Porcine epidemic diarrhea virus mRNA vaccine and application

Abstract

The invention belongs to the technical field of biology, and provides a porcine epidemic diarrhea virus mRNA vaccine and application. The mRNA molecule comprises a coding region for coding the S protein of the PEDV; the PEDV is of a G2a type, and two proline mutations I1076P and L1077P are introduced into an S protein. The invention further discloses a DNA molecule. An S protein of PEDV; a recombinant plasmid; recombining the host cell; an mRNA vaccine formulation which can be used to prepare an antibody against the S protein of PEDV after immunizing an animal; the invention further discloses application of the mRNA molecule, the DNA molecule, the recombinant plasmid or the host cell to preparation of a medicine for preventing PEDV (Porcine Epidemic Diarrhea Virus) infection. The vaccine prepared by the invention is fast in antibody generation and high in protection rate, can generate high-level neutralizing antibodies in serum and breast milk of sows after the pregnant sows are immunized before parturition, has a remarkable protection effect on piglets, and can effectively prevent the piglets from being infected with PEDV (Porcine Epidemic Diarrhea Virus).

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a porcine epidemic diarrhea virus mRNA vaccine and uses thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious, and fatal infectious disease caused by infection with the porcine epidemic diarrhea virus (PEDV) of the alphacoronavirus genus of the Coronaviridae family, characterized by vomiting, acute diarrhea, dehydration, and hemorrhagic enteritis. Since its emergence in 2010, PEDV has been very active in my country, showing a high positive rate and genetic diversity. The strains continue to mutate, seriously threatening the healthy development of my country's pig industry. Currently, PEDV has become an important pathogen affecting the healthy development of my country's pig industry. In particular, the disease shows different susceptibility in different age groups. Very young pigs are most susceptible to the disease due to their incomplete immune development and weak body resistance.

[0003] PEDV contains 7 open reading frames (ORFs), namely ORF1a, ORF1b and ORF2-6. Among them, ORF2 encodes S protein. S protein is a type I transmembrane protein located on the surface of virus particles, which plays a key role in mediating virus invasion, inducing the production of neutralizing antibodies, and determining the virulence of the virus. According to the homology of the S gene of PEDV or the S1 subunit sequence of its N-terminal hypervariable region, PEDV can be divided into two genotypes, G1 and G2, of which G1 is further divided into G1a and G1b subtypes, and G2 is further divided into G2a and G2b subtypes. Genetic analysis and source evolution analysis of PEDV strains found that the PEDV currently prevalent in my country is mainly G2a (mutated strong strain) and G2b (recombinant strong strain) subtype strains. S protein is considered to be the main antigen for vaccine design. However, S protein is very unstable and undergoes obvious conformational changes during receptor binding, changing from an active state to an inactive state. The active state is usually called the "prefusion conformation" and the inactive state is called the "postfusion conformation". The prefusion conformation of the S protein has better immunogenicity and can therefore be considered an ideal target antigen.

[0004] At present, there is no specific drug for treating PEDV-infected pigs, especially piglets, and there is no good way to protect them. PED vaccination is an important means to prevent and control the spread of PED. At present, commonly used PED vaccines include inactivated vaccines, live attenuated vaccines and subunit vaccines, which play an important role in preventing and controlling PED. However, the immunogenicity of inactivated vaccines is low, and multiple vaccinations are usually required to enhance the immune effect; live attenuated vaccines have the risk of reversion to virulence and the production of new recombinant strains; subunit vaccines have low antibody levels, weak immunogenicity, and a short protection period. In addition, the continuous recombination and mutation of PEDV strains during the epidemic may cause the key antigens of traditional vaccine strains to not match those of the epidemic strains, thereby reducing the immune protection efficacy of the vaccine.

[0005] In short, the current PED vaccine prevention and control effect is not ideal. Summary of the invention

[0006] The present disclosure provides a porcine epidemic diarrhea virus mRNA vaccine and its use, so as to at least solve the above technical problems existing in the prior art.

