mRNA Vaccine for Preventing Porcine Reproductive and Respiratory Syndrome and Preparation Method

Abstract

The present invention relates to the technical field of mRNA vaccines, and particularly to an mRNA vaccine for preventing porcine reproductive and respiratory syndrome and a preparation method thereof. The present invention utilizes the structural proteins and non-structural proteins of porcine reproductive and respiratory syndrome virus to develop an mRNA vaccine for preventing porcine reproductive and respiratory syndrome. After being used, the vaccine can simulate the process of natural infection, be translated and modified in the body cells, and can be presented by MHC class I molecules and MHC class II molecules, inducing stronger cellular immune and humoral immune responses, that is, dual effects. Moreover, the mRNA provided by the present invention has a self-adjuvant effect, eliminating the need for adjuvant screening, which simplifies the process and is powerful in function.

Description

Technical Field

[0001] The present invention relates to the technical field of mRNA vaccines, and in particular to an mRNA vaccine for preventing porcine reproductive and respiratory syndrome and a preparation method thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease characterized by reproductive disorders such as abortion, stillbirth, and mummified fetuses in sows caused by porcine reproductive and respiratory syndrome virus, and respiratory symptoms in pigs of all ages. The main harm of the virus is that pigs of all ages are infected, and different breeds, genders, and ages of pigs are affected. The virus has a long excretion time after infection, so the virus can be repeatedly transmitted in the pig population through horizontal and vertical transmission methods and is difficult to cure completely. The main harms to sows are reproductive disorders, low immunity, delayed estrus, etc.

[0003] PRRSV has an envelope, and the particles are round, usually with a diameter of 40 - 60 nm. The envelope surface contains structural proteins encoded by different genes. Among them, GP3, GP4, GP5, and M proteins are the main envelope proteins. These proteins all contain neutralizing antigenic epitopes, can induce the body to produce neutralizing antibodies, and are all used as candidate antigens for diagnosis and vaccine research, and have important research value.

[0004] Currently, mRNA vaccines have achieved rapid development in human vaccines, namely novel coronavirus vaccines, influenza vaccines, Zika vaccines, etc. Some vaccines have been used in human experiments and achieved good results. Facing the complex problems that appear in the clinical application of various current PRRSV vaccines, choosing mRNA vaccines for PRRS prevention and control is a good choice.

[0005] mRNA is an intermediate step in the transcription of encoded DNA and the production of proteins by cytoplasmic ribosomes. The core principle of mRNA vaccines is to transfer the gene sequence encoding one or more immunogens into the cytoplasm of host cells, and then express and translate the proteins, that is, antigens, within the cells, making them located inside the cell membrane or secreted outside the cell membrane, and then presented on the major histocompatibility complex (MHC), namely MHC class I and MHC class II, and then recognized by CD8+ and CD4+ T cells to initiate an adaptive immune response. mRNA vaccines produce antigens within target cells, mimicking the natural infection process of the virus in the human body, can effectively induce humoral immunity and cellular immunity, and have relatively high safety. mRNA vaccines can bypass the limitations in obtaining virus strains, bacterial strains, etc., and support the provision of any specific antigen, whether from viruses, bacteria, or parasites, and can support the development of vaccines against multiple pathogens.

[0006] mRNA vaccines have certain relative advantages over subunit, inactivated virus, live attenuated virus, and DNA vaccines. (1) High safety: Since mRNA vaccines are a non-infectious, non-integrating platform technology, there is no risk of infection or insertional mutations and potential integration of the genome with host chromosomes. In addition, mRNA can be naturally degraded by normal cells, and its half-life in the body can be regulated by various modifications and delivery systems. (2) High efficiency: Various modifications make mRNA more stable and easier to translate. By synthesizing mRNA into a carrier molecule and allowing it to be rapidly taken up and expressed in the cytoplasm, efficient in vivo administration can be achieved. mRNA is the smallest genetic carrier, so anti-carrier immunity is avoided and mRNA vaccines can be repeatedly administered. (3) It can be mass-produced at low cost and does not require amplification in bacteria or cell culture, so mRNA vaccines have the potential for rapid, inexpensive, and scalable production.

[0007] At present, there is no porcine reproductive and respiratory syndrome virus mRNA vaccine developed and marketed in China. Therefore, an mRNA vaccine is developed to prevent porcine reproductive and respiratory syndrome virus infection.

[0008] In summary, the problems with existing PRRSV vaccines are:

[0009] (1) Inactivated PRRSV vaccine: It is safe, easy to store and transport, and insensitive to the interference of maternal antibodies. However, it requires a large immunization dose, many immunizations, a long blank period, and produces a large amount of non-neutralizing antibodies. It has almost no protective effect against heterologous strains, making it difficult to control and eradicate the presence and spread of PRRSV in pig farms, and it cannot produce a cellular immune response.

[0010] (2) PRRSV attenuated vaccine: Attenuated vaccines produce antibodies faster and induce cellular immune responses, but there is a risk that the attenuated vaccine strain will become more virulent, leading to virus recombination and mutation, which is not conducive to disease elimination.

[0011] (3) DNA vaccine: There are reports on the research of PRRSV DNA vaccine. The problem is that DNA can be integrated into the DNA of cells, causing the activation of oncogenes and autoimmune reactions in the body, which poses a potential biosafety risk.

[0012] (4) The production process of PRRSV vaccine is complex, the production scale is large, and the land occupied is large, which invisibly increases the production cost.

[0013] In view of this, the present invention is proposed. Summary of the Invention

[0014] The first object of the present invention is to develop a preventive porcine reproductive and respiratory syndrome mRNA vaccine by using the structural proteins and non-structural proteins of porcine reproductive and respiratory syndrome virus. After administration, the vaccine can simulate the process of natural infection, be translated and modified in the body cells, and can be presented by MHC class I molecules and MHC class II molecules, inducing stronger cellular immune and humoral immune responses, that is, dual effects.

[0015] Another object of the present invention is to provide a method for preparing the above mRNA vaccine, with a view to achieving rapid and safe production and being suitable for popularization and application.

[0016] In order to solve the above technical problems and achieve the above objects, the present invention provides the following technical solutions:

[0017] In the first aspect, the present invention provides the application of a porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope in the preparation of an mRNA vaccine, and the structural protein is selected from at least one of (i) to (iii);

[0018] (i) membrane protein or its derivative;

[0019] (ii) M protein or its derivative;

[0020] (iii) N protein or its derivative.

[0021] Preferably, the membrane protein includes at least one of GP3, GP4 or GP5.

[0022] In the second aspect, the present invention provides the application of the porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope described in the foregoing embodiment in combination with a non-structural protein having antigen activity in the preparation of an mRNA vaccine, and the non-structural protein includes Nsp protein or its derivative;

[0023] Preferably, the Nsp protein includes at least one of Nsp2, Nsp9 or Nsp10.

[0024] In the third aspect, the present invention provides a first mRNA molecule, and the first mRNA molecule encodes a first fusion protein, and the first fusion protein includes at least two polypeptide fragments derived from a porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope connected by a flexible linker.

[0025] Preferably, the porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope is selected from the structural proteins described in the foregoing embodiment.

[0026] Preferably, the nucleotide sequence of the open reading frame ORF3 encoding the GP3 protein is SEQ ID No.1.

[0027] Preferably, the nucleotide sequence of the open reading frame ORF4 encoding the GP4 protein is SEQ ID No.2.

[0028] Preferably, the nucleotide sequence of the open reading frame ORF5 encoding the GP5 protein is SEQ ID No.3 or SEQ ID No.4.

[0029] Preferably, the nucleotide sequence of the open reading frame ORF6 encoding the M protein is SEQ ID No.5.

[0030] Preferably, the nucleotide sequence of the open reading frame ORF7 encoding the N protein is SEQ ID No.6.

[0031] In a fourth aspect, the present invention provides a second mRNA molecule, which encodes a second fusion protein, and the second fusion protein includes at least one polypeptide fragment described in the foregoing embodiments connected by a flexible linker and at least one polypeptide fragment derived from a non-structural protein with antigenic activity.

[0032] Preferably, the non-structural protein with antigenic activity is selected from the non-structural proteins described in the foregoing embodiments.

[0033] Preferably, the nucleotide sequence of the open reading frame ORF1 encoding the Nsp2 protein is selected from any one of SEQ ID No.18 to SEQ ID No.20.

[0034] Preferably, the nucleotide sequence of the open reading frame ORF1 encoding the Nsp9 protein is SEQ ID No.21.

[0035] Preferably, the nucleotide sequence of the open reading frame ORF1 encoding the Nsp10 protein is SEQ ID No.22.

