Polynucleotide encoding porcine epidemic diarrhea virus S protein and application thereof

Abstract

The invention discloses polynucleotide for coding porcine epidemic diarrhea virus S protein and application of the polynucleotide. The amino acid sequence of the porcine epidemic diarrhea virus S protein is as shown in SEQ ID NO. 4; a nucleotide sequence for coding the S protein of the porcine epidemic diarrhea virus is as shown in SEQ ID NO.7; the nucleotide sequence of the circular RNA for coding the porcine epidemic diarrhea virus S protein is shown as SEQ ID NO. 9. The circular RNA for coding the porcine epidemic diarrhea virus S protein is ultra-long RNA, the cyclization efficiency of preparing the circular RNA according to a conventional method is low, and industrial amplification application cannot be carried out. By optimizing the amino acid sequence and the nucleotide sequence of the S protein, the cyclization efficiency is improved by 2.4 times (from 23% to 55%), the yield is effectively improved, the preparation cost is greatly reduced, and industrial amplification can be carried out. The RNA vaccine containing the circular RNA is safe and efficient to inject target animal pregnant sows.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid vaccines, and in particular relates to a polynucleotide encoding porcine epidemic diarrhea virus S protein and a use thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea in newborn piglets, which is associated with high mortality. Therefore, the development of effective and safe vaccines remains a top priority for controlling PEDV infection. Invention Patent CN 115820679 A discloses a circular RNA vaccine against porcine epidemic diarrhea virus and its construction method and application. In the scheme, the circular RNA sequence comprises an internal ribosome entry site sequence 1 (IRES1), a human tissue plasminogen activator signal peptide sequence (SP), a porcine epidemic diarrhea virus COE sequence, a flexible connecting 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 the scheme is an artificial chimeric antigen, which, from N-terminus to C-terminus, comprises the porcine epidemic diarrhea virus COE sequence (amino acids 507-640 of the natural S protein, wherein the natural S protein is a homotrimer, and each monomer molecule comprises 1386 amino acids), a flexible connecting peptide GS sequence (GGGGS sequence), and the Folden sequence at the C-terminus of the fibrin of the T4 phage (composed of 27 amino acids (GYIPEAPRDGQAYVRKDGEWVLLSTFL), and this domain can be connected with the viral protein to form an oligomeric homotypic protein, thereby significantly increasing the immune activity of the protein). However, this antigen is an artificial chimeric antigen, and a new antigenic epitope is formed at the chimeric site, which can easily stimulate an excessively strong inflammatory response and lead to side effects. Therefore, the safety of the mRNA vaccine expressing this antigen in vivo needs further investigation.

[0003] Injection of a linear mRNA vaccine expressing the full length of porcine epidemic diarrhea virus S protein (1386aa, 4158bp) within a reasonable dosage range has been shown to have good safety and efficacy (see invention patent CN 118147173 B and document doi:10.1128 / mbio.02958-23). ​​However, the mRNA used in this scheme is linear and has an unstable structure. Cap and Poly (A) tail design and modification are required to improve the expression efficiency and stability of the linear mRNA molecules. Base modification is required to reduce the immune response to the mRNA molecules themselves. These modifications lead to high vaccine manufacturing costs. According to statistics, the cost of linear mRNA capping and base modification 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 are continuous covalent closed loop structures that do not require complex and expensive Cap and Poly (A) tail modifications, nor do they require the use of modified nucleotides (N1-methyl-pseudouridine), and can have high stability and protein expression efficiency. According to research, the half-life of circular RNA in vitro is 1.5 times that of linear RNA, and the half-life in cells is 2.5 times longer than that of linear RNA. The duration of circular RNA antigen expression is more than doubled, which is conducive to the large-scale preparation, storage and transportation of RNA and the activation of stronger immune responses. In addition, vaccines developed using circular RNA can significantly reduce manufacturing costs by at least 50%, which is conducive to the implementation and promotion of this product in the animal health market.

