein and the porcine rotavirus PoRV VP4 optimized protein. Aiming at PEDV S protein, 17th phenylalanine of a signal peptide is mutated into alanine, 1097th phenylalanine of HR1 and 1263rd threonine of HR2 in a heptapeptide repeat region are respectively mutated into cysteine to form a disulfide bond, the protein expression quantity and the trimer proportion are obviously improved, the expression quantity in CHO cells reaches 1530mu g / mL, and the trimer proportion reaches 85%. For PoRV VP4 protein, a flexible peptide GSSG is inserted in front of a C-terminal 6 * His tag, the problem that a nickel column cannot be combined due to C-terminal negative charge interference is solved, and efficient purification is achieved. The bivalent vaccine is prepared by equivalently mixing the two proteins and adding an M108L adjuvant, animal experiments show that the bivalent vaccine can induce piglets to generate high-titer neutralizing antibodies, the protection rates for PEDV and PoRV challenge both reach 100%, and the bivalent vaccine is good in safety. The invention provides an efficient and safe novel vaccine for prevention and control of porcine viral diarrhea, and the application value is remarkable.

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-porcine rotavirus bivalent subunit vaccine and a preparation method and application thereof. Background Art

[0002] Porcine viral diarrhea, a highly contagious disease affecting the swine industry, spreads rapidly and widely, posing a serious threat to pig health and the sustainable development of the industry. Statistics show that diarrhea-related mortality accounts for 39.8% of all piglet deaths in my country. Failure to effectively prevent and control the disease will not only severely damage the livestock industry economy but also pose a potential risk to public health. Clinically, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine transmissible gastroenteritis virus (TGEV) are the primary pathogens causing the disease. These can infect pigs of all ages, presenting with typical symptoms such as watery diarrhea, vomiting, dehydration, and abnormal body temperature. Co-infection with two or more viruses is common, significantly increasing the difficulty of disease prevention and control. Co-infection with PEDV and PoRV is the most prominent in the current epidemic situation in my country.

[0003] In recent years, genetic variation has driven significant evolution in the epidemiological characteristics of porcine diarrheal viruses. Monitoring of 2,987 diarrheal samples from five southern provinces in my country from 2012 to 2018 revealed that PEDV was the predominant strain, with a detection rate of 50.21%-62.10% and a prevalence rate of 96.43% on pig farms. PDCoV was second, with a detection rate of 19.62%-29.19% and a prevalence rate of 70.24%. TGEV and PoRV both had detection rates below 3%. A 2018-2021 survey of 7,107 samples across eight provinces revealed that PEDV maintained the highest average positive rate (56.09%), while the PoRV positive rate showed an increasing trend, reaching 10.45% in 2021. PEDV-PoRV co-infections accounted for 77.6% of all co-infection cases. Analysis of 1,791 samples from five provinces in South China from 2021 to 2023 further confirmed that the PEDV detection rate remained at 47.40%-52.22%, while the PoRV detection rate increased to 25.81%-50.81%, with G9 being the predominant strain. The prevalence of mixed infections of the two strains increased annually from 4.64% to 9.87%, with PEDV / PDCoV / PoRV triple infections occurring (average positivity rate 1.90%). These data suggest that PEDV monoinfection remains the primary cause, PDCoV prevalence is slowing, but PoRV and PEDV-PoRV mixed infections have shown a significant upward trend. This is consistent with findings that mixed infections dominated the 2015-2016 piglet diarrhea outbreak in Northeast my country.

[0004] Porcine epidemic diarrhea (PED) caused by PEDV has no obvious seasonality and is most prevalent in cold seasons. It has acute onset and is highly contagious through contact. The mortality rate of infected piglets within one week of age is as high as 100%, and the virus can survive in manure storage tanks for four months, leading to repeated outbreaks of PED on pig farms. The virus was first discovered in 1971 and successfully isolated in 1978. my country first reported a case of the virus in 1973 and completed its identification in 1984. A highly pathogenic PED outbreak in South China in 2010 quickly spread throughout the country, causing nearly 100% mortality in piglets and affecting 29 provinces and regions. A similar outbreak also occurred in North America from 2013 to 2014, killing nearly 7 million piglets. Although PEDV has only one serotype, it can be divided into GⅠ and GⅡ types based on the evolution of the S gene. Before 2010, the prevalent strains in my country were all GⅠ type, while the current dominant prevalent strains have evolved into GⅡ type, which has reduced the protective efficacy of traditional vaccines prepared based on the classic strain CV777. The development of new vaccines is imminent.

