RNA vaccine for porcine epidemic diarrhea and preparation method therefor

Abstract

Provided are an RNA vaccine for porcine epidemic diarrhea and a preparation method therefor. The RNA vaccine is obtained by means of gene synthesis, in-vitro transcription, and lipid nanoparticle encapsulation, which can rapidly respond to the currently prevalent new PEDV strains, reduces the research and production cycle of new vaccine development, and provides an effective technical approach for the prevention and control of newly mutated highly virulent strains. The prepared RNA vaccine for porcine epidemic diarrhea has high production efficiency, can induce relatively high levels of porcine epidemic diarrhea virus neutralizing antibodies and binding antibodies in vivo, effectively reducing the occurrence of diarrhea in piglets, and thus holds great promotional value for the prevention and control of porcine epidemic diarrhea.

Description

RNA vaccine for porcine epidemic diarrhea and preparation method thereof Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an RNA vaccine for porcine epidemic diarrhea and a preparation method thereof. Background Art

[0002] Since 2010, porcine epidemic diarrhea (PED) has become widespread in my country. It is a major cause of diarrhea in piglets, severely jeopardizing the healthy development of my country's swine industry. PEDV is divided into two genotypes—G1 and G2. G1 includes subtypes G1a and G1b, while G2 includes subtypes G2a and G2b. Classic strains, such as CV777, are classified as the G1a subtype. G1b, G2a, and G2b subtypes are all newly emerged strains since 2010. G1b is a low-virulence variant, while G2a and G2b are highly pathogenic variants, with G2b being a highly pathogenic recombinant variant. Since 2010, identification and sequencing analysis of PEDV (porcine epidemic diarrhea virus) strains have demonstrated that the G2 genogroup of variant PEDV strains is highly prevalent in China, with the majority of isolates belonging to the G2 genogroup. Currently, a series of epidemic strains that are significantly different from traditional PEDV strains have appeared in many areas of China, including G2c subtype and G2d subtype.

[0003] Compared with inactivated vaccines, nucleic acid vaccines can induce a stronger immune response in the body. The key to developing vaccines in the research and development of inactivated vaccines is to select low-toxicity, high-titer, and highly immunogenic strains, while mRNA vaccines are not restricted by strains. As long as the structure of the virus is clear, the vaccine can be designed according to the genotype. The continuous mutation of the PEDV genome, mixed infection with other viruses and huge economic losses all require the development of an effective vaccine within a very short development cycle. Therefore, mRNA vaccines can help to develop vaccines against emerging infectious disease pandemics in a relatively short period of time. mRNA vaccines mainly express antigens, antibodies or other functional proteins. When mRNA is used as a vaccine, mRNA needs to translate the target protein to induce appropriate immunogenicity to activate the human immune system, but many mRNA molecules are currently unstable.

[0004] PEDV is an intestinal disease of pigs caused by coronavirus. After infection through fecal-oral transmission, PEDV replicates in the epithelial cells of the small intestine, quickly leading to destruction and necrosis of infected cells. This causes acute, highly contagious and severe intestinal diseases in pigs, resulting in a high mortality rate in piglets. Currently, there is no specific drug for the treatment of porcine epidemic diarrhea. Therefore, vaccination reduces the risk of PEDV infection, thereby achieving the purpose of disease prevention and control, becoming the most economical, effective and reliable method. Among them, the S protein expressed on the PEDV viral envelope has receptor binding activity and membrane fusion activity. It is a key protein for coronavirus to infect cells and plays an important role in infection. It is also an important site for host neutralizing antibodies and a key target for the current design of mRNA antiviral vaccines. The present invention uses the S protein on the PEDV viral envelope as a key target for mRNA vaccine design. By designing and optimizing the CDS region sequence, the efficiency of exogenous protein expression is improved, inducing the body to produce more neutralizing antibodies.

[0005] Summary of the Invention

[0006] In response to the problems existing in the existing technology, the present invention provides an RNA vaccine for porcine epidemic diarrhea virus and its preparation method and application, aiming to solve some of the problems in the existing technology. The porcine epidemic diarrhea RNA vaccine and its preparation method provided by the present invention have a short research and development time, high safety, can produce immune protection against the current major PEDV epidemic strains, and can provide technical support for the prevention and control of porcine epidemic diarrhea.

[0007] The present invention provides an RNA vaccine for porcine epidemic diarrhea. The RNA vaccine is synthesized by using a 5'UTR nucleic acid sequence, a porcine epidemic diarrhea virus CDS region sequence, and a 3'UTR nucleic acid sequence.

[0008] As a preferred embodiment, the CDS region sequence of porcine epidemic diarrhea virus is selected from the spike protein antigens of porcine epidemic diarrhea virus of G2a and G2b genotypes.

[0009] As a preferred embodiment, the spike protein antigen of porcine epidemic diarrhea virus of the G2a gene subtype includes a sequence encoded by the nucleic acid sequence of any one of SEQ ID NO1-22 and translated into a protein, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO1-22.

[0010] As a preferred embodiment, the spike protein antigen of porcine epidemic diarrhea virus of the G2b gene subtype includes a sequence encoded by the nucleic acid sequence of any one of SEQ ID NO23-45 and translated into a protein, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO23-45.

[0011] As a preferred embodiment, the 5'UTR nucleic acid sequence includes a nucleic acid sequence encoded by any one of SEQ ID NOs 46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 46-118.

