MRNA freeze-dried vaccine of porcine epidemic diarrhea virus as well as construction method and application of mRNA freeze-dried vaccine

Abstract

The invention belongs to the technical field of veterinary biological products, and relates to an mRNA freeze-dried vaccine of a porcine epidemic diarrhea virus as well as a preparation method and application of the mRNA freeze-dried vaccine. When the mRNA vaccine is prepared, firstly, the S gene sequences of the porcine epidemic diarrhea virus GIIa type and the porcine epidemic diarrhea virus GIIc type which are separated and stored in a laboratory are selected; the preparation method comprises the following steps: firstly, carrying out codon optimization, then respectively connecting with 5 'UTR, 3' UTR, a termination codon and a polyA tail nucleic acid sequence in series, connecting to a vector, carrying out in-vitro template transcription, carrying out in-vitro transcription, capping and purifying to obtain mRNA, and finally, encapsulating and freeze-drying to prepare the mRNA vaccine. The invention further provides application of the vaccine to immunization of pigs, cellular immunity and humoral immunity in the pigs can be stimulated, the immunogenicity and protective efficacy of the vaccine are superior to those of commercially available bivalent live vaccines, and the vaccine can be used for preventing porcine diarrhea caused by infection of currently epidemic PEDV GIIa and GIIc type strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of veterinary biological products, and relates to a mRNA freeze-dried vaccine of porcine epidemic diarrhea virus and a construction method and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea is a highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV), which is mainly characterized by watery diarrhea, vomiting, dehydration and death in piglets. Multiple laboratory studies have shown that genetic variation of PEDV is the main reason for immune failure and high mortality in piglets, mainly manifested as: compared with the classic strain CV777 (GI type), the S gene of the clinical isolate (GII type) has multiple deletions and insertions. Currently, GII type strains are mainly prevalent in farms. With the epidemic variation of the virus, PEDV is further divided into GⅠa, GⅠb, GⅠc, GⅠd, GⅡa, GⅡb, GⅡc, GⅡd and GⅢ, etc. 9 subtypes, different strains lack complete cross protection, increasing the difficulty of PED prevention and control. It is worth noting that a high-morbidity PEDV outbreak in 2010 is a new PEDV variant strain, which is classified as GII type through whole genome phylogenetic analysis. GII type has higher pathogenicity than GI type, and the mortality rate of infected GII type strains in suckling piglets is as high as 100%, seriously endangering the development of the pig industry. Through monitoring and analysis of the causes of diarrhea in pig farms from 2011 to 2021, it can be seen that among the causes of diarrhea in pig farms, GII type PEDV strains have become the main epidemic strains in current pig farms. Another study found that all strains were distributed in 3 subgroups, i.e. 81.5% of GIIa type subgroup, 4.3% of GIIb type subgroup and 14.1% of GIIc type subgroup, indicating that the dominant genotype of the epidemic PEDV strain has changed from GIIb type to GIIa type and GIIc type.

[0003] Currently, commercial PEDV vaccines are mainly inactivated vaccines and attenuated live vaccines, and traditional vaccines cannot meet the current prevention and control needs of PEDV in terms of protection efficacy, safety, etc. Therefore, various new PEDV vaccines, such as subunit vaccines, recombinant live vector vaccines, mRNA vaccines, etc. have become a research hotspot, however, subunit vaccines have poor immunogenicity, short protection duration, recombinant live vector vaccines have antigen genes prone to integration, safety deficiencies, and virulence prone to strengthen, etc. mRNA vaccines do not enter the nucleus, only translate and express antigens in the cytoplasm, foreign genes will not recombine with host genes, and have high safety, etc. With the in-depth research, PEDV mRNA new vaccines will be applied in clinic.

