G9 type porcine rotavirus mRNA vaccine and preparation method thereof

Abstract

The invention discloses a G9 type porcine rotavirus mRNA vaccine and a preparation method thereof, and relates to the technical field of biological medicines. The preparation method comprises the step of carrying out coating treatment on VP7-mRNA by utilizing lipidosome to prepare the mRNA vaccine, the VP7-mRNA comprises a 5UTR (Untranslated Region), a Kozak sequence, a PoRV G9 type VP7 sequence of which the nucleotide sequence is shown as SEQ ID NO.2, three termination codons, a 3UTR (Untranslated Region) and a polyA tail. The mRNA vaccine of the G9 type porcine rotavirus provides an effective means for prevention and control of the porcine rotavirus, after target animals are immunized, high-level neutralizing antibodies resisting the porcine rotavirus can be generated in the bodies of the target animals, piglet diarrhea caused by the porcine rotavirus can be effectively reduced, and the mRNA vaccine of the G9 type porcine rotavirus has important popularization value for prevention and control of the porcine rotavirus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a mRNA vaccine of G9 type porcine rotavirus and a preparation method thereof. BACKGROUND

[0002] Porcine rotavirus (PoRV) is an acute enteric infectious disease pathogen that seriously endangers the health of piglets, mainly causing watery diarrhea, vomiting and dehydration in piglets. The disease has a high incidence and a certain lethal effect on newborn piglets. In addition, it has strong resistance to the environment and is difficult to completely eliminate, and is a common hidden killer in pig farms. PoRV belongs to the Rotavirus genus of Reoviridae family, and its genome is segmented double-stranded RNA, with 11 segments. According to the different VP6 protein antigens, it can be divided into 9 groups, including A-D and F-J, among which the A group rotavirus is the most harmful and the most widespread. Rotavirus encodes 6 structural proteins (VP1-4, VP6 and VP7) and 5 or 6 non-structural proteins (NSP1-5 / 6). The VP7 protein has been proven to be the main protective antigen that can induce neutralizing antibodies.

[0003] The latest epidemiological data shows that G9 type porcine rotavirus accounts for more than 70%. Vaccination is still the most effective method to prevent and control infectious diseases. At present, the types of vaccines mainly include inactivated vaccine, attenuated live vaccine, protein subunit vaccine, genetic engineering live vector vaccine and nucleic acid vaccine, etc. There is no commercial vaccine for PoRV (G9 type) at present, and further research and development of safe and effective vaccines to control PoRV infection are of great significance for the prevention and control of PoRV. mRNA vaccine belongs to nucleic acid vaccine, which has been proven to be an effective means to control viral diseases. It can introduce mRNA containing antigen protein coding into human or animal body, directly translate to produce corresponding antigen protein, and induce specific immune response of the body to achieve the effect of preventing diseases. In addition, mRNA vaccine has good safety, only uses the gene sequence of the virus but not the virus itself, therefore, mRNA vaccine has the advantages of not containing virus components and no infection risk. At the same time, the production process of mRNA vaccine is simple, does not require cell culture and animal source matrix, has strong specificity, higher effectiveness, short research and development cycle, and can quickly develop new candidate vaccines to respond to virus variation. However, mRNA vaccine involves knowledge of biological information, biochemistry, material chemistry, immunology and many other disciplines, and has great research and development difficulty. Therefore, there are few reports on mRNA vaccines for porcine rotavirus at present, especially the development of mRNA vaccines for G9 type porcine rotavirus is still blank. SUMMARY

[0004] The application aims to provide a G9 type porcine rotavirus mRNA vaccine and a preparation method thereof to solve the problems of the prior art.

[0005] To achieve the above-mentioned purpose, the application provides the following solutions.

[0006] The application provides a preparation method of a G9 type porcine rotavirus mRNA vaccine, characterized by comprising the following steps: coating and treating VP7-mRNA by using liposomes to prepare the mRNA vaccine.

[0007] The VP7-mRNA comprises a 5'UTR with a nucleotide sequence as shown in SEQ ID NO. 1, a Kozak sequence with a nucleotide sequence of GCCACC, a PoRV G9 type VP7 sequence with a nucleotide sequence as shown in SEQ ID NO. 2, three stop codons with a nucleotide sequence of UGAUAGUAA, a 3'UTR with a nucleotide sequence as shown in SEQ ID NO. 5, and a polyA tail with a nucleotide sequence as shown in SEQ ID NO. 6.