[0005] PEDV, a member of the coronavirus genus, is a single-stranded, positive-sense RNA virus with a diameter of 95-190 nm and a 28kb genome. It encodes four structural proteins, S, N, E, and M, and the ORF3 accessory protein, along with 16 non-structural proteins (nsp1-nsp16). The S protein, a transmembrane glycoprotein distributed as a trimer on the viral surface, contains multiple neutralizing epitopes that mediate viral attachment and host cell invasion, making it a core target for the development of subunit and live attenuated vaccines.

[0006] PoRV belongs to the genus Rotavirus and is an important pathogen of intestinal diarrhea in young animals. It is transmitted through the fecal-oral route and is most prevalent in winter and spring. Piglets aged 2-8 weeks are susceptible, while adult pigs are mostly latently infected. Group A rotavirus (PoRVA) is the most prevalent and has the risk of cross-species transmission. It has been confirmed that it can be transmitted between pigs and humans, posing a threat to public health. The virus is divided into G (24 types) and P (33 types). Common types in pigs are G3, G4, G5, G9, G11 and P

[13] , P

[19] , and P

[23] . In Asia, the prevalence of RVA is mainly G5, G3, G9 and P[7] and P[6]. In recent years, the detection rate of G9 type PoRV in my country has increased significantly. The existing vaccines are mainly targeted at G5 type, and the pressure of prevention and control is prominent. The VP4 protein of PoRV, as the outer capsid spike protein, has key biological functions: it is cleaved by trypsin into VP8 (which mediates host cell adsorption) and VP5 (involved in viral penetration), and it also regulates plaque formation and viral virulence. Studies have demonstrated that recombinantly expressed VP4 protein has excellent antigenicity and immunogenicity. A bivalent subunit vaccine prepared with it can induce high-titer neutralizing antibodies in mice. After immunization of sows, slgA antibodies are transferred through colostrum, effectively reducing intestinal lesions in piglets. Therefore, it has been selected as a vaccine target antigen.

[0007] Based on the above research, the present research selects PEDV S protein and PoRV VP4 protein to construct a double subunit vaccine. Among them, the trimer conformation of the S protein has strong immunogenicity, and a mammalian cell expression system needs to be used to maintain its natural structure; the VP4 protein selects an E. coli expression system, which realizes large-scale production by using the advantages of high efficiency and low cost. SUMMARY

[0008] The present application solves the problems of low expression, difficult purification and insufficient immunogenicity in the prior art by optimizing the structure of PEDV S protein and PoRV VP4 protein, and the specific technical solutions are as follows:

[0009] In one aspect, the present application discloses a design of PEDV S mutant protein: through the analysis of the structure and function of PEDV S protein, the present application performs 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 extracellular secretion of the protein is promoted; the 1097th phenylalanine and the 1263rd threonine in the heptad repeat region HR1 and HR2 domains are mutated to cysteine, respectively, to enhance the stability of the trimer interface by forming disulfide bonds; at the same time, the C-terminal transmembrane region and intracellular region are removed, and a 6×His tag is added for purification. The amino acid sequence of the mutated protein is shown as SEQ ID NO. 2, and the corresponding codon-optimized nucleotide sequence is shown as SEQ ID NO. 3. The present application also discloses a preparation method of PEDV S mutant protein: the optimized mutant protein gene is cloned into a pCDNA3.1 expression vector, and after transformation of CHO-K1 cells, a stable high-expression monoclonal cell strain is obtained by G418 screening. After serum-free fermentation culture, when the cell density reaches 7-8×10 6 mg / mL, the protein is purified by nickel column affinity chromatography to obtain PEDV S mutant protein with a purity of more than 95% and a concentration of 6.89 mg / mL. The preparation method realizes high expression of the mutant protein in CHO cells, which can reach 1.25-1.5 mg / mL, and the expression amount fluctuates by no more than 10% after continuous passage of the cell strain for 20 generations.