[0012] As a preferred embodiment, the 3'UTR nucleic acid sequence includes a sequence encoded by any one of SEQ ID NOs 119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 119-130.

[0013] As a preferred embodiment, the 5'UTR nucleic acid sequence is encoded by the nucleic acid sequence of any one of SEQ ID NOs 46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NOs 46-118; the 3'UTR nucleic acid sequence is encoded by the nucleic acid sequence of SEQ ID NOs 119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NOs 119-130; the spike protein antigen of the porcine epidemic diarrhea virus of the G2a or G2b genotype, the 5'UTR nucleic acid sequence and the 3'UTR nucleic acid sequence are any combination.

[0014] As a preferred embodiment, the invention further comprises nanolipid particles, wherein the nanolipid particles encapsulate the RNA vaccine.

[0015] The present invention also provides a method for preparing a porcine epidemic diarrhea RNA vaccine, comprising the following steps: selecting a 5'UTR nucleic acid sequence, a 3'UTR nucleic acid sequence, a porcine epidemic diarrhea virus CDS region sequence, a stop codon, and a polyA synthetic RNA vaccine nucleic acid sequence, constructing the RNA vaccine nucleic acid sequence on a vector, performing enzyme digestion, transcription, and purification to obtain the RNA vaccine.

[0016] As a preferred embodiment, an adjuvant is also added to the RNA vaccine of porcine epidemic diarrhea virus.

[0017] The RNA vaccines of the above technical solutions can be administered to animals that need preventive treatment for virus-related infections, diseases, or conditions. Preferably, the animals include livestock such as pigs, cattle, poultry (e.g., chickens, ducks, geese, or turkeys), goats, and sheep, as well as domestic animals such as mice, rabbits, dogs, cats, and horses. Further preferred animals include pigs, rodents, horses, rabbits, and cattle. Most preferably, the vaccines are used to stimulate an immune response in pigs or to protect pigs against PEDV-related diseases.

[0018] The present invention also provides a porcine epidemic diarrhea virus CDS region sequence, wherein the porcine epidemic diarrhea virus CDS region is selected from the spike protein antigen of porcine epidemic diarrhea virus of G2a or G2b genotype;

[0019] The spike protein antigen of porcine epidemic diarrhea virus of the G2a genotype is encoded by the nucleic acid sequence of any one of SEQ ID NOs. 1-22 and translated into a protein sequence, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs. 1-22;

[0020] The spike protein antigen of porcine epidemic diarrhea virus of the G2b gene subtype is encoded by the nucleic acid sequence of any one of SEQ ID NO23-45 and translated into a protein sequence, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO23-45.

[0021] The present invention also provides a 5'UTR nucleic acid sequence, which comprises a polynucleotide of any one of SEQ ID NOs 46-118.

[0022] As a preferred embodiment, the 5'UTR nucleic acid sequence comprises any one of SEQ ID NOs 46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NOs 1-73.

[0023] As a preferred embodiment, the 5'UTR nucleic acid sequence has a GC content of no less than 30%.

[0024] As a preferred embodiment, the 5'UTR nucleic acid sequence includes a Kozak sequence.

[0025] As a preferred solution, an expression vector or host cell comprising the 5'UTR nucleic acid sequence described in the above technical solution.

[0026] As a preferred solution, a DNA or RNA vaccine comprising the 5'UTR nucleic acid sequence described in the above technical solution.

[0027] As a preferred solution, it includes the nucleic acid product for treatment or prevention of the 5'UTR nucleic acid sequence, or the protein product for treatment or prevention as described in the above technical solution.

[0028] As a preferred solution, it includes the use of the 5'UTR nucleic acid sequence described in the above technical solution in the expression of the encoded protein.

[0029] The present invention also provides a 3'UTR nucleic acid sequence, comprising a polynucleotide containing any one of SEQ ID NOs 119-130.

[0030] As a preferred embodiment, the 3'UTR nucleic acid sequence comprises a sequence of any one of SEQ ID NOs 119-130, or a sequence having at least 80% homology to any one of SEQ ID NOs 119-130.

[0031] As a preferred solution, it includes an expression vector, host cell, DNA or RNA vaccine containing the 3'UTR nucleic acid sequence described in the above technical solution.

[0032] As a preferred solution, it includes the nucleic acid product for treatment or prevention of the 3'UTR nucleic acid sequence, or the protein product for treatment or prevention as described in the above technical solution.

[0033] As a preferred solution, the 3'UTR nucleic acid sequence described in the above technical solution is used in the expression of encoded proteins.

[0034] The vaccine can be administered using a number of methods, including intranasal, oral, or intramuscular administration.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] Existing vaccines against porcine epidemic diarrhea virus are all inactivated vaccines or attenuated vaccines prepared based on virus isolation and culture, with long R&D cycles. Furthermore, porcine epidemic diarrhea virus disease genotypes are diverse and prone to genetic mutation. The antigenic sites of currently prevalent strains have undergone significant changes, and there are genetic differences between strains. Traditional vaccines are no longer suitable for the prevention and control of modern porcine epidemic diarrhea virus, resulting in the failure of traditional vaccines and the spread of the disease. The present invention discloses a method for preparing a porcine epidemic diarrhea RNA vaccine and its application, which reduces the vaccine R&D and production cycle, provides an effective technical means for the prevention and control of porcine epidemic diarrhea, can produce a high level of porcine epidemic diarrhea virus neutralizing antibodies, can effectively reduce the incidence of diarrhea in piglets, and has significant promotion value for the prevention and control of porcine epidemic diarrhea.