[0008] Further, the liposomes comprise ionizable liposomes, distearoylphosphatidylcholine, cholesterol and polyethylene glycol conjugated lipids.

[0009] Further, the ionizable liposomes are (6Z, 9Z, 28Z, 31Z)-heptadeca-6, 9, 28, 31-tetraenoic acid-19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA).

[0010] Further, the polyethylene glycol conjugated lipids are 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-2K-DMG).

[0011] Preferably, the molar ratio of the ionizable liposomes, the distearoylphosphatidylcholine, the cholesterol and the polyethylene glycol conjugated lipids is 50:10:38.5:1.5.

[0012] Further, the coating treatment comprises the step of mixing and reacting a buffer solution containing the VP7-mRNA with an ethanol solution of the liposomes.

[0013] Further, the buffer solution is a citrate buffer solution.

[0014] Further, the volume ratio of the buffer solution containing the VP7-mRNA to the ethanol solution of the liposome is 3:1.

[0015] The application further provides an mRNA vaccine of the G9 type porcine rotavirus prepared by the preparation method.

[0016] The application discloses the following technical effects:

[0017] The application develops an mRNA vaccine of the G9 type porcine rotavirus, reduces the vaccine development and production cycle, and provides an effective means for the prevention and control of the porcine rotavirus.

[0018] The mRNA vaccine has good stability and reduces the generation of by-products, and is optimized in the following ways: (1) the 5'UTR sequence is optimized; (2) the 3'UTR sequence and the length of the polyA tail are optimized; (3) modified nucleotides are added to the sequence; (4) the sequences of the 5'UTR and 3'UTR untranslated regions are optimized; (5) the CDS region sequence is optimized for host codon preference; and (6) the secondary structure of the CDS region of the target antigen VP7 is optimized.

[0019] The design and optimization of the CDS region sequence of the G9 type porcine rotavirus coat protein VP7 can improve the efficiency of expression of the exogenous protein and increase the expression amount of the antigen, so as to induce a strong immune response of the body and generate high-level neutralizing antibodies. The design and optimization of the CDS region sequence of the G9 type porcine rotavirus coat protein VP7 are performed in the following ways: (1) using the codons with higher frequency in the pig body to replace the rare codons in the sequence, so as to increase the protein expression amount; (2) optimizing the GC content of the protein coding sequence, so as to improve the stability of the RNA and increase the protein expression level; (3) optimizing the secondary structure of the coding region, so as to reduce the free energy of the RNA, and the lower the free energy of the RNA, the higher the protein expression amount; (4) optimizing the sequences of the 5'UTR and 3'UTR untranslated regions in the RNA chain; (5) optimizing the restriction endonuclease sites in the RNA chain; and (6) optimizing the repeat region sequence in the RNA chain to remove the sequences that cause instability of the RNA.

[0020] The 5'UTR sequence obtained by optimization can improve the translation efficiency of the target protein, thereby increasing the expression level of the target protein. The 3'UTR sequence obtained by optimization has stronger functions of promoting the expression of the target gene, can play the function of promoting translation in various host cells, has broad spectrum, and shows a wide application prospect in RNA vaccines.

[0021] This invention produces an RNA vaccine through gene synthesis, in vitro transcription, and lipid nanoparticle encapsulation, which can resist diarrhea caused by G9 porcine rotavirus and improve economic efficiency. The mRNA vaccine prepared by this invention, when injected intramuscularly into pregnant sows, enables the sows to produce high levels of neutralizing antibodies before farrowing. Simultaneously, piglets acquire high levels of neutralizing antibodies through suckling, effectively reducing the incidence of diarrhea, dehydration, and vomiting in piglets. This has significant promotional value for the prevention and control of porcine diarrhea and provides an important technical means for the prevention and control of PoRV. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a gel electrophoresis image of the mRNA from Example 1;

[0024] Figure 2 This is a diagram showing the in vitro expression screening results of LNP in Example 3;

[0025] Figure 3 The image shows the particle size and monodispersity characterization of VP7-mRNA-LNP in Example 4.

[0026] Figure 4 The image shows the indirect immunofluorescence detection results of Example 5; where Mock represents the negative control; pCAGGS-VP7 represents the plasmid expressing VP7 in eukaryotes; and mRNA VP7 represents the mRNA-VP7 lipid nanoparticle LNP1.