[0036] In a fifth aspect, the present invention provides a construct, which includes the first mRNA molecule or the second mRNA molecule, and the nucleotide sequence of the construct further includes a UTR sequence, a Kozak sequence, a signal peptide coding sequence, a promoter sequence, and a poly(A) sequence.

[0037] Preferably, the UTR sequence includes a 5'UTR sequence with a nucleotide sequence as shown in SEQ ID No.13 and a 3'UTR sequence with a nucleotide sequence as shown in SEQ ID No.16.

[0038] Preferably, the Kozak sequence is as shown in SEQ ID No.15.

[0039] Preferably, the nucleotide sequence encoding the signal peptide is as shown in SEQ ID No.14.

[0040] Preferably, the promoter includes the T7 promoter, and the nucleotide sequence is as shown in SEQ ID No. 12.

[0041] Preferably, the base length of the poly(A) is 50 - 150 bps, and more preferably 129 bps.

[0042] In a sixth aspect, the present invention provides a biological material, including:

[0043] (a) A recombinant vector, including the construct and plasmid vector described in the foregoing embodiments;

[0044] (b) A transformant, including a host cell containing the construct described in the foregoing embodiments, or the recombinant vector in (a).

[0045] Preferably, the plasmid vector includes pcDNA3.1, CET 1019 HS-puro, pIRES or pRL-SV40, and more preferably pcDNA3.1.

[0046] Preferably, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0047] More preferably, the prokaryotic cell includes DH5α, JM109 or BL21, and even more preferably DH5α.

[0048] More preferably, the eukaryotic cell includes a yeast cell, Marco-145, PK-15, Hela or an insect cell, and even more preferably Marco-145.

[0049] In a seventh aspect, the present invention provides a preparation method of the construct described in the foregoing embodiments, and the preparation method includes:

[0050] (1) Synthesizing gene fragments of the expression construct, and the nucleotide sequence of the construct sequentially includes, from the 5'-end to the 3'-end, a promoter sequence - 5'UTR sequence - Kozak sequence - signal peptide coding sequence - the first mRNA molecule or the second mRNA molecule - 3'UTR sequence - poly(A) sequence;

[0051] (2) Inserting the gene fragment obtained in step (1) into a plasmid vector to obtain a recombinant vector, then transforming the recombinant vector into a host cell to obtain a transformant, extracting the recombinant vector after the transformant proliferates, and obtaining the construct after in vitro transcription.

[0052] In an eighth aspect, the present invention provides the application of the first mRNA molecule, the second mRNA molecule, the construct, the biological material described in the foregoing embodiments, or the preparation method described in the foregoing embodiments in the preparation of an mRNA vaccine.

[0053] In a ninth aspect, the present invention provides a method for preparing an mRNA vaccine. The preparation method includes mixing the construct described in the foregoing embodiments, or the construct obtained by using the preparation method described in the foregoing embodiments, with a vector, and then obtaining the mRNA vaccine through dialysis, concentration, and sterile filtration in sequence.

[0054] Preferably, the vector includes lipid nanoparticles.

[0055] Preferably, the lipid nanoparticle components include D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG.

[0056] Preferably, the molar ratio of D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG is (50):(14 - 9):(35 - 40):1, and more preferably 50:10:37.5:2.5.

[0057] Preferably, the preparation method includes mixing D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG in an organic solvent according to the molar ratio to obtain lipid nanoparticles, then mixing with the construct, and then obtaining the mRNA vaccine through dialysis, concentration, and sterile filtration in sequence.

[0058] Preferably, the organic solvent is selected from ethanol, isopropanol, or methanol, and more preferably ethanol.

[0059] Preferably, the lipid nanoparticles and the construct are mixed at a volume flow rate of 1:(1 - 10), and more preferably, the volume flow rate is 1:3.

[0060] In a tenth aspect, the present invention provides an mRNA vaccine prepared by using the preparation method described in the foregoing embodiments. The mRNA vaccine has a particle size of 90 - 100 nm, a Zeta potential of -20 - -7.5 mV, and an encapsulation efficiency of more than 90%.

[0061] The beneficial effects of the porcine reproductive and respiratory syndrome mRNA vaccine and its preparation method provided by the present invention include:

[0062] (1) By using the preparation method of the mRNA vaccine provided by the present invention, rapid research and development and production can be achieved, standardization can be realized, mass production and quality control are easy, and the same production process is applicable to multiple different products, that is, the development cycle is short and the product is controllable.

[0063] (2) After administration, the mRNA vaccine provided by the present invention can mimic the process of natural infection, be translated and modified within the body cells, and can be presented by MHC class I molecules and MHC class II molecules, inducing stronger cellular and humoral immune responses, that is, dual efficacy, and there is no risk of infection and genomic integration, that is, biosafety.

[0064] (3) The mRNA provided by the present invention has an adjuvant self - effect, eliminating the need for adjuvant screening, which simplifies the process and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0066] Figure 1 It is a flowchart for the design and construction of the PRRSV mRNA vaccine provided by the embodiment of the present invention;

[0067] Figure 2 It is a schematic diagram for the construction of the pcDNA3.1 - PRRSV - C plasmid provided by the embodiment of the present invention;

[0068] Figure 3 It is an enzyme digestion electrophoresis diagram of the pcDNA3.1 - PRRSV - C plasmid provided by the embodiment of the present invention;

[0069] Figure 4A It is the detection result of CD4+ cells after immunizing mice with a 10μg dose of the PRRSV mRNA vaccine;

[0070] Figure 4B It is the detection result of CD4+ cells after immunizing mice with a 5μg dose of the PRRSV mRNA vaccine;

[0071] Figure 4C It is the detection result of CD4+ cells after immunizing mice with a 2μg dose of the PRRSV mRNA vaccine;

[0072] Figure 5A It is the detection result of CD8+ cells after immunizing mice with a 10μg dose of the PRRSV mRNA vaccine;

[0073] Figure 5B It is the detection result of CD8+ cells after immunizing mice with a 5μg dose of the PRRSV mRNA vaccine;

[0074] Figure 5CDetection results of CD8+ cells in mice immunized with 2 μg dose of PRRSV mRNA vaccine;

[0075] Figure 6 Cytokine detection results in mice immunized with PRRSV mRNA vaccine. Specific implementation manners

[0076] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Generally, the components of the embodiments of the present invention described and illustrated herein can be arranged and designed in various different configurations.

[0077] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0078] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0079] In a specific implementation manner, in a first aspect, the present invention provides the application of a porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope in the preparation of an mRNA vaccine, and the structural protein is selected from at least one of (i) to (iii);

[0080] (i) Membrane protein or its derivative;

[0081] (ii) M protein or its derivative;

[0082] (iii) N protein or its derivative.

[0083] In an alternative implementation manner, the membrane protein includes at least one of GP3, GP4 or GP5.

[0084] GP3, GP4, GP5 or M protein contains the main neutralizing epitopes and can induce a high level of neutralizing antibody titer.

[0085] In a second aspect, the present invention provides the application of the porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing antigen epitope described in the foregoing implementation manner in combination with a non-structural protein having antigen activity in the preparation of an mRNA vaccine, and the non-structural protein includes an Nsp protein or its derivative.

[0086] In an alternative embodiment, the Nsp protein comprises at least one of Nsp2, Nsp9 or Nsp10.

[0087] In a third aspect, the present invention provides a first mRNA molecule encoding a first fusion protein, the first fusion protein comprising at least two polypeptide fragments derived from a porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing epitope, which are linked by a flexible linker.

[0088] In an alternative embodiment, the porcine reproductive and respiratory syndrome virus structural protein containing a neutralizing epitope is selected from the structural proteins described in the foregoing embodiments.

[0089] In an alternative embodiment, the nucleotide sequence of the open reading frame ORF3 encoding the GP3 protein is SEQ ID No.1; the nucleotide sequence of the open reading frame ORF4 encoding the GP4 protein is SEQ ID No.2; the nucleotide sequence of the open reading frame ORF5 encoding the GP5 protein is SEQ ID No.3 or SEQ ID No.4; the nucleotide sequence of the open reading frame ORF6 encoding the M protein is SEQ ID No.5; the nucleotide sequence of the open reading frame ORF7 encoding the N protein is SEQ ID No.6.

[0090] In a fourth aspect, the present invention provides a second mRNA molecule encoding a second fusion protein, the second fusion protein comprising at least one polypeptide fragment described in any one of the foregoing embodiments, which is linked by a flexible linker, and at least one polypeptide fragment derived from a non-structural protein having antigenic activity.

