RNA vaccine for porcine epidemic diarrhea and preparation method thereof

Abstract

According to the RNA vaccine for porcine epidemic diarrhea and the preparation method of the RNA vaccine, the RNA vaccine is obtained through gene synthesis, in-vitro transcription and lipid nanoparticle wrapping, the current epidemic new PEDV strain can be rapidly dealt with, the research and development and production cycle of new vaccine development is shortened, and an effective technical means is provided for prevention and control of new variant virulent strains. The RNA vaccine for porcine epidemic diarrhea prepared by the invention is high in production efficiency, can generate a high-level porcine epidemic diarrhea virus neutralizing antibody and binding antibody in vivo, can effectively reduce the occurrence of piglet diarrhea, and has great popularization value for prevention and control of porcine epidemic diarrhea.

Description

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] PED (Porcine Epidemic Diarrhea) is the main disease that causes diarrhea in piglets, which seriously endangers the healthy development of my country's pig farming industry. PEDV is divided into two genotypes - G1 and G2. G1 includes G1a and G1b subtypes, G2 includes G2a and G2b subtypes, and the classic strain represented by CV777 strain is classified as G1a subtype. G1b, G2a and G2b subtypes are all new strains that have emerged since 2010, among which G1b is a low-pathogenic variant strain, G2a and G2b are highly pathogenic variant strains, and G2b is a highly pathogenic recombinant variant strain. Since 2010, the identification and sequencing analysis of PEDV (Porcine Epidemic Diarrhea Virus) strains have shown that the G2 genogroup of variant PEDV strains is highly prevalent in China, and most of the isolated strains are members of the G2 genogroup. At present, a series of epidemic strains that are significantly different from traditional PEDV strains have appeared in many regions of China, including G2c subtype and G2d subtype.

[0003] Compared with inactivated vaccines, nucleic acid vaccines can induce stronger immune responses 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 virus structure is clear, vaccines 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 effective vaccines in a very short development cycle. Therefore, mRNA vaccines can help 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, which can cause appropriate immunogenicity to activate the human immune system, but many mRNA molecules are currently unstable themselves.

[0004] PEDV is an intestinal disease of pigs caused by coronavirus. After fecal-oral transmission, PEDV replicates in the epithelial cells of the small intestine, quickly leading to the destruction and necrosis of infected cells. This causes acute, highly contagious and severe intestinal diseases in pigs, and leads to a high mortality rate in piglets. There is currently no specific drug for the treatment of porcine epidemic diarrhea, so vaccination is used to reduce the risk of PEDV infection, thereby achieving the purpose of disease prevention and control, and 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, is a key protein for coronavirus-infected 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 the design of mRNA vaccines. The efficiency of exogenous protein expression is improved by designing and optimizing the CDS region sequence, and the body is induced to produce more neutralizing antibodies. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an RNA vaccine of porcine epidemic diarrhea virus and a preparation method and application thereof, aiming to solve some of the problems in the prior art. The porcine epidemic diarrhea RNA vaccine and the preparation method thereof 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.

[0006] The 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.

[0007] 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 gene subtypes.

[0008] As a preferred embodiment, the spike protein antigen of porcine epidemic diarrhea virus of the G2a gene subtype includes a sequence encoded by and translated into a protein by the nucleic acid sequence of any one of SEQ ID NO1-22, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO1-22.

[0009] As a preferred embodiment, the spike protein antigen of porcine epidemic diarrhea virus of the G2b gene subtype includes a sequence encoded by and translated into a protein by the nucleic acid sequence of any one of SEQ ID NO23-45, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO23-45.

[0010] 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 with any one of SEQ ID NOs 46-118.

[0011] As a preferred embodiment, the 3'UTR nucleic acid sequence includes a sequence encoded by any of SEQ ID NOs 119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to any of SEQ ID NOs 119-130.