[0037] The mRNA vaccine of the present invention has good stability and avoids the production of by-products, which is mainly achieved through improvements and optimization in the following aspects: (1) optimizing the cap structure of the 5'UTR nucleic acid sequence; (2) adjusting the length of the Poly (A) tail of the 3'UTR nucleic acid sequence; (3) adding modified nucleotides to the sequence; (4) optimizing the sequences in the 5'UTR and 3'UTR untranslated regions; (5) optimizing according to the codon preference of the host; and (6) optimizing the secondary structure of the CDS region of porcine epidemic diarrhea virus.

[0038] The porcine epidemic diarrhea virus CDS region sequence of the present invention is designed and optimized to improve the efficiency of exogenous protein expression, increase protein expression, and induce the body to produce more neutralizing antibodies. The porcine epidemic diarrhea virus CDS region of the present invention is designed and optimized in the following ways: (1) using common codons that appear frequently in pigs to replace rare codons, thereby increasing protein production and improving protein expression; (2) the GC content (i.e., the ratio of G to C in the four bases of the sequence) is high, and at a high GC content, the protein expression amount is higher; (3) the secondary structure (free energy) is small, and at the minimum free energy, the protein expression amount is higher; (3) the sequence in the 5' and 3' untranslated regions is optimized, (4) the restriction endonuclease site is optimized, and (5) the repetitive sequence is optimized to remove sequences that make RNA unstable.

[0039] The optimized 5'UTR sequence obtained in the present invention improves the translation efficiency of the target protein. The optimized 5'UTR sequence can be used to construct and prepare high-efficiency expression vectors, increase the expression level of the target protein, and be applied to the development and application of RNA vaccines. It can be used to regulate the expression of various proteins and treat various diseases.

[0040] The 3'UTR provided by the present invention has a stronger function of promoting mRNA expression. The 3'UTR fragment provided by the present invention plays a translation-promoting function in different types of host cells and has a broad spectrum of effects, showing application prospects in RNA therapeutic drugs or mRNA vaccines.

[0041] The optimized 3'UTR of the present invention can improve the translation efficiency of the target protein and can be used to construct and prepare high-efficiency expression vectors, thereby increasing the expression level of the target protein and being applied to the development and application of RNA vaccines. In addition, it can also be used to regulate the expression of various proteins and treat various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is an electrophoresis diagram of G2a mRNA;

[0043] Figure 2 is an electrophoresis diagram of G2b mRNA;

[0044] Figure 3 is an electrophoresis diagram of mRNA constructed from G2b, 3'UTR, and 5'UTR;

[0045] Figure 4 is a graph showing the fluorescence expression results of 5'UTR sequence optimization;

[0046] Figure 5 is a graph showing the fluorescence expression results of 3'UTR sequence optimization;

[0047] FIG6 is a diagram showing the hEPO expression results of 3'UTR sequence optimization;

[0048] FIG7 is a diagram showing the results of in vitro expression of G2a mRNA;

[0049] FIG8 is a diagram showing the results of in vitro expression of G2b mRNA;

[0050] Figure 9 is a diagram showing the in vitro expression results of the mRNA vaccine constructed with G2b type and 5'UTR and 3'UTR;

[0051] Figure 10 is a graph showing in vivo antibody level detection;

[0052] Figure 11 is a graph showing the titer of IgG antibodies in colostrum;

[0053] Figure 12 is a graph showing the titer of IgA antibodies in colostrum;

[0054] Figure 13 shows the levels of binding IgG antibodies in piglet serum;

[0055] Figure 14 shows the levels of binding IgA antibodies in piglet serum;

[0056] FIG15 is a diagram showing the results of expressing the S protein of the G2b strain in tandem with the G2a sequence and the G2b sequence;

[0057] Figure 16 shows the results of expressing the S protein of the G2a strain in tandem with the G2a sequence and the G2b sequence. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0059] Example 1: Design of porcine epidemic diarrhea virus CDS region sequence

[0060] By designing and optimizing the CDS region sequence of the G2a and G2b subtypes of porcine epidemic diarrhea virus, the efficiency of exogenous protein expression is improved, and the body is induced to produce more neutralizing antibodies. The CDS region sequence of porcine epidemic diarrhea virus is designed and optimized by the following methods:

[0061] (1) Changing the codon preference and replacing the rare codons of the viral S protein with codons that appear frequently in mammals (especially pigs) can increase the expression of the protein in pigs.

[0062] Refer to the human codon preference table;

[0063] The codon bias of a single protein was calculated and used for optimization based on the codon bias of highly expressed proteins in pigs.

[0064] Refer to the mouse codon preference table;

[0065] Universal codon preference tables for humans, mice, and pigs: https: / / www.kazusa.or.jp / codon / ;

[0066] Database of pig highly expressed proteins: https: / / www.rnaatlas.org / ;

[0067] Calculation of protein codon bias: EMBOSS (European Molecular Biology Open Software Suite).

[0068] (2) Changing the GC content. Experimental studies have found that too low a GC content is more likely to cause mRNA degradation. When the GC content is appropriately increased, the mRNA is more stable and the protein can be continuously expressed, thereby increasing the expression level.