[0010] The present application also discloses a design and preparation process of PoRV VP4 optimized protein: the VP4 protein of PoRV G9P23 type (the original sequence is shown as SEQ ID NO. 4) is selected, because the C-terminal contains continuous acid residues (the last 1-2 positions are negatively charged aspartic acid D), and the C-terminal 6×His tag cannot be combined with the nickel column due to steric hindrance and charge repulsion, so a flexible peptide GSSG is inserted between the C-terminal of the VP4 protein and the 6×His tag, which can be combined with the nickel column through the flexible peptide GSSG, and the C-terminal of the VP4 protein is removed to avoid the steric hindrance and charge repulsion of the C-terminal 6×His tag. The negatively charged region was isolated by a spatial distance to eliminate charge interference, so that the His tag was efficiently exposed. The optimized sequence is shown in SEQ ID NO.5. In terms of expression and purification process, the optimized VP4 gene was inserted into the NcoI / XhoI restriction site of the pET28a vector to construct the recombinant vector pET28a-VP4opt. After transformation of E. coli BL21 (DE3) competent cells, the cells were cultured at 37°C until OD600 = 0.6 and 0.5 mM IPTG was added for induction for 4 hours. The protein mainly existed in the form of inclusion bodies. After the bacteria were ultrasonically disrupted, the precipitate was renatured in renaturation buffer (50 mM Tris-HCl + 300 mM NaCl + 4 M urea, pH 8.0) and then purified using a nickel column (equilibrium buffer: 50 mM Tris-HCl + 300 mM NaCl + 20 mM imidazole + 4 M urea, pH 8.0; elution buffer: 50 mM Tris-HCl + 300 mM NaCl+300mM imidazole+4M urea, pH8.0), the column hanging rate increased from 0 to more than 95%. The eluate was filtered through a membrane exchange solution to remove urea and imidazole, and sterilized through a 220nm filter membrane. The purity reached more than 95% and the concentration was 1.253mg / mL.

[0011] The present invention also discloses a porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine and a preparation method thereof: the vaccine comprises an effective amount of a PEDV S mutant protein, an effective amount of a PoRV VP4 optimized protein, and an adjuvant; the concentrations of the PEDV S mutant protein and the PoRV VP4 optimized protein are both 50 μg / mL; the adjuvant is M108L, and the mass ratio of the antigen solution to the adjuvant is 9:1.

[0012] The PEDV S mutant protein of the present invention increases its secretion efficiency in CHO cells by 9.2 times (from 150 μg / mL to 1530 μg / mL) through signal peptide optimization, solving the problem of low native protein expression. Disulfide bond-mediated trimer stabilization increases the trimer ratio from 50% to 85%, retaining more neutralizing antigen epitopes and significantly enhancing immunogenicity (the antibody titer in mice after secondary immunization reached 1:12750, twice that of the wild type). The PoRV VP4 protein completely resolves the problem of negative charge interference at the C-terminus by inserting the flexible peptide GSSG, increasing the nickel column attachment rate from 0 to over 95%, improving purification efficiency by 40 times, and maintaining intact antigenicity of the purified protein (specifically binding to PoRV-positive serum, with clear Western blot bands). The bivalent vaccine prepared in this way is highly effective and safe: the protection rate against PEDV G2a, G2c and PoRV G9P23 types reaches 100%, the piglets have no diarrhea or vomiting symptoms after infection, and the fecal viral load is significantly reduced; it can simultaneously induce high-titer neutralizing antibodies (PEDV ≥ 1:64, PoRV ≥ 1:64) and Th1 / Th2 cytokines (IFN-γ 256pg / mL, IL-4 187pg / mL), synergistically enhancing humoral immunity and cellular immunity; 3-5 day old piglets had no abnormalities in body temperature, spirit and injection site within 14 days after being vaccinated with a double dose, with excellent safety, and the CHO cell serum-free fermentation and Escherichia coli prokaryotic expression processes are mature, and the large-scale production cost is reduced by more than 30% compared with inactivated vaccines, making it suitable for industrial production.

[0013] In summary, the present invention provides a new type of vaccine that is efficient, safe, and low-cost for the prevention and control of porcine viral diarrhea (especially mixed infection of PEDV and PoRV), and has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] 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.

[0016] Figure 3 SDS-PAGE results of porcine epidemic diarrhea virus S mutant protein; M is marker; 1 is S protein.

[0017] Figure 4 SDS-PAGE identification results of wild-type PEDV S protein; M is a marker; 1 is the supernatant of the collected cells.

[0018] Figure 5 Detection of optimized protein expression of PoRV VP4; M is a marker, 1 is the whole bacteria before induction, 2 is the whole bacteria after induction, 3 is the supernatant after bacterial cell crushing, and 4 is the precipitate after bacterial cell crushing.

[0019] Figure 6 SDS-PAGE and western blot detection results of PoRV VP4 optimized protein, where 1 is the PoRV VP4 optimized protein. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] Example 1: Preparation and testing of PEDV S mutant protein

[0023] 1. Sequence Analysis and Mutation

[0024] 1. Signal peptide optimization

[0025] 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.

[0026] 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:

[0027] Mutation site: phenylalanine (F) at position 17 → alanine (A) (F17A).