[0005] However, the preparation of circular RNA for ultra-long nucleotide sequences (greater than 5000bp) is still a major difficulty in the industry. Circular RNA is obtained by cyclization of linear RNA circular intermediates. R. Alexander Wesselhoeft et al. (doi:10.1038 / s41467-018-05096-6) found that the cyclization efficiency gradually decreases with the length of RNA. In this scheme, the longest RNA length of 4800bp can be cyclized to form circular RNA, but the cyclization efficiency is very low at this time (estimated to be less than 15%). In the course of the research of the present invention, it was found that the full-length nucleotide sequence (4158 bp) of the S protein of porcine epidemic diarrhea virus disclosed in patent CN 118147173 B was inserted into the circular element disclosed in patent CN 114574483 B to construct and prepare a template plasmid, and the plasmid was used to perform transcription and circularization reactions according to the process of patent CN 117305328 A. The obtained circular RNA was 5070 bp in size, which was an ultra-long RNA. The circularization efficiency was only 23%, and the product loss rate was as high as 77%, which could not be industrially amplified and applied. Summary of the invention

[0006] The object of the present invention is to provide a porcine epidemic diarrhea virus S protein and a circular RNA encoding the protein in view of the deficiencies in the above-mentioned prior art. The present invention provides a circular RNA molecule expressing the porcine epidemic diarrhea virus S protein, the circular RNA is 5070bp, which is an ultra-long RNA, and the circular RNA is obtained from a circular intermediate through a cyclization reaction. The extremely low efficiency of the cyclization reaction of ultra-long RNA is a difficulty in the industry. The present invention improves the cyclization efficiency by 2.4 times (from 23% to 55%) by optimizing the amino acid sequence and nucleotide sequence of the S protein, effectively improving the yield and greatly reducing the preparation cost, and the process can be industrially scaled up. The RNA vaccine containing the circular RNA is safe and efficient for injection into the target animal, pregnant sows.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] <First aspect>

[0009] The present invention relates to a porcine epidemic diarrhea virus S protein, and the amino acid sequence of the porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.4.

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

[0011] <Second Aspect>

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

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

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

[0015] <Third Aspect>

[0016] The present invention relates to a method for preparing a circular RNA molecule expressing the 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 plasmid is transcribed in vitro to form a circular intermediate;

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

[0021] <Fourth Aspect>

[0022] The present invention relates to a circular RNA vaccine of porcine epidemic diarrhea virus S protein, comprising the aforementioned circular RNA molecules expressing porcine epidemic diarrhea virus S protein and lipid nanoparticles.

[0023] <Fifth Aspect>

[0024] The present invention relates to a recombinant engineering bacterium, which is obtained by connecting a gene encoding the porcine epidemic diarrhea virus S protein to a plasmid vector and then transforming Escherichia coli.

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

[0026] <Sixth Aspect>

[0027] The invention relates to Escherichia coli PS012, whose preservation number is CCTCC NO: M20242332.

[0028] The Escherichia coli PS012 of the present invention has been submitted to the China Center for Type Culture Collection for preservation on October 25, 2024, with the preservation address being Wuhan University, Wuhan, China, and the preservation number being CCTCCNO: M20242332. It is used to prepare the S-D3 plasmid, and then to prepare the circular RNA vaccine.

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

[0030] (1) The present invention solves the problem of low circularization efficiency and inability to industrialize and scale up circular RNA when preparing circular RNA from S protein by optimizing the amino acid and nucleotide sequences of S protein of porcine epidemic diarrhea virus. The circular RNA is prepared under optimized conditions, and the circularization efficiency is increased from 23% to 55%, the reaction efficiency is increased by 2.4 times, the product feed ratio is greatly improved, and the process can be industrialized and scaled up.

[0031] (2) The RNA vaccine was prepared using optimized circular RNA and was safe for pregnant sows. After vaccination, the pigs' spirits, feeding, drinking, and behavior were normal, and no abnormal reactions such as redness, swelling, and nodules were observed at the injection site. After vaccination, no abnormalities were observed during the pregnancy stage of the sows, and the litters were born normally. There was no significant difference in the number of healthy piglets, stillbirths, and weak fetuses compared with the PBS control group.

[0032] (3) The RNA vaccine was prepared using optimized circular RNA and was highly effective in the target animal, pregnant sows. The vaccine induced high levels of virus neutralizing antibodies in the blood of sows, with a titer 1.6 times that of similar circular RNA vaccines disclosed in the patent. In addition, the antibodies produced by the vaccine prepared in this study can be transferred to newborn piglets through breast milk, thereby producing effective passive immunity and effectively preventing newborn piglets from being infected with PEDV. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 1 Agarose gel electrophoresis diagram of each plasmid DNA;

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

[0036] Figure 3 Capillary electrophoresis diagram of each circular RNA molecule;

[0037] Figure 4 This is the expression identification diagram of each circular RNA molecule in mammalian cells (indirect immunofluorescence method);

[0038] Figure 5 This is an electron microscope morphology of the mRNA vaccine. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to 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 PEDV epidemic strain downloaded from GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) (GenBank: MZ161075.1), 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 SA protein amino acid sequence (see SEQ ID NO.1). The S protein gene coding sequence was codon optimized and further designed according to conventional methods to replace the rare codons of pigs in the sequence; on the premise of avoiding specific restriction sites and unfavorable motifs such as EcoRI, HindIII, and SpeI, the sequence was modified to appropriately increase the content of codons with the third base being G or C (GC 3%), while taking into account the balance of GC distribution of the entire sequence, and the SA protein nucleotide sequence (see SEQ ID NO.2) was obtained.