[0004] At present, scholars have done a lot of work on PEDV mRNA vaccine, such as CN113274491A provides an RNA vaccine for swine epidemic diarrhea and a construction method thereof, and CN119491005A provides a swine epidemic diarrhea virus mRNA vaccine and use, but the above patents are only for single genotype, and cannot provide comprehensive and effective immune protection for the current widely prevalent GIIa and GIIc PEDV strains of PEDV, and cannot meet the prevention and control needs of the pig industry for the main epidemic strains; in addition, the vaccines provided by the above patents are in liquid state, and the liquid vaccine is sensitive to temperature, light and other environmental conditions during storage and transportation, and needs strict cold chain guarantee. In actual application, any omission of the cold chain link may lead to reduced vaccine quality, reduced activity, affect the immune effect, increase the cost and risk. At the same time, the liquid vaccine has a short shelf life, compared with the freeze-dried vaccine, the liquid vaccine is not conducive to long-term storage and large-scale popularization and application, and it is difficult to meet the vaccine storage needs of pig farms in different scenarios. SUMMARY

[0005] The purpose of the present scheme is to provide an mRNA (GIIa and GIIc) freeze-dried vaccine for preventing swine epidemic diarrhea virus. The swine epidemic diarrhea virus GIIa and swine epidemic diarrhea virus GIIc S gene sequences isolated and preserved in the laboratory are selected, and after codon optimization, they are connected with 5'UTR, 3'UTR, stop codon and polyA tail nucleic acid sequences in series, and connected to a pUC57 vector in vitro transcription template. After in vitro transcription, capping and purification, the mRNA vaccine is prepared by freeze-drying after being encapsulated by lipid nanoparticles, which can be used to immunize pigs and can stimulate cellular immunity and humoral immunity in pigs, thereby preventing piglet diarrhea caused by swine epidemic diarrhea virus GIIa and GIIc infection.

[0006] In order to achieve the above target, the present application adopts the following technical scheme.

[0007] Firstly, an mRNA freeze-dried vaccine for swine epidemic diarrhea virus is provided, which comprises: a LNP composition of a first mRNA, wherein the first mRNA is a 5'UTR, a RNA sequence encoding a full-length S protein of swine epidemic diarrhea virus GIIa, a stop codon, a 3'UTR and a polyA tail nucleic acid connected in series; a LNP composition of a second mRNA, wherein the second mRNA is a 5'UTR, a RNA sequence encoding a truncated S protein of swine epidemic diarrhea virus GIIc, a stop codon, a 3'UTR and a polyA tail nucleic acid connected in series; and a pharmaceutically acceptable freeze-drying protective agent; The LNP composition of the first mRNA and the LNP composition of the second mRNA are mixed, and then mixed with a freeze-drying protective agent to form a single-dose vaccine. All uridine residues in the RNA sequence encoding the full-length S protein of porcine epidemic diarrhea virus GIIa type and the RNA sequence encoding the truncated S protein of porcine epidemic diarrhea virus GIIc type are replaced by pseudouridine Ψ.

[0008] Further, the RNA sequence encoding the full-length S protein of porcine epidemic diarrhea virus GIIa type is codon-optimized and shown as SEQ ID NO. 1; the RNA sequence encoding the truncated S protein of porcine epidemic diarrhea virus GIIc type is codon-optimized and shown as SEQ ID NO. 2; the nucleic acid sequences of the 5'UTR and the 3'UTR are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the nucleic acid sequence of the polyA tail is shown as SEQ ID NO. 5.

[0009] Further, the lipid nanoparticles in the LNP composition of the first mRNA and the LNP composition of the second mRNA are prepared by mixing cationic lipids, neutral lipids, cholesterol and polyethylene glycol lipids, and the molar ratio of the cationic lipids, neutral lipids, cholesterol and polyethylene glycol lipids is 48:10:39.8:2.2.

[0010] Further, the particle size of the LNP composition of the first mRNA is 77.9 nm, and the particle size of the LNP composition of the second mRNA is 87.2 nm.

[0011] Further, the first mRNA-LNP composition and the second mRNA-LNP composition are mixed in a volume ratio of 1:1, and then mixed with an equal volume of a freeze-drying protective agent solution, which contains 12 w / v% sucrose, 8 w / v% trehalose and 0.5 w / v% mannitol.

[0012] Then, a preparation method of an mRNA freeze-dried vaccine of porcine epidemic diarrhea virus is provided, comprising the following steps: Step 1, the purpose gene sequence encoding the first mRNA fragment and the purpose gene sequence of the second mRNA fragment are respectively connected to a vector to construct an in vitro transcription vector; Step 2, the two in vitro transcription vectors obtained in step 1 are linearized plasmids, and then in vitro transcription, capping and purification are performed to obtain the first mRNA and the first mRNA, respectively; Step 3, the first mRNA and the first mRNA are respectively mixed with an ethanol solution of lipid nanoparticles by microfluidic mixing to form the LNP composition of the first mRNA and the LNP composition of the second mRNA. Step 4, the LNP composition of the first mRNA and the LNP composition of the second mRNA in step 3 are mixed in a volume ratio of 1:1, then an equal volume of a lyophilization protectant solution is added for mixing, and after filling, lyophilization is performed to obtain a lyophilized mRNA vaccine.