[0091] In an alternative embodiment, the non-structural protein having antigenic activity is selected from the non-structural proteins described in the foregoing embodiments.

[0092] In an alternative embodiment, the nucleotide sequence of the open reading frame ORF1 encoding the Nsp2 protein is selected from any one of the sequences SEQ ID No.18 to SEQ ID No.20;

[0093] The nucleotide sequence of the open reading frame ORF1 encoding the Nsp9 protein is SEQ ID No.21;

[0094] The nucleotide sequence of the open reading frame ORF1 encoding the Nsp10 protein is SEQ ID No.22.

[0095] It has been confirmed that the expression products of the above first mRNA molecule and second mRNA molecule can induce the production of high levels of neutralizing antibodies and cytokines in the body.

[0096] In a fifth aspect, the present invention provides a construct, which comprises a first mRNA molecule or a second mRNA molecule, and the nucleotide sequence of the construct further comprises a UTR sequence, a Kozak sequence, a signal peptide coding sequence, a promoter sequence and a poly(A) sequence.

[0097] In an alternative embodiment, the UTR sequence comprises a 5'UTR sequence with a nucleotide sequence as shown in SEQ ID No. 13 and a 3'UTR sequence with a nucleotide sequence as shown in SEQ ID No. 16;

[0098] The Kozak sequence is as shown in SEQ ID No. 15;

[0099] The signal peptide coding sequence is as shown in SEQ ID No. 14;

[0100] The promoter comprises a T7 promoter with a nucleotide sequence as shown in SEQ ID No. 12;

[0101] The base length of the poly(A) is 50 - 150 bps, preferably 129 bps.

[0102] In a sixth aspect, the present invention provides a biological material, comprising:

[0103] (a) A recombinant vector, comprising the construct described in the foregoing embodiment and a plasmid vector;

[0104] (b) A transformant, comprising a host cell containing the construct described in the foregoing embodiment, or the recombinant vector in (a).

[0105] In an alternative embodiment, the plasmid vector comprises pcDNA3.1, CET 1019 HS-puro, pIRES or pRL-SV40, preferably pcDNA3.1;

[0106] The host cell comprises a prokaryotic cell or a eukaryotic cell;

[0107] Preferably, the prokaryotic cell comprises DH5α, JM109, or BL21, more preferably DH5α;

[0108] Preferably, the eukaryotic cell comprises a yeast cell, Marco-145, PK-15, Hela or an insect cell, more preferably Marco-145.

[0109] In a seventh aspect, the present invention provides a method for preparing the construct described in the foregoing embodiment, the preparation method comprising:

[0110] (1) Synthesize the gene fragment of the expression construct, the nucleotide sequence of which sequentially includes, from the 5'-end to the 3'-end, a promoter sequence - 5'UTR sequence - Kozak sequence - signal peptide coding sequence - the first mRNA molecule or the second mRNA molecule - 3'UTR sequence - poly(A) sequence;

[0111] (2) Insert the gene fragment obtained in step (1) into a plasmid vector to obtain a recombinant vector, then transform the recombinant vector into a host cell to obtain a transformant. After the transformant proliferates, extract the recombinant vector and obtain the construct through in vitro transcription.

[0112] The mRNA provided by the PRRSV mRNA gene design, synthesis and mRNA vaccine and its preparation method of the present invention contains mRNAs of porcine reproductive and respiratory syndrome virus antigens of different lineages and can provide cross-immune protection against heterologous strains.

[0113] In a specific embodiment, the preparation method of the mRNA provided by the present invention includes the following steps:

[0114] Screen the positive recombinant plasmid, digest it with the restriction enzyme BamH I and recover the linearized plasmid, and then obtain PRRSV mRNA through in vitro transcription.

[0115] The positive recombinant plasmid is a plasmid with the gene encoding the above-mentioned PRRSV fusion protein.

[0116] The positive recombinant plasmid is a recombinant plasmid containing the DNA molecule shown in SEQ ID No.8 of the sequence listing or a recombinant plasmid containing the DNA molecule shown in SEQ ID No.9 of the sequence listing or the recombinant plasmid shown in SEQ ID No.10 of the sequence listing.

[0117] In a specific embodiment, the preparation method of PRRSV mRNA sequentially includes the following steps:

[0118] (1) Take the positive recombinant plasmid, digest it with the restriction enzyme BamH I, and recover the linearized plasmid;

[0119] (2) Transcribe to obtain RNA;

[0120] (3) After completing step (2), purify it using an RNA purification kit to obtain purified RNA;

[0121] (4) Digest the linear DNA;

[0122] (5) After completing step (4), purify it using an RNA purification kit to obtain purified RNA;

[0123] (6) Carry out capping;

[0124] (7) After completing step (6), purification is carried out using an RNA purification kit to obtain purified RNA, which is the PRRSV mRNA.

[0125] The method of step (4): Take the purified RNA, add DNase I and incubate at 37 °C for 1 h. Specifically, 1 U of DNase I is added per 1 μg of RNA.

[0126] In an eighth aspect, the present invention provides the use of the first mRNA molecule, the second mRNA molecule, the construct, the biological material described in the foregoing embodiments, or the preparation method described in the foregoing embodiments in the preparation of an mRNA vaccine.

[0127] In a ninth aspect, the present invention provides a method for preparing an mRNA vaccine, the preparation method comprising mixing the construct described in the foregoing embodiments, or the construct obtained by using the preparation method described in the foregoing embodiments with a carrier, and then successively performing dialysis, concentration and sterile filtration to obtain the mRNA vaccine.

[0128] In an alternative embodiment, the carrier includes lipid nanoparticles.

[0129] In an alternative embodiment, the lipid nanoparticle components include D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG.

[0130] In an alternative embodiment, the molar ratio of D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG is (50):(14 - 9):(35 - 40):1, preferably 50:10:37.5:2.5.

[0131] In an alternative embodiment, the preparation method includes mixing D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG in an organic solvent according to the molar ratio to obtain lipid nanoparticles, and then mixing with the construct, and then successively performing dialysis, concentration and sterile filtration to obtain the mRNA vaccine.

[0132] Preferably, the organic solvent is selected from ethanol, isopropanol or methanol, and more preferably ethanol.

[0133] In an alternative embodiment, the lipid nanoparticles and the construct are mixed at a flow rate of 1:(1 - 10).

[0134] In a specific embodiment, the method for preparing the lipid nanoparticles provided by the present invention specifically includes the following steps:

[0135] Take PRRSV mRNA and dilute it with 100 mM citric acid buffer at pH 4.4 to obtain Solution I; take D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG, dissolve them in 95% ethanol to obtain Solution II; simultaneously load Solution I and Solution II into a microfluidic mixer, with the volume ratio of Solution I to Solution II being 3:1, collect the effluent, dialyze, concentrate, and then filter, and collect the filtrate, which is the PRRSV mRNA LNPs solution. The molar ratios of the four components of D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG are 50:10:37.5:2.5 in sequence.

[0136] In a specific embodiment, the preparation method of the lipid nanoparticles encapsulating PRRSV mRNA provided by the present invention has the overall steps as Figure 1 shown, and specifically includes the following steps:

[0137] As described in the preparation method of lipid nanoparticles, simultaneously load Solution I and Solution II into a microfluidic mixer, with the volume ratio of Solution A to Solution B being 3:1 and the total flow rate being 5 - 8 ml / min; collect the effluent, transfer the effluent into a dialysis bag, place it in a phosphate buffer at pH 7.4 for dialysis at 4°C for 24 h, take the liquid phase in the dialysis bag, concentrate it to an RNA concentration of 2 mg / ml using a centrifugal filter, and then filter it through a 0.22 μm filter membrane, and collect the filtrate, which is the PRRSV mRNA LNPs solution.

[0138] In the tenth aspect, the present invention provides an mRNA vaccine prepared by using the preparation method described in any one of the foregoing embodiments. The particle size of the mRNA vaccine is 90 - 100 nm, the Zeta potential is -20 to -7.5 mV, and the encapsulation efficiency is above 90%.

[0139] The following will describe in detail some embodiments of the present invention with reference to the accompanying drawings. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0140] Example 1

[0141] This example provides a preparation method for three fusion mRNAs, which is specifically as follows:

[0142] 1.1 Gene sequence design and synthesis

[0143] Log in to the NCBI GenBank, search for and select the main membrane protein gene sequences of porcine reproductive and respiratory syndrome virus (PRRSV) of several different lineages, select the related genes encoding the main non-structural proteins Nsp2, Nsp9, Nsp10 or the main membrane proteins GP3, GP4, GP5, M protein and N protein, preferably the related genes of GP3, GP4, GP5, M protein and N protein. Through the analysis of DNAStar and DNAMan biological software, screen and find the antigenic sites with neutralizing effects on the protein surface. According to the relevant literature, select the appropriate antigenic epitopes with neutralizing activity, and through different combination methods, link the selected genes containing antigenic epitopes through a linker for subsequent fusion expression. Analyze and optimize the codons for them.

