Polynucleotide encoding porcine epidemic diarrhea virus s protein and uses thereof

Abstract

The application discloses a polynucleotide encoding a porcine epidemic diarrhea virus S protein and application thereof; the amino acid sequence of the porcine epidemic diarrhea virus S protein is shown as SEQ ID NO. 4; the nucleotide sequence of the porcine epidemic diarrhea virus S protein is shown as SEQ ID NO. 7; and the nucleotide sequence of the circular RNA encoding the porcine epidemic diarrhea virus S protein is shown as SEQ ID NO. 9. The circular RNA encoding the porcine epidemic diarrhea virus S protein is super-long RNA, and the cyclization efficiency of the circular RNA prepared by a conventional method is low, and the industrial amplification application cannot be carried out. In the application, the S protein amino acid sequence and the nucleotide sequence are optimized, the cyclization efficiency is increased by 2.4 times (from 23% to 55%), the yield is effectively improved, the preparation cost is greatly reduced, and the industrial amplification can be carried out. The RNA vaccine containing the circular RNA is safe and efficient for injection of target animals, i.e., pregnant sows.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid vaccine technology, specifically relating to a polynucleotide encoding the S protein of porcine epidemic diarrhea virus and its uses. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a porcine enteric pathogenic coronavirus that causes severe diarrhea in newborn piglets, which is associated with high mortality. Therefore, developing an effective and safe vaccine remains a top priority in controlling PEDV infection. (Invention Patent CN 115820679) A discloses a circular RNA vaccine against porcine epidemic diarrhea virus (PEDV), its construction method, and its application. The circular RNA sequence includes an internal ribosome entry site sequence 1 (IRES1), a human tissue plasminogen activator signal peptide sequence (SP), a PEDV COE sequence, a flexible linker peptide GS sequence, a T4 phage fibrin Folden sequence, and an internal ribosome entry site sequence 2 (IRES2). The antigen expressed in vivo by the circular RNA used in this scheme is an artificial chimeric antigen. From the N-terminus to the C-terminus, it consists of the PEDV COE sequence (amino acids 507-640 of the natural S protein, where the natural S protein is a homotrimer, with each monomer containing 1386 amino acids), the flexible linker peptide GS sequence (GGGGS sequence), and the Folden sequence at the C-terminus of the T4 phage fibrin (composed of 27 amino acids (GYIPEAPRDGQAYVRKDGEWVLLSTFL). This domain can link with the viral protein to form an oligomeric homoprotein, significantly increasing the protein's immunogenicity. However, this antigen is an artificial chimeric antigen, which forms a new antigenic epitope at the chimeric site. This can easily trigger an excessively strong inflammatory response, leading to adverse reactions. Therefore, the safety of mRNA vaccines expressing this antigen in vivo still needs further investigation.

[0003] Injecting a linear mRNA vaccine expressing the full-length (1386aa, 4158bp) S protein of porcine epidemic diarrhea virus within a reasonable dosage range has been shown to have good safety and efficacy (see invention patent CN 118147173 B and literature doi:10.1128 / mbio.02958-23). ​​However, the scheme uses only linear mRNA, which is structurally unstable. Cap and poly(A)tail design and modification are required to improve expression efficiency and the stability of linear mRNA molecules. Base modification is also required to reduce the immune response against the mRNA molecule itself. These modifications lead to high vaccine manufacturing costs. According to statistics, the cost of capping and base modification of linear mRNA accounts for up to 67% of the total raw material cost, which is not conducive to the practical application of mRNA vaccines in the field of animal health.

[0004] Circular RNA molecules have a continuous covalently closed circular structure, requiring neither complex and expensive cap and poly(A)tail modifications nor the use of modified nucleotides (N1-methyl-pseudouridine), thus exhibiting high stability and protein expression efficiency. Studies have shown that the half-life of circular RNA in vitro is 1.5 times that of linear RNA, and its half-life in cells is 2.5 times longer. Furthermore, the duration of antigen expression by circular RNA is more than doubled, which is beneficial for the large-scale preparation, preservation, and transportation of RNA, as well as for activating stronger immune responses. In addition, vaccines developed using circular RNA can significantly reduce manufacturing costs by at least 50%, facilitating the application and promotion of this product in the animal health market.