[0007] According to the first aspect of the present disclosure, an mRNA molecule is provided, which comprises a coding region, wherein the coding region encodes the S protein of porcine epidemic diarrhea virus (PEDV); the porcine epidemic diarrhea virus is of G2a type, and the S protein introduces two proline mutations I1076P and L1077P.

[0008] Specifically, in the above mRNA molecule, the coding region encoding the S protein of porcine epidemic diarrhea virus is based on the amino acid sequence of the G2a type PEDV epidemic strain (GenBank: PP461398.1), and the codon degeneracy is used to replace the synonymous codons in the original sequence with the optimal codons for pigs, ensuring that the coding sequence of the S protein is more consistent with the usage preference of pig codons and avoiding the appearance of rare codons; at the same time, the GC content of the coding sequence (40-60%) and the secondary structure of the mRNA are optimized (the folding free energy is optimized from -1162.60 kcal / mol to -1255.00 kcal / mol). In addition, based on the cryo-electron microscopy structure study of the PEDV S protein, the present disclosure introduces two proline mutations (I1076P and L1077P) into the S protein to achieve the effect of stabilizing the pre-fusion conformation of the S protein.

[0009] In one embodiment, the mRNA molecule further comprises a 5'UTR, a Kozak sequence, a 3'UTR and PolyA.

[0010] Specifically, porcine epidemic diarrhea virus includes highly pathogenic variant strains, which are mainly G2 type, and also include multiple G1 type strains and classical strains.

[0011] According to the second aspect of the present disclosure, a DNA molecule for transcribing the above-mentioned mRNA molecule is provided, wherein the nucleic acid sequence of the coding region encoding the S protein of porcine epidemic diarrhea virus is as shown in SEQ ID NO:1, or the nucleic acid sequence of the coding region encoding the S protein of porcine epidemic diarrhea virus has at least 70% sequence identity with SEQ ID NO:1.

[0012] Specifically, the above-mentioned nucleic acid sequence encoding the S protein of porcine epidemic diarrhea virus is a sequence obtained by sequencing in high-incidence areas in my country. Vaccine preparations, monoclonal antibodies, drugs, etc. prepared based on this sequence have a significant effect in preventing porcine epidemic diarrhea virus infection.

[0013] Preferably, the nucleic acid sequence of the coding region encoding the S protein of porcine epidemic diarrhea virus has at least 75% sequence identity with SEQ ID NO: 1; preferably, at least 80% sequence identity; preferably, at least 85% sequence identity; preferably, at least 90% sequence identity; preferably, at least 95% sequence identity; more preferably, at least 99% sequence identity.

[0014] In one embodiment, the DNA molecule further comprises a T7 promoter.

[0015] In one embodiment, the nucleic acid sequence of the T7 promoter in the DNA molecule is shown in SEQ ID NO:2, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:3, the nucleic acid sequence of the Kozak sequence is shown in SEQ ID NO:4, the nucleic acid sequence of the 3'UTR is shown in SEQ ID NO:5, and the nucleic acid sequence of PolyA is shown in SEQ ID NO:6.

[0016] In one embodiment, the nucleic acid sequence of the Kozak sequence can also be shown as SEQ ID NO: 7 (GCC), SEQ ID NO: 8 (ACC), SEQ ID NO: 9 (GCCACC) or SEQ ID NO: 10 (GCCANN, N represents A, T / U, C or G).

[0017] According to a third aspect of the present disclosure, there is provided an S protein of porcine epidemic diarrhea virus expressed by the above mRNA molecule or the above DNA molecule.

[0018] According to a fourth aspect of the present disclosure, a recombinant plasmid is provided, comprising the above-mentioned DNA molecule.

[0019] According to a fifth aspect of the present disclosure, a recombinant host cell is provided, comprising the above-mentioned DNA molecule or the above-mentioned recombinant plasmid.

[0020] According to the sixth aspect of the present disclosure, an mRNA vaccine preparation is provided, comprising the above-mentioned mRNA molecule and a vector.

[0021] In one embodiment, the carrier is a lipid nanoparticle or a polymer nanoparticle.