[0144] The design and synthesis of the fusion protein gene include: (1) (PRRSV-A): ORF3—linker—ORF4—linker—ORF5—linker—ORF6—linker—ORF7; (2) (PRRSV-B): ORF3—inker—ORF4—linker—ORF6—linker—ORF5—linker—ORF7; (3) (PRRSV-C): ORF3—linker—ORF4—linker—3’ORF5—linker—ORF6—linker—ORF7—linker—5’ORF5. The PRRSV ORF3 sequence is SEQ ID No.1, the PRRSV ORF4 sequence is SEQ ID No.2, and the 5’ORF5 and 3’ORF5 sequences are SEQ ID No.3 and SEQ ID No.4 respectively. The PRRSV ORF6 sequence is SEQ ID No.5, and the PRRSV ORF7 sequence is SEQ ID No.6. The 5’ORF5 sequence is repetitively designed at the 3’ end of the fusion protein gene, and the functional sequence SEQ ID No.7 is inserted in the middle. The functional sequence connects two antigenic epitopes and plays a role in ensuring that the antigenic epitopes do not interfere with each other. The designed fusion protein genes are shown as SEQ ID No.8~No.10 in the sequence listing.

[0145] The design of the fusion protein A gene sequence is shown in SEQ ID No.8, and the translated amino acid sequence is as follows:

[0146] LEPGKSFWCRIGHDRCSENGPGPGPETMRWATVLACLLPSLLAIGPGPGSHIQLIYNLGPGPGLAALICFVIRLAKNCLYRWRSPVIVEKGGKVEVEGHLIDLKRVVLDGSAATPLTRVSAELWGRLGPGPGCNDSTAPQKVLLAFSALKVSRGRLLGLLHLGPGPGVRHHFTPSEVRLIRATAS* (as shown in SEQ ID No. 23).

[0147] The gene sequence design of fusion protein B is shown in SEQ ID No. 9, and the translated amino acid sequence is as follows:

[0148] LEPGKSFWCRIGHDRCSENGPGPGPETMRWATVLACLLPSLLAIGPGPGCNDSTAPQKVLLAFSALKVSRGRLLGLLHLGPGPGSHIQLIYNLGPGPGLAALICFVIRLAKNCLYRWRSPVIVEKGGKVEVEGHLIDLKRVVLDGSAATPLTRVSAELWGRLGPGPGVRHHFTPSEVRLIRATAS* (as shown in SEQ ID No. 24).

[0149] The gene sequence design of fusion protein C is shown in SEQ ID No. 10, and the translated amino acid sequence is as follows:

[0150] LEPGKSFWCRIGHDRCSENGPGPGPETMRWATVLACLLPSLLAIGPGPGLAALICFVIRLAKNCGPGPGLYRWRSPVIVEKGGKVEVEGHLIDLKRVVLDGSAATPLTRVSAELWGRLGPGPGCNDSTAPQKVLLAFSGPGPGALKVSRGRLLGLLHLGPGPGVRHHFTPSEGPGPGVRLIRATASGPGPGSHIQLIYNLAKFVAAWTLKAAASHIQLIYNL* (as shown in SEQ ID No. 25).

[0151] Add non-coding sequences (UTRs) upstream and downstream of the DNA molecule shown in SEQ ID No. 10 in the sequence listing, and add Kozak sequence, signal peptide, T7 promoter sequence, and 129 As downstream to obtain the DNA molecule shown in SEQ ID No. 11. In SEQ ID No. 8, nucleotides 1 to 19 form the T7 promoter (the sequence shown is SEQ ID No. 12), nucleotides 20 to 77 form the 5' UTR (the sequence shown is SEQ ID No. 13), nucleotides 84 to 144 form the signal peptide sequence (the sequence shown is SEQ ID No. 14), nucleotides 78 to 83 form the Kozak sequence (the sequence shown is SEQ ID No. 15), nucleotides 84 to 812 encode the fusion protein (the sequence shown is SEQ ID No. 10), nucleotides 813 to 944 form the 3' UTR (the sequence shown is SEQ ID No. 16), and nucleotides 945 to 1073 form poly A (the sequence shown is SEQ ID No. 17). After codon optimization of the above gene, it was sent to Nanjing Genscript Biotechnology Co., Ltd. for synthesis.

[0152] According to the method of SEQ ID No. 10, add the corresponding modification sequences and perform codon optimization on SEQ ID No. 8 and SEQ ID No. 9 in the sequence listing, then perform gene synthesis. Select pcDNA3.1 as the vector and insert it into the vector. The recombinant plasmids were named pcDNA3.1-PRRSV-A, pcDNA3.1-PRRSV-B, and pcDNA3.1-PRRSV-C respectively (as Figure 2 shown).

[0153] 1.2 Transformation of Recombinant Plasmids

[0154] (1) Streak the DH5a strain stored at ~70 °C on an LB agar plate without antibiotics. After culturing in an incubator at 37 °C for 12 - 16 h, pick a single well-grown colony and inoculate it into 5 mL of LB liquid medium. Culture it with shaking at 250 - 300 r / min at 37 °C for 5 - 6 h.

[0155] (2) Transfer an appropriate amount of the activated bacterial solution aseptically into a saline bottle containing 5 mL of LB, and continue culturing with shaking for about 2.5 - 3 h until the OD600nm value reaches 0.5 - 0.6. Then, transfer the bacteria aseptically to a sterile 50 mL polypropylene centrifuge tube pre-cooled with ice and place it on ice for 30 min.

[0156] (3) Centrifuge at 4000 r / min for 10 min at 4°C, discard the supernatant. Invert the centrifuge tube for 1 min to allow the remaining culture medium to drain completely. Then add 10 mL of ice-precooled 0.1 mol / L CaCl2 to resuspend the pellet and incubate on ice for 30 min.

[0157] (4) Centrifuge at 4000 r / min for 10 min at 4°C, discard the supernatant. Add approximately 2 min of ice-precooled 0.1 mol / L CaCl2 to the pellet and gently resuspend the cells. The prepared competent cells are ready. A small amount can be directly taken for transformation or stored at 4°C in an ice bath for a short time. 15% sterile glycerol can be added at a final concentration, aliquoted at 0.1 mL / tube, and stored at -70°C in the refrigerator for later use.

[0158] (5) Take 100 μL of the prepared competent cells in a sterile 1.5 mL EP tube, add the ligation product (about 10 μL), mix well, incubate on ice for 30 min, then heat shock in a 42°C water bath for 90 s, and then incubate on ice for 2 - 5 min. Add 400 μL of LB, resuscitate and culture at 37°C on a shaker at 200 - 220 r / min for 45 - 60 min. Centrifuge at 5000 r / min for 3 min, discard 400 μL of the supernatant, gently resuspend the bacterial pellet with the remaining supernatant, and evenly spread on the resistant plate. First, place it upright in a 37°C incubator for 1 - 2 h until the liquid on the plate surface is fully absorbed into the agar (when there is no liquid flow on the surface of the medium is appropriate), then place it upside down and continue to culture for 12 - 18 h to observe the appearance of transformed single colonies.

[0159] If it is to transform a ready-made plasmid, heat shock in a 42°C water bath for 90 s, return to ice for 2 min, then directly take 30 - 50 μL of the bacterial solution and spread it directly on an LB plate containing the corresponding antibiotic, and then it can be cultured in a 37°C incubator.

[0160] 1.3 Screening and Identification of Recombinant Plasmids

[0161] 1.3.1 Small-scale Preparation of Plasmids

[0162] (1) Use a sterilized toothpick to pick a single colony on the plate and inoculate it into 4 mL of LB culture medium containing the corresponding antibiotic, shake and culture overnight at 37°C and 300 r / min.

[0163] (2) Transfer the bacterial solution into a 1.5 mL EP tube, centrifuge at 12000 r / min for 1 min, discard the supernatant, invert the centrifuge tube to drain the liquid completely, and collect the cells. (Note: Reserve 0.5 mL of the bacterial solution for each sample, add an appropriate amount of sterilized glycerol, and store at -20°C for later use. After the small-scale extraction and identification are completed, keep the positive recombinant bacterial solution).

[0164] (3) Add 100 μL of ice-precooled Solution I and vortex until the cells are fully suspended.