[0005] However, the preparation of circular RNA from ultra-long nucleotide sequences (greater than 5000 bp) remains a major challenge in the industry. Circular RNA is obtained by circularization of linear RNA intermediates. Studies by R. Alexander Wesselhoeft et al. (doi:10.1038 / s41467-018-05096-6) found that circularization efficiency gradually decreases with RNA length. Their method can circularize RNA up to 4800 bp in length, but at this point, the circularization efficiency is already very low (estimated to be below 15%). During the research process of this invention, it was discovered that inserting the full-length nucleotide sequence (4158bp) of the porcine epidemic diarrhea virus S protein disclosed in patent CN 118147173 B into the circular element disclosed in patent CN 114574483 B to construct and prepare a template plasmid, and using this plasmid to perform transcription and circularization reactions according to the process of patent CN 117305328 A, the obtained circular RNA was 5070bp in size, which is an ultra-long RNA. The circularization efficiency was only 23%, and the product loss rate was as high as 77%, which could not be used for industrial scale-up applications. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the existing technology by providing a porcine epidemic diarrhea virus (PEDV) S protein and a circular RNA encoding the protein. This invention provides a circular RNA molecule expressing the PEDV S protein. This circular RNA is 5070 bp, belonging to the ultra-long RNA category, and is obtained from a circularization intermediate via a cyclization reaction. The extremely low efficiency of cyclization reactions for ultra-long RNAs is a major challenge in the industry. This invention optimizes the amino acid and nucleotide sequences of the S protein, increasing the cyclization efficiency by 2.4 times (from 23% to 55%), effectively improving yield and significantly reducing preparation costs. The process can be scaled up industrially. RNA vaccines containing this circular RNA are safe and highly effective when injected into pregnant sows.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] <First Aspect>

[0009] This invention relates to a porcine epidemic diarrhea virus (PEDV) S protein, the amino acid sequence of which is shown in SEQ ID NO.4.

[0010] As one embodiment, the nucleotide sequence of the porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.7.

[0011] <Second aspect>

[0012] This invention relates to a circular RNA molecule expressing the aforementioned porcine epidemic diarrhea virus S protein.

[0013] As one embodiment, the nucleotide sequence of the circulatory intermediate forming the circular RNA is shown in SEQ ID NO.8.

[0014] As one embodiment, the nucleotide sequence of the circular RNA molecule is shown in SEQ ID NO.9.

[0015] <Third aspect>

[0016] This invention relates to a method for preparing a circular RNA molecule expressing the aforementioned porcine epidemic diarrhea virus S protein, the method comprising the following steps:

[0017] S1, gene synthesis and gene cloning;

[0018] S2, bacterial culture and plasmid DNA extraction, purification and linearization;

[0019] S3, linearized plasmids are transcribed in vitro to form circular intermediates;

[0020] S4. The cyclization intermediate undergoes a cyclization reaction to form circular RNA.

[0021] <Fourth Aspect>

[0022] This invention relates to a circular RNA vaccine for porcine epidemic diarrhea virus S protein, comprising the aforementioned circular RNA molecule expressing porcine epidemic diarrhea virus S protein and lipid nanoparticles.

[0023] <Fifth Aspect>

[0024] This invention relates to a recombinant engineered bacterium, which is obtained by ligating a gene containing the encoding the aforementioned porcine epidemic diarrhea virus S protein into a plasmid vector and then transforming it into Escherichia coli.

[0025] As one implementation, the nucleotide sequence of the gene encoding the porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.7.

[0026] <Sixth Aspect>

[0027] This invention relates to a strain of Escherichia coli PS012, with accession number CCTCC NO:M20242332.