[0022] In a preferred embodiment, the carrier is a lipid nanoparticle.

[0023] In one embodiment, the mRNA vaccine preparation is a vaccine preparation for preventing porcine epidemic diarrhea virus infection.

[0024] In particular, the mRNA vaccine preparation can protect piglets from being infected with the porcine epidemic diarrhea virus by immunizing sows.

[0025] In one embodiment, the mRNA vaccine preparation is a vaccine preparation for preparing antibodies against the S protein of porcine epidemic diarrhea virus after immunizing animals.

[0026] According to a seventh aspect of the present disclosure, there is provided use of the above-mentioned mRNA molecule, the above-mentioned DNA molecule, the above-mentioned recombinant plasmid or the above-mentioned recombinant host cell in the preparation of a medicament for preventing porcine epidemic diarrhea virus infection.

[0027] In one possible embodiment, the drug is a vaccine.

[0028] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:

[0029] The mRNA vaccine disclosed in the present invention can transfer the porcine epidemic diarrhea virus antigen encoded by mRNA into the target cells of the host, thereby inducing the host cells to produce corresponding antibodies. When the pathogen carrying the antigen invades again, the host's immune system can quickly trigger humoral and cellular immune responses, thereby preventing the occurrence of porcine epidemic diarrhea disease. This vaccine is designed based on the S protein (G2a type) of the porcine epidemic diarrhea virus strain prevalent in the local area. It has fast antibody production, high protection rate, and can effectively protect against mutant strains. After the pregnant sows are immunized with the above-mentioned mRNA vaccine before delivery, high levels of neutralizing antibodies can be produced in the serum and breast milk of the sows, which has a significant protective effect on piglets and can effectively prevent piglets from being infected with porcine epidemic diarrhea virus.

[0030] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0032] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0033] Figure 1 The electrophoresis result diagram in Example 2 of the present disclosure is shown; wherein, M-Marker, PEDV-S-PEDV S protein;

[0034] Figure 2 The development result diagram of mRNA in Example 2 and mRNA-LNP in Example 3 of the present disclosure is shown; wherein, - represents mRNA and mRNA-LNP without transfection of PEDV S protein in cells, PEDV-S-mRNA represents mRNA transfected with PEDV S protein, and PEDV-S-mRNA-LNP represents mRNA-LNP transfected with PEDV S protein;

[0035] Figure 3 The negatively stained electron microscope observation image of mRNA-LNP in Example 3 of the present disclosure is shown;

[0036] Figure 4 The cryo-electron microscopy observation image of mRNA-LNP in Example 3 of the present disclosure is shown;

[0037] Figure 5 The figure shows the particle size analysis result of mRNA-LNP in Example 3 of the present disclosure;

[0038] Figure 6 The figure shows the antibody titer test result of mRNA-LNP in Example 4 of the present disclosure;

[0039] Figure 7 The figure shows the titer detection result of IgG of PEDV S protein after immunization with mRNA-LNP in Example 4 of the present disclosure;

[0040] Figure 8 The figure shows the titer detection result of neutralizing antibodies in the serum of piglets 28 days after immunization with the mRNA-LNP vaccine in Example 4 of the present disclosure;

[0041] Fig. 9 Shown are photos of symptoms of 3 groups of piglets in Example 5 of the present disclosure after 1 day of infection;

[0042] Fig.10 The photo of feces of piglets in the immune challenge group about one week after the challenge in Example 5 of the present disclosure is shown;

[0043] Fig.11 The anatomical diagrams of the intestines and stomachs of the piglets in the challenge control group and the immune challenge group one week after the challenge in Example 5 of the present disclosure are shown; wherein the first column is the anatomical diagram of the intestines, and the second and third columns are the anatomical diagrams of the stomach. DETAILED DESCRIPTION

[0044] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0045] Example 1

[0046] This embodiment prepares an mRNA vaccine for preventing porcine epidemic diarrhea virus (PEDV) infection, which specifically includes the following steps:

[0047] (1) Sequence design. The core components of the PEDV-mRNA vaccine include 5'UTR, Kozak sequence, S protein encoding PEDV (G2a type, with two proline mutations I1076P and L107

[0048] 7P, whose amino acid sequence is as shown in SEQ ID NO:11: MKSLTYFWLFLPVLSTLSLPQDVTRCSAKT

[0049] NFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFLSHIRGGHGFEIGISQE

[0050] PFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTADNDVTTGRNCLFNKAIPAHMSEHSVVGI

[0051] TWDNDRVTVFSDKIYHFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCT

[0052] ANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAMPKIYGLGQFFS

[0053] FNQTIDGVCNGAAVQRAPEALRFNINDTSVILAEGSIVLHTALGTNLSFVCSNSSDPHLATFTIPLGAT

[0054] QVPYYCFLKVDTYNSTVYKFLALLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDD

[0055] VSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDDGFYPISSRNLLSHEQPISFVT

[0056] LPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNC

[0057] PFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKSLEGVTDV

[0058] SFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYV

[0059] DDDIVGVISSLSSTFNSTRELPGFFYHSNDGSNCTEPVLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVT

[0060] GNISIPTNFSMSIRTEYLQLYNTPVSVDCATYVCNGNSHCKQLLTQYTAACKTIESALQLSARLESVE

[0061] VNSMLTISEEALQLATISSFNGDGYNFTNVLGVSVYDPARGRVVQKRSFIEDLLFNKVVTNGLGTVD

[0062] EDYKRCSNGRSVADLVCAQYYSGVMVLPGVVDAEKLHMYSASLIGGMVLGGFTSAAALPFSYAVQ

[0063] ARLNYLALQTDVLQRNQQMLAESFNSAIGNITSAFESVKEAISQTSKGLNTVAHALTKVQEVVNSQG

[0064] AALTQLTVQLQHNFQAISSSIDDIYSRLDPPSADVQVDRLITGRLSALNAFVAQTLTKYTEVQASRKL

[0065] AQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVNVIAIAGLCVNDEIALT

[0066] LREPGLVLFTHELQDTATEYFVSSRRMYEPRKPTVGDFVQIESCVVTYVNLTRDQLPEVIPDYIDVNK

[0067] TLDEILASLPNRTGPSLSLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNR

[0068]