[0069] GC content calculation: https: / / www.novopro.cn / tools / gc-content.html;

[0070] Adjusting GC content: Optimization of codons and GC content is generally performed simultaneously, and there is no need to adjust GC content separately.

[0071] (3) mRNA secondary structure (free energy). The smaller the free energy, the more compact the mRNA structure and the tighter the folding, which reduces the possibility of degradation and thus increases protein expression.

[0072] RNA secondary structure prediction tools: Mfold; RNAfold;

[0073] RNA tertiary structure prediction tools: 3dRNA; MC-SYM.

[0074] (4) Optimization of restriction endonuclease sites: At the DNA level, unnecessary endonuclease sites are reduced, the probability of DNA template degradation is reduced, the stability of the DNA template is improved, and the process of transcription to obtain mRNA is more stable.

[0075] Non-essential restriction endonuclease sites: EcoRI, NcoI, SacI, BglII, HindIII, BsaI.

[0076] (5) Optimization of repetitive sequences. Experimental studies have found that excessive or overly complex repetitive sequences may make the structure of mRNA too complex, causing unexpected immune responses and thus inhibiting protein expression. Therefore, removing unnecessary repetitive sequences can reduce the risk of mRNA entering the animal body and causing autoimmunity.

[0077] Repetitive sequences cannot be removed using a single software package; comprehensive optimization software is required. Comprehensive optimization software includes: GeneArt Instant Designer; mRNAid.

[0078] Through the above design and optimization methods, the porcine epidemic diarrhea virus CDS region sequences of the G2a genotype of SEQ ID NOs. 1-22 and the porcine epidemic diarrhea virus CDS region sequences of the G2b genotype of SEQ ID NOs. 23-45 were finally designed as shown in the following table.

[0079] Example 2: 5'UTR sequence design

[0080] Novel 5'UTR sequences were independently designed, produced, and tested in this study. All of these sequences are random, and multiple nucleic acid sequences of 50 nt in length are generated. The ribosome load of the sequence generated in the first step is predicted by calculation. Sequences with appropriate ribosome loads are selected for mutation, and the ribosome load is calculated again. This process is repeated until the target ribosome load is reached, and the sequences that reach the target ribosome load are output. The GC content of the sequence is calculated using the Biopython toolkit, and the RNA secondary structure and minimum free energy are calculated using the ViennaRNA toolkit, and the corresponding secondary structure diagram is generated. Suitable sequences are selected by filtering conditions such as GC content, minimum free energy, and RNA secondary structure. The sequence table is shown in the 5'UTR sequence below.

[0081] Example 3: 3'UTR sequence design

[0082] AU-rich sequences (Adenylate-uridylate-rich elements (AREs)) in the 3'UTR of mRNAs can induce translational repression, dealkenylation (poly(A) tail shortening), decapping, or cleavage, leading to mRNA decay. The presence of adenylate / uridylate (AU)-rich sequences (AREs) in native 3'UTRs is implicated in regulating mRNA stability and serves as cis-acting sequences that activate rapid mRNA decay. Tristetraprolin (TTP) and its paralogs, butyrate response factors 1 and 2 (BRF-1 / 2), bind to AU-rich elements through their two zinc finger domains and promote mRNA degradation. The number and position of the AUUUA repeats within the AREs critically influence poly(A) tail shortening and RNA degradation. Another GU-rich sequence (GRE) within this region, in mammalian cells, binds to the CELF1 protein, accelerating mRNA decay. Some AU-rich elements are primarily located within a class of mRNAs encoding short-term factors, including cytokines, lymphokines, growth factors, and oncogenes. MiRNAs often target miRNA binding sequences within the 3'UTR of mRNAs to promote mRNA decay. It is generally believed that shorter 3'UTRs are more stable, likely due to a lack of microRNA (miRNA) binding sites, thereby protecting the mRNA from degradation. Studies have shown that cancer cell lines often express a greater number of mRNA isoforms with shorter 3'UTRs compared to similarly proliferating, untransformed cell lines. These shorter isoforms are often caused by alternate cleavage and polyadenylation (APA). These shorter mRNA isoforms exhibit greater stability and typically produce tenfold more protein, in part due to a loss of miRNA-mediated repression. MiRNAs, or miRNA binding sites as defined above, can be removed from or introduced into the 3'UTR to tailor nucleic acid expression.

[0083] When designing IVT mRNAs, predict and eliminate such sequences that interfere with mRNA stability. TargetScan 7.2 (http: / / www.targetscan.org / vert_72 / ), miRanda (http: / / www.microma.org / microma / home.do), DIANATools (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=site / page&view=software), miRDB (http: / / www.mirdb.org / miRDB / ), star Base (http: / / starbase.sysu.edu.cn / index.php), and miRWalk (http: / / mirwalk.umm.uni-heidelberg.de / ) can be used to detect miRNA-targeted sites in the 3' UTR.

[0084] When designing 3'UTR sequences, screen the 3'UTRs of genes with high expression efficiency or high protein molecular weight from the UTR database UTRdb (http: / / utrdb.ba.itb.cnr.it / ) or AURA (http: / / aura.science.unitn.it / about / ). Use the website to detect sites targeted by 3'UTR miRNAs and identify AU-rich sequences (AREs) and GU-rich sequences (GREs). Avoid or delete AU-rich sequences (AREs) and GU-rich sequences (GREs) in the 3'UTR. The following table lists the novel 3'UTR sequences generated and tested in this study.