[0165] (4) Add 200 μL of freshly prepared Solution II, invert to mix well, and incubate on ice for 5 min.

[0166] (5) Add 150 μL of pre-chilled Solution III, invert the centrifuge tube several times to mix well, and then incubate on ice for 5 min.

[0167] (6) Centrifuge at 12,000 r / min for 5 min, aspirate the supernatant into another 1.5 mL centrifuge tube, add an equal volume of isopropanol, vortex to mix well, and let it stand at room temperature for 10 min to precipitate.

[0168] (7) Centrifuge at 12,000 r / min for 10 min, discard the supernatant, wash the precipitate with 75% cold ethanol, then air-dry or let it dry naturally. Dissolve the precipitate in 200 μL of TE (pH 8.0) containing an appropriate amount of RNase (20 μg / mL) or ddH₂O, and incubate in a water bath at 56 °C for 30 min or 37 °C for 1 h to remove RNA.

[0169] (8) Add 100 μL of ice-pre-chilled 7.5 mol / L NH₄Ac, which is 1 / 2 the volume, mix well, and incubate on ice for 10 min.

[0170] (9) Centrifuge at 12,000 r / min at 4 °C for 10 min, aspirate the supernatant into another 1.5 mL centrifuge tube, add 1 volume of isopropanol or 2 volumes of absolute ethanol, and let it precipitate at room temperature for 30 min.

[0171] (10) Centrifuge at 12,000 r / min for 5 min, discard the supernatant, wash the precipitate with 75% cold ethanol, air-dry and then dissolve it in an appropriate amount of ddH₂O or TE (pH 8.0). Take 1 - 2 μL of the sample for electrophoresis to observe the quality of the extracted plasmid, and store the rest at -2 °C for later use.

[0172] (11) Use Sac II and BamH I to perform double digestion identification on the extracted plasmid (see Figure 3 ), at 37 °C for 3 h. Electrophorese the digestion products on a 1% agarose gel, and send the plasmid with correct bands to GenScript Biotech Corporation for sequencing.

[0173] 1.3.2 Large-scale extraction of recombinant plasmid

[0174] (1) Inoculate the identified positive recombinant bacterial liquid stored at -20 °C into 100 mL of LB liquid medium containing the corresponding antibiotic, and culture overnight at 37 °C with a rotation speed of 250 - 300 r / min.

[0175] (2) Transfer the bacterial liquid to a 50 mL centrifuge tube, centrifuge at 6000 r / min for 5 - 10 min to collect the bacterial precipitate.

[0176] (3) Add 3 mL of ice-precooled Solution I to the precipitate, blow it to disperse and vortex until the cells are fully suspended.

[0177] (4) Add 6 mL of freshly prepared Solution II, invert the centrifuge tube several times, and incubate on ice for 7 - 10 min.

[0178] (5) Add 4.5 mL of ice-precooled Solution III, invert the centrifuge tube several times, and then incubate on ice for 7 - 10 min.

[0179] (6) Centrifuge at 10,000 r / min at 4 °C for 15 - 20 min, transfer the supernatant to another 50 mL centrifuge tube, add 0.6 volume of isopropanol, mix well, and incubate at -20 °C or room temperature for 30 min.

[0180] (7) Centrifuge at 10,000 r / min at room temperature for 15 - 20 min, discard the supernatant, wash the precipitate with 75% cold ethanol, then dry it under vacuum or air-dry it naturally. Add 1.5 mL of TE (pH 8.0) to fully dissolve the precipitate, and then add an equal volume (1.5 mL) of ice-precooled 5 mol / L NH4Ac, and mix well.

[0181] (8) Centrifuge at 10,000 r / min at 4 °C for 15 - 20 min, discard the precipitate, transfer the supernatant to a 7 mL centrifuge tube, add an equal volume (3 mL) of isopropanol, mix well, and incubate at -20 °C or room temperature for 30 min.

[0182] (9) Centrifuge at 10,000 r / min for 15 - 20 min, discard the supernatant, wash the precipitate with ice-precooled 75% ethanol, dry it under vacuum or air-dry it naturally. Add 500 μL of TE (pH 8.0) to fully dissolve it and then transfer it to a 1.5 mL centrifuge tube.

[0183] (10) Add an appropriate amount of RNase, incubate at 37 °C for 1 h or 56 °C for 30 min to remove RNA.

[0184] (11) Add an equal volume (500 μL) of freshly prepared 13% PEG8000 (containing 1.6 mo1 / L NaCl), mix well, and incubate at -20 °C or room temperature for 30 min.

[0185] (12) Centrifuge at 12,000 r / min at 4 °C for 10 min, discard the supernatant, and resuspend the precipitate with 400 μL of TE (pH 8.0).

[0186] (13) Extract twice with an equal volume (400 μL) of phenol:chloroform:isoamyl alcohol (25:24:1), and extract once with chloroform:isoamyl alcohol. Centrifuge at 12,000 r / min for 10 min each time for extraction.

[0187] (14)Carefully aspirate the upper aqueous phase (try to avoid aspirating the lower oil phase) and transfer it to another 1.5 mL centrifuge tube. Add 100 μL of 10 mol / L NH4Ac, mix well, then add 2 volumes (usually 1 mL) of ice-cold absolute ethanol and mix. Incubate at -20 °C for 30 min.

[0188] (15)Centrifuge at 12000 r / min for 5 - 10 min at 4 °C, discard the supernatant, wash the pellet with ice-cold 75% ethanol, vacuum dry or air dry, and dissolve the pellet in 50 - 100 μL TE (pH 8.0) or sterilized ddH2O.

[0189] (16)Take 0.5 μL and electrophorese it on a 0.8% agarose gel. Observe the quality of the extracted plasmid under ultraviolet light, and store the remaining part at -20 °C for later use.

[0190] 1.4 mRNA Preparation

[0191] 1.4.1 Linearization of Recombinant Plasmid

[0192] After extracting a large amount of recombinant plasmid pcDNA3.1 - PRRSV - A / B / C, use a ultra - micro spectrophotometer to measure its concentration and purity. The single - enzyme digestion reaction system is shown in Table 1, and the reaction conditions are: 37 °C for 3 h.

[0193] Table 1 Enzyme Digestion Reaction System

[0194]

[0195] 1.4.2 In Vitro Transcription Reaction of Recombinant Plasmid

[0196] Select T7 - Flash ScribeTM Transcription Kit for in vitro transcription. During the preparation of the in vitro transcription system, replace UTP with N1 - Methylpseudouridine - 5’ - Triphosphate. The reaction system is shown in Table 2, and the reaction conditions are: for the first step of the reaction, 35 °C for 30 min; after the reaction, add 1 μL of Rnase - Free DNaseI enzyme to 20 μL of the above product and continue the reaction at 35 °C for 15 min.

[0197] Table 2 In Vitro Transcription Reaction

[0198]

[0199] 1.4.3 Capping of Transcription Products

[0200] Operate using the ScriptCapTM Cap 1 Capping System kit. The reaction system is shown in Table 3. Incubate the capping reaction solution at 65 °C for 15 min.

[0201] Table 3 Capping Reaction (Step1)

[0202]

[0203] Step2

[0204]

[0205] Step3

[0206]

[0207] 1.4.4 Cap-mRNA Purification

[0208] Use the Purification for Large Scale Transcription Reactions kit for the purification reaction. Pay attention to cleaning the workbench, using RNase-free tips, and changing gloves during the operation. The specific operation steps are as follows:

[0209] (1) Dissolve the RNA in 100 μL of Elution Solution and gently mix with a pipette;

[0210] (2) Add 350 μL of Binding Solution Concentrate to the sample and gently mix with a pipette;

[0211] (3) Then add 250 μL of absolute ethanol and gently mix with a pipette;

[0212] (4) Add the mixed sample to the filter column, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0213] (5) Add 500 μL of Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0214] (6) Repeat step 5 once;

[0215] (7) Put the filter column back and continue to centrifuge empty to remove all Wash Buffer, centrifuge at 12000 rpm for 1 min;

[0216] (8) Transfer the column to a new collection tube, add 50 μL of Eluent Solution, and incubate at 65 - 70 °C for 5 - 10 min;

[0217] Centrifuge at room temperature at 12,000 rpm for 1 min;

[0218] To increase the RNA recovery, an additional 50 μL can be added for elution and collected in the same tube.