[0027] Figure 5 This is a Western blotting electrophoresis image of Example 5; where M: standard DNA molecule; Mock: negative control; pCAGGS-VP7 represents a plasmid expressing VP7 in eukaryotes; mRNA VP7 represents mRNA-VP7 lipid nanoparticle LNP1;

[0028] Figure 6 This is a graph showing the detection results of IgG antibody levels in mouse serum in Example 6;

[0029] Figure 7 This is a graph showing the detection results of neutralizing antibody levels in mice in Example 6;

[0030] Figure 8 This is a graph showing the detection results of IgG antibody levels in sow serum in Example 7;

[0031] Figure 9 This is a graph showing the detection results of neutralizing antibody levels in sow serum in Example 7;

[0032] Figure 10 This is a graph showing the detection results of IgG antibody levels in the serum of piglets from immunized sows after 5 days of lactation in Example 8;

[0033] Figure 11 This is a graph showing the detection results of neutralizing antibody levels in the serum of piglets from immunized sows after 5 days of lactation in Example 8;

[0034] Figure 12 This is a schematic diagram of the clinical symptoms of piglets after viral challenge in Example 9;

[0035] Figure 13 This is a statistical chart of diarrhea scores in piglets after passive immunization challenge in Example 9;

[0036] Figure 14 This is a statistical chart showing the viral shedding in the feces of piglets after passive immunization challenge in Example 9. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0041] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous with each other, i.e., to include, but not limited to.

[0042] The present application develops an mRNA vaccine of G9 type porcine rotavirus, which comprises a modified mRNA chain; as shown in Table 1, the modified mRNA chain comprises: a 5'UTR with a nucleotide sequence as shown in SEQ ID NO. 1, a Kozak sequence with a nucleotide sequence of GCCACC, a PoRV G9 type VP7 sequence with a nucleotide sequence as shown in SEQ ID NO. 2, 3 stop codons with a nucleotide sequence of UGAUAGUAA, a 3'UTR with a nucleotide sequence as shown in SEQ ID NO. 5, and a polyA tail with a nucleotide sequence as shown in SEQ ID NO. 6.

[0043] Table 1 Sequences of each segment of the mRNA vaccine of G9 type porcine rotavirus

[0044]

[0045] Example 1

[0046] Design of mRNA vaccine:

[0047] Taking the VP7 glycoprotein of PoRV NJ2012 (GenBank: MT874991.1) as the reference amino acid sequence, the mRNA vaccine was designed. The DNA sequence corresponding to the mRNA shown in Table 1 was synthesized, and the DNA corresponding to SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 was used as the CDS region sequence, respectively, to construct the corresponding three plasmid templates, and then the T7 RNA polymerase and linearized plasmid DNA template were used to synthesize mRNA in vitro, which were named VP7-mRNA SEQ ID NO. 2, VP7-mRNA SEQ ID NO. 3 and VP7-mRNA SEQ ID NO. 4. The above mRNA was detected by agarose gel electrophoresis for band size and purity. The results are as follows Figure 1As shown: the band sizes of VP7-mRNA SEQ ID NO.2, VP7-mRNA SEQ ID NO.3, and VP7-mRNA SEQ ID NO.4 are all around 1300bp. VP7-mRNA SEQ ID NO.2 has the highest purity and no obvious banding (this part of the experiment was completed at Suzhou Huiliao Biomedical Technology Co., Ltd.).

[0048] Example 2

[0049] Preparation of mRNA vaccines:

[0050] The DNA sequences corresponding to the mRNAs shown in Table 1 were synthesized and ligated into an expression vector to construct a linearized plasmid DNA template. Then, using T7 RNA polymerase and the linearized plasmid DNA template, mRNA was synthesized in vitro via transcription and named VP7-mRNA (commissioned to Suzhou Huiliao Biomedical Technology Co., Ltd.).

[0051] (6Z,9Z,28Z,31Z)-docosahexaenoic acid-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (Dlin-MC3-DMA; an ionizable lipid), distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-2K-DMG; a polyethylene glycol-conjugated lipid) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (to bring the total concentration of all liposomes to 6.56 mM) to obtain a lipid ethanol solution. The three VP7-mRNAs were then diluted separately in 50 mM citrate buffer (pH=4) to obtain three mRNA solutions (VP7-mRNA SEQ ID NO.2, VP7-mRNA SEQ ID NO.3, and VP7-mRNA SEQ ID NO.4). Three mRNA-VP7 lipid nanoparticles (LNPs) were prepared by mixing three mRNA solutions with lipid ethanol solutions at a volume ratio of 3:1 using a microfluidic device (Mianna I NanoL). After removing the ethanol, the mixtures were filtered through a 0.22 μm sterile filter. The lipid nanoparticles prepared using VP7-mRNA SEQ ID NO.2, VP7-mRNA SEQ ID NO.3, and VP7-mRNA SEQ ID NO.4 were named LNP1, LNP2, and LNP3, respectively, and stored at 4°C for later use.