[0085] The effects of 3'UTR structure on mRNA translation and stability were further investigated, and subsequent experiments were conducted. The control, Control-BioNTech, was selected from the sequence of AES-mtRNR1, and its sequence information is shown below.

[0086] Example 4: RNA vaccine for porcine epidemic diarrhea and its construction and preparation

[0087] 1. Amplification of target fragment and construction and preparation of recombinant plasmid.

[0088] (1) Prepare the reaction system according to the table below:

[0089] (2) Perform PCR (polymerase chain reaction) amplification according to the following table for 30 cycles:

[0090] (3) Digest the target DNA fragment and prepare the reaction system according to the table below;

[0091] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0092] (4) Enzyme digestion of the vector and preparation of the reaction system according to the table below;

[0093] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0094] (5) Prepare the reaction system according to the table below and carry out the ligation reaction;

[0095] (6) Place the above ligation reaction system in a water bath at 22°C and incubate for 1 hour.

[0096] 5. Connection product conversion and plate coating

[0097] (1) Add 10 μl of recombinant product to 100 μl of DH5a competent cells and incubate on ice for 30 min;

[0098] (2) Heat shock in a 42°C water bath for 90 seconds, then remove and place in an ice bath for 3 minutes;

[0099] (3) Add 600 μl of antibiotic-free LB medium and incubate at 37°C in a shaker for 1 h;

[0100] (4) Centrifuge at 6000 rpm for 4 min, remove 500 μl of supernatant, gently pipette the precipitate, and take 100 μl of bacterial solution to coat the corresponding resistance plate;

[0101] (5) Place in a constant temperature incubator at 37°C and culture for 16-20 hours;

[0102] (6) Pick a single clone and place it in a shaking incubator at 37°C for 16-17 hours.

[0103] 6. Plasmid extraction and identification

[0104] (1) Plasmids were extracted by alkaline lysis, eluted with DEPC-treated water, and the concentration, 260 / 280 ratio, and 260 / 230 ratio of the extracted plasmids were measured using a UV spectrophotometer. Enzyme-digested agarose gel electrophoresis was used to determine whether the size of the extracted plasmids was correct.

[0105] Prepare the enzyme digestion reaction system according to the table below;

[0106] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0107] 7. Plasmid linearization and purification

[0108] (1) Prepare HindIII single enzyme digestion system according to the table below:

[0109] (2) The above reaction solutions were mixed evenly, centrifuged briefly, and incubated at 37°C for 2.5 h before column purification.

[0110] 8. Co-transcriptional capping experiment

[0111] (1) Prepare the co-transcription capping system according to the following table:

[0112] (2) The reaction mixture was mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h;

[0113] (3) After the transcription reaction is completed, 1 μL of DNase I is added to each tube, mixed, centrifuged briefly, and incubated at 37°C for 1 h to remove the template DNA;

[0114] (4) RNA purification was performed using lithium chloride precipitation.

[0115] The method for preparing an RNA vaccine for porcine epidemic diarrhea first selects the 5'UTR, 3'UTR, and the spike protein antigen of the G2a gene subtype or the spike protein antigen of the G2b gene subtype of porcine epidemic diarrhea virus and constructs them on a vector. The recombinant plasmid is then subjected to enzyme digestion, transcription, and purification steps as described above to finally obtain an RNA nucleic acid vaccine.

[0116] According to the above steps, the G2a and G2b genotypes in the table were prepared into RNA nucleic acid vaccines and identified by gel electrophoresis, as shown in Figure 1, where 1 represents PEDV-G2a-1, 2 represents PEDV-G2a-2, 3 represents PEDV-G2a-3, 4 represents PEDV-G2a-4, 5 represents PEDV-G2a-5, 6 represents PEDV-G2a-6, and 7 represents PEDV-G2a-7. As shown in Figure 2, where 1 represents PEDV-G2b-23, 2 represents PEDV-G2b-24, 3 represents PEDV-G2b-25, 4 represents PEDV-G2b-26, 5 represents PEDV-G2b-27, 6 represents PEDV-G2b-28, 7 represents PEDV-G2b-29, and 8 represents PEDV-G2b-30.

[0117] Select SEQ ID No: 57, 62, 75 in the 5'UTR sequence list, SEQ ID No: 57, 62, 75 in the 3'UTR sequence list The nucleic acid sequences of the RNA vaccine synthesized by combining No. 124, 126, and 128 with PEDV-G2b-3 were constructed on a vector, and enzyme digestion, transcription, and purification were performed to obtain the RNA vaccine nucleic acid, as shown in Figure 3. 1 represents 57-PEDV-G2b-3-124, 2 represents 57-PEDV-G2b-3-126, 3 represents 57-PEDV-G2b-3-128, 4 represents 62-PEDV-G2b-3-124, 5 represents 62-PEDV-G2b-3-126, 6 represents 62-PEDV-G2b-3-128, 7 represents 75-PEDV-G2b-3-124, 8 represents 75-PEDV-G2b-3-126, and 9 represents 75-PEDV-G2b-3-128.