[0012] As a preferred embodiment, the 5'UTR nucleic acid sequence is encoded by the nucleic acid sequence of any one of SEQ ID NO46-118, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO46-118; the 3'UTR nucleic acid sequence is encoded by the nucleic acid sequence of SEQ ID NO119-130, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with any one of SEQ ID NO119-130; the spike protein antigen of porcine epidemic diarrhea virus of the G2a or G2b genotype, the nucleic acid sequence of the 5'UTR and the nucleic acid sequence of the 3'UTR are any combination.

[0013] As a preferred embodiment, it also includes nanolipid particles, which encapsulate the RNA vaccine.

[0014] 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 cutting, transcription and purification to obtain the RNA vaccine.

[0015] As a preferred embodiment, an adjuvant is also added to the RNA vaccine of porcine epidemic diarrhea virus.

[0016] The RNA vaccine of the above technical solution can be administered to animals that need preventive treatment of 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, and household animals such as mice, rabbits, dogs, cats, and horses. Further preferred animals include pigs, rodents, horses, rabbits, and cattle. Most preferably, an immune response is stimulated in pigs or used to protect pigs against PEDV-related diseases.

[0017] 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 the porcine epidemic diarrhea virus of the G2a or G2b gene subtype;

[0018] The spike protein antigen of porcine epidemic diarrhea virus of G2a gene subtype 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 with any one of SEQ ID NOs 1-22;

[0019] 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.

[0020] The present invention also provides a 5'UTR nucleic acid sequence, which comprises a polynucleotide of any one of SEQ ID NOs 46-118.

[0021] 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.

[0022] As a preferred embodiment, the 5'UTR nucleic acid sequence has a GC content of no less than 30%.

[0023] As a preferred embodiment, the 5'UTR nucleic acid sequence includes a Kozak sequence.

[0024] As a preferred solution, an expression vector or host cell comprising the 5'UTR nucleic acid sequence described in the above technical solution.

[0025] As a preferred embodiment, a DNA or RNA vaccine comprising the 5'UTR nucleic acid sequence described in the above technical solution.

[0026] As a preferred embodiment, it comprises a nucleic acid product for treatment or prevention comprising the 5'UTR nucleic acid sequence, or a protein product for treatment or prevention as described in the above technical solution.

[0027] 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.

[0028] The present invention also provides a 3'UTR nucleic acid sequence, comprising a polynucleotide containing any one of SEQ ID NOs 119-130.

[0029] 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 with any one of SEQ ID NOs 119-130.

[0030] As a preferred embodiment, it comprises an expression vector, a host cell, a DNA or RNA vaccine containing the 3'UTR nucleic acid sequence described in the above technical solution.

[0031] As a preferred embodiment, it comprises 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.

[0032] As a preferred embodiment, the 3'UTR nucleic acid sequence described in the above technical solution is used in the expression of encoded proteins.

[0033] The vaccine can be administered using a number of methods including intranasal, oral, or intramuscular administration.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] Existing vaccines against porcine epidemic diarrhea virus are all inactivated vaccines or attenuated vaccines prepared based on virus isolation and culture, with a long research and development cycle. In addition, porcine epidemic diarrhea virus disease genotypes are numerous and prone to gene mutation. The antigenic sites of the current epidemic 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 viruses, causing the failure of traditional vaccines and the spread of epidemics. The preparation method and application of a porcine epidemic diarrhea RNA vaccine disclosed in the present invention reduce the vaccine research and development and production cycle, provide effective technical means for the prevention and control of porcine epidemic diarrhea, can produce a higher level of porcine epidemic diarrhea virus neutralizing antibodies, can effectively reduce the occurrence of piglet diarrhea, and have great promotion value for the prevention and control of porcine epidemic diarrhea.

[0036] The mRNA vaccine of the present invention has excellent stability and avoids the production of by-products, which is mainly achieved through the following improvements and optimizations: (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.