[0069] ACTCTGTCACCCCATGCTCCTTCAGTGAGCAAGCTGCTTACGTGGATGACGACATCGTAGGGGT

[0070] GATATCTTCTTTAAGCAGCTCAACTTTCAATTCCACACGGGAGCTGCCAGGATTCTTCTACCATA

[0071] GCAATGATGGCTCCAACTGTACAGAACCTGTGTTAGTCTATAGCAATATTGGGGTTTGTAAATCT

[0072] GGGTCAATCGGATACGTCCCATCGCAGTCCGGACAGGTGAAAATTGCCCCCACGGTAACAGGG

[0073] AACATCTCAATCCCAACCAACTTCAGCATGTCCATAAGGACCGAGTACCTGCAGCTGTACAACA

[0074] CTCCAGTGAGTGTGGACTGCGCGACTTATGTGTGTAATGGGAATAGCCACTGCAAGCAATTGCT

[0075] CACCCAGTACACCGCTGCCTGCAAGACTATTGAATCAGCCCTACAGCTGAGCGCGCGATTGGAG

[0076] AGTGTGGAGGTTAACTCCATGCTCACTATTTCTGAGGAAGCGTTACAGCTTGCAACAATTTCGA

[0077] GCTTCAACGGGGACGGTTATAACTTCACCAATGTGCTGGGCGTTAGTGTGTATGACCCGGCCAG

[0078] GGGCAGAGTTGTGCAAAAAAGAAGCTTCATCGAAGACTTGTTATTTAACAAGGTGGTTACTAAC

[0079] GGGCTGGGAACTGTTGATGAAGATTATAAGCGCTGTTCAAATGGACGCAGCGTGGCTGACCTGG

[0080] TCTGCGCCCAGTATTATTCAGGTGTGATGGTCTTGCCTGGCGTCGTAGACGCCGAGAAGCTGCA

[0081] TATGTACAGTGCTAGCCTCATTGGCGGCATGGTGCTGGGAGGGTTCACTTCTGCGGCTGCATTAC

[0082] CCTTCTCCTATGCAGTTCAGGCCAGACTGAACTACCTGGCGTTGCAGACAGATGTGCTCCAACG

[0083] AAATCAACAGATGCTGGCAGAAAGCTTTAATTCTGCTATAGGCAATATTACCTCCGCATTTGAA

[0084] AGCGTAAAAGAAGCCATAAGTCAGACTTCGAAAGGCCTGAATACAGTCGCACATGCATTGACC

[0085] AAAGTTCAGGAGGTTGTCAATTCTCAAGGTGCTGCACTGACACAGTTGACCGTACAACTACAGC

[0086] ACAACTTTCAGGCAATCTCAAGCTCCATTGATGATATTTACAGTAGGCTTGACCCGCCCTCTGCT

[0087] GATGTGCAGGTCGACAGATTGATTACGGGAAGGTTAAGTGCCTTGAACGCCTTCGTGGCTCAGA

[0088] CGCTGACGAAATACACAGAGGTGCAGGCTTCGCGGAAACTAGCACAGCAGAAGGTGAATGAAT

[0089] GTGTGAAGTCTCAGAGCCAGCGATACGGGTTTTGCGGCGGTGATGGAGAGCACATCTTCAGCTT

[0090] GGTTCAGGCCGCCCCGCAGGGGCTGCTGTTCCTGCACACTGTTCTGGTGCCTGGTGATTTTGTTA

[0091] ATGTCATCGCCATCGCGGGGTTGTGCGTTAATGATGAAATCGCGCTGACCCTTCGGGAACCTGG

[0092] TCTGGTACTGTTCACGCACGAGCTTCAGGACACGGCTACAGAGTATTTTGTGTCGTCGCGCCGG

[0093] ATGTATGAGCCCCGCAAGCCCACAGTGGGGGACTTCGTGCAGATTGAGTCTTGCGTCGTCACCT

[0094] ATGTCAATCTCACAAGGGACCAGCTACCAGAAGTGATCCCAGACTACATTGACGTCAACAAAAC

[0095] TCTCGACGAGATCCTGGCCAGCCTGCCCAACCGAACTGGACCATCACTAAGCTTAGACGTGTTT

[0096] AACGCCACTTACCTCAACCTGACAGGCGAGATCGCCGATCTTGAACAGCGGTCAGAATCCCTGA

[0097] GGAACACCACCGAGGAGTTACAAAGCCTGATTTATAATATAAACAATACCCTGGTTGATCTGGA

[0098] GTGGCTGAACAGAGTGGAAACCTACATCAAATGGCCCTGGTGGGTGTGGTTGATCATTTTTATT

[0099] GTGCTGATATTTGTGGTCTCGCTACTGGTGTTCTGTTGCATTTCAACAGGCTGTTGTGGCTGCTGT

[0100] GGGTGTTGCGGGGCTTGCTTTTCCGGTTGCTGTAGAGGACCCCGTCTGCAGCCCTACGAGGCCTTTGAGAAAGTCCACGTGCAATGA as shown), 3'UTR (nucleic acid sequence as SEQ ID NO:5: CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGC AATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC) and PolyA (the nucleic acid sequence is shown in SEQ ID NO:6:AAAAAAAAAAAAAAAAAAAAAAAA

[0101] AAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA).

[0102] (2) Linearization of plasmid template. Gene synthesize the DNA molecule designed above and clone it into the plasmid by recombination to obtain a recombinant plasmid; the sequence is verified to be correct by sequencing. Extract the plasmid and purify the DNA to obtain a linearized template.