[0052] Example 3

[0053] mRNA-VP7 in vitro expression screening:

[0054] The LNP1, LNP2, and LNP3 prepared in Example 2 above were respectively administered at 4 μg / 1×10 6 Cells were transfected into 293T cells cultured in 6-well plates. Cells were collected 24 hours later, and in vitro expression was detected using Western blotting. The specific steps for detecting LNP target protein expression were as follows: Transfected 293T cells were collected, lysis buffer and SDS-loading were added, and the cells were boiled for denaturation; 10 μL of sample was loaded, and electrophoresis was performed at 80 V for 30 min, then at 120 V for 60 min; transfer to a membrane at 200 mA for 90 min; the membrane was washed three times with TBST, blocked with milk; washed three times with TBST, incubated with Anti-His-HRP antibody; and developed after three washes with TBST. Experimental results are as follows: Figure 2 As shown in the figure. The results showed that LNP1 had significant expression of the target protein, while LNP2 and LNP3 showed weaker expression.

[0055] Example 4

[0056] The LNP1, LNP2, and LNP3 particles prepared in Example 2 were measured using a Malvern nanoparticle size analyzer. The particle size and polydispersity index (PDI) were determined by dynamic light scattering. The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK). The results are shown in Table 2: The prepared mRNA-VP7 lipid nanoparticles, with LNP1 having a size of 77.60 nm and a polydispersity index varying around 0.1, exhibited an encapsulation efficiency >95%, demonstrating good physicochemical properties. For vaccine efficacy evaluation, they were named mRNA-VP7 vaccine (e.g., ...). Figure 3 (as shown in the figure). LNP2 and LNP3 have particle sizes >100nm, PDI >0.2, and encapsulation efficiency <80%, which are poor physicochemical properties and will not be included in subsequent studies.

[0057] Table 2 Characterization of mRNA-VP7 lipid nanoparticles

[0058]

[0059] Example 5

[0060] In vitro transfection and antigen expression analysis:

[0061] HEK293T cells were used at a rate of 5 × 10⁻⁶ 5Cells were seeded at a density of 100 μg / well in 24-well plates and cultured overnight at 37°C with 5% CO2. Cells were transfected with mRNA-VP7 lipid nanoparticles (1 μg / well) and a eukaryotic VP7 expression plasmid (1 μg / well) using Lip3000 and cultured for another 24 h. After culture, the culture supernatant was discarded, and the cells were washed once with 1×PBS. Untreated cells served as the control group. Cells were fixed with cold anhydrous ethanol, and their expression was detected using indirect immunofluorescence (IFA). Cells were lysed with 80 μL of cell lysis buffer (RIPA) per well, and 5×Loading Buffer was added according to the specified ratio. The cells were boiled at 100°C for 10 min, and Western blotting was performed to detect mRNA expression. The primary antibody was a murine monoclonal antibody against VP7, and the secondary antibodies were FITC-labeled goat anti-mouse fluorescent secondary antibody and HRP-labeled goat anti-mouse antibody. IFA results are shown below. Figure 4 As shown, the mRNA-VP7 vaccine can be efficiently expressed in 293T cells. Western blotting results are as follows: Figure 5 As shown, this indicates that mRNA-VP7 can effectively express the target protein in vitro.

[0062] Example 6

[0063] Lipid nanoparticle-encapsulated RNA vaccine immunization in mice and antibody level detection:

[0064] Ten 6-week-old BALB / c female mice were randomly divided into two groups of five each. The experimental group (VP7-mRNA group) received a subcutaneous injection of the mRNA vaccine prepared in Example 2 at a dose of 10 μg per mouse. The control group (Buffer group) received a subcutaneous injection of an equal volume of PBS. Vaccinated mice received a booster dose 14 days after vaccination, at the same dose as the initial immunization. Serum samples were collected before immunization and at 2, 4, and 6 weeks after the initial immunization. The levels of neutralizing antibodies in the serum were detected using an indirect ELISA method and a neutralization assay.