[0013] Further, the specific steps of the microfluidic mixing in step 3 are as follows: First, the cationic lipid SM102, neutral lipid DSPC, cholesterol and polyethylene glycol lipid DMG-PEG2K are prepared into a mixed lipid with a molar ratio of 48:10:39.8:2.2 using anhydrous ethanol, and the mixed lipid organic phase solution is prepared into a 20mg / ml mixed lipid organic phase solution by adding anhydrous ethanol; Then, the mRNA in the aqueous phase and the mixed lipid organic phase solution are mixed by a microfluidic mixer at a flow rate ratio of 3:1 to obtain a lipid nanoparticle wrapped with mRNA, i.e. the first mRNA LNP composition and the second mRNA LNP composition; wherein the mRNA end flow rate is 9ml / min and the mixed lipid end flow rate is 3ml / min.

[0014] Further, the lyophilization procedure in step 4 is as follows: (1) pre-freezing, 4℃, 30min, -50℃, 4h; (2) primary drying: -40℃, 0.1pa, 15h; -30℃, 0.1pa, 2h; -20℃, 0.1pa, 2h; -10℃, 0.1pa, 30min; (3) secondary drying: 0℃, 0.1pa, 30min; 4℃, 0.2pa, 6h.

[0015] The use of the above mRNA lyophilized vaccine in the preparation of a medicine for preventing diarrhea caused by PEDV GIIa and GIIc strains in piglets is also proposed.

[0016] Further, the medicine is administered by intramuscular injection to piglets aged 3-5 days, and the same dose is administered again after 21 days, and the injection dose of the medicine for each piglet is 1 mL of the liquid after reconstitution of the lyophilized mRNA vaccine, and the liquid contains 100 μg mRNA.

[0017] Compared with the prior art, the beneficial effects of the present application are as follows: 1. The vaccine is designed for two major epidemic strains of porcine epidemic diarrhea GIIa and GIIc, breaking through the limitations of traditional single strain vaccines, and providing a more comprehensive solution for preventing porcine epidemic diarrhea; 2. The S genes of porcine epidemic diarrhea virus GIIa and GIIc are designed to be different lengths, facilitating subsequent identification and detection; 3. The complete mRNA construction strategy and freeze-drying process are adopted to improve the stability and immune effect of the mRNA vaccine, and solve the key technical problems of the mRNA vaccine in the production and application process; 4. The vaccine using freeze-dried LNP-mRNA form to express PEDV GIIa-S and GIIc-S respectively can induce 3-5 day-old piglets to induce high-titer neutralizing antibodies and achieve 100% homologous challenge protection. The immunogenicity and protective efficacy are superior to the commercial bivalent live vaccine, and can be used for preventing PEDV GIIa and GIIc strain infection. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the linearization result of the fusion plasmid. Among them, M, Marker 5000; 1, pUC57-PEDV-GIIa S plasmid; 2, pUC57-PEDV-GIIa S linearized plasmid; 3, pUC57-PEDV-GIIc S plasmid; 4, pUC57-PEDV-GIIc S linearized plasmid.

[0019] Figure 2 is the mRNA transcription detection result. Among them, A, GIIa S mRNA; B, GIIc S mRNA.

[0020] Figure 3 is the mRNA-LNP cell level expression detection result. Among them, M, protein Marker; 1, GIIa S; 2, cell control; 3, GIIc S.

[0021] Figure 4 is the morphology of the vaccine after freeze-drying.

[0022] Figure 5 is the safety test result of the porcine epidemic diarrhea virus mRNA vaccine (GIIa type and GIIc type) on the minimum use day-old piglets. Among them, A, temperature monitoring result; B, daily gain change result.