[0037] 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 with high frequency of occurrence in pigs to replace rare codons, increasing protein production and improving protein expression; (2) GC content (i.e., the ratio of G and C in the four bases of the sequence) is high, and the protein expression level is higher at a high GC content; (3) The secondary structure (free energy) is small, and the protein expression level is higher at the minimum free energy; (3) Sequence optimization in the 5' and 3' untranslated regions, (4) Restriction endonuclease site optimization, (5) Optimization of repeated sequences, and removal of sequences that make RNA unstable.

[0038] The 5'UTR sequence optimized in the present invention improves the translation efficiency of the target protein. The optimized 5'UTR sequence can be used for the construction and preparation of a high-efficiency expression vector, improve 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 be used to treat various diseases.

[0039] 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.

[0040] The optimized 3'UTR of the present invention can improve the translation efficiency of the target protein, can be used for the construction and preparation of efficient expression vectors, improve the expression level of the target protein, and be applied to the development and application of RNA vaccines. In addition, it can also be used to regulate the expression of various proteins and be used to treat various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the mRNA electrophoresis diagram of G2a;

[0042] Figure 2 is the mRNA electrophoresis diagram of G2b;

[0043] Figure 3 Construct mRNA electropherogram for G2b and 3'UTR and 5'UTR;

[0044] Figure 4 Optimize the fluorescence expression result graph for the 5'UTR sequence;

[0045] Figure 5 Optimize fluorescence expression results for 3'UTR sequence;

[0046] Figure 6 hEPO expression result diagram for 3'UTR sequence optimization;

[0047] Figure 7 This is the result of in vitro expression of G2a mRNA;

[0048] Figure 8 This is the result of in vitro expression of G2b mRNA;

[0049] Fig. 9 The results of in vitro expression of mRNA vaccine constructed for G2b type with 5'UTR and 3'UTR;

[0050] Fig.10 This is a graph showing the level of antibodies in the body;

[0051] Fig.11 This is a graph of the binding IgG antibody titer in colostrum;

[0052] Fig.12 This is a graph of the binding IgA antibody titer in colostrum;

[0053] Fig.13 is the level of binding IgG antibodies in piglet serum;

[0054] Fig.14 is the level of binding IgA antibodies in piglet serum;

[0055] Fig.15 This is the result of expressing the S protein of the G2b strain in tandem with the G2a sequence and the G2b sequence;

[0056] Fig.16 This is the result of expressing the S protein of the G2a strain by tandem expression of the G2a sequence and the G2b sequence. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0058] Example 1: Design of porcine epidemic diarrhea virus CDS region sequence

[0059] 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 porcine epidemic diarrhea virus CDS region sequence is designed and optimized in the following ways:

[0060] (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.

[0061] Refer to the human codon preference table;

[0062] Referring to the codon preference of highly expressed proteins in pigs, the codon preference of a single protein was calculated and used for optimization;

[0063] Refer to the codon preference table for mouse;

[0064] Query the universal codon preference table for humans, mice, and pigs: https: / / www.kazusa.or.jp / codon / ;

[0065] Database of pig highly expressed proteins: https: / / www.rnaatlas.org / ;

[0066] Calculate protein codon bias: EMBOSS (European Molecular Biology Open Software Suite).

[0067] (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, mRNA is more stable and the protein can be continuously expressed, thereby increasing the expression level.

[0068] GC content calculation: https: / / www.novopro.cn / tools / gc-content.html ;

[0069] Adjusting GC content: Optimization of codons and GC content is generally performed simultaneously, and there is no need to adjust GC content separately.

[0070] (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.

[0071] RNA secondary structure prediction tools: Mfold; RNAfold;

[0072] RNA tertiary structure prediction tools: 3dRNA; MC-SYM.

[0073] (4) Optimization of restriction endonuclease sites: reducing unnecessary endonuclease sites at the DNA level, lowering the probability of DNA template degradation, and improving the stability of the DNA template, making the process of transcribing to obtain mRNA more stable.

[0074] Non-essential restriction endonuclease sites: EcoRI, NcoI, SacI, Bgl II, HindIII, BsaI.