[0118] Example 5: Preparation and characterization of lipid nanoparticles

[0119] Cationic lipid SM102, DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and the mRNA from Example 4 was diluted in 50 mM citrate buffer, pH 4. LNPs were prepared by mixing the lipid ethanol solution with the mRNA aqueous solution from Example 4 at a volume ratio of 1:3 using a microfluidic device. The alcohol was removed, and the lipid nanoparticles were filtered through a 0.22 μm sterile filter. The prepared lipid nanoparticle mRNA vaccine was transferred to a 4°C refrigerator for storage until ready for use.

[0120] Example 6: 5'UTR sequence optimization to regulate the translation efficiency of the FLuc gene

[0121] HEK-293T cells were seeded at 30,000 cells per well in a 96-well culture plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The next day, RNA was constructed using a portion of the 5'UTR sequence designed in Example 2 above, along with the CDS and 3'UTR sequences encoding the Luc gene. The resulting RNA, Control-BioNtech, Control-Moderna, and Lipo3000 transfection reagent (hereinafter referred to as Lipo3000) were diluted in Opti-MEM medium. The diluted Lipo3000 and RNA were mixed directly at room temperature, mixed thoroughly, and incubated at room temperature for 10 minutes. After discarding the culture medium from the overnight cells, 50 μL of complete medium was added to each well, followed by 50 μL of the incubated Lipo3000 and RNA mixture, and the cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours of incubation, 100 μL of Bright-Light reagent was added to each well, mixed by vortexing for 3 minutes, and the luminescence value of each well was measured using a microplate reader. In Figure 4, Control-BioNtech is the preferred 5'UTR for Biontech's COVID vaccine mRNA BNT162b2. Control-Moderna is the 5'UTR selected for Moderna's COVID vaccine mRNA 1273. Figure 4 shows that compared to the 5'UTR sequence of Control-BioNtech's BNT162b2, the novel 5'UTR sequences 9, 10, 12, 23, 29, and 30 exhibit higher translation efficiency.

[0122] Example 7: Optimization of 3'UTR sequence to regulate the translation efficiency of Fluc gene (FLuc refers to FLuc coding sequence, T7 promoter coding sequence)

[0123] 293T cells were cultured to 50-60% confluence for transfection. Lipofectamine 3000 reagent was diluted with MEM medium according to the design instructions and mixed thoroughly. RNA containing the 3'UTR sequence and the Control-BioNTech RNA control were diluted to a certain concentration using MEM medium respectively to prepare an RNA premix. The mixture was mixed thoroughly and the diluted RNA was added to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio). Incubate at room temperature for 15 minutes, add the RNA-liposome complex to the cells, culture the infected 293T cells in a CO2 incubator, and detect RNA expression after 16 hours of culture. As shown in Figure 5, Control-BioNTech is the 3'UTR of Biontech. Compared with Biontech's 3'UTR, the 3'UTR sequence of the present invention significantly improves the translation efficiency of the coding sequence. Figure 5 shows that the new 3'UTR sequence has a higher translation efficiency than the control sequence. The insertion of 3'UTR-3 and 3'UTR-6 fragments increased the luciferase expression level in 293T cells by about 2 times.

[0124] Example 8: 3'UTR sequence optimization to regulate hEPO protein expression

[0125] A549 cells were seeded at 10,000 cells per well in a 24-well culture plate and cultured overnight in an incubator at 37°C and 5% CO2. Lipo3000, 1200 ng of Control-BioNTech control RNA, and 1200 ng of RNA containing the 3'UTR sequence were diluted in Opti-MEM medium (or serum-free DMEM medium). The diluted Lipo3000 and RNA were mixed directly at room temperature, mixed thoroughly, and incubated at room temperature for 10 minutes. The medium was discarded from the overnight cultured cells, and the mixture of Lipo3000 and RNA was added. The volume was supplemented with Opti-MEM medium (or serum-free DMEM medium) to 1 mL and incubated in an incubator at 37°C and 5% CO2 for 4 hours. After transfection, the medium was discarded from the cells, 1 ml of complete medium was added, and the cells were cultured for another 18-20 hours. After the incubation period, the cell culture supernatant was aspirated and the hEPO protein concentration in the supernatant was measured using an hEPO ELISA kit. As shown in Figure 6, Control-BioNTech is a 3'UTR from Biontech. Compared to the Biontech 3'UTR, the 3'UTR sequence of the present invention significantly improved the translation efficiency of the coding sequence. Figure 6 shows higher translation efficiency than the control sequence, with the insertion of 3'UTR-6 and 3'UTR-10 increasing protein expression by approximately 2-fold compared to the control.