[0028] The *Escherichia coli* PS012 strain of this invention was deposited with the China Center for Type Culture Collection (CCTCC) on October 25, 2024, at Wuhan University, Wuhan, China, with accession number CCTCCNO:M20242332. It is used to prepare the S-D3 plasmid, which is then used in the preparation of a circular RNA vaccine.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) This invention solves the problem of low circularization efficiency and inability to scale up industrial applications when preparing circular RNA from porcine epidemic diarrhea virus (PEDV) S protein by optimizing the amino acid and nucleotide sequences of the S protein. By using optimized conditions to prepare circular RNA, the circularization efficiency is increased from 23% to 55%, the reaction efficiency is increased by 2.4 times, the product feed ratio is significantly improved, and the process can be scaled up industrially.

[0031] (2) An RNA vaccine prepared using optimized circular RNA was safe for use in pregnant sows. After immunization, the pigs showed normal behavior, appetite, and water intake, and no abnormal reactions such as redness or induration were observed at the injection site. After vaccination, no abnormalities were observed during the gestation period, and the sows gave birth normally. The number of healthy piglets, stillbirths, and weak piglets were not significantly different from the PBS control group.

[0032] (3) An RNA vaccine prepared using optimized circular RNA was highly effective in inoculating pregnant sows, the target animals. The vaccine induced high levels of virus-neutralizing antibodies in the sow's blood, with titers 1.6 times higher than those of similar circular RNA vaccines disclosed in the patent. Furthermore, the antibodies generated by the vaccine prepared in this study could be transferred to newborn piglets through milk, thereby generating effective passive immunity and effectively preventing PEDV infection in newborn piglets. Attached Figure Description

[0033] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0034] Figure 1 Agarose gel electrophoresis images of the DNA from each plasmid;

[0035] Figure 2 Chromatograms of each cyclization product analyzed by HPLC;

[0036] Figure 3 Capillary electrophoresis images of various circular RNA molecules;

[0037] Figure 4 Diagram showing the expression and identification of various circular RNA molecules in mammalian cells (indirect immunofluorescence assay);

[0038] Figure 5 This is an electron microscope image of the morphology of an mRNA vaccine. Detailed Implementation

[0039] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0040] Example 1: Construction of gene template plasmid

[0041] Based on the S gene sequence of the G2b type PEDV epidemic strain (GenBank: MZ161075.1) downloaded from GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), two proline mutations (I1076P and L1077P) were introduced into the S protein, and a 6×His tag was added to the 3' end to obtain the amino acid sequence of the SA protein (see SEQ ID NO.1). The codons of the S protein gene coding sequence were optimized and further designed using conventional methods, replacing rare codons of pigs in the sequence. Under the premise of avoiding specific restriction enzyme sites such as EcoRI, HindIII, and SpeI and unfavorable motifs, the sequence was modified to appropriately increase the content of codons with G or C at the third base (GC 3%), while taking into account the balance of GC distribution throughout the sequence, to obtain the nucleotide sequence of the SA protein (see SEQ ID NO.2).

[0042] The recombinant nucleic acid molecule and recombinant expression vector sequence for constructing circular RNA are referenced in patent CN 114574483 B. The circular intermediate for preparing circular RNA is composed of intron fragment II, translation initiation element truncated fragment II, coding region, translation initiation element truncated fragment I, and intron fragment I in sequence. The intron is derived from the T4 phage td gene, and the translation initiation element is a mutant sequence obtained by mutating G to T at position 13, T to C at position 19, and G to T at position 20 of Echovirus E29 IRES. The coding region is then incorporated into the SA protein nucleotide sequence to obtain the SA circular intermediate nucleotide sequence (see SEQ ID NO. 3).

[0043] This invention further optimizes and screens the amino acid sequence of the SA protein based on changes in viral epidemiological characteristics, high-order protein structure analysis, and B-cell and T-cell epitopes of the S protein, obtaining an optimized SD protein amino acid sequence (see SEQ ID NO.4). Using pigs as the expression host, codon optimization of the SD protein was performed using a publicly available platform (Thermofisher) to obtain the nucleotide sequence S-D1 (see SEQ ID NO.5). Using pigs as the expression host, nucleotide sequence optimization and further design of the SD protein yielded two novel nucleotide sequences, S-D2 (see SEQ ID NO.6) and S-D3 (see SEQ ID NO.7). Replacing the coding region nucleotides (positions 947-5125 bp) in the SA cyclic intermediate with the S-D1, S-D2, and S-D3 nucleotide sequences respectively yielded the S-D1 cyclic intermediate, S-D2 cyclic intermediate, and S-D3 cyclic intermediate sequentially.