[0075] (5) Optimization of repetitive sequences. Experimental studies have found that excessive or overly complex repetitive sequences may make the structure of mRNA too complicated, 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.

[0076] Repeated sequences cannot be removed using a single software, and comprehensive optimization software is required. Comprehensive optimization software: GeneArt Instant Designer; mRNAid.

[0077] Through the above design and optimization methods, the porcine epidemic diarrhea virus CDS region sequences of the G2a gene subtype of SEQ ID NO1-22 and the porcine epidemic diarrhea virus CDS region sequences of the G2b gene subtype of SEQ ID NO23-45 were finally designed as shown in the following table.

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[0123]

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[0146]

[0147] Example 2: 5'UTR sequence design

[0148] Novel 5'UTR sequences designed, produced and tested independently 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, and the sequence with the appropriate ribosome load is 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. The appropriate sequence is 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 in the following table.

[0149]

[0150]

[0151]

[0152] Example 3: 3'UTR sequence design

[0153] AU-rich sequences (Adenylate-uridylate-rich elements, AREs) in the 3'UTR of mRNA can induce translational inhibition, dealkenylation (shortening of the poly-A tail), decapping or cleavage, and lead to mRNA decay. The presence of adenylate / uridylate (AU)-rich sequences (AREs) in the natural 3'UTR is associated with the regulation of mRNA stability and is a cis-acting sequence that activates 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 AUUUA repeats on the ARE have a key influence on the shortening of the poly(A) tail and RNA degradation, while another GU-rich sequence (GRE) in this region can bind to the CELF1 protein in mammalian cells to accelerate mRNA decay. Some AU-rich elements are mainly located in a certain class of mRNAs, which mainly encode short-term factors including cytokines, lymphokines, growth factors and oncogenes. MiRNAs usually target miRNA binding sequences in the 3'UTR of mRNAs to promote mRNA decay. It is generally believed that shorter 3'UTRs are more conducive to transcript stability, which may be due to the lack of microRNA (miRNA) binding sites, thereby avoiding mRNA degradation. Studies have shown that compared with similarly proliferating untransformed cell lines, cancer cell lines usually express a large number of mRNA isoforms with shorter 3'UTRs. These shorter isoforms are usually caused by alternate cleavage and polyadenylation (APA). These shorter mRNA isoforms show higher stability and typically produce ten times more protein, partly due to the loss of miRNA-mediated repression. MiRNAs or binding sites for miRNAs as defined above can be removed from the 3'-UTR or introduced into the 3'-UTR to customize the expression of nucleic acids.

[0154] When designing IVT mRNA, the interference of such sequences on mRNA stability can be predicted and excluded. Online software such as TargetScan7.2 (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 the sites targeted by 3'UTR miRNA.

[0155] When designing 3'UTR sequences, screen out 3'UTRs of genes that may have high expression efficiency or express proteins with large molecular weight from the UTR database UTRdb (http: / / utrdb.ba.itb.cnr.it / ) or AURA (http: / / aura.science.unitn.it / about / ), detect the sites targeted by 3'UTR miRNA through the website, find AU-rich sequences (ARE) and GU-rich sequences (GREs), avoid or delete AU-rich sequences (ARE) and GU-rich sequences (GREs) on 3'UTR. The following table lists the novel 3'UTR sequences produced and tested in this study.

[0156] The 3'UTR structure further screened the 3'UTR sequence and conducted subsequent experiments based on the effect of the 3'UTR structure on the translation and stability of mRNA. Control-BioNTech was used as the control, and the sequence was selected from the source AES-mtRNR1. The sequence information is as follows.

[0157] cTggTacTgcaTgcacgcaaTgcTagcTgccccTTTcccgTccTgggTaccccgagTcTcccccgaccTcgggTcccaggTaTgcTcccaccTccaccTgccccacTcaccaccTcTgcTagTTccagacaccTcccaa gcacgcagcaaTgcagcTcaaaacgcTTagccTagccacacccccacgggaaacagcagTgaTTaaccTTTagcaaTaaacgaaagTTTaacTaagcTaTacTaaccccagggTTggTcaaTTTcgTgccagccacacc

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[0161] Example 4: RNA vaccine for porcine epidemic diarrhea and its construction and preparation

[0162] 1. Amplification of target fragment and construction and preparation of recombinant plasmid.