[0028] 2. Trimer Optimization

[0029] The heptad repeat regions (HR1 and HR2) of PEDV S protein are the key regions for forming the trimer. The hydrophobic amino acid residues of the HR1 region are essential for the stability of the trimer. Based on this, the F at position 1097 at the C-terminal of the HR1 domain is mutated to C, and the T at position 1263 at the N-terminal of the HR2 domain is mutated to C, so that the two cysteine residues can form a disulfide bond, thereby enhancing the HR1-HR2 interaction, stabilizing the trimer interface, and increasing the proportion of the trimer. Specifically as follows:

[0030] Mutant site: phenylalanine (F) at position 1097 to cysteine (C) (F1097C);

[0031] Mutant site: threonine (T) at position 1263 to cysteine (C) (T1263C);

[0032] The PEDV S protein after the above mutation treatment (remove the transmembrane region and intracellular region at the C-terminal, and add 6His at the C-terminal for subsequent purification) is marked as PEDV S mutant protein, and its amino acid sequence is shown in SEQ ID NO. 2.

[0033] II. Preparation of PEDV S mutant protein

[0034] (I) Experimental materials

[0035] 1. Cell line: CHO-K1 cells.

[0036] 2. Vector: pCDNA3.1 expression vector.

[0037] 3. Strain: E. coli DH5α competent cells.

[0038] 4. Tool enzyme: restriction enzymes EcoRI, XhoI, T4 DNA ligase.

[0039] 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)

[0040] 6. Other reagents: Lipofectamine 3000 transfection reagent (Thermo Fisher); PureLink TM HiPure Plasmid Maxiprep Kit (Thermo Fisher); Trypsin-EDTA solution (Gibco); Cell freezing solution: 90% FBS + 10% DMSO (Sigma).

[0041] (2) Experimental steps and results

[0042] 1. Plasmid Construction

[0043] According to the amino acid sequence of the PEDV S mutant protein (SEQ ID NO.2), codon optimization was performed (the optimized nucleotide sequence is shown in SEQ ID NO.3), and primers containing restriction enzyme sites (EcoRI and XhoI restriction enzyme sites) were designed.

[0044] PCR amplification was performed using the PEDV S mutant protein gene sequence (SEQ ID NO. 3) as a template. The reaction volume was 50 μL, containing 1 μL template DNA (100 ng / μL), 2 μL of upstream and downstream primers (10 μM each), 25 μL of 2× Phusion Master Mix, and 20 μL of ddH2O. Reaction conditions were: initial denaturation at 98°C for 30 seconds, followed by 35 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 5 minutes, followed by a final extension at 72°C for 10 minutes. The amplified product was confirmed by 1% agarose gel electrophoresis and was consistent with the expected size.

[0045] After PCR product purification using the GeneJET PCR Purification Kit, the vector and target gene were double-digested. Each digestion system consisted of 50 μL of the vector digestion system, containing 5 μg of vector, 5 μL of 10× CutSmart Buffer, and 2 μL each of EcoRI and XhoI. The target gene digestion system consisted of 3 μg of PCR product, with all other components being identical. After digestion at 37°C for 2 hours, the vector fragment (approximately 5.4 kb) and the target gene fragment (approximately 4.1 kb) were recovered by 1% agarose gel electrophoresis using the GeneJET Gel Extraction Kit.

[0046] Ligate the digested vector and target gene at a 1:3 molar ratio using a 20 μL reaction system 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. Ligate overnight at 16°C. Transform 10 μL of the ligation product into competent E. coli DH5α cells, spread onto LB plates containing 100 μg / mL ampicillin, and incubate at 37°C overnight.

[0047] After single colony was picked and inoculated, plasmid was extracted and then subjected to enzyme digestion and PCR identification. Figure 1). After double digestion of the recombinant plasmid with EcoRI / XhoI, an approximately 5.4 kb vector fragment and an approximately 4.1 kb target gene fragment were observed on an agarose gel, consistent with the expected size. Positive clones were sequenced for verification, and sequencing results confirmed that the inserted target gene sequence was identical to the designed PEDV S mutant protein sequence, with no base mutations or frameshifts.

[0048] The correct single clone was inoculated into 500 mL of LB medium containing ampicillin and cultured at 37°C for 16 hours. TM High-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.

[0049] 2. CHO cell transfection and monoclonal screening

[0050] 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.

[0051] One day before transfection, CHO-K1 cells were plated at 1×10 5 Cells were seeded in a 24-well plate at a density of 100 cells / well and transfected when the cell confluence reached 70-80%. Each well of the transfection system included: Tube A contained 50 μL Opti-MEM medium and 1 μg plasmid DNA, and Tube B contained 50 μL Opti-MEM medium and 2.5 μL Lipofectamine 3000. 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°C 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 that ( 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).

[0052] One day before transfection, CHO-K1 cells were plated at 5 × 10 5Cells 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.