[0103] (3) mRNA in vitro transcription. Construct the following reaction system:

[0104]

[0105] After thorough mixing, centrifuge at low speed and incubate at 37°C for 2-4 hours. After terminating the reaction, wash, dry, and dissolve in DNase / RNase-free water to obtain mRNA, which is then stored at -20°C.

[0106] (4) Preparation of mRNA-LNP (lipid nanoparticles).

[0107] Preparation of mRNA aqueous phase: The total volume of the mRNA aqueous phase is about 667 μL (26 μL of mRNA + 320.5 μL, 100 mM sodium citrate (pH 4.0) + 320.5 μL of DNase / RNase-free water). The volume ratio of mRNA phase: Lipid phase is 3:1.

[0108] Preparation of Lipid phase: Take 36.36 μL of ALC-0315, 24.60 μL of DSPC, 55.89 μL of Chole and 3.14 μL of ALC-0159, add anhydrous ethanol to make up the total volume to 222 μL, and mix well.

[0109] Mixing: Set the program: the flow rate of the Lipid channel is 3 mL / min, the flow rate of the mRNA channel is 9 mL / min, use the volume of Lipid phase 0.2 mL, mRNA phase 0.6 mL, and mix the Lipid phase and mRNA phase.

[0110] Calculate the final concentration: After mixing, the measured volume is 550 mL. If the encapsulation efficiency is 90%, the final concentration of mRNA is 0.10125 μg / mL, and the concentration is approximately 100 ng / μL.

[0111] Alcohol removal: dilute with 1×PBS in an ultrafiltration tube and centrifuge to a concentration of approximately 100 ng / μL.

[0112] After filtering with a 0.22μm filter membrane, it can be directly used for animal administration.

[0113] Example 2

[0114] In this example, the quality of the mRNA in Example 1 was evaluated.

[0115] 1. Denaturing PAGE identification of mRNA:

[0116] (1) Add the mRNA loading system to the wells of the denaturing gel and perform electrophoresis at 100 V for 30 min.

[0117] The electrophoresis results are as follows Figure 1 shown. Figure 1 It was shown that the size of the electrophoresis band was consistent with the size of the S protein coding sequence of PEDV, indicating that the preparation of the above mRNA molecule was successful.

[0118] 2. Detection of mRNA expression:

[0119] (1) Prepare 8 EP tubes, add 100 mL of OPTI-MEM medium to each tube, and divide 4 EP tubes into two groups, namely mRNA group and lipo group;

[0120] (2) Add 2 μg of mRNA to the mRNA group and 4 μL of transfection reagent to the lipo group, and mix well; add the mixture of the mRNA group to the lipo group, mix well, and let stand for 20 min;

[0121] (3) Add all the solutions in each tube to a 12-well plate and shake evenly to spread the cells evenly (the cells cultured in the 12-well plate are HEK293T cells); replace the solution or replenish the solution after 4 to 6 hours;

[0122] (4) 24 h after transfection, cells were harvested, placed on ice for 15 min, centrifuged at 13,000 rpm for 15 min, and the supernatant was collected;

[0123] (5) Take 15 μL of the supernatant to prepare the electrophoresis sample, add the sample to the well of SDS-PAGE, and perform electrophoresis at 90 V for 30 min and 180 V for 40 min;

[0124] (6) After transfer, blocking, incubation with primary antibody (PEDV antibody) and secondary antibody, development is performed.

[0125] The development results are as follows Figure 2 shown. Figure 2 The results showed that compared with the control group, a band of about 130 kDa was clearly detected in the cells transfected with PEDV S protein mRNA, indicating that the mRNA was successfully transferred into the cells and expressed in the cells.

[0126] Example 3

[0127] This example evaluates the quality of the mRNA-LNP in Example 1.

[0128] 1. Observation of mRNA-LNP by negative staining electron microscopy:

[0129] Glow discharge, set the discharge time to 60s, current to 15mA, and continue to discharge for 10s; place the mRNA-LNP sample on the grid, add uranyl acetate to stain, dry it, and then use it for negative staining electron microscopy observation. The results are as follows Figure 3 shown. Figure 3 It was shown that small round particles with clear overall contours were observed under an electron microscope, which was consistent with the morphology of lipid nanoparticles.