[0163] (1) Prepare the reaction system according to the following table:

[0164] Components volume <![CDATA[RNase-free H2O]]> Up to 50ul 2x Phanta Max Buffer 25ul Primers 1ul each dNTP Mix 1ul Phanta Max Super-Fidelity DNA Polymerase 1ul

[0165] (2) Perform PCR (polymerase chain reaction) amplification according to the following table for 30 cycles:

[0166] Pre-denaturation 95℃3min transsexual 95℃30s annealing 61℃30s extend 72℃3min30s Final extension 72℃5min

[0167] (3) Digest the target DNA fragment and prepare the reaction system according to the table below;

[0168] Components volume Target DNA fragment 15ul MqI 2ul HindIII 2ul 10x Buffer 5ul <![CDATA[RNase-free H2O]]> Up to 50ul

[0169] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0170] (4) Vector digestion, and the reaction system was prepared as shown in the table below;

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[0172]

[0173] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0174] (5) Prepare the reaction system according to the table below to carry out the ligation reaction;

[0175] Components volume 10×T4 DNA Ligase Buffer 1ul T4 DNA Ligase 1ul Carrier 1ul Target DNA fragment after enzyme digestion 4ul <![CDATA[RNase-free H2O]]> Up to 10ul

[0176] (6) Place the above ligation reaction system in a water bath and incubate at 22°C for 1 h.

[0177] 5. Connection product conversion and plate coating

[0178] (1) Add 10ul of the recombinant product to 100ul of DH5a competent cells and incubate on ice for 30min;

[0179] (2) Heat shock in a 42°C water bath for 90 seconds, then remove and place in an ice bath for 3 minutes;

[0180] (3) Add 600 μl of antibiotic-free LB medium and incubate at 37°C in a shaker for 1 h;

[0181] (4) Centrifuge at 6000 rpm for 4 min, remove 500 μl of supernatant, gently blow up the precipitate with a pipette, and take 100 μl of bacterial solution to coat the corresponding resistance plate;

[0182] (5) Place in a constant temperature incubator at 37°C for 16-20 hours;

[0183] (6) Pick a single clone and place it in a shaking incubator and culture at 37°C for 16-17 h.

[0184] 6. Plasmid extraction and identification

[0185] (1) Plasmids were extracted by alkaline lysis method, eluted with DEPC-treated water, and the concentration, 260 / 280 ratio, and 260 / 230 ratio of the extracted plasmids were measured by UV spectrophotometer. Enzyme-cutting agarose gel electrophoresis was used to determine whether the size of the extracted plasmids was correct.

[0186] Prepare the enzyme digestion reaction system according to the table below;

[0187]

[0188]

[0189] The enzyme digestion reaction system was placed in a water bath and incubated at 37°C for 1 h.

[0190] 7. Plasmid linearization and purification

[0191] (1) Prepare HindIII single enzyme digestion system as shown in the table below:

[0192] Components <![CDATA[RNase-free H2O]]> Up to 100ul 10×Buffer 10ul HindIII 12ul pDNA 10ug

[0193] (2) The above reaction solutions were mixed evenly, centrifuged briefly, incubated at 37°C for 2.5 h, and then purified by column.

[0194] 8. Co-transcriptional capping experiment

[0195] (1) Prepare the co-transcription capping system according to the following table:

[0196] Components Volume(uL) Final concentration <![CDATA[RNase-free H2O]]> Up to 20 —— 10×Transcription Buffer 2 1× ATP / CTP / GTP / UTP (100 mM each) 2each 10mM each Cap1-GAG (100mM) 2 10mM Template DNA 0.5ug —— T7 RNA Polymerase Mix 2 ——

[0197] (2) The reaction mixture was mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h;

[0198] (3) After the transcription reaction is completed, add 1 μL DNase I to each tube, mix well, centrifuge briefly, and incubate at 37°C for 1 h to remove the template DNA;

[0199] (4) RNA purification was performed using lithium chloride precipitation.