[0044] The genes of the four circular intermediates (numbered SA, S-D1, S-D2, and S-D3) were synthesized by Genscript Biotech and cloned into the BAMHI and XHOI restriction sites of the pUC57 vector to form four template plasmid DNAs (numbered SA, S-D1, S-D2, and S-D3) using homologous recombination. These DNAs were then transformed into E. coli DH5α and sequenced to verify their correctness.

[0045] Example 2: Escherichia coli fermentation and preparation of recombinant plasmids

[0046] Escherichia coli fermentation: Escherichia coli DH5α strains (numbered SA, S-D1, S-D2, and S-D3) were inoculated into 500 mL of LB medium containing antibiotics (100 μg / mL ampicillin). The cultures were incubated with shaking at 28–32 °C until the OD600 value reached approximately 2.0–4.0. Then, at an inoculation rate of 0.5–3%, the seed culture was transferred to a 5 L fermenter for fermentation. When the OD600 value of the bacterial cells reached approximately 80–100, the cells were collected by centrifugation. The wet weights were 1106 g, 978 g, 1094 g, and 997 g respectively, meeting the requirements.

[0047] Plasmid DNA extraction: (1) Take 200g of harvested E. coli fermentation cells and add 1.6L of Solution I (preserved at 4℃) at a mass-to-volume ratio of 1:8 (cells to Solution I (50mM Tris-HL, 10mM EDTA, pH 8.0)). Stir until dissolved. (2) After dissolution, add 1.6L of Solution II (0.2M NaOH, 1.0% SDS), stir slowly, and then let stand for 3-10 minutes. (3) After the reaction is complete, add 1.6L of Solution III (3M KAC, pH 5.5, pre-cooled at 4℃), shake well, and let stand for 30 minutes. (4) After the reaction is complete, add 1.6L of 2M CaCl2 solution, shake gently, and let stand for 1 hour. The lysate solution will separate into two layers: the upper layer is solid and the lower layer is liquid. Use a peristaltic pump to pump out the liquid and filter it through a 1.0μm pore size filter to remove impurities. (5) The lysis buffer was filtered through a Quickstand ultrafiltration system using a 300KD hollow fiber ultrafiltration membrane for 1.5 hours, with a concentration factor of 5-10 times the volume. (6) The plasmid was purified using an AKTA purifier system. The DEAE (GE Healthcare Life Science, USA) was used to purify the plasmid. Five column volumes were equilibrated with loading buffer (0.55M NaCl, 50mM Tris-HCl, 10mM EDTA, pH 8.0). Elution buffer (0.65M NaCl, 50mM Tris-HCl, 10mM EDTA, pH 8.0) was used for gradient elution, and the eluted sample was collected. (7) The Capto™ core 700 (GE Healthcare Life Science, USA) column was first equilibrated with PBS for three column volumes. Then, the sample collected after purification by the ion exchange column was loaded. After the sample was loaded, elution was performed with PBS, and the eluted sample was collected. (8) Concentration and percolation were performed using a 100KD hollow fiber ultrafiltration membrane (GE Healthcare Life Science, USA, membrane area 500cm2, membrane fiber tube diameter 1mm) through a Quickstand ultrafiltration separation system. The membrane negative pressure (TMP) was 1psi, the concentration ratio was 5-10 times the volume, and the percolation ratio was 4-12 times.

[0048] Four template plasmid DNAs were prepared (numbered SA, S-D1, S-D2, and S-D3, with masses of 154 mg, 138 mg, 169 mg, and 172 mg, respectively). The A260 / A280 ratio was between 1.8 and 2.0, and the supercoil ratio was greater than 90% as determined by agarose gel electrophoresis (see [link to study]). Figure 1 The plasmid DNA was analyzed by agarose gel electrophoresis. After single enzyme digestion, the size was confirmed to be correct by agarose gel electrophoresis, and the prepared plasmid DNA met the requirements.