[0042] The sequences of recombinant nucleic acid molecules and recombinant expression vectors for constructing circular RNA refer to patent CN 114574483 B. The circular intermediate used to prepare circular RNA is composed of intron fragment II, translation initiation element truncation fragment II, coding region, translation initiation element truncation fragment I, and intron fragment I in sequence, wherein the intron is derived from the T4 phage td gene, and the translation initiation element is a mutant sequence obtained by mutating the 13th G to T, the 19th T to C, and the 20th G to T of Echovirus E29 IRES. The SA protein nucleotide sequence is introduced into the coding region to obtain the SA circular intermediate nucleotide sequence (see SEQ ID NO.3).

[0043] The present invention further optimizes and screens the amino acid sequence of SA protein based on changes in viral epidemiological characteristics, high-level structural analysis of proteins, and B cell epitopes and T cell epitopes of S protein to obtain an optimized SD protein amino acid sequence (see SEQ ID NO.4). Using pigs as expression hosts, the SD protein is codon optimized using an open platform (thermofisher) to obtain a nucleotide sequence S-D1 (see SEQ ID NO.5). Using pigs as expression hosts, the SD protein is nucleotide sequence optimized and further designed to obtain two new nucleotide sequences S-D2 (see SEQ ID NO.6) and S-D3 (see SEQ ID NO.7). The coding region nucleotides (947-5125bp positions) in the SA cyclic intermediate are replaced with S-D1, S-D2, and S-D3 nucleotide sequences to obtain S-D1 cyclic intermediates, S-D2 cyclic intermediates, and S-D3 cyclic intermediates in sequence.

[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 through homologous recombination to form four template plasmid DNAs (numbered SA, S-D1, S-D2, and S-D3), which were then transformed into Escherichia coli DH5α and verified to be correct by sequencing.

[0045] Example 2: E. coli fermentation and preparation of recombinant plasmid

[0046] E. coli fermentation: E. coli DH5α strains (numbered SA, S-D1, S-D2, S-D3) were inoculated into 500 mL of LB medium containing antibiotics (100 μg / ml ampicillin) respectively, and cultured under shaking at 28-32°C until the OD600 value was about 2.0-4.0. The seed liquid was inoculated into a 5L fermentation tank at an inoculation amount of 0.5-3% for fermentation culture. When the OD600 value of the bacteria reached about 80-100, the bacteria were collected by centrifugation, and the wet weights were 1106 g, 978 g, 1094 g, and 997 g, respectively, which met the requirements.

[0047] Plasmid DNA extraction: (1) Take 200g of the harvested E. coli fermentation cells, add 1.6L of solution I stored at 4°C according to the mass volume ratio of 1:8 between the cells and solution I (50mMTris-HL, 10mM EDTA, pH8.0), and stir until dissolved. (2) After dissolution, add 1.6L of solution II (0.2M NaOH, 1.0% SDS), stir slowly, and then stand for reaction for 3-10min. (3) After the reaction is completed, add 1.6L of solution III (3M KAC, pH5.5, pre-cooled at 4°C), shake well, and stand for reaction for 30min. (4) After the reaction is completed, finally add 1.6L of 2M CaCl2 solution, shake gently, and stand for reaction for 1h. The lysis solution is divided into two layers, the upper layer is solid and the lower layer is liquid. The liquid is discharged with a peristaltic pump and filtered through a filter element with a pore size of 1.0μm to remove impurities. (5) The lysate was filtered through a Quickstand ultrafiltration separation system using a 300KD hollow fiber ultrafiltration membrane, the operation time was 1.5 hours, and the concentration multiple was 5-10 times the volume. (6) The plasmid was purified by an AKTA purifier purification system, DEAE (GE Healthcare Life Science, USA) was used to purify the plasmid, the loading buffer (0.55M NaCl, 50mM Tris-HCL, 10mm EDTA, pH 8.0) was balanced for 5 column volumes, the eluent (0.65M NaCl, 50mM Tris-HCl, 10Mm EDTA, pH8.0), the solution was gradient eluted, and the eluted sample was collected. (7) The CaptoTM core 700 (GE Healthcare LifeScience, USA) column was first balanced with PBS for 3 column volumes, and then the sample collected by the ion exchange column was loaded. After the sample loading was completed, PBS was used for elution, and the eluted sample was collected. (8) The product was concentrated and diafiltrated using a Quickstand ultrafiltration separation system using a 100 KD hollow fiber ultrafiltration membrane (GE Healthcare Life Science, USA, membrane area 500 cm2, membrane fiber tube diameter 1 mm). The membrane negative pressure (TMP) was 1 psi, the concentration multiple was 5-10 times the volume, and the diafiltration multiple was 4-12 times.