[0130] 2. mRNA-LNP cryo-electron microscopy observation:

[0131] Glow discharge, gold mesh (Au, 300mesh, R1.2 / 1.3) current was set to 20mA, glow discharge time was 180s, and duration was 10s;

[0132] After the frozen sample was prepared, the sample was observed under a 200kV cryo-electron microscope. Figure 4 shown. Figure 4 It was shown that small round particles were also observed under cryo-electron microscopy, with clear overall contours, which was consistent with the results of negative staining electron microscopy observations.

[0133] 3.DLS (Malvern) particle size characterization:

[0134] Dynamic light scattering was used to measure the average particle size and polydispersity index (PDI) of mRNA-LNP. Figure 5 As shown, the average particle size of mRNA-LNP is 75 nm and PDI=0.04.

[0135] 4. Expression detection of mRNA-LNP:

[0136] Add the mRNA-LNP solution to a 6-well plate and shake evenly to spread the cells (the cells cultured in the 6-well plate are HEK293T cells). Replace the medium or replenish the solution after 4 to 6 hours. After 24 hours of culture, use the WB method to detect the expression of PEDV S protein. The results are as follows: Figure 2 shown. Figure 2 The results showed that compared with the control group, a band of about 130 KDa was clearly detected in the cells transfected with PEDV S protein mRNA-LNP, which was consistent with the band size detected by transfecting PEDV S protein mRNA, indicating that the mRNA-LNP was successfully transferred into the cells and expressed in the cells.

[0137] Example 4

[0138] This example evaluates the immune effect of the mRNA-LNP in Example 1.

[0139] 1. Antibody titer detection of mRNA-LNP:

[0140] In order to detect the antibody level of porcine epidemic diarrhea virus in mouse serum, purified PEDV was used as the coating antigen, and the IgG antibody in the serum of immunized mice (the control group mice were injected with PBS, and the experimental group mice were injected with 7μg of PEDV S protein mRNA-LNP. Two weeks after the first immunization, the same dose was used for booster immunization once 14 days later) was detected by ELISA. The results are shown in the figure. Figure 6 and Figure 7 shown. Figure 6 , Figure 7 It was shown that after mice were immunized with mRNA-LNP, they were induced to produce higher levels of antibodies, that is, a high level of humoral immune response; and the antibody titer was 1:10 to 10000.

[0141] 2. Antibody neutralization test:

[0142] Vero cells were plated into 12-well plates at 0.8-1.0×10 5cells / well; about 8 hours later, Vero cells were infected with PEDV: PEDV was mixed with an equal volume of serum to be tested and incubated, the virus was diluted to 50 PFU / well, and incubated at 37°C, 5% CO2 for 1 hour; 100 μL of PEDV virus and virus / antibody mixture was added to the wells to be infected, and 100 μL of serum-free DMEM medium was added to the control wells. Incubated in a cell culture incubator for 1.5 hours, the virus was recovered, and the cells were cultured for another 12 hours. The pathological changes of the cells were observed. The results are as follows Figure 8 shown. Figure 8 The results showed that compared with PBS injection, the neutralizing antibody titer in the piglet serum increased significantly 28 days after the injection of mRNA-LNP vaccine, which increased by nearly 4 times. This shows that the mRNA-LNP vaccine prepared by the present disclosure can efficiently produce high-titer neutralizing antibodies.

[0143] Example 5

[0144] This example evaluates the protective effect of the mRNA-LNP in Example 1.

[0145] Three healthy primiparous pregnant sows were selected, and serum and anal swabs were collected to detect ASFV, PEDV, and rotavirus A infection. Pregnant sows that were not immunized with PEDV vaccine, ASFV antigen negative and PEDV antigen negative, and in good health were selected for the experiment. The specific experiments are shown in Table 1.

[0146] Table 1

[0147]

[0148]

[0149] After the above test, the test results are shown in Table 2.