[0053] When monoclonal cells reached 80-90% confluence, the supernatant was collected for ELISA analysis of target protein expression. The cells were then digested and frozen for backup. ELISA results showed that 12 of the 48 resistant clones expressed the PEDV S mutant protein, with expression levels ranging from 50 to 820 μg / mL. The three clones with the highest expression levels (S8, S14, and S22) were selected for limiting dilution purification.

[0054] After digestion of the high-expressing monoclonal cells, dilute them to 1 cell / mL with screening medium and inoculate 100 μL per well (i.e., 0.1 cell / well) into a 96-well plate, ensuring that most wells contain only single cells. After 7-10 days of culture, observe and mark the wells where single cells have grown under a microscope, expand the single cell clones, and re-test the expression level. Repeat the limiting dilution method 2-3 times to obtain three stable, high-expressing monoclonal cell lines (S8-3, S14-2, and S22-5), with expression levels of 1250 μg / mL, 1530 μg / mL, and 1470 μg / mL, respectively.

[0055] Monoclonal cell lines were serially passaged for 20 generations, with protein expression levels measured every five generations. The results showed that expression levels in all three cell lines remained stable during the passage process, with fluctuations of no more than 10%. Cryopreservation and resuscitation experiments demonstrated that the cell lines maintained good growth and protein expression levels after cryopreservation and resuscitation.

[0056] 3. Purification of PEDV S mutant protein

[0057] The selected cell lines were inoculated into CD-CHO medium containing 10% FBS and cultured in a shaker at 37°C, 5% CO2, and 120 rpm. The FBS concentration was gradually reduced until the cells were fully adapted to the serum-free culture environment. 6Fermentation begins when the cell count reaches 90% cells / ml and the viability is greater than 90%, marking Day 0. Adjust the shaker temperature to 33°C and the rotation speed to 80 rpm, conditions that are conducive to protein expression. Record cell density, cell viability, and glucose content daily, maintaining the glucose content at 6-8 g / L. Feed the medium daily with 2% Feed WM2 to provide adequate nutrients for cell growth and protein expression. Fermentation ends on Day 10, and the fermentation broth is harvested. Centrifuge the fermentation medium at 4°C and 5000 rpm for 20 minutes to remove cells and cell debris, and collect the supernatant. Purification was performed using a nickel column. First, the nickel column was equilibrated with an equilibration buffer (20mMTris-HCl, 500mM NaCl, 20mM imidazole, pH 7.9). The supernatant was then loaded and washed with a wash buffer (20mM Tris-HCl, 500mM NaCl, 50mM imidazole, pH 7.9) to remove contaminants. Finally, the target protein was eluted with an elution buffer (20mM Tris-HCl, 500mM NaCl, 250mM imidazole, pH 7.9). The elution peak was collected and the membrane was used for buffer exchange. The protein solution was replaced with PBS buffer and then sterilized by passing through a 220nm filter membrane. The cells were then stored at -80°C in separate devices until ready for use.

[0058] The purified PEDV S mutant protein was detected by SDS-PAGE, and the results showed that ( Figure 3 ), only a single bright band appeared at approximately 180 kDa on the Coomassie brilliant blue-stained gel. Grayscale analysis showed that the purity of the PEDV S mutant protein was above 95%. The protein content was determined using a BCA concentration detection kit, and the result was 6.89 mg / ml.

[0059] 4. Preparation of PEDV S Protein

[0060] According to the above method for preparing PEDV S mutant protein, after removing the transmembrane region and intracellular region, PEDV S protein (not subjected to mutation treatment, i.e., wild-type PEDV S protein) was prepared. Under the same culture conditions and purification process, it was found that its expression level in CHO cells was generally lower than that of PEDV S mutant protein. ELISA detection showed that the protein expression level of the highest expressing clone was only 150 μg / mL, and the purity after purification was ( Figure 4 ) was approximately 92%. Furthermore, the trimer formation ratio was detected using the same HPLC method as for the mutant protein, and the results showed that the trimer formation ratio was approximately 50%, significantly lower than the 85% for the PEDV S mutant protein. This indicates that signal peptide optimization and trimer interface optimization mutations of the PEDV S protein significantly increased protein expression and trimer formation ratio in CHO cells.

[0061] Example 2: Analysis of immunogenicity of PEDV S mutant protein

[0062] 1. Experimental Materials

[0063] 1. Experimental animals: 6-8 week old SPF-grade BALB / c female mice, weighing 18-22 g.