[0048] Four template plasmid DNAs (numbered SA, S-D1, S-D2, and S-D3) were prepared, 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 Figure 1 Plasmid DNA agarose gel electrophoresis), after single enzyme digestion, agarose gel electrophoresis identified the size was correct, and the prepared plasmid DNA met the requirements.

[0049] Table 1 Plasmid DNA preparation

[0050] serial number Mass / 200g of bacteria Purity (A260 / A280) Supercoil ratio / % S 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 by restriction endonuclease SpeI (nearshore protein), and the digestion system was as follows: 10× buffer: 2 ml, plasmid: 100 mg, SpeI: 3 ml, water was added to 20 ml, and digestion was carried out at 37°C overnight. After digestion, Capto Q ImpRes (Source 30Q) (Cytiva) column was used for chromatography purification (loading buffer was 10 mM PBS, pH 7.4; elution buffer was 10 mM PBS, 500 mM NaCl, pH 7.4) to obtain linearized plasmid.

[0054] (2) In vitro transcription: The transcription system is configured 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, GTP / ATP / CTP / UTP final concentration 16 mM / 32 mM / 8 mM / 8 mM, T7 RNA polymerase (RNA Polymerase) final concentration 1000 KU, constant temperature shaking reaction at 37 ° C / 220 rpm for 2 hours, then add 100 ml of deoxyribonuclease I (DNaseI) (1000 U / ml) to the transcription system, and constant temperature shaking reaction at 37 ° C / 220 rpm for 15 minutes.

[0059] (3) Precipitation of mRNA of the transcription product: Add LiCl solution to the above transcription product and place it at -20°C for overnight precipitation; centrifuge the overnight treated solution to obtain mRNA precipitate, dry it at room temperature and resuspend the precipitate in enzyme-free water to obtain a linear mRNA aqueous solution (i.e., a circular 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 mixed thoroughly and uniformly. The cyclization conditions were as follows: the linear mRNA feed amount was 3.5 g, the final magnesium ion concentration was 8 mM, the cyclization reaction temperature was 52°C, and the cyclization reaction time was 15 min.

[0064] (5) Analysis of cyclization reaction products

[0065] The cyclization products of each group were analyzed by HPLC method, and the chromatograms are shown in Figure 2 , 2 to 3 chromatographic peaks were obtained on each chromatogram, of which the chromatographic peak at the position of 20-24min was the target circular RNA molecule, and the rest were impurities. The cyclization efficiency of the cyclization reaction was obtained by calculating the content ratio of the target circular RNA and the impurity according to the peak area ratio. After analysis, the cyclization efficiency of SA, S-D1, S-D2, and S-D3 circular RNAs were 23%, 38%, 47%, and 55%, respectively, and the yields were 16.1g, 20.7g, 37.4g, and 41.8g, respectively. The cyclization efficiency and yield of the cyclization reaction of the cyclized intermediate molecule SA based on the published S protein nucleotide sequence were both low, and production amplification could not be performed. After optimization, the cyclization efficiency of the cyclized intermediate molecule S-D1 was increased by 1.7 times, while the cyclization efficiencies of the cyclized intermediate molecules S-D2 and S-D3 were further increased by 2.0 times and 2.4 times, respectively, which greatly increased the product feed ratio, improved production efficiency, and could be amplified. The nucleotide sequences of the circular intermediate and 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] The SA, S-D1, S-D2, and S-D3 circular RNA molecules were concentrated by TFF Capsule (LV Centramate, PALL), where the membrane area of ​​TFF capsule was 0.02 m 2 , the flow rate was 120 mL / min, and the ultrafiltered liquid was 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, S-D3) are shown in Figure 3 , the size is correct and the purity is above 95%, which meets the requirements.