[0219] 1.5 In vitro cell transfection assay of Cap-mRNA

[0220] 1.5.1 Cell transfection

[0221] Use LipofectamineTM 3000 Reagent for the cell transfection assay of Cap-mRNA. The specific operation steps are as follows:

[0222] (1) Seed Marco-145 cells in a 12-well plate at a density of 1×10 4 cells / well and culture them in MEM medium containing 10% fetal bovine serum. Incubate in a 37 °C incubator with 5% CO2 until the cell confluence is nearly 80%;

[0223] (2) Replace with serum-free MEM medium and add 500 μL to each well;

[0224] (3) Refer to the transfection reagent instruction manual, mix Lipofectamine 3000 Reagent and Cap-mRNA at a volume-to-mass ratio of 3 μL / 2 μg, let stand at room temperature for 15 min, then add dropwise to the wells. Gently shake the liquid in the well plate evenly and continue to culture in the incubator;

[0225] (4) If the culture time exceeds 12 h, complete medium needs to be supplemented at 6 h, 500 μL per well.

[0226] 1.5.2 Detection after mRNA transfection

[0227] 1.5.2.1 Immunoblotting assay After PRRSV-A / B / C transfected Marco-145 cells for 12 h, discard the original medium in the wells, wash twice with PBS to remove residual medium, add RIPA cell lysis buffer (add protease inhibitor PMSF at a ratio of 100:1), fully lyse the cells and collect them into a 1.5 mL EP tube, centrifuge at 14,000 rpm for 10 min. Transfer the supernatant to a new 1.5 mL EP tube, take 2 μL for protein concentration determination. The specific determination method refers to the instruction manual of the BCA protein concentration determination kit.

[0228] Take protein samples of the same mass, add water to make the volume the same, add 1 / 4 volume of 5×SDS loading buffer, mix well, boil in water for 5 min, take 25 μg of the sample for loading, and perform 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The specific operation steps are as follows:

[0229] (1)Electrophoresis: First, run at 80 V for 30 min to allow the protein samples to uniformly enter the separating gel, then adjust the voltage to 120 V and end the electrophoresis when the bands are close to the bottom of the gel block;

[0230] (2)Transfer: Transfer the proteins in the gel to a 0.45-μm nitrocellulose membrane (NC membrane) using the semi-dry method, with the parameters set at 15 V for 31 min;

[0231] (3)Blocking: Place the NC membrane in 5% non-fat milk and block for 2 h;

[0232] (4)Washing: After blocking, wash once with TBST to remove the residual non-fat milk on the membrane surface;

[0233] (5)Primary antibody incubation: Prepare a primary antibody diluted 1:2000 with 5% BSA, rabbit-derived PRRSV GP5 protein positive serum, incubate at room temperature for 3 h, or incubate overnight at 40 °C;

[0234] (6)Washing: Wash 3 times with TBST, 8 minutes each time;

[0235] (7)Secondary antibody incubation: Prepare a secondary antibody diluted 1:4000 with TBST, HRP-labeled goat anti-rabbit IgG antibody, incubate at room temperature for 30 min;

[0236] (8)Washing: Wash 3 times with TBST, 10 minutes each time;

[0237] (9)Development: Drop ECL developing solution on the membrane, after 1 minute, place it in the instrument for imaging.

[0238] 1.5.2.2 Indirect immunofluorescence assay

[0239] (1)After 3 days of cell culture, take out the cell plate, discard the supernatant, add 200 μl of PBST (0.1 mol / L PBS with 0.05% Tween 20, the same below) to each well, wash 3 times, then add 100 μl of 80% pre-cooled acetone to each well, fix at room temperature for 10 minutes. Discard the fixing solution, pat dry the remaining liquid in the cell plate, and place it in a biosafety cabinet with the blower on for about 5 minutes to allow the cell plate to dry naturally. Add 200 μl of PBST to each well and wash 4 times. After pouring out the PBST, store the antigen plate at 4 °C or -20 °C for later use.

[0240] (2) Add 75 μl of 3% PBA to each well in the antigen plate. Vortex 25 μl of PRRSV negative serum and positive serum (diluted 1:200) evenly. Incubate overnight at 4°C.

[0241] (3) After removing the primary antibody, add 200 μl of PBST to each well and wash 4 times. Add 50 μl of FITC-labeled goat anti-rabbit IgG secondary antibody diluted 200-fold with 1% PBA to each well and incubate at room temperature for 4 hours. After pouring out the secondary antibody, then add 200 μl of PBST to each well and wash 4 times.

[0242] (4) After pouring out the PBST, add another 50 μl of PBST and read the antibody titer under a 200-fold field of view of an inverted fluorescence microscope. In the positive control wells, typical specific bright green fluorescence should be observed in the cytoplasm or nucleus of positive cells, while there is no specific green fluorescence in the negative control wells.

[0243] Example 2 Preparation of mRNA Vaccine

[0244] 2.1 Raw Material Preparation

[0245] Dissolve and mix the four components of the cationic lipids D-Lin-MC3-DMA, distearoylphosphatidylcholine, cholesterol, and PEGylated lipid PEG-DMG in ethanol at a molar ratio of 50:10:37.5:2.5.

[0246] 2.2 Experimental Procedures

[0247] At a lipid mixture:mRNA volume flow rate ratio of 1:3, mix and package in a nanoparticle preparation instrument of Precision Nanosystems to obtain packaged mRNA-LNP, abbreviated as PRRSV-A mRNA LNP, PRRSV-B mRNA LNP, and PRRSV-C mRNA LNP respectively. Dialyze and ultrafiltrate and concentrate the packaged mRNA-LNP into DPBS, and obtain samples for subsequent animal experiments after sterile filtration.

[0248] Take the PRRSV-A / B / C mRNA LNPs solution and measure the particle size, PDI, and zeta potential of the LNPs using a Malvern instrument. The particle size is between 90 nm and 100 nm, as shown in Table 4. PDI: 0.129. Zeta potential: -7.5 Mv. Take the PRRSV-A / B / C mRNA LNPs solution respectively and calculate the encapsulation efficiency of the LNPs using an RNA analysis kit, and the encapsulation efficiency is not less than 90%.

[0249] Table 4 Parameters Related to Vaccine Preparation

[0250]

[0251] Example 3 Immune Evaluation of mRNA Vaccine

[0252] 3.1 Experimental Operation

[0253] Prepare porcine reproductive and respiratory syndrome virus mRNA vaccines with different components by the methods provided in Examples 1 and 2. Select 18 - 25 g BALB / c mice and immunize the mice according to the grouping method shown in the following table. The grouping is shown in Table 5. Fourteen days after the first immunization, a second immunization is carried out. Twenty-eight days after the first immunization, blood is collected to detect cytokine and membrane protein antibody levels.

[0254] Table 5 Experimental Grouping

[0255]

[0256] 3.2 Experimental Results

[0257] 3.2.1 Detection Results of Cytokines

[0258] Through collecting whole blood of mice for blood analysis, the detection results of Th1 - type cellular immune responses of IFN - γ, IL - 2, CD4+ T, and CD8+ cells are shown in Figures 4 - 6. The results show that under different immunization doses, the antigen combination of the PRRSV - C - LNP group has certain advantages in cellular immune response compared with other combinations. Further verified the superiority of the combined design of antigen peptides in the PRRSV - C - LNP group in vaccine application.

[0259] 3.2.2 Detection Results of Neutralizing Antibodies

[0260] Neutralization tests were carried out on the specific antibodies produced after the first and second immunizations, and different lineage virus strains were selected to react with the specific antibodies. The specific detection results are shown in Table 6, and there are no significant differences among the groups. Twenty-eight days after immunization, the average neutralizing antibody titers of all 10 μg dose groups are not lower than 1:16. According to relevant literature reports, a PRRSV neutralizing antibody titer not lower than 1:16 can 100% prevent the occurrence of viremia and the transplacental infection of PRRSV.

[0261] Table 6 Antibody Detection and Analysis of Vaccinated Mice

[0262]

[0263] Among the immunized groups, the antigen combination of the PRRSV - C - LNP group has certain advantages in neutralizing antibody titer compared with other combinations, and the antibody titer is between 1:16 and 1:32. It shows that reasonable splitting and optimization of the GP5 antigen and removing the "shielding effect" between antigen epitopes have a significant effect on the production of neutralizing antibodies.