[0023] Figure 6 is the efficacy test of the porcine epidemic diarrhea virus mRNA vaccine (GIIa type and GIIc type). Among them, A, PEDV GIIa antibody detection result; B, PEDV GIIc antibody detection result. DETAILED DESCRIPTION

[0024] The scheme of the present application is explained in detail below through the following examples.

[0025] Example 1 An mRNA freeze-dried vaccine (GIIa type and GIIc type) for porcine epidemic diarrhea virus and its construction. An mRNA freeze-dried vaccine (GIIa type and GIIc type) for porcine epidemic diarrhea virus and its construction.

[0026] 1. Construction of recombinant plasmid According to the porcine epidemic diarrhea virus GIIa type and porcine epidemic diarrhea virus GIIc type S gene sequence isolated and preserved in the laboratory, the full-length GIIa type S gene (4287 bp) and the truncated GIIc type S gene (2526 bp) mRNA transcription templates were designed, and the codon preference for expression in pigs was adjusted and the GC content was increased (the adjusted GIIa type S gene sequence is shown in SEQ ID NO. 1 and the truncated GIIc type S gene sequence is shown in SEQ ID NO. 2).

[0027] The adjusted GIIa type S gene sequence and the truncated GIIc type S gene sequence were respectively connected with 5'UTR (SEQ ID NO. 3), 3'UTR (SEQ ID NO. 4), stop codon, and polyA tail nucleic acid sequence (SEQ ID NO. 5) in series to construct an in vitro transcription template to pUC57 vector, and pUC57-PEDV-GIIa S and pUC57-PEDV-GIIc S were obtained. When preparing RNA, uracil (U) was replaced with pseudouridine (Ψ) to reduce immunogenicity.

[0028] 2. In vitro transcription, capping and purification Selection of restriction enzyme B spQ I was used to linearize pUC57-PEDV-GIIa and pUC57-PEDV-GIIc plasmids, and the linearization conditions were: 50°C for 1h; the linearization system was: B spQ I 20μl; plasmid 200μg; 10×Buffer 100μl; supplemented with H2O to 1ml. The linearized plasmid was recovered by isopropanol precipitation. The linearization results showed that pUC57-PEDV-GIIa S and pUC57-PEDV-GIIc S were completely linearized. Figure 1 )。

[0029] Subsequently, the linearized plasmid DNA was subjected to in vitro transcription reaction, and the reaction system was as shown in Table 1.

[0030] Table 1 Transcription reaction system and conditions

[0031] After reaction in a 37°C constant temperature shaker for 3h, 10μl Rnase-Free DNase I enzyme was added to the system to remove DNA, and the reaction was carried out in a 37°C constant temperature shaker for 15~30min.

[0032] The product after in vitro transcription was added with LiCl to make its final concentration 2.5M, and was put into -20℃ for precipitation for 30min, centrifuged at 12000rpm for 15min at 4℃, the supernatant was aspirated, 75% ethanol pre-cooled at -20℃ was added into the centrifuge tube, and the RNA was washed gently by up-down overturning, and the operation was repeated for 2 times. After the last time, the excess ethanol was aspirated, and the mRNA was dissolved after air drying at room temperature. The mRNA was detected, and the results showed that the mass concentrations of GIIa S mRNA and GIIc S mRNA were 2443ng / μl and 2293ng / μl respectively, the OD 260 / 280 ratios were all between 1.8~2.0, and the capillary gel electrophoresis results showed that there were single peaks near 4636bp and 2696bp respectively, and there was no degradation. It was shown that the mRNA had high purity and good integrity. Figure 2

[0033] 3, mRNA encapsulation The cationic lipid (SM102), neutral lipid (DSPC), cholesterol and polyethylene glycol lipid (DMG-PEG2K) were prepared into mixed lipids according to the molar ratio of 48:10:39.8:2.2 using anhydrous ethanol, and then anhydrous ethanol was added to prepare an organic phase solution of 20mg / ml. The organic phase solution was filtered through a 0.22μm filter membrane and stored at -20℃. Before use, it was fully recovered to room temperature and completely dissolved.