[0126] Example 9: In vitro transfection and antigen expression analysis

[0127] HEK-293T cells were plated at 5 × 10 6 Cells were seeded into 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Lipo3000 and 8 μg of mRNA were diluted in Opti-MEM medium. The diluted Lipo3000 and mRNA were mixed directly at room temperature, incubated for 10 minutes, and then the mixture of Lipo3000 and mRNA was added to the wells. The volume was made up to 3 mL of Opti-MEM medium and the cells were incubated at 37°C in a 5% CO2 incubator for 4 hours. After transfection, the medium was removed from the cells, 3 mL of complete medium was added, and the cells were cultured for an additional 18 hours. After incubation, the medium was removed from the cells, washed once with 1 mL of 1× PBS, and thoroughly lysed with cell lysis buffer. 20 μl of loading buffer was added, and the cells were heated at 100°C for 15 minutes before Western blotting. The primary antibody used in Western blotting was a mouse monoclonal antibody, and the secondary antibody was a goat anti-mouse antibody. As shown in Figure 7, PEDV-G2a can be efficiently expressed in HEK-293T cells, among which PEDV-G2a-1, PEDV-G2a-3, PEDV-G2a-4, and PEDV-G2a-5 have the highest expression levels. 1 represents PEDV-G2a-1, 2 represents PEDV-G2a-2, 3 represents PEDV-G2a-3, 4 represents PEDV-G2a-4, 5 represents PEDV-G2a-5, 6 represents PEDV-G2a-6, and 7 represents PEDV-G2a-7. As shown in Figure 8 , PEDV-G2b can also be efficiently expressed in HEK-293T cells, where 1 represents PEDV-G2b-1, 2 represents PEDV-G2b-2, 3 represents PEDV-G2b-3, 4 represents PEDV-G2b-4, 5 represents PEDV-G2b-5, 6 represents PEDV-G2b-6, 7 represents PEDV-G2b-7, and 8 represents PEDV-G2b-8. As shown in Figure 9 , the 5'UTR and 3'UTR constructed on G2b can also be efficiently expressed in HEK-293T cells. 1 represents 57-PEDV-G2b-3-124, 2 represents 57-PEDV-G2b-3-126, 3 represents 57-PEDV-G2b-3-128, 4 represents 62-PEDV-G2b-3-124, 5 represents 62-PEDV-G2b-3-126, 6 represents 62-PEDV-G2b-3-128, 7 represents 75-PEDV-G2b-3-124, 8 represents 75-PEDV-G2b-3-126, and 9 represents 75-PEDV-G2b-3-128.

[0128] Example 10: Immunization of mice with lipid nanoparticle-encapsulated RNA vaccines and detection of antibody levels

[0129] The PEDV-G2b-3 sequence was selected and an RNA vaccine was prepared according to the above steps. Male BALB / c mice aged 6-8 weeks were randomly divided into three groups. Intramuscular immunization was used, with the primary immunization performed on day 0 and the booster immunization performed on day 14. Each group received the same immunization dose of 100 μL per immunization. The blank injection group received an equal volume of PBS. Tail blood was collected 7 and 21 days after the second immunization, and serum was collected. Antibody levels were determined by indirect ELISA. As shown in Figure 10, the results showed that the experimental group produced higher antibody levels. A serum neutralization test was performed on the serum on day 28, and the neutralizing antibody titer in the serum neutralization test was 1:30. This indicates that the porcine epidemic diarrhea RNA vaccine prepared by the present invention can provide an effective prevention and control measure for PEDV.

[0130] Example 11: Immunization of pigs with lipid nanoparticle-encapsulated RNA vaccines and detection of antibody levels

[0131] The PEDV-G2b-1 sequence was selected and prepared using the aforementioned procedures. Antibody responses in sows were assessed, with sows vaccinated at two time points: 30 and 16 days before farrowing. PEDV antibody levels were assessed in serum and colostrum. As shown in Table 1, neutralizing antibody titers in serum and colostrum reached 1:32 and 1:64, respectively, after the second vaccination. Figure 11 shows the colostrum-binding IgG antibody titer, and Figure 12 shows the colostrum-binding IgA antibody titer.

[0132] Table 1 Vaccine neutralizing antibody titer

[0133] Example 12: Detection of the passive immunity level provided by colostrum of immune sows to suckling piglets

[0134] Colostrum from immunized sows has a passive protective effect against PEDV on suckling piglets. Using sows vaccinated as in Example 11, each sow's newborn piglets were breastfed for 5 days, and then each newborn piglet was challenged with the virus. The piglets were killed on the 3rd and 10th days after the challenge. Piglet serum was collected on the 5th day to detect PEDV-binding IgG and Ig antibodies. The results showed that PEDV-binding IgG and IgA antibodies were detected in the serum of piglets born to immunized sows, but not in the serum of piglets born to sows in the control group, indicating that antibodies can be transferred to the piglets through breastfeeding. In summary, the PEDV mRNA vaccine can provide effective passive immunity to newborn piglets and protect them against PEDV infection. Figure 13 represents the level of binding IgG antibodies in serum, and Figure 14 represents the level of binding IgA antibodies in serum.

[0135] Example 13: Active Immunization Efficacy Test of Porcine Epidemic Diarrhea mRNA Vaccine in Piglets

[0136] The PEDV mRNA vaccine prepared according to the above steps using the PEDV-G2b-1 sequence was inoculated into the neck muscles of 5 healthy susceptible piglets aged 3-5 days. A PBS control group of 5 piglets was also established. Each group was boosted with the same dose 2 weeks later. 14 days after the second vaccination, serum was collected from all the experimental pigs and then challenged with the virus. Each pig in the first and second groups was orally administered 20 ml of PEDV (AH2012 / 12 strain) cytotoxicity (virus content 10 5.0 TCID 50 / ml) and observed for 10 consecutive days. The results showed that after challenge with porcine epidemic diarrhea virus, all mice in the mRNA vaccine-immunized group (5 / 5) were protected, while all mice in the non-immunized control group (5 / 5) developed disease. Neutralizing antibody titers in the immunized group were all above 1:32, while those in the control group were all ≤1:4. Specific results are shown in Table 2.