[0150] Table 2

[0151]

[0152] The above clinical symptoms can be seen from Fig. 9 , Fig.10 Further confirmation, Fig. 9 The results showed that the piglets in the third group (the challenge control group) had the most severe symptoms one day after the challenge, with diarrhea appearing the earliest, and severe diarrhea and dehydration. All of them refused to eat, and all died within 5 days after the challenge. Some of the piglets in the first and second groups (the immune challenge group) vomited milk and had diarrhea one day after the challenge, and the symptoms appeared later. Their appetite was always good, and all the piglets survived. Some of the piglets with symptoms stopped having diarrhea after one week and had formed feces. Fig.10The results show that the buttocks of the first group of piglets were clean and no diarrhea was observed; the second group of piglets had formed feces. It can be seen that about one week after the challenge, all the piglets in the immune challenge group stopped having diarrhea and other symptoms, had formed feces, and had a good appetite. The above results show that the mRNA vaccine (mRNA-LNP) of the PEDV S protein prepared in Example 1 has a good immune effect. After the sows were immunized with the vaccine, the protection rate of the piglets born to the challenge reached 100%. After the challenge, all the control group died, and all the immunized group survived. After the challenge, some piglets in the immunized group had transient mild symptoms, which recovered after one week.

[0153] In addition, all the piglets in the immune challenge group were killed one week after the challenge, and the intestines and stomachs of the piglets in the challenge control group and the immune challenge group were dissected to observe the changes in the three groups. Fig.11 shown. Fig.11 The results showed that the intestines and stomachs of the piglets in the immunization group were normal without any lesions one week after the infection; however, all the piglets in the infection control group had died one week after the infection, and their intestines and stomachs also had lesions.

[0154] The above results show that injection of the mRNA vaccine prepared in Example 1 can effectively protect piglets.

[0155] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.

[0156] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0157] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. An mRNA molecule, characterized in that The mRNA molecule comprises a coding region, wherein the coding region encodes the S protein of porcine epidemic diarrhea virus; the porcine epidemic diarrhea virus is of G2a type, and two proline mutations I1076P and L1077P are introduced into the S protein.

2. The mRNA molecule according to claim 1, characterized in that The mRNA molecule further comprises a 5'UTR, a Kozak sequence, a 3'UTR and PolyA.

3. A DNA molecule for transcribing the mRNA molecule according to any one of claims 1 to 2, characterized in that: The nucleic acid sequence of the coding region encoding the S protein of porcine epidemic diarrhea virus is shown in SEQ ID NO:1, or the nucleic acid sequence of the coding region encoding the S protein of porcine epidemic diarrhea virus has at least 70% sequence identity with SEQ ID NO:

1.

4. The S protein of porcine epidemic diarrhea virus expressed by the mRNA molecule according to any one of claims 1 to 2 or the DNA molecule according to claim 3.

5. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the DNA molecule according to claim 3.

6. A recombinant host cell, characterized in that The recombinant host cell comprises the DNA molecule according to claim 3 or the recombinant plasmid according to claim 5.

7. An mRNA vaccine preparation, characterized in that: The mRNA vaccine preparation comprises the mRNA molecule according to any one of claims 1 to 2 and a vector.

8. The mRNA vaccine preparation according to claim 7, characterized in that The carrier is a lipid nanoparticle or a polymer nanoparticle; Preferably, the mRNA vaccine preparation is a vaccine preparation for preventing porcine epidemic diarrhea virus infection; Preferably, the mRNA vaccine preparation is a vaccine preparation that protects piglets from being infected with the porcine epidemic diarrhea virus by immunizing sows.

9. The mRNA vaccine preparation according to claim 7, characterized in that The mRNA vaccine preparation is a vaccine preparation for preparing antibodies against the S protein of porcine epidemic diarrhea virus after immunizing animals.

10. Use of the mRNA molecule according to any one of claims 1 to 2, the DNA molecule according to claim 3, the recombinant plasmid according to claim 5 or the recombinant host cell according to claim 6 in the preparation of a medicament for preventing porcine epidemic diarrhea virus infection.

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