[0034] According to the amount of encapsulated RNA, the volume of the water phase (mRNA) and the organic phase (mixed lipids) was calculated (water phase volume: organic phase volume = 3:1), and mixed through a microfluidic mixer at a flow rate ratio of 3:1, with the mRNA end flow rate being 9ml / min and the mixed lipid end flow rate being 3ml / min. After the sample was collected, a certain volume of dialysis solution (20mM Tris+10.7mM sodium acetate pH=8.5) was added, and the sample was dialyzed at 4℃ for 2h, and then the dialysis solution was replaced. After dialysis, the sample was concentrated by ultrafiltration tube, and sucrose solution was added as a protective agent at a final concentration of 12%, and named as GIIa S mRNA-LNP and GIIc S mRNA-LNP. The particle size, PDI, isoelectric potential, encapsulation rate and other indicators of the above preparations were detected, and the results are shown in Table 2.

[0035] Table 2 mRNA-LNP preparation detection results

[0036] 4, Western blot verification of mRNA-LNP expression ​293T cells were inoculated in 6-well plates, and when the cells grew to 90%, 2 μg of GIIa S mRNA-LNP and GIIc S mRNA-LNP were added dropwise, respectively, and cultured in a 37°C, 5% CO2 incubator for 24 h, and the cells were collected. Subsequently, Western blot was performed, using a mouse PEDV S protein antibody as a primary antibody and an HRP-labeled goat anti-mouse IgG (H+L) as a secondary antibody, to specifically detect and analyze the target protein.

[0037] The results show that the GIIa S target protein appears specific bands near 180 kDa, and the GIIc S target protein appears specific bands near 100 kDa-150 kDa, while the cell control has no corresponding bands. Figure 3 The above results show that the LNP-wrapped GIIa S mRNA-LNP and GIIc S mRNA-LNP can be effectively protected and delivered into cells and complete translation.

[0038] 5. Vaccine preparation and freeze-drying The qualified GIIa S mRNA-LNP and GIIc S mRNA-LNP were diluted to 200 μg / ml, mixed at a ratio of 1:1, and then mixed with a freeze-drying protective agent (12% sucrose, 8% trehalose, and 0.5% mannitol) at the same ratio to prepare the vaccine, and then uniformly mixed and quantitatively packaged.

[0039] After packaging, rapid freezing and vacuum drying were performed. The freeze-dried vaccine presents a white cake-like loose body. Figure 4

[0040] Table 3 Freeze-drying program

[0041] Example 2Safety test of porcine epidemic diarrhea virus mRNA vaccine (GIIa type and GIIc type) on minimum age piglets.

[0042] Three to five-day-old piglets were selected and randomly divided into two groups, with five piglets in each group. The mRNA vaccine prepared in Example 2 was reconstituted with a diluent to 200 μg / 2 ml per piglet, and healthy piglets were inoculated by neck muscle, and a blank control group was set up at the same time.

[0043] All piglets were clinically observed from 3 days before inoculation to 14 days after inoculation, and the spirit, respiration, and feeding of the piglets were recorded. The inoculation site was observed for redness and swelling. Specific symptoms such as diarrhea, vomiting, and dehydration were observed. The temperature of all piglets was measured for 10 days, with the basal body temperature being the average of the temperature on the 2 days before inoculation and the day of inoculation. The body weight changes of all piglets were statistically analyzed from the day of inoculation and 14 days after inoculation.​

[0044] Results show that: the trial of mRNA vaccine over-dose vaccination of 3-5 day-old piglets, all piglets have no abnormal reaction in the whole observation period of spirit, breathing, feeding; and the absorption of the vaccination site is good, no red and swollen situation, all piglets are healthy and alive after vaccination, and no specific symptoms such as diarrhea, vomiting and dehydration appear (Table 4); after vaccination of 3-5 day-old piglets, the body temperature is slightly elevated on the first day, but all of them are not more than 0.5℃ of the basic body temperature (A); the piglets grow normally, and the average daily gain of the vaccine inoculation group has no significant difference compared with the blank control group (B). The above test results confirm that the porcine epidemic diarrhea virus mRNA vaccine (GIIa and GIIc) is safe for over-dose vaccination of 3-5 day-old piglets. Figure 5 Figure 5

[0045] Table 4 Clinical symptom observation results of over-dose vaccination of 3-5 day-old piglets

[0046] Example 3 Porcine epidemic diarrhea virus mRNA vaccine (GIIa and GIIc) efficacy test.

[0047] 1. Experimental animals and grouping and inoculation.