[0064] 2. Reagents: M903 adjuvant, produced by Hangzhou Yisikang Pharmaceutical Technology Co., Ltd.; anti-PEDV S protein monoclonal antibody (produced by Zhejiang Hongsheng Biotechnology Co., Ltd.); HRP-labeled goat anti-mouse IgG secondary antibody and TMB colorimetric solution (produced by Beijing Solebao Technology Co., Ltd.); 2M H2SO4 stop solution (prepared in-house);

[0065] Mouse IFN-γ ELISA detection kit (Beijing Solebold Technology Co., Ltd.); mouse IL-4 ELISA detection kit (Beijing Solebold Technology Co., Ltd.).

[0066] 2. Experimental steps and results

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 4.ELISA antibody test and results

[0071] 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;

[0072] Blocking: 5% BSA-PBS, 200 μL / well, 37°C for 1 hour;

[0073] 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;

[0074] Add secondary antibody: HRP-labeled goat anti-mouse IgG (1:5000), 100 μL / well, 37°C for 1 hour;

[0075] Color development: TMB substrate, 100 μL / well, room temperature in the dark for 15 minutes;

[0076] Stop: 2M H2SO4, 50 μL / well;

[0077] Detection: Read OD with microplate reader 450 nm value.

[0078] Antibody titer determination standard: The highest dilution with an OD value ≥ 2.1 times that of the negative control is the antibody titer.

[0079] 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.

[0080] Table 1 Antibody detection results

[0081]

[0082] 5. Cytokine detection and results

[0083] 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;

[0084] 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;

[0085] 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.

[0086] Collect the supernatant: Centrifuge at 1200 rpm for 10 minutes, collect the supernatant, and store at -80°C until testing;

[0087] ELISA test: Strictly follow the kit instructions and set up 3 replicate wells for each sample;

[0088] 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.

[0089] Table 2 Cytokine detection results

[0090] Group IFN-γ (pg / mL) IL-4 (pg / mL) PEDV S mutant protein group 256±32 187±25 PEDV S protein group 156±24 125±16 Adjuvant control group 32±8 28±6 PBS control group 25±7 22±5

[0091] Example 3: Evaluation of the protective effect of PEDV S mutant protein vaccine on animals

[0092] 1. Experimental Materials

[0093] 1. Experimental animals: 2-5 day-old SPF Duroc×Landrace×Large White hybrid piglets.

[0094] 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.

[0095] 3. Vaccines

[0096] PEDV S mutant protein vaccine: the PEDV S mutant protein was diluted to 55.5 μg / ml with PBS, and then mixed with M108L adjuvant (produced by Hangzhou Yiskang Pharmaceutical Technology Co., Ltd.) at a mass ratio of 9:1. After mixing, it was divided and stored at 2-8°C for standby, and the content of the target protein was 50 μg / ml.

[0097] Wild-type PEDV S protein vaccine: the preparation method was the same as that of the PEDV S mutant protein vaccine, and the content was consistent.

[0098] Commercial PEDV inactivated vaccine: a certain well-known brand of commercially available vaccine, 2 ml per head;

[0099] PBS control group: sterile PBS solution.

[0100] 4. Reagents: viral RNA extraction kit (Beijing Solabio Technology Co., Ltd.) and PEDV fluorescent quantitative PCR detection kit (Guangzhou Weibao Xin Biological Technology Co., Ltd.).

[0101] II. Experimental procedures and results

[0102] 1. Grouping and immunization: 25 piglets were randomly divided into 5 groups, 5 in each group, and the grouping is as follows:

[0103] Group 1: PEDV S mutant protein vaccine group;

[0104] Group 2: wild-type PEDV S protein vaccine group;

[0105] Group 3: commercial inactivated vaccine group;

[0106] Group 4: adjuvant control group (PBS+M108L adjuvant);

[0107] Group 5: PBS control group

[0108] 2. Immunization method: intramuscular injection, 1 mL per piglet, and secondary immunization was performed 2 weeks after the first immunization.

[0109] 3. Serological detection and results

[0110] Blood sampling 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);

[0111] Detection method: detection was performed according to the neutralizing antibody detection method.

[0112] 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.

[0113] Table 3 Results of porcine epidemic diarrhea virus neutralizing antibody test

[0114]

[0115]

[0116] 4. Virus challenge experiment

[0117] Time of challenge: 1 week after the second vaccination (21 days);

[0118] Challenge dose: Oral inoculation of 10 6.0 TCID 50 / mL of porcine epidemic diarrhea virus JH strain, inoculation volume 10mL;

[0119] Observation period: Observe continuously for 7 days after the challenge, and record the following indicators every day:

[0120] Clinical symptoms: diarrhea score (0-4 points), vomiting, and mental state (1-3 points);

[0121] Fecal shedding: Fecal samples are collected daily to test for viral RNA levels.

[0122] 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.

[0123] Table 4 Statistics of clinical observation results after challenge

[0124] 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)

[0125] 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.