[0069] The levels of porcine diarrhea virus S protein expressed by four circular RNA molecules (numbered SA, S-D1, S-D2, and S-D3) in mammalian cells were further detected. The four circular RNA molecules were transfected into HEK-293T cells respectively using Lipofectamine 2000 reagent (Invitrogen), and untransfected cells were used as blank controls. 48 hours after transfection, the cells were fixed with 80% cold ethanol and blocked with 5% BCA, and diluted mouse anti-S protein monoclonal antibody (Shenlian Bio) was added as the first antibody and incubated at 37°C for 2 hours. The cells were washed three times with PBS for 5 minutes each. Donkey anti-mouse IgG secondary antibody-AlexaFluor 488 (Invitrogen) was added and incubated at 37°C for 1 hour. The cells were then washed three times with PBS and recorded and analyzed using a Zeiss AxioVertA1 inverted fluorescence microscope. The results are shown in the figure. Figure 4 As shown, no fluorescence was detected in the control, while obvious fluorescence was detected in the four experimental groups, indicating that all of them had obvious S protein expression. Among them, the fluorescence intensity of S-D3 was significantly higher than that of SA, S-D1 and S-D2, indicating that this group of molecules had the highest expression level in mammalian cells under the same conditions.

[0070] Example 4. Preparation and analysis of lipid nanoparticles (LNP)

[0071] (1) LNP-mRNA preparation

[0072] The preferred S-D3 circular RNA molecule was dissolved in a 10 mM citric acid / sodium citrate buffered saline solution at pH 4.0, with a final RNA molecule concentration of 200 μg / mL to prepare solution I. CMX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoic acid butyl ester) and cholesterol (5-cholesten-3β-ol), DSPC (distearoylphosphatidylcholine), and PEG-DMG (polyethylene glycol-dimyristylglycerol) were dissolved in ethanol at a molar ratio of 50:38.5:10:1.5 to obtain solution II. Solution I and solution II were mixed at an N / P ratio of 7:1 using a microfluidic device, and the flow rate during mixing was 20 ml / min. The mixed solution was immediately diluted with a Tris-HCl solution (pH 7.4, containing 8% sucrose (w / v)), and the ethanol component in the solution was removed by tangential flow filtration and concentrated to an RNA concentration of 50 μg / mL, and the mRNA vaccine (numbered mRNA-S) was obtained by sterile filtration.

[0073] (2) Inspection

[0074] The average particle size of LNP particles was measured by dynamic light scattering on a Zeta potential laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern), and the polydispersity index PDI was less than 0.3, and the surface potential was negative. TM RiboGreen TM RNA Assay Kit (Invitrogen TM The encapsulation efficiency of R11490 was 92%, and the physical parameters of the sample met the requirements (the results are shown in Table 4).

[0075] The morphology was observed by transmission electron microscopy. Figure 5 Under an electron microscope, the annular mRNA lipid nanoparticles have uniform morphology, consistent particle distribution, obvious particle structural characteristics, and their size and morphology meet the requirements.

[0076] Table 4 Characterization of LNP-mRNA samples

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

[0078] Example 5, pregnant sow immunity test

[0079] (1) Animal Immunization Experiment

[0080] Six sows one month before farrowing were randomly divided into two groups (n=3), namely the vaccine group (mRNA-S group) and the control group (PBS group). The vaccines were inoculated intramuscularly with 1 ml of mRNA-S vaccine (the optimal dose after exploration, 1 ml containing 50 μg of mRNA), and the control group was inoculated with 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 the 0th, 14th and 28th days after immunization, and milk was collected on the 5th day after farrowing.

[0081] (2) Analysis of sow immune safety

[0082] After vaccination, the pigs were in good spirits, eating, drinking, and behaving normally, and no abnormal reactions such as redness, swelling, or nodules were observed at the injection site. After vaccination, no abnormalities were observed during the pregnancy stage of the sows, and the litters were normal. The number of healthy piglets, stillbirths, and weak fetuses were not significantly different from those in the PBS control group (see Table 5), which preliminarily indicated that the vaccine provided in this study was safe for pregnant sows.

[0083] Table 5 Farrowing after vaccination

[0084]

[0085] (3) Analysis of immune effectiveness

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

[0087] ELISA was used to detect the specific IgG antibody in sow serum and secretory IgA antibody (sIgA) in milk. The recombinant S protein antigen of porcine epidemic diarrhea virus (Shenlian Biopharmaceuticals (Shanghai) Co., Ltd.) expressed and purified was coated on a 96-well plate, and the S antigen coating amount was 25 ng / well. During the detection, serum and milk were diluted 100 times, incubated for 30 minutes, HRP-labeled mouse anti-swine secondary antibody was 1:8000 (Solabo Company), incubated for 30 minutes, TMB substrate was used for 20 minutes, and finally the 450nm absorbance value (OD 450nm ).