[0048] 30 healthy susceptible piglets of 3-5 days old were divided into 6 groups, 5 in each group. The first and third groups were immunized with porcine epidemic diarrhea virus mRNA vaccine (GIIa and GIIc), and the vaccine was diluted to 100 μg / head portion / ml, and 1.0 head portion was injected into the neck muscle; the second and fourth groups were immunized with commercial porcine infectious gastroenteritis and porcine epidemic diarrhea bivalent live vaccine (WH-1R strain + AJ1102-R strain) by neck muscle injection, 1.0 head portion was immunized; the fifth group was PEDV HB17 strain (GIIa) challenge control group; the sixth group was PEDV HBX24 (GIIc) challenge control group.

[0049] The second immunization was performed 21 days after the first immunization.

[0050] 2. Neutralizing antibody detection Blood was collected 21 days after the second immunization, and serum was separated. PEDV neutralizing antibody detection was performed, and the strains used in the neutralizing antibody detection test were PEDV HB17 strain (GIIa) and PEDV HBX24 (GIIc), respectively.

[0051] 3. Challenge strain and challenge procedure ​​On day 21 after the second immunization (the day of blood collection for the detection of neutralizing antibodies), the piglets in groups 1, 2 and 5 were orally administered 10.0 ml of PEDV HB17 (GIIa type) strain virus solution (virus content 10 6.0 TCID 50 / ml), and the piglets in groups 3, 4 and 6 were orally administered 10.0 ml of PEDV HBX24 (GIIc type) strain virus solution (virus content 10 6.0 TCID 50 / ml) (Table 5). The incidence and protection of each group were recorded and counted.

[0052] Table 5 Grouping of test animals for challenge

[0053] 4、Results (1) The results of the detection of neutralizing antibodies showed that the porcine epidemic diarrhea virus mRNA vaccine (GIIa type and GIIc type) and the commercial vaccine could induce the production of neutralizing antibodies against different PEDV genotypes (GIIa type and GIIc type), and the neutralizing antibody titers of the mRNA vaccine were higher than those of the commercial bivalent live vaccine (as shown in Table 7). Figure 6

[0054] (2) The results of the PEDV challenge protection test showed that the mRNA vaccine achieved 5 / 5 protection after challenge with PEDV HB17 strain and PEDV HBX24 strain; the commercial bivalent live vaccine achieved 5 / 5 protection after challenge with PEDV HB17 strain and 3 / 5 protection after challenge with PEDV HBX24 strain, and the different challenge control groups all had 5 / 5 incidence (as shown in Table 6).

[0055] The above results show that the porcine epidemic diarrhea virus mRNA vaccine (GIIa type and GIIc type) against different representative PEDV strains (GIIa type and GIIc type) has better immunoprotection effect than the commercial bivalent live vaccine.

[0056] Table 6 Challenge results of mRNA vaccine and commercial bivalent live vaccine immunized piglets against different PEDV strains

[0057] Although the present application has been described in detail in the foregoing description with general principles and specific embodiments thereof, it should be further appreciated that modifications and improvements to the application herein disclosed will occur to those skilled in the art. Accordingly, the modifications and improvements are intended to be included within the scope of the application.​

Claims

1. A freeze-dried mRNA vaccine for porcine epidemic diarrhea virus, characterized in that, The vaccines include, The LNP composition of the first mRNA, wherein the first mRNA is a 5'UTR, an RNA sequence encoding the full-length S protein of porcine epidemic diarrhea virus type GIIa, a stop codon, a 3'UTR, and a polyA tail nucleic acid connected in sequence; The LNP composition of the second mRNA, wherein the second mRNA is a 5'UTR, an RNA sequence encoding a truncated S protein of porcine epidemic diarrhea virus GIIc type, a stop codon, a 3'UTR, and a polyA tail nucleic acid connected in sequence; And pharmaceutically acceptable lyophilization protectants; The LNP composition of the first mRNA and the LNP composition of the second mRNA are mixed and then mixed with a lyophilization protectant to form a single-dose vaccine. In the RNA sequences encoding the full-length S protein of porcine epidemic diarrhea virus type GIIa and the truncated S protein of porcine epidemic diarrhea virus type GIIc, all uridine residues are replaced by pseudouridine Ψ.