[0126] Table 5 Fecal viral load test results after challenge (CT value)

[0127]

[0128] 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.

[0129] Example 4: Design, preparation and testing of porcine rotavirus VP4 protein

[0130] The VP4 protein of the G9P23 type porcine rotavirus, which is currently more popular, was selected as a candidate protein. Its original amino acid sequence is shown in SEQ ID NO.4. Using a prokaryotic expression system to directly add a 6His tag to the C-terminus, it was found that it was basically completely unable to be purified by a nickel column during purification. Based on this, we analyzed the amino acid sequence and found that there were continuous negatively charged residues just upstream of the C-terminal His-tag, especially DD (the second and first positions from the bottom are D, D); this caused a strong local negative charge environment and / or steric hindrance, which seriously interfered with the His-tag and Ni 2 To this end, we inserted a flexible peptide GSSG between the C-terminus of the protein and the His-tag to physically separate the His-tag from the upstream acidic residues (especially the adjacent D), providing a space with low charge interference. The amino acid sequence of the porcine rotavirus VP4 protein designed in this way (i.e., the porcine rotavirus VP4 optimized protein) is shown in SEQ ID NO. 5.

[0131] Construction of BL21-VP4 (DE3) prokaryotic expression vector: (1) Take out a competent cell of expression bacteria E.coilBL21 (DE3) from the -80℃ ultra-low temperature freezer and gently place it on ice. (2) When the competent cells have just thawed, take 1μL of the recombinant plasmid that has been identified and add it to the activated competent cells, and gently pipette it several times to mix the plasmid and competent cells thoroughly. (3) Place the above mixture on ice for 30 minutes, then quickly and steadily place it in a 42℃ water bath and heat shock for 90 seconds to promote its transformation. (4) After the heat shock is completed, place the mixture on ice again for 2 minutes to terminate the reaction. Slowly add 1ml of resistance-free LB medium to the mixture, and then place it in a constant temperature shaker at 37℃ and 180r / min for incubation for 1 hour. (5) Take out the mixture from the shaker, centrifuge at 3500r / min for 3 minutes, discard the supernatant, suspend it with 100μL LB culture medium, and spread it on the prepared kanamycin-resistant plate. (6) After spreading, let it stand at room temperature for 10 minutes until the liquid to be spread is completely absorbed, put the plate in a 37℃ incubator for inversion culture, and take it out when the colony is clearly visible. (7) Pick a monoclonal colony with a clear edge from the plate and transfer it to LB liquid culture medium containing kanamycin resistance, culture at 37℃, 220r / min until the exponential growth phase and freeze it. (8) Both PCR and double enzyme digestion identification are positive, which means that the construction of the prokaryotic expression vector is completed. The recombinant plasmid pET28a-VP4 was transformed into BL21 (DE3) competent cells, and after a small amount of induced expression, the PoRV VP4 optimized protein in the lysate was detected by SDS-PAGE. The results are shown in Figure 2. Figure 5 The results showed that the optimized PoRV VP4 protein was expressed in the lysate. The protein size was consistent with the expected result and was basically in inclusion bodies.

[0132] Protein expression and purification: BL21-VP4 (DE3) bacterial liquid was amplified and cultured at 37°C. When OD600 = 0.6, 0.5 mM IPTG was added for induction for 4 hours. The protein mainly existed in the form of inclusion bodies. After the bacteria were ultrasonically disrupted, the precipitate was renatured with renaturation buffer (50 mM Tris-HCl + 300 mM NaCl + 4 M urea, pH 8.0) and then purified using a nickel column (equilibrium buffer: 50 mM Tris-HCl + 300 mM NaCl + 20 mM imidazole + 4 M urea, pH 8.0; elution buffer: 50 mM Tris-HCl + 300 mM NaCl + 300 mM imidazole + 4 M urea, pH 8.0). The column attachment rate increased from 0 to more than 95%. The eluate was subjected to membrane exchange solution to remove urea and imidazole, and finally filtered through a 0.22 μm low protein adsorption filter to obtain the optimized porcine rotavirus VP4 protein (PoRV VP4 optimized protein), BCA concentration was 1.253 mg / ml. The purified PoRV VP4 optimized protein was analyzed by SDS-PAGE and western blot with porcine rotavirus positive serum, and a specific band with a molecular weight of about 60 kDa was observed (see Figure 6 ), and the purity of SDS-PAGE was above 95%.