[0065] Indirect ELISA method:

[0066] (1) The purified VP7-1a protein was coated with carbonate buffer at pH 9.6 at a concentration of 1 μg / mL and added to a polystyrene microtiter plate at 100 μL / well. The plate was then incubated overnight at 4°C.

[0067] (2) On the second day, discard the liquid and wash three times with 200 μL of PBST buffer per well, each time for 5 min.

[0068] (3) Prepare 5% (m / v) skim milk with PBS buffer, 200 μL / well, and block at 37℃ for 2 h. After discarding the blocking solution, wash three times with PBST buffer, 5 min each time.

[0069] (4) Dilute the serum with PBS buffer, take the serum collected after three immunizations, and the negative serum is the serum collected after immunizing mice with PBS. Take 100 μL / well at a ratio of 1:100, incubate at 37℃ for 1 h, discard the primary antibody and wash three times with PBST buffer for 5 min each time.

[0070] (5) Dilute goat anti-mouse HRP-IgG with PBS buffer at a ratio of 1:4000, 100 μL / well, incubate at 37℃ for 1 h, discard the secondary antibody and wash three times with PBST buffer, 5 min each time.

[0071] (6) Add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) per well and incubate at room temperature in the dark for 10 min.

[0072] (7) Add 50 μL of stop solution (2 M H2SO4) per well to terminate the reaction, and read the OD using a microplate reader. 450nm value.

[0073] Neutralization test:

[0074] (1) All serums were aliquoted and placed in a 56℃ water bath for inactivation treatment for 30 min;

[0075] (2) In a 96-well cell culture plate, the inactivated serum was serially diluted to 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280 and 1:2560, with a final volume of 50 μL per well and two replicates for each dilution.

[0076] (3) PoRV virus solution with known titer (AHFY2022 strain, Genbank accession number: OQ979280-OQ979291) was added with trypsin to a final concentration of 10 μg / mL and activated in a 37℃ water bath or metal bath for 60 min. The virus solution was then diluted with maintenance solution to 200 TCID. 50 / 50 μL;

[0077] (4) Add 50 μL of virus dilution to the above serum dilution plate and incubate at 37°C and 5% CO2 for 1 h;

[0078] (5) Once the 96-well plate is covered with a monolayer of MA104 cells, discard the cell culture medium, wash twice with DMEM, transfer 100 μL of the incubated serum and virus mixture obtained in step (4) to the corresponding well, incubate at 37°C and 5% CO2, observe and record the cytopathic effect, and observe for 72 h.

[0079] Negative and positive serum controls were also set up. The neutralizing titer of the serum was defined as the highest dilution that completely suppressed the cytopathic effect (CPE).

[0080] Indirect ELISA test results as follows Figure 6 As shown, the serum of mice showed a high IgG antibody response 2 weeks after the second immunization. At 4 weeks after the second immunization, the immunized mice still maintained a high level of IgG antibodies.

[0081] Neutralization test results as follows Figure 7 As shown, two weeks after the first immunization, the titer of neutralizing antibodies in the serum of immunized mice increased, ranging from 1:40 to 1:80, while the titer of neutralizing antibodies in the serum of control group (Buffer group) mice was less than 1:20. Two weeks after the second immunization, the titer of neutralizing antibodies increased significantly, ranging from 1:320 to 1:640. Four weeks after the second immunization, the titer of neutralizing antibodies was still maintained above 1:320.

[0082] The above results indicate that the mRNA-VP7 vaccine can induce a strong humoral immune response in mice, suggesting that the porcine rotavirus mRNA vaccine prepared in this invention is a candidate vaccine for preventing and controlling diarrhea caused by porcine rotavirus.

[0083] Example 7

[0084] Detection of antibody levels in sows immunized with RNA vaccines encapsulated in lipid nanoparticles:

[0085] Sows were divided into two groups, and were vaccinated with the mRNA-VP7 vaccine prepared in Example 2 and PBS buffer, respectively. The experimental group (mRNA-VP7 group) was immunized with 20 μg of mRNA-VP7 vaccine at two time points, 40 days and 20 days before the expected farrowing date; the control group (Buffer group) was given the same dose of PBS buffer.

[0086] Sow serum was collected before the first vaccination (40 days before farrowing), before the second vaccination (20 days before farrowing), and on the day of farrowing to detect anti-PoRV antibody levels. The results are as follows: Figure 8 As shown in the results, high levels of IgG antibodies were detected in the serum of sows immunized with the mRNA-VP7 vaccine, which were higher than those in the Buffer group.