[0049] Table 1 Plasmid DNA Preparation

[0050] serial number Mass / 200g of bacterial cells Purity (A260 / A280) Superspiral ratio / % SA 154mg 1.89 91％ S-D1 138mg 1.87 92％ S-D2 169mg 1.90 90％ S-D3 172mg 1.89 92％

[0051] Example 3: Preparation and Analysis of Circular RNA

[0052] (1) Plasmid linearization and purification

[0053] The recombinant plasmid was digested and linearized using the restriction endonuclease SpeI (nearshore protein). The digestion system was as follows: 10× buffer: 2 ml, plasmid: 100 mg, SpeI: 3 ml, water to a final volume of 20 ml, digested overnight at 37°C. After digestion, the plasmid was purified by chromatography using a Capto Q ImpRes (Source 30Q) column (Cytiva) (loading buffer: 10 mM PBS, pH 7.4; elution buffer: 10 mM PBS, 500 mM NaCl, pH 7.4) to obtain the linearized plasmid.

[0054] (2) In vitro transcription: The transcription system was prepared as shown in Table 2 below:

[0055] Table 2 In vitro transcription reaction system

[0056]

[0057]

[0058] In vitro transcription conditions: linearized plasmid template concentration 50 mg, final GTP / ATP / CTP / UTP concentrations 16 mM / 32 mM / 8 mM / 8 mM, final T7 RNA polymerase concentration 1000 KU, incubation at 37℃ / 220 rpm for 2 h with constant temperature shaking, then 100 ml deoxyribonuclease I (DNase I) (1000 U / ml) was added to the transcription system, and incubation at 37℃ / 220 rpm for 15 min with constant temperature shaking.

[0059] (3) Transcription product mRNA precipitation: LiCl solution was added to the above transcription product and the mixture was placed at -20℃ overnight for precipitation; the overnight treatment solution was centrifuged to obtain mRNA precipitate, dried at room temperature and then resuspended in enzyme-free water to obtain linear mRNA aqueous solution (i.e., cyclic intermediate).

[0060] (4) mRNA cyclization and concentration: The cyclization reaction system is shown in Table 3 below:

[0061] Table 3 In vitro cyclization reaction system

[0062]

[0063] The above solutions were thoroughly mixed. The cyclization conditions were as follows: linear mRNA feed amount was 3.5 g, magnesium ion final concentration was 8 mM, cyclization reaction temperature was 52 °C, and cyclization reaction time was 15 min.

[0064] (5) Analysis of cyclization reaction products

[0065] The cyclization reaction products of each group were analyzed by HPLC, and the chromatograms are shown in the figure. Figure 2 Two to three chromatographic peaks were obtained on each chromatogram, with the peak at 20-24 min representing the target circular RNA molecule and the rest representing impurities. The cyclization efficiency was obtained by calculating the ratio of target circular RNA to impurities based on the peak area ratio. Analysis showed that the cyclization efficiencies of SA, S-D1, S-D2, and S-D3 circular RNAs were 23%, 38%, 47%, and 55%, respectively, with yields of 16.1 g, 20.7 g, 37.4 g, and 41.8 g. The cyclization efficiency and yield of the cyclization intermediate molecule SA based on the published S protein nucleotide sequence were both low, making scale-up impossible. After optimization, the cyclization efficiency of the cyclization intermediate molecule S-D1 increased by 1.7 times, while the cyclization efficiencies of the cyclization intermediates S-D2 and S-D3 further increased by 2.0 times and 2.4 times, respectively, significantly improving the product feed ratio and production efficiency, thus enabling scale-up. The nucleotide sequences of the cyclic intermediate and the circular RNA molecule of S-D3 are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0066] (6) Concentration and analysis of purified circular RNA

[0067] SA, S-D1, S-D2, and S-D3 circular RNA molecules were concentrated using TFF capsules (LV Centramate, PALL), with a membrane area of ​​0.02 m². 2 The flow rate was 120 mL / min. The ultrafiltered liquid was then transferred to a sterile sample bottle after sterile filtration.