[0200]

[0201]

[0202] The method for preparing an RNA vaccine for porcine epidemic diarrhea first selects 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, and then performs enzyme cutting, transcription, and purification steps on the recombinant plasmid to finally obtain an RNA nucleic acid vaccine.

[0203] 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. Figure 1 As shown, 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. Figure 2 As shown, 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.

[0204] Select SEQ ID No: 57, 62, 75 in the 5'UTR sequence table, SEQ ID No: 124, 126, 128 in the 3'UTR sequence table and arrange and combine with PEDV-G2b-3 to synthesize the nucleic acid sequence of RNA vaccine, construct on the vector, perform enzyme digestion, transcription, purification to obtain RNA vaccine nucleic acid, such as Figure 3 As shown, 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.

[0205] Example 5: Preparation and characterization of lipid nanoparticles

[0206] The 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 in Example 4 was diluted in 50mM citrate buffer pH=4. The lipid ethanol solution was mixed with the mRNA aqueous solution in Example 4 at a volume ratio of 1:3 by a microfluidic device to prepare LNP, and the alcohol was removed and the lipid nanoparticles were filtered through a 0.22μm sterile filter. The prepared lipid nanoparticle mRNA vaccine was transferred to a 4°C refrigerator for storage.

[0207] Example 6: 5'UTR sequence optimization to regulate the translation efficiency of FLuc gene

[0208] HEK-293T cells were inoculated in a 96-well culture plate at a number of 30,000 cells per well and cultured in an incubator at 37°C and 5% CO2 for 24 hours. On the second day, part of the 5'UTR sequence designed in Example 2 above was combined with the CDS region and 3'UTR sequence encoding the Luc gene to construct RNA, and the obtained RNA, Control-BioNtech, Control-Moderna and Lipo3000 transfection reagent (hereinafter referred to as Lipo3000) were diluted with opti-MEM culture medium respectively; the diluted Lipo3000 and RNA were directly mixed at room temperature, mixed evenly and incubated at room temperature for 10 minutes; after discarding the culture medium of the cells cultured overnight, 50 μL of complete culture medium was added to each well, and then 50 μL of the mixture of incubated Lipo3000 and RNA was added, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. After culturing for 24 hours, 100 μL of Bright-Light reagent was added to each well, shaken and mixed for 3 minutes, and the luminescence value of each well was measured using an enzyme marker. Figure 4 In the data, 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 mRNA1273. Figure 4 It was shown that the new 5'UTR sequences 9, 10, 12, 23, 29, and 30 showed higher translation efficiency compared with the 5'UTR sequence of Control-BioNtech's BNT162b2.

[0209] 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)

[0210] Transfect 293T cells when they are cultured to 50-60% confluence, dilute Lipofectamine 3000 reagent with MEM medium according to the design instructions, mix thoroughly, dilute RNA containing 3'UTR sequence and Control-BioNTech RNA control sample with MEM medium to a certain concentration, prepare RNA premix, mix thoroughly, add diluted RNA to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio). Incubate at room temperature for 15 minutes, add RNA-liposome complexes to cells, culture the infected 293T cells in a CO2 incubator, and detect RNA expression after 16 hours of culture. Figure 5As shown, Control-BioNTech is the 3'UTR of Biontech. Compared with the 3'UTR of Biontech, the 3'UTR sequence of the present invention significantly improves the translation efficiency of the coding sequence. Figure 5 The results showed that the new 3'UTR sequence had a higher translation efficiency than the control sequence. The insertion of 3'UTR-3 and 3'UTR-6 fragments increased the luciferase expression in 293T cells by about 2 times.