[0088] The results of IgG antibody reaction are shown in Table 6. The OD 450nm The mean value was less than 0.2. There was no antibody response on the 0th day of immunization in the vaccine group, and OD on the 14th day 450nm The mean value is 0.725 (positive rate 100%). One injection of mRNA vaccine can induce antibody production. OD on the 28th day 450nm The mean value reached 3.199 (positive rate 100%), and high levels of antibodies were produced after booster immunization, which is in line with the rules of vaccination, indicating that circular RNA vaccines induce high levels of humoral immune responses.

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

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

[0091]

[0092] Table 7 IgA antibody reaction detection in 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 two-fold dilutions starting from 1:2, the serum was diluted with PEDV G2b strain (200TCID 50 / 0.1mL) in equal volumes and incubate at 37°C for 1 hour. Then, the mixture was inoculated onto the Vero cell monolayer of a 96-well tissue culture plate, incubated at 37°C for 2.5h, the mixture was discarded, and washed 3 times with DMEM. Then, DMEM trypsin (10μg / mL) required for the virus to enter the cells was added to each well and incubated at 37°C for 3 to 5 days. The Vero cell pathological changes were observed daily until they stabilized. Under the premise that the control test was effective, if the virus and serum mixture was inoculated into Vero cells without cytopathic changes, it means that the serum of this dilution has the ability to neutralize the virus, and the virus neutralization titer of each serum to be tested was calculated using the Reed-Muench method.

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

[0097] Table 8 Virus neutralizing antibody titer in sow serum

[0098]

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

[0100] The farrowing piglets were free to suckle breast milk for 5 days. Six piglets were randomly selected from the immunization group and the PBS control group, and serum was collected to test the neutralizing antibody titer against the PEDV G2b strain. According to the study (doi: 10.1128 / jvi.01309-24), the virus neutralizing antibody titer of piglet serum higher than 1:16 can effectively protect. In this study, the mean virus neutralizing antibody titer in piglet serum was 1:40, and the highest was 1:64 (see Table 9). The antibodies produced by the vaccine prepared in this study can be transferred to newborn piglets through milk, thereby producing effective passive immunity, which can effectively prevent newborn piglets from infection with PEDV.

[0101] Table 9 Virus neutralizing antibody titers in serum of 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 Escherichia coli DH5α competent cells using the calcium chloride method, and spread on LB medium plates containing the corresponding antibiotics (100 μg / ml ampicillin), cultured at 37°C, and when the colonies on the plates were clearly visible, a single colony with a full head was picked and placed in 3 ml liquid LB medium containing the corresponding antibiotics (100 μg / ml ampicillin), cultured at 37°C until OD600 was 0.6-0.8, 1 ml of the bacterial solution was added to a final concentration of 8% glycerol, and stored at -80°C to obtain the transformed S-D3 Escherichia coli strain, which was verified to be correct by sequencing. The S-D3 Escherichia coli strain has been submitted to the China Center for Type Culture Collection for preservation, with the preservation address being Wuhan University, Wuhan, China, and the preservation number being CCTCC NO:M 20242332.

[0105] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may 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 the porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.

4.

2. The porcine epidemic diarrhea virus S protein according to claim 1, characterized in that The nucleotide sequence encoding the porcine epidemic diarrhea virus S protein 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 forming 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 according to claim 3, characterized in that: The method comprises the following steps: S1, gene synthesis and gene cloning; S2, bacterial culture and plasmid DNA extraction, purification and linearization; S3, linearized plasmid is transcribed in vitro to form a circular intermediate; S4. The circular intermediate undergoes a cyclization reaction to form a circular RNA.

7. A circular RNA vaccine of porcine epidemic diarrhea virus S protein, characterized in that: It comprises the circular RNA molecule and lipid nanoparticles as described in claim 3.

8. A recombinant engineered bacterium, characterized in that: The gene encoding the porcine epidemic diarrhea virus S protein as claimed in claim 1 is connected to a plasmid vector and then transformed into Escherichia coli to obtain the recombinant engineered bacteria.

9. The recombinant engineered bacterium 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, whose deposit number is CCTCC NO: M 20242332.

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