[0087] The level of neutralizing antibodies in the serum of sows immunized with mRNA-VP7 was detected, and the results are shown in [the table]. Figure 9 The results showed that after two immunizations before farrowing, the neutralizing antibody titer in the sow's serum at farrowing could reach over 1:1024.

[0088] Example 8

[0089] Colostrum from immunized sows provides a means of detecting passive immune antibody levels in suckling piglets.

[0090] To evaluate the passive immunization effect of the vaccine, sows were vaccinated using the experimental grouping and immunization method described in Example 8. After each sow's piglets were breastfed for 5 days, serum samples were collected from the piglets for antibody testing.

[0091] like Figure 10 As shown, the level of anti-PoRV IgG in piglet serum was high. Antibodies were detected in the serum of piglets born to immunized sows, but not in the serum of piglets born to control sows, indicating that antibodies are transferred through colostrum.

[0092] like Figure 11 As shown, the neutralizing antibody level in 5-day-old suckling piglets immunized with mRNA vaccine can reach as high as 1:2048 or more, which is higher than that in the non-immunized control group.

[0093] In summary, mRNA vaccines can provide effective passive immunity to newborn piglets, protecting them against PoRV infection.

[0094] Example 9

[0095] The efficacy of colostrum from immunized sows as passive immunization for suckling piglets:

[0096] To evaluate the passive immunization effect of the vaccine, sows were vaccinated using the experimental grouping and immunization method described in Example 8. After the sows gave birth, piglets from 5 sows vaccinated with mRNA vaccine and piglets from 5 sows in the Buffer group were selected for the experiment. After 5 days of breastfeeding, the piglets were orally administered 2 mL of wild-type PoRV (AHFY2022 strain G9P

[23] ) (Genbank accession number: OQ979280-OQ979291) with a viral titer of 10. 5 TCID 50 ( / mL), observe for 5 consecutive days. For example Figure 12 As shown, the immunized piglets were in good spirits and had clean and tidy coats; the non-immunized challenge group piglets had rough and messy coats and were accompanied by diarrhea symptoms.

[0097] The diarrhea score of piglets after viral challenge is as follows: Figure 13As shown in the figure. The results showed that after challenge with porcine rotavirus, 80% of the piglets born to sows immunized with the mRNA vaccine did not have diarrhea (4 / 5), while all piglets born to sows in the Buffer group had diarrhea (5 / 5).

[0098] Results of fecal virus excretion testing in piglets after viral challenge: Figure 14 As shown in the results, passive immunization with mRNA vaccines can significantly reduce fecal virulence in piglets.

[0099] In summary, mRNA vaccines can provide effective passive immunity to newborn piglets, protecting them against PoRV infection while reducing viral shedding.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an mRNA vaccine against G9 porcine rotavirus, characterized in that, The process includes the step of encapsulating VP7-mRNA with liposomes to prepare the mRNA vaccine. The VP7-mRNA includes a 5ʹUTR as shown in SEQ ID NO.1, a Kozak sequence with the nucleotide sequence GCCACC, a PoRV G9 type VP7 sequence as shown in SEQ ID NO.2, three stop codons with the nucleotide sequence UGAUAGUAA, a 3ʹUTR as shown in SEQ ID NO.5, and a polyA tail as shown in SEQ ID NO.

6.

2. The preparation method according to claim 1, characterized in that, The liposomes include ionizable liposomes, distearate phosphatidylcholine, cholesterol, and polyethylene glycol conjugated lipids.

3. The preparation method according to claim 2, characterized in that, The ionizable liposome is (6Z,9Z,28Z,31Z)-heptadecanoic acid-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate.

4. The preparation method according to claim 2, characterized in that, The polyethylene glycol conjugated lipid is 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the ionizable liposome, the distearate phosphatidylcholine, the cholesterol, and the polyethylene glycol conjugated lipid is 50:10:38.5:1.

5.

6. The preparation method according to claim 1, characterized in that, The coating process includes the step of mixing a buffer solution containing the VP7-mRNA with an ethanol solution of the liposomes.

7. The preparation method according to claim 6, characterized in that, The buffer solution is a citrate buffer.

8. The preparation method according to claim 6, characterized in that, The volume ratio of the buffer solution containing the VP7-mRNA to the ethanol solution of the liposomes is 3:

1.

9. An mRNA vaccine for G9 porcine rotavirus prepared by the preparation method according to any one of claims 1-8.

Images