[0137] Table 2 Results of challenge test with porcine epidemic diarrhea virus AH2012 / 12 strain after piglet vaccination

[0138] Example 14: G2a and G2b tandem transfection in vitro and antigen expression analysis

[0139] According to the sequence design method in Example 1, the G2b and G2a sequences were optimized in tandem to obtain the SEQ ID NOs 131-139 shown in the following table. According to the RNA vaccine for porcine epidemic diarrhea and its construction and preparation method in Example 4, the recombinant plasmid was subjected to enzyme digestion, transcription, and purification steps to finally obtain RNA nucleic acid.

[0140] HEK-293T cells were plated at 5 × 10 6Cells were seeded into 6-well plates and cultured overnight in an incubator at 37°C, 5% CO2. Lipo3000 and 8 μg of mRNA were diluted in Opti-MEM medium. The diluted Lipo3000 and mRNA (SEQ ID 131, 133, 135) were mixed directly at room temperature and incubated for 10 minutes. The incubated Lipo3000 and mRNA mixture was then added, and the volume was made up to 3 mL with Opti-MEM medium. The cells were then incubated in an incubator at 37°C, 5% CO2 for 4 hours. After transfection, the medium was removed from the cells, 3 ml of complete medium was added, and the cells were cultured for an additional 18 hours. After the culture, the culture medium was discarded from the cells, and 1 ml of 1× PBS was added to wash them once. 80 μl of cell lysis buffer was used to fully lyse the cells, and 20 μl of loading buffer was added. The cells were heated at 100°C for 15 min, and Western Blot analysis was performed. Figure 15 shows the S protein expression results of the incubated G2b strain, and Figure 16 shows the S protein expression results of the incubated G2a strain. It can be seen from the figures that the tandem expression of G2a and G2b can successfully express the G2b antigen, and can also successfully express the G2a antigen.

[0141] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention, and that those skilled in the art may make changes, modifications and variations to the above embodiments within the scope of the present invention.

Claims

1. An RNA vaccine for porcine epidemic diarrhea, characterized in that: The RNA vaccine is synthesized using a 5'UTR nucleic acid sequence, a porcine epidemic diarrhea virus CDS region sequence, and a 3'UTR nucleic acid sequence; The porcine epidemic diarrhea virus CDS region is selected from the spike protein antigen of porcine epidemic diarrhea virus of G2a or G2b genotype; The spike protein antigen of the porcine epidemic diarrhea virus of the G2a gene subtype includes a sequence encoded by the nucleic acid sequence of any one of SEQ ID NOs 1-22 and translated into a protein, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NOs 1-22; The spike protein antigen of the porcine epidemic diarrhea virus of the G2b gene subtype is encoded by the nucleic acid sequence of any one of SEQ ID NO23-45 and translated into a protein sequence, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO23-45.

2. The RNA vaccine for porcine epidemic diarrhea according to claim 1, characterized in that: The 5'UTR is encoded by a nucleic acid sequence of any one of SEQ ID NOs 46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 46-118.

3. The RNA vaccine for porcine epidemic diarrhea according to claim 1, characterized in that: The 3'UTR nucleic acid sequence includes a sequence encoded by any one of SEQ ID NOs 119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 119-130.

4. The RNA vaccine for porcine epidemic diarrhea according to claim 1, characterized in that: Also included are nanolipid particles that encapsulate RNA vaccines.

5. The RNA vaccine for porcine epidemic diarrhea according to claim 1, characterized in that: The 5'UTR nucleic acid sequence comprises a nucleic acid sequence encoded by any one of SEQ ID NOs 46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 46-118; The 3'UTR nucleic acid sequence is encoded by any one of the nucleic acid sequences in SEQ ID NOs 119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any one of SEQ ID NOs 119-130; The spike protein antigen, 5'UTR nucleic acid sequence and 3'UTR nucleic acid sequence of the porcine epidemic diarrhea virus of the G2a or G2b gene subtype are any combination.

6. A method for preparing an RNA vaccine for porcine epidemic diarrhea, characterized in that: The steps include: The 5'UTR nucleic acid sequence, 3'UTR nucleic acid sequence, porcine epidemic diarrhea virus CDS region sequence, stop codon and polyA synthetic RNA vaccine nucleic acid sequence are selected, the RNA vaccine nucleic acid sequence is constructed on a vector, and enzyme digestion, transcription and purification are performed to obtain the RNA vaccine.

7. The method for preparing an RNA vaccine for porcine epidemic diarrhea according to claim 6, further comprising adding an adjuvant to the porcine epidemic diarrhea virus vaccine.

8. The RNA vaccine according to any one of claims 1 to 5, characterized in that The subjects of administration include animals in need of preventive treatment of virus-related infections, diseases, or conditions. Animals in which immune responses are stimulated by the use of RNA vaccines include livestock such as pigs, cattle, poultry, goats, and sheep, and household animals such as mice, rabbits, dogs, cats, and horses. Preferred animals include pigs, rodents, horses, rabbits, and cattle. Most preferably, RNA vaccines stimulate immune responses in pigs.

9. The RNA vaccine according to any one of claims 1 to 5 is administered by intramuscular injection, subcutaneous injection, intradermal injection, nasal administration, oral administration, anal administration or vaginal administration.

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