[0068] The capillary gel electrophoresis results of four circular RNA molecules (numbered SA, S-D1, S-D2, and S-D3) are shown in the figure. Figure 3 The sizes are correct and the purity is above 95%, meeting the requirements.

[0069] Further investigation was conducted to determine the expression levels of porcine diarrhea virus (S) protein by four circular RNA molecules (SA, S-D1, S-D2, and S-D3) in mammalian cells. HEK-293T cells were transfected with the four circular RNA molecules using Lipofectamine 2000 reagent (Invitrogen), with untransfected cells serving as a blank control. Forty-eight hours after transfection, cells were fixed with 80% cold ethanol and blocked with 5% BCA. Diluted mouse anti-S protein monoclonal antibody (Shenlian Bio) was added as the primary antibody, and the cells were incubated at 37°C for 2 hours. Cells were washed three times with PBS for 5 minutes each time. Donkey anti-mouse IgG secondary antibody – Alexa Fluor 488 (Invitrogen) – was added, and the cells were incubated at 37°C for 1 hour. Cells were then washed three times with PBS, and the results were recorded and analyzed using a Zeiss Axio Vert A1 inverted fluorescence microscope. Results are shown below. Figure 4 As shown, no fluorescence was detected in the control group, while obvious fluorescence was detected in all four experimental groups, indicating that there was obvious expression of S protein. Among them, the fluorescence intensity of S-D3 was significantly higher than that of SA, S-D1 and S-D2, indicating that under the same conditions, this group of molecules had the highest expression level in mammalian cells.

[0070] Example 4: Preparation and Analysis of Lipid Nanoparticles (LNPs)

[0071] (1) LNP-mRNA preparation

[0072] The preferred S-D3 circular RNA molecule was dissolved in a 10 mM citrate / sodium citrate buffer solution at pH 4.0, resulting in a final RNA concentration of 200 μg / mL, to prepare solution I. Butyl 2-octanoate butyl ester (CMX4) was dissolved in ethanol with cholesterol (5-cholesterol-3β-ol), DSPC (distearate phosphatidylcholine), and PEG-DMG (polyethylene glycol dimyristic acid glyceride) at a molar ratio of 50:38.5:10:1.5 to obtain solution II. Solution I and Solution II were mixed using a microfluidic device at an N / P ratio of 7:1 at a flow rate of 20 ml / min. The mixture was immediately diluted with Tris-HCl solution (pH 7.4, containing 8% sucrose (w / v)), and ethanol was removed by tangential flow filtration. The solution was then concentrated to an RNA concentration of 50 μg / mL and sterilely filtered to obtain the mRNA vaccine (designated mRNA-S).

[0073] (2) Inspection

[0074] The average particle size of LNP particles was measured to be 103 nm, with a polydispersity index (PDI) of less than 0.3 and a negative surface potential using a dynamic light scattering method on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) potential-laser particle size analyzer. The results were analyzed using Quant-iT... TM RiboGreen TM RNA Assay Kit (Invitrogen) TM The encapsulation rate determined by R11490 was 92%, and the physical parameters of the sample met the requirements (see Table 4 for results).

[0075] Its morphology was observed using transmission electron microscopy, and the results are shown in [Figure number missing]. Figure 5 The cyclic mRNA lipid nanoparticles exhibited uniform morphology, consistent particle distribution, and distinct particle structure characteristics under an electron microscope, with their size and morphology meeting the requirements.

[0076] Table 4 Characterization of LNP-mRNA samples

[0077] serial number Particle size (nm) PDI Surface potential (mV) Encapsulation efficiency (%) mRNA-S 103 0.14 -4.2 92

[0078] Example 5: Immunization test in pregnant sows

[0079] (1) Animal Immunization Experiment

[0080] Six sows one month before farrowing were randomly divided into two groups (n=3): a vaccine group (mRNA-S group) and a control group (PBS group). The vaccine was administered intramuscularly. The vaccine group received 1 ml of mRNA-S vaccine (the optimal dose after trial and error, 1 ml containing 50 μg mRNA), while the control group received 1 ml of PBS. All sows were immunized 30 days before farrowing and received a second immunization 15 days before farrowing. Sow serum was collected on days 0, 14, and 28 after immunization, and milk was collected on day 5 after farrowing.