[0211] Example 8: 3'UTR sequence optimization to regulate hEPO protein expression

[0212] A549 cells were seeded in 24-well culture plates at a number of 10,000 cells per well and cultured overnight in an incubator at 37°C and 5% CO2. Lipo3000, 1200ng of Control-BioNTech control RNA and 1200ng of RNA containing 3'UTR sequence were diluted with Opti-MEM medium (or serum-free DMEM medium) respectively; the diluted Lipo3000 and RNA were directly mixed at room temperature, and incubated at room temperature for 10 minutes after mixing evenly; after the cells were cultured overnight, the culture medium was discarded, and the mixture of incubated Lipo3000 and RNA was added, and Opti-MEM medium (or serum-free DMEM medium) was supplemented to 1mL, and cultured in an incubator at 37°C and 5% CO2 for 4 hours. After transfection, the cells discarded the culture medium, added 1ml of complete culture medium, and continued to culture for 18-20 hours. After the culture was completed, the cell culture supernatant was aspirated, and the concentration of hEPO protein in the supernatant was detected using the hEPO Elisa detection kit. As Figure 6 As shown, Control-BioNTech is the 3'UTR of Biontech. Compared with the 3'UTR of Biontech, the 3'UTR sequence of the present invention significantly improves the translation efficiency of the coding sequence. Figure 6 All of them showed higher translation efficiency than the control sequence, and the insertion of 3'UTR-6 and 3'UTR-10 increased the protein expression by about 2 times compared with the control group.

[0213] Example 9: In vitro transfection and antigen expression analysis

[0214] HEK-293T cells were plated at 5 × 10 6100 cells were inoculated into a 6-well culture plate and cultured overnight in an incubator at 37°C and 5% CO2. Lipo3000 and 8μg of mRNA were diluted with Opti-MEM culture medium respectively; the diluted Lipo3000 and mRNA were directly mixed at room temperature, incubated at room temperature for 10 minutes after mixing evenly, and then the mixture of incubated Lipo3000 and mRNA was added, and Opti-MEM culture medium was supplemented to 3mL, and cultured in an incubator at 37°C and 5% CO2 for 4 hours. After transfection, the cells discarded the culture medium, added 3ml of complete culture medium, and continued to culture for 18 hours. After the culture was completed, the cells discarded the culture medium, added 1ml of 1×PBS to wash once, used cell lysis buffer to fully lyse the cells, added 20μl of Loading buffer, heated at 100°C for 15 minutes, and performed Western Blot detection. The primary antibody used in Western Blot is a mouse monoclonal antibody, and the secondary antibody is a goat anti-mouse antibody. Figure 7 As shown, 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 higher 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. Figure 8 As shown, 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. Fig. 9 As shown, 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.

[0215] Example 10: Immunization of mice with lipid nanoparticle-encapsulated RNA vaccines and detection of their antibody levels

[0216] The PEDV-G2b-3 sequence was selected and RNA vaccines were prepared according to the above steps. Male BALB / c mice aged 6-8 weeks were randomly divided into 3 groups. Intramuscular immunization was used, with the first immunization on day 0 of the experiment and the booster immunization on day 14 of the experiment. The immunization dose of each group was the same, with a volume of 100uL. The blank injection group was injected with an equal volume of PBS. Blood was collected from the tail 7 and 21 days after the second immunization, and serum was collected. The antibody level was determined by indirect ELISA. Fig.10 As shown, the results showed that the antibody level produced by the experimental group was higher. The serum on the 28th day was subjected to a serum neutralization test, and the neutralizing antibody titer in the serum neutralization test was 1:30. This shows that the porcine epidemic diarrhea RNA vaccine prepared by the present invention can provide an effective prevention and control means for PEDV.

[0217] Example 11: Immunization of pigs with lipid nanoparticle-encapsulated RNA vaccines and detection of antibody levels

[0218] The PEDV-G2b-1 sequence was selected and RNA vaccines were prepared according to the above steps. The antibody response in sows was detected, and sows were immunized at two time points, 30 days and 16 days before farrowing. The PEDV antibody levels were detected in sow serum and colostrum. As shown in Table 1, the neutralizing antibody titers in serum and colostrum reached 1:32 and 1:64, respectively, after the second immunization. Fig.11 Represents the binding IgG antibody titer in colostrum, Fig.12 Represents the titer of binding IgA antibodies in colostrum.