[0264] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. SEQUENCE LISTING <110> Tiankang Pharmaceutical Co., Ltd. <120> mRNA Vaccine for Preventing Porcine Reproductive and Respiratory Syndrome and Preparation Method <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF3 encoding GP3 protein <400> 1 cttgaacccg gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaac 57 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF4 encoding GP4 protein <400> 2 cctgagacca tgaggtgggc aaccgtttta gcctgtcttt tgccatccct actggcaatt 60 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF5 encoding GP5 protein - I <400> 3 tctcatattc agttgattta taactta 27 <210> 4 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF5 encoding GP5 protein - II <400> 4 ctggctgcgc tgatttgctt tgtcattagg cttgcgaaga actgcctcta tcgttggcgg 60 tcgcccgtca ttgtggagaa agggggtaag gttgaggtcg aaggtcacct gatcgacctc 120 aagagagttg tgcttgatgg ttccgcggca acccctttaa ccagagtttc agcggaacta 180 tggggtcgtc tc 192 <210> 5 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF6 encoding M protein <400> 5 tgcaatgata gcacagctcc acagaaggtg cttttggcgt tttccgctct aaaggtaagt 60 cgcggccgac tgctagggct tctgcacctt 90 <210> 6 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF7 encoding N protein <400> 6 gtcaggcatc actttacccc tagtgaggtg cgtctgatcc gcgccacagc atca 54 <210> 7 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Functional sequence <400> 7 gcgaagttcg tcgcggcgtg gactttgaag gcggcggcg 39 <210> 8 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein A gene sequence <400> 8 cttgaacccg gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaacggt 60 ccaggcccag gtcctgagac catgaggtgg gcaaccgttt tagcctgtct tttgccatcc 120 ctactggcaa ttggtccagg cccaggttct catattcagt tgatttataa cttaggtcca 180 ggcccaggtc tggctgcgct gatttgcttt gtcattaggc ttgcgaagaa ctgcctctat 240 cgttggcggt cgcccgtcat tgtggagaaa gggggtaagg ttgaggtcga aggtcacctg 300 atcgacctca agagagttgt gcttgatggt tccgcggcaa cccctttaac cagagtttca 360 gcggaactat ggggtcgtct cggtccaggc ccaggttgca atgatagcac agctccacag 420 aaggtgcttt tggcgttttc cgctctaaag gtaagtcgcg gccgactgct agggcttctg 480 caccttggtc caggcccagg tgtcaggcat cactttaccc ctagtgaggt gcgtctgatc 540 cgcgccacag catcatga 558 <210> 9 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein B gene sequence <400> 9 cttgaacccg gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaacggt 60 ccaggcccag gtcctgagac catgaggtgg gcaaccgttt tagcctgtct tttgccatcc 120 ctactggcaa ttggtccagg cccaggttgc aatgatagca cagctccaca gaaggtgctt 180 ttggcgtttt ccgctctaaa ggtaagtcgc ggccgactgc tagggcttct gcaccttggt 240 ccaggcccag gttctcatat tcagttgatt tataacttag gtccaggccc aggtctggct 300 gcgctgattt gctttgtcat taggcttgcg aagaactgcc tctatcgttg gcggtcgccc 360 gtcattgtgg agaaaggggg taaggttgag gtcgaaggtc acctgatcga cctcaagaga 420 gttgtgcttg atggttccgc ggcaacccct ttaaccagag tttcagcgga actatggggt 480 cgtctcggtc caggcccagg tgtcaggcat cactttaccc ctagtgaggt gcgtctgatc 540 cgcgccacag catcatga 558 <210> 10 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein C gene sequence[[ID=:19]] <400> 10 cttgaacccg gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaacggt 60 ccaggcccag gtcctgagac catgaggtgg gcaaccgttt tagcctgtct tttgccatcc 120 ctactggcaa ttggtccagg cccaggtctg gctgcgctga tttgctttgt cattaggctt 180 gcgaagaact gcggtccagg cccaggtctc tatcgttggc ggtcgcccgt cattgtggag 240 aaagggggta aggttgaggt cgaaggtcac ctgatcgacc tcaagagagt tgtgcttgat 300 ggttccgcgg caaccccttt aaccagagtt tcagcggaac tatggggtcg tctcggtcca 360 ggcccaggtt gcaatgatag cacagctcca cagaaggtgc ttttggcgtt ttccggtcca 420 ggcccaggtg ctctaaaggt aagtcgcggc cgactgctag ggcttctgca ccttggtcca 480 ggcccaggtg tcaggcatca ctttacccct agtgagggtc caggcccagg tgtgcgtctg 540 atccgcgcca cagcatcagg tccaggccca ggttctcata ttcagttgat ttataactta 600 gcgaagttcg tcgcggcgtg gactttgaag gcggcggcgt ctcatattca gttgatttat 660 aacttatga 669 <210> 11 <211> 1073 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein gene after adding expression components <400> 11 taatacgact cactataggg cgaattgaca tttgcttctg acacaactgt gttcactagc 60 aacctcaaac agacaccgcc accatggaac aagagaaaga gcacaagaca gtggaaatga 120 aagcgagggg gacacggagg aatcttgaac ccggcaagtc tttttggtgc aggatagggc 180 atgaccgatg tagtgagaac ggtccaggcc caggtcctga gaccatgagg tgggcaaccg 240 ttttagcctg tcttttgcca tccctactgg caattggtcc aggcccaggt ctggctgcgc 300 tgatttgctt tgtcattagg cttgcgaaga actgcggtcc aggcccaggt ctctatcgtt 360 ggcggtcgcc cgtcattgtg gagaaagggg gtaaggttga ggtcgaaggt cacctgatcg 420 acctcaagag agttgtgctt gatggttccg cggcaacccc tttaaccaga gtttcagcgg 480 aactatgggg tcgtctcggt ccaggcccag gttgcaatga tagcacagct ccacagaagg 540 tgcttttggc gttttccggt ccaggcccag gtgctctaaa ggtaagtcgc ggccgactgc 600 tagggcttct gcaccttggt ccaggcccag gtgtcaggca tcactttacc cctagtgagg 660 gtccaggccc aggtgtgcgt ctgatccgcg ccacagcatc aggtccaggc ccaggttctc 720 atattcagtt gatttataac ttagcgaagt tcgtcgcggc gtggactttg aaggcggcgg 780 cgtctcatat tcagttgatt tataacttat gagctcgctt tcttgctgtc caatttctat 840 taaaggttcc tttgttccct aagtccaact actaaactgg gggatattat gaagggcctt 900 gagcatctgg attctgccta ataaaaaaca tttattttca ttgcaaaaaa aaaaaaaaaa 960 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 1073 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T7 promoter <400> 12 taatacgact cactatagg 19 <210> 13 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> 5'UTR sequence <400> 13 gcgaattgac atttgcttct gacacaactg tgttcactag caacctcaaa cagacacc 58 <210> 14[[ID=*37]] <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Coding sequence of signal peptide <400> 14 atggaacaag agaaagagca caagacagtg gaaatgaaag cgagggggac acggaggaat 60 <210> 15 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Kozak sequence Note: There seems to be a formatting or encoding issue with the original text which has some repeated lines and some lines that might not be in a standard format. The translation attempts to maintain the integrity of the provided text as much as possible. Also, the tag was left as is as it's not clear if there's a specific translation rule for it. If it's a custom or misformatted tag, more context would be needed for a more accurate translation.<400> 15 gccaccgcca cc 12 <210> 16 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> 3'UTR sequence <400> 16 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60 taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120 tattttcatt gc 132 <210> 17 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> poly A sequence <400> 17 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120 aaaaaaaaa 129 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> The nucleotide sequence of the open editing frame ORF1 encoding Nsp2 protein <400> 18 gctggaaaaa gagcgaga 18 <210> 19 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence two of open reading frame ORF1 encoding Nsp2 protein <400> 19 gagagagtga gaccttcaga cgactgggcc actgacgagg acctagtgaa cact 54 <210> 20 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence three of open reading frame ORF1 encoding Nsp2 protein <400> 20 aaggccaacc cggtcactcc gggagaggta aaggaaaaga ttgaccagta tctc 54 <210> 21 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF1 encoding Nsp9 protein <400> 21 ggtaggtgtc ttgaagctga tctagcatct tgcgatcgga gcacccct 48 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of open reading frame ORF1 encoding Nsp10 protein <400> 22 gcgattcaac cggattaccg ggacaagctg atgtctatg 39 <210> 23 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of fusion protein A <400> 23 Leu Glu Pro Gly Lys Ser Phe Trp Cys Arg Ile Gly His Asp Arg Cys 1 5 10 15 Ser Glu Asn Gly Pro Gly Pro Gly Pro Glu Thr Met Arg Trp Ala Thr 20 25 30 Val Leu Ala Cys Leu Leu Pro Ser Leu Leu Ala Ile Gly Pro Gly Pro 35 40 45 Gly Ser His Ile Gln Leu Ile Tyr Asn Leu Gly Pro Gly Pro Gly Leu 50 55 60 Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala Lys Asn Cys Leu Tyr 65 70 75 80 Arg Trp Arg Ser Pro Val Ile Val Glu Lys Gly Gly Lys Val Glu Val 85 90 95 Glu Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala 100 105 110 Ala Thr Pro Leu Thr Arg Val Ser Ala Glu Leu Trp Gly Arg Leu Gly 115 120 125 Pro Gly Pro Gly Cys Asn Asp Ser Thr Ala Pro Gln Lys Val Leu Leu 130 135 140 Ala Phe Ser Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu 145 150 155 160 His Leu Gly Pro Gly Pro Gly Val Arg His His Phe Thr Pro Ser Glu 165 170 175 Val Arg Leu Ile Arg Ala Thr Ala Ser 180 185 <210> 24 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of fusion protein B <400> 24 Leu Glu Pro Gly Lys Ser Phe Trp Cys Arg Ile Gly His Asp Arg Cys 1 5 10 15 Ser Glu Asn Gly Pro Gly Pro Gly Pro Glu Thr Met Arg Trp Ala Thr 20 25 30 Val Leu Ala Cys Leu Leu Pro Ser Leu Leu Ala Ile Gly Pro Gly Pro 35 40 45 Gly Cys Asn Asp Ser Thr Ala Pro Gln Lys Val Leu Leu Ala Phe Ser 50 55 60 Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Gly 65 70 75 80 Pro Gly Pro Gly Ser His Ile Gln Leu Ile Tyr Asn Leu Gly Pro Gly 85 90 95 Pro Gly Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala Lys Asn 100 105 110 Cys Leu Tyr Arg Trp Arg Ser Pro Val Ile Val Glu Lys Gly Gly Lys 115 120 125 Val Glu Val Glu Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp 130 135 140 Gly Ser Ala Ala Thr Pro Leu Thr Arg Val Ser Ala Glu Leu Trp Gly 145 150 155 160 Arg Leu Gly Pro Gly Pro Gly Val Arg His His Phe Thr Pro Ser Glu 165 170 175 Val Arg Leu Ile Arg Ala Thr Ala Ser 180 185 <210> No. 25 <211> No. 222 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of fusion protein C <400> No. 25 Leu Glu Pro Gly Lys Ser Phe Trp Cys Arg Ile Gly His Asp Arg Cys 1 5 10 15 Ser Glu Asn Gly Pro Gly Pro Gly Pro Glu Thr Met Arg Trp Ala Thr 20 25 30 Val Leu Ala Cys Leu Leu Pro Ser Leu Leu Ala Ile Gly Pro Gly Pro 35 40 45 Gly Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala Lys Asn Cys 50 55 60 Gly Pro Gly Pro Gly Leu Tyr Arg Trp Arg Ser Pro Val Ile Val Glu 65 70 75 80 Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu Lys Arg 85 90 95 Val Val Leu Asp Gly Ser Ala Ala Thr Pro Leu Thr Arg Val Ser Ala 100 105 110 Glu Leu Trp Gly Arg Leu Gly Pro Gly Pro Gly Cys Asn Asp Ser Thr 115 120 125 Ala Pro Gln Lys Val Leu Leu Ala Phe Ser Gly Pro Gly Pro Gly Ala 130 135 140 Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Gly Pro 145 150 155 160 Gly Pro Gly Val Arg His His Phe Thr Pro Ser Glu Gly Pro Gly Pro 165 170 175 Gly Val Arg Leu Ile Arg Ala Thr Ala Ser Gly Pro Gly Pro Gly Ser 180 185 190 His Ile Gln Leu Ile Tyr Asn Leu Ala Lys Phe Val Ala Ala Trp Thr 195 200 205 Leu Lys Ala Ala Ala Ser His Ile Gln Leu Ile Tyr Asn Leu 210 215 220