[0133] Example 5: Preparation and testing of a combined porcine epidemic diarrhea virus-porcine rotavirus subunit vaccine

[0134] 1. Preparation of Bivalent Subunit Vaccine

[0135] The PEDV S mutant protein and PoRV VP4 optimized protein were diluted to 111 μg / ml with PBS respectively, and then the diluted PEDV S mutant protein and PoRV VP4 optimized protein were mixed in equal volumes to obtain the antigen solution. Finally, the mixed antigen solution was mixed with M108L adjuvant at a mass ratio of 9:1, mixed and packaged, and stored at 2-8°C for future use. The content of PEDVS mutant protein and PoRV VP4 optimized protein in the vaccine was 50 μg / ml.

[0136] 2. Safety testing

[0137] Safety test of the vaccine on 3-5 day-old healthy susceptible piglets (piglets born from sows with no more than 1:4 of neutralizing antibody against PEDV and no more than 1:8 of neutralizing antibody against PoRV, and no positive results of PCR detection of TGEV, PEDV, porcine deltacoronavirus and PoRV) was conducted. The piglets were observed for 2 days before vaccination, and the body temperature was measured every morning to obtain the average value as the basal body temperature. Each piglet was inoculated with 2.0 ml of the vaccine in the neck muscle, and the body temperature was measured every morning after inoculation. The piglets were observed for 14 days, and the mental state, appetite and reaction at the inoculation site were observed every day. The results are shown in Table 6. After vaccination, the body temperature, mental state and appetite of the piglets were normal, and the inoculation site was normal, and no abnormality was observed. Therefore, the safety of the vaccine is good.

[0138] Table 6. Results of safety test of the vaccine on 3-5 day-old piglets

[0139]

[0140] 3. Effectiveness test

[0141] Twenty 3-5 day-old healthy susceptible piglets were divided into 4 groups, with 5 piglets in each group. The first and second groups were inoculated with 1.0 ml of the vaccine in the neck muscle, and the same dose was used for booster immunization after 2 weeks. The third and fourth groups were used as controls. Seven days after the second immunization, the serum of all the test piglets was collected, and then the piglets were subjected to challenge. Each piglet of the first and third groups was orally administered with 10.0 ml of PEDV JH strain cytotoxin (virus content: 10 6.0 TCID 50 / ml), and each piglet of the second and fourth groups was orally administered with 10.0 ml of PoRV SX strain tissue toxin (prepared and preserved by Zhejiang Hongsheng Biological Technology Co., Ltd.) (virus content: 100 ID 50 / 10.0 ml). The piglets were continuously observed for 10 days after challenge. At the same time, the anal swabs of all the piglets were collected at 1, 3, 5, 7 and 10 days after challenge, and subjected to PCR detection. The results are shown in Tables 7-10. After challenge with the two viruses respectively, the immune groups were all 5 / 5 protected, and the control groups of the corresponding viruses were all 5 / 5 sick. The neutralizing antibody against PEDV of the immune groups should be ≥1:32, and the neutralizing antibody against PoRV should be ≥1:64. The neutralizing antibody against PEDV of the control group challenged with PEDV was ≤1:4, and the neutralizing antibody against PoRV of the control group challenged with PoRV was ≤1:8. Therefore, the vaccine has good immunization effect.

[0142] Table 7. Results of challenge test of three batches of vaccines with PEDV JH strain

[0143]

[0144] Table 8. Results of fluorescence quantitative PCR detection of anal swabs at different times after challenge with PEDV

[0145]

[0146] Table 9 Results of challenge test of three batches of porcine rotavirus SX strain

[0147]

[0148]

[0149] Table 10 Fluorescence quantitative PCR test results of anal swabs at different times after porcine rotavirus challenge

[0150]

[0151] 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 amino acid sequence of the PEDV S mutant protein is shown in SEQ ID NO.

2.

2. 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.

3. A recombinant expression vector, characterized in that: The vector comprises the nucleotide sequence according to claim 2, and the vector is a pCDNA3.1 expression vector.

4. A recombinant cell line, characterized in that The cell line contains the recombinant expression vector according to claim 3, and the cell line is a CHO-K1 cell.

5. A porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine, characterized in that: The vaccine comprises an effective amount of the PEDV S mutant protein according to claim 1, an effective amount of the PoRV VP4 optimized protein and an adjuvant.

6. The vaccine according to claim 5, characterized in that The amino acid sequence of the optimized PoRV VP4 protein is shown in SEQ ID NO.

5.

7. The vaccine according to claim 5, characterized in that The concentrations of the PEDV S mutant protein and the PoRV VP4 optimized protein were both 50 μg / mL.

8. The vaccine according to claim 5, characterized in that The adjuvant is M108L, and the mass ratio of the antigen solution to the adjuvant is 9:

1.

9. Use of the PEDV S mutant protein according to claim 1 in the preparation of a porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine.

10. Use of the optimized PoRV VP4 protein according to claim 6 in the preparation of a porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine.

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