2. The mRNA freeze-dried vaccine according to claim 1, characterized in that, The RNA sequence encoding the full-length S protein of porcine epidemic diarrhea virus GIIa, after codon optimization, is shown in SEQ ID NO.1; the RNA sequence encoding the truncated S protein of porcine epidemic diarrhea virus GIIc, after codon optimization, is shown in SEQ ID NO.2; the nucleic acid sequences of the 5'UTR and 3'UTR are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and the polyA-tailed nucleic acid sequence is shown in SEQ ID NO.

5.

3. The mRNA freeze-dried vaccine according to claim 1, characterized in that, The lipid nanoparticles in the LNP composition of the first mRNA and the LNP composition of the second mRNA are formulated by mixing cationic lipids, neutral lipids, cholesterol and polyethylene glycol-modified lipids, with a molar ratio of 48:10:39.8:2.

2.

4. The mRNA freeze-dried vaccine according to claim 3, characterized in that, The particle size of the LNP composition of the first mRNA is 77.9 nm, and the particle size of the LNP composition of the second mRNA is 87.2 nm.

5. The mRNA freeze-dried vaccine according to claim 4, characterized in that, The first mRNA-LNP composition and the second mRNA-LNP composition are mixed at a volume ratio of 1:1, and then mixed with an equal volume of a lyophilization protectant solution, wherein the lyophilization protectant solution contains 12 w / v% sucrose, 8 w / v% trehalose and 0.5 w / v% mannitol.

6. A method for preparing a freeze-dried mRNA vaccine of porcine epidemic diarrhea virus as described in any one of claims 1 to 5, characterized in that, Includes the following steps, Step 1: Connect the target gene sequence encoding the first mRNA fragment as described in claim 1 and the target gene sequence encoding the second mRNA fragment to a vector respectively to construct an in vitro transcription vector; Step 2: Linearize the two in vitro transcription vectors obtained in Step 1 into plasmids, then perform in vitro transcription, capping, and purification to obtain the first mRNA and the first mRNA, respectively. Step 3: Mix the first mRNA and the first mRNA with an ethanol solution of lipid nanoparticles via microfluidics to form an LNP composition of the first mRNA and an LNP composition of the second mRNA. Step 4: Mix the LNP composition of the first mRNA and the LNP composition of the second mRNA from Step 3 at a volume ratio of 1:1, then add an equal volume of lyophilization protectant solution, mix, fill, and lyophilize to obtain the lyophilized mRNA vaccine.

7. The preparation method according to claim 6, characterized in that, The specific steps of microfluidic mixing in step 3 are as follows: First, cationic lipid SM102, neutral lipid DSPC, cholesterol, and polyethylene glycol-modified lipid DMG-PEG2K were mixed with anhydrous ethanol at a molar ratio of 48:10:39.8:2.2 to prepare a mixed lipid organic phase solution. Then, anhydrous ethanol was added to prepare a mixed lipid organic phase solution of 20 mg / ml. Then, the aqueous mRNA and the lipid-mixed organic phase solution were mixed at a flow rate ratio of 3:1 using a microfluidic mixer to prepare lipid nanoparticles encapsulated with mRNA, namely, the LNP composition of the first mRNA and the LNP composition of the second mRNA; wherein the flow rate of the mRNA end was 9 ml / min and the flow rate of the lipid-mixed end was 3 ml / min.

8. The preparation method according to claim 6, characterized in that, The freeze-drying process is as follows: (1) Pre-freeze at 4℃ for 30 min, then at -50℃ for 4 h; (2) Single drying: -40℃, 0.1pa, 15 h; -30℃, 0.1pa, 2 h; -20℃, 0.1pa, 2 h; -10℃, 0.1pa, 30 min; (3) Secondary drying: 0℃, 0.1pa, 30min; 4℃, 0.2pa, 6h.

9. Use of the mRNA freeze-dried vaccine according to any one of claims 1 to 5 in the preparation of a medicament for preventing diarrhea in piglets caused by infection with PEDV strains GIIa and GIIc.

10. The use according to claim 9, characterized in that, The drug is administered intramuscularly to piglets aged 3-5 days, and the same dose is given again 21 days later. The drug injection dose for each piglet is 1 mL of the reconstituted lyophilized mRNA vaccine liquid, which contains 100 μg mRNA.

Images