[0219] Table 1 Vaccine neutralizing antibody titer

[0220]

[0221]

[0222] Example 12: Detection of passive immunity level provided by colostrum of immune sows to suckling piglets

[0223] The colostrum of the immunized sows has a passive protective effect against PEDV for suckling piglets. Using the sows immunized as in Example 11, after the newborn piglets of each sow were breastfed for 5 days, each newborn piglet was challenged with the virus, and was killed on the 3rd day and the 10th day after the challenge. The 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 the antibodies can be transferred to the piglets through lactation. In summary, the PEDV mRNA vaccine can provide effective passive immunity for newborn piglets and protect them against PEDV infection. Fig.13Represents the level of binding IgG antibodies in serum, Fig.14 Represents the amount of IgA antibodies bound to water in serum.

[0224] Example 13: Active immunization efficacy test of porcine epidemic diarrhea mRNA vaccine on piglets

[0225] The PEDV mRNA vaccine prepared according to the above steps was selected from the PEDV-G2b-1 sequence and inoculated into the neck muscles of 5 healthy susceptible piglets aged 3-5 days. A PBS control group of 5 piglets was set up. Each group was boosted with the same dose after an interval of 2 weeks. 14 days after the second immunization, serum was collected from all experimental pigs and then challenged with viruses respectively; each pig in the first and second groups was orally administered 20 ml of PEDV (AH2012 / 12 strain) cytotoxic drug (virus content of 10 5.0 TCID 50 / ml), and observed for 10 consecutive days. The results showed that after the porcine epidemic diarrhea virus challenge, all the mRNA vaccine immunization groups were protected (5 / 5), and all the non-immunized control groups became ill (5 / 5). The neutralizing antibody titers of the immunized groups were all higher than 1:32, and the neutralizing antibody titers of the control groups were all ≤1:4. The specific results are shown in Table 2.

[0226] Table 2 Results of challenge test with porcine epidemic diarrhea virus AH2012 / 12 strain after piglet vaccination

[0227]

[0228]

[0229] Example 14: G2a and G2b tandem in vitro transfection and antigen expression analysis

[0230] According to the sequence design method in Example 1, the G2b and G2a sequences were optimized in series to obtain the SEQ ID NO131-139 sequences 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 cutting, transcription, and purification steps to finally obtain RNA nucleic acid.

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] HEK-293T cells were plated at 5 × 10 6 100 cells were inoculated into a 6-well culture plate and cultured overnight in an incubator at 37°C and 5% CO2. Lipo3000 and 8 μg of mRNA were diluted with Opti-MEM medium respectively. The diluted Lipo3000 and mRNA (SEQ ID131, 133, 135) were directly mixed at room temperature. After mixing evenly, they were incubated at room temperature for 10 minutes. Then, the mixture of incubated Lipo3000 and mRNA was added, and Opti-MEM medium was added to 3 mL. The cells were cultured in an incubator at 37°C and 5% CO2 for 4 hours. After transfection, the cells were discarded from the culture medium, 3 ml of complete culture medium was added, and the culture was continued for 18 hours. After the culture was completed, the cells were discarded from the culture medium, 1 ml of 1×PBS was added to wash once, 80 μl of cell lysis buffer was used to fully lyse the cells, 20 μl of Loading buffer was added, and the cells were heated at 100°C for 15 minutes. Western Blot detection was performed. Fig.15 This is the result of incubating the S protein expression of the G2b strain. Fig.16 This is a graph showing the expression results of the S protein of the incubated G2a strain. It can be seen from the figure that the tandem connection of G2a and G2b can successfully express the G2b antigen, and can also successfully express the G2a antigen.

[0248] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify and vary 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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