[0081] (2) Analysis of the immune safety of sows

[0082] After vaccination, the pigs showed normal behavior, appetite, and water intake, and no abnormal reactions such as redness, swelling, or induration were observed at the injection site. After vaccination, the sows showed no abnormalities during gestation, gave birth normally, and the number of healthy piglets, stillbirths, and weak piglets was not significantly different from the PBS control group (see Table 5), preliminarily indicating that the vaccine provided in this study is safe for pregnant sows.

[0083] Table 5. Farrowing situation after vaccination

[0084]

[0085] (3) Immunoefficacy analysis

[0086] 1) Serum IgG / milk sIgA antibody detection

[0087] Specific IgG antibodies in sow serum and secretory IgA antibodies (sIgA) in sow milk were detected by ELISA. The purified porcine epidemic diarrhea virus (PEDV) recombinant S protein antigen (Shenlian Biopharmaceutical (Shanghai) Co., Ltd.) was coated onto 96-well plates at a coating concentration of 25 ng / well. For detection, serum and milk were diluted 100-fold and incubated for 30 min. HRP-labeled mouse anti-pig secondary antibody (Solepro) was added at a dilution of 1:8000 (Solepro) and incubated for 30 min. TMB substrate was then added for 20 min. Finally, the absorbance (OD) at 450 nm was measured using a multi-mode microplate reader. 450nm ).

[0088] The results of IgG antibody reaction detection are shown in Table 6. OD in the PBS group 450nm The mean value was less than 0.2. No antibody response was observed in the vaccine group on day 0 of immunization, and the OD value was [missing value] on day 14. 450nm The mean value was 0.725 (positive rate 100%). One dose of the mRNA vaccine induced antibody production, with OD on day 28. 450nm The mean value reached 3.199 (positive rate 100%), and high levels of antibodies were produced after booster immunization, which is consistent with the vaccination pattern, indicating that the circular RNA vaccine induces a high level of humoral immune response.

[0089] The results of sIgA antibody response detection are shown in Table 7. The vaccine group successfully induced the production of sIgA and OD in the milk of sows. 450nm The mean value was 0.451 (positive rate 100%). The production of sIgA is key to the protection of newborn piglets by the swine epidemic diarrhea vaccine. Piglets obtain protection against the virus by consuming breast milk.

[0090] Table 6. Detection of serum IgG antibody reaction (OD) 450nm )

[0091]

[0092] Table 7 Detection of IgA antibody reaction in breast milk (OD) 450nm )

[0093]

[0094] 2) Detection of virus neutralizing antibody titer in sow blood

[0095] The collected sow serum was inactivated at 56°C for 30 min. After continuous 2-fold dilutions starting at a 1:2 ratio, the serum was compared with PEDV G2b strain (200 TCID50). 50Mix equal volumes of the virus and serum (0.1 mL) and incubate at 37°C for 1 hour. Then, seed the mixture onto a 96-well Vero cell monolayer and incubate at 37°C for 2.5 hours. Discard the mixture and wash three times with DMEM. Next, add 10 μg / mL of DMEM trypsin for virus entry into each well and incubate at 37°C for 3–5 days. Observe the Vero cell pathogenesis daily until stable. If no cytopathic effect occurs when the virus-serum mixture is inoculated into Vero cells, provided the control experiment is effective, then the serum at that dilution has the ability to neutralize the virus. Calculate the virus neutralizing titer of each test serum using the Reed-Muench method.

[0096] According to publicly available research, a neutralizing titer of 1:64 or higher against porcine epidemic diarrhea virus in serum can produce a strong virus neutralization effect. In this study, no neutralizing antibodies were found in the serum of the vaccine group on day 0 of immunization, the average neutralizing antibody titer on day 14 was 1:53 (reaching a maximum of 1:64), and the average neutralizing antibody titer on day 28 reached 1:171 (reaching a maximum of 1:256) (see Table 8), indicating that a strong humoral immune response was stimulated. Furthermore, the circular RNA vaccine targeting the full length of the porcine epidemic diarrhea virus (PEDV) S protein provided in this study is significantly more effective than the circular RNA vaccine targeting the COE region of the PEDV S protein (see patent CN 115820679 A). When immunized with the same dose in pregnant sows, the average serum neutralizing antibody titer of the sows on day 14 was only 1:32 (maximum 1:32), and the average serum neutralizing antibody titer on day 28 was only 1:107 (maximum 1:128). The average serum neutralizing antibody titer induced by this vaccine is 1.6 times that of the published PEDV circular RNA vaccines, indicating a stronger and more durable immune effect.

[0097] Table 8. Serum neutralizing antibody titers in sows

[0098]

[0099] 3) Analysis of passive immune protection in piglets

[0100] Five days after farrowing, piglets were allowed free access to breast milk. Six piglets were randomly selected from the immunized group and the PBS control group, and serum samples were collected to detect the neutralizing antibody titer against PEDV G2b strain. According to previous research (doi: 10.1128 / jvi.01309-24), a serum neutralizing antibody titer higher than 1:16 in piglets provides effective protection. In this study, the average serum neutralizing antibody titer in piglets was 1:40, with a maximum of 1:64 (see Table 9). The antibodies produced by the vaccine prepared in this study can be transferred to newborn piglets through breast milk, thereby generating effective passive immunity and effectively preventing PEDV infection in newborn piglets.

[0101] Table 9. Serum neutralizing antibody titers in farrowing piglets.

[0102] Vaccine Group 1:64 1:32 1:16 1:32 1:64 1:32 PBS group <1:4 <1:4 <1:4 <1:4 <1:4 <1:4

[0103] Example 6: Construction and preservation of S-D3 Escherichia coli strain

[0104] The recombinant plasmid S-D3 was transformed into *E. coli* DH5α competent cells using the calcium chloride method. The transformed cells were then plated on LB agar plates containing the corresponding antibiotic (100 μg / ml ampicillin) and incubated at 37°C. Once colonies were clearly visible, plump single colonies were picked and incubated in 3 ml of liquid LB agar containing the corresponding antibiotic (100 μg / ml ampicillin) at 37°C until the OD600 reached 0.6–0.8. One ml of this bacterial culture was then added to 8% glycerol and frozen at -80°C to obtain the transformed *E. coli* strain S-D3, which was verified by sequencing. The *E. coli* strain S-D3 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242332.

[0105] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A porcine epidemic diarrhea virus S protein, the amino acid sequence of which is shown in SEQ ID NO.

4.

2. A nucleic acid molecule encoding the porcine epidemic diarrhea virus S protein as described in claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

7.

3. A circular RNA molecule expressing the porcine epidemic diarrhea virus S protein as described in claim 1.

4. The circular RNA molecule according to claim 3, characterized in that, The nucleotide sequence of the circular intermediate that forms the circular RNA is shown in SEQ ID NO.

8.

5. The circular RNA molecule according to claim 3, characterized in that, The nucleotide sequence of the circular RNA molecule is shown in SEQ ID NO.

9.

6. A method for preparing a circular RNA molecule as described in claim 3, characterized in that, The method includes the following steps: S1, gene synthesis and gene cloning; S2, bacterial culture and plasmid DNA extraction, purification and linearization; S3, linearized plasmids are transcribed in vitro to form circular intermediates; S4. The cyclization intermediate undergoes a cyclization reaction to form circular RNA.

7. A circular RNA vaccine containing the S protein of porcine epidemic diarrhea virus, characterized in that, Includes the circular RNA molecules and lipid nanoparticles as described in claim 3.

8. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria are obtained by ligating the gene encoding the S protein of porcine epidemic diarrhea virus as described in claim 1 into a plasmid vector and then transforming it into Escherichia coli.

9. The recombinant engineered bacteria according to claim 8, characterized in that, The nucleotide sequence of the gene encoding the porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.

7.

10. An Escherichia coli PS012 strain, with accession number CCTCC NO: M 20242332.

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