Claims

1. Use of porcine reproductive and respiratory syndrome virus structural proteins containing neutralizing epitopes in the preparation of mRNA vaccines, wherein the structural proteins include membrane protein, M protein and N protein; The membrane protein includes GP3, GP4 and GP5; The nucleotide sequence of the open reading frame ORF3 encoding GP3 protein is SEQ ID No.1; The nucleotide sequence of the open reading frame ORF4 encoding GP4 protein is SEQ ID No.2; The nucleotide sequence of the open reading frame 5’ORF5 encoding GP5 protein is SEQIDNo.3; or, the nucleotide sequence of the open reading frame 3’ORF5 encoding GP5 protein is SEQID No.4; The nucleotide sequence of the open reading frame ORF6 encoding M protein is SEQIDNo.5; The nucleotide sequence of the open reading frame ORF7 encoding N protein is SEQIDNo.6; The combination order of the structural proteins is ORF3, ORF4, ORF5, ORF6, ORF7; or, ORF3, ORF4, ORF6, ORF5, ORF7; or, ORF3, ORF4, 3’ORF5, ORF6, ORF7, 5’ORF5.

2. The first mRNA molecule, characterized in that, The first mRNA molecule encodes a first fusion protein, and the first fusion protein includes at least two polypeptide fragments derived from porcine reproductive and respiratory syndrome virus structural proteins containing neutralizing epitopes connected by a flexible linker; The polypeptide fragment order of the porcine reproductive and respiratory syndrome virus structural proteins containing neutralizing epitopes is selected from the combination order of the structural proteins described in claim 1; The nucleotide sequence of the first fusion protein is as shown in SEQIDNo.8~SEQIDNo.

10.

3. Construct, characterized in that, The construct includes the first mRNA molecule described in claim 2, and the nucleotide sequence of the construct further includes UTR sequence, Kozak sequence, signal peptide coding sequence, promoter sequence and poly(A) sequence; The UTR sequence includes a 5’UTR sequence with a nucleotide sequence as shown in SEQ ID No.13 and a 3’UTR sequence with a nucleotide sequence as shown in SEQIDNo.16; The nucleotide sequence of the Kozak sequence is as shown in SEQ ID No.15; The nucleotide sequence encoding the signal peptide is as shown in SEQ ID No.14; The promoter includes a T7 promoter, and the nucleotide sequence is as shown in SEQ ID No.12; The base length of the poly(A) is 50~150bps.

4. The construct according to claim 3, wherein The base length of the poly(A) is 129bps.

5. A biomaterial, characterized in that, Including: (a) A recombinant vector, including the construct described in claim 3 and a plasmid vector; (b) A transformant, including a host cell containing the construct described in claim 3, or the recombinant vector in (a).

6. The biomaterial according to claim 5, characterized in that, The plasmid vector includes pcDNA3.1, CET1019 HS-puro, pIRES or pRL-SV40.

7. The biomaterial according to claim 6, wherein The plasmid vector is pcDNA3.

1.

8. The biomaterial according to claim 5, characterized in that, The host cell includes a prokaryotic cell or a eukaryotic cell.

9. The biomaterial according to claim 8, wherein, The prokaryotic cells include DH5α, JM109 or BL21.

10. The biomaterial according to claim 9, wherein The prokaryotic cell is DH5α.

11. The biological material according to claim 8, characterized in that, The eukaryotic cells include yeast cells, Marco-145, PK-15, Hela or insect cells.

12. The biomaterial according to claim 11, characterized in that, The eukaryotic cell is Marco-145.

13. The method for preparing the construct according to claim 3, characterized in that, The preparation method includes: (1) Synthesizing a gene fragment of an expression construct, the nucleotide sequence of the construct sequentially includes, from the 5'-end to the 3'-end, a promoter sequence - 5'UTR sequence - Kozak sequence - signal peptide coding sequence - the first mRNA molecule - 3'UTR sequence - poly(A) sequence; (2) Inserting the gene fragment obtained in step (1) into a plasmid vector to obtain a recombinant vector, then transforming the recombinant vector into a host cell to obtain a transformant, extracting the recombinant vector after the transformant proliferates, and obtaining the construct after in vitro transcription.

14. Use of the first mRNA molecule according to claim 2, the construct according to claim 3, the biological material according to any one of claims 5 to 12, or the preparation method according to claim 13 in the preparation of an mRNA vaccine.

15. A method for preparing an mRNA vaccine, characterized in that, The preparation method includes mixing the construct according to claim 4, or the construct obtained by using the preparation method according to claim 13 with a carrier, and then obtaining the mRNA vaccine after dialysis, concentration and sterile filtration in sequence.

16. The preparation method according to claim 15, wherein, The carrier includes lipid nanoparticles.

17. The preparation method according to claim 16, wherein, The components of the lipid nanoparticles include D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG.

18. The preparation method according to claim 17, wherein The molar ratio of D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG is (50):(14 - 9):(35 - 40):

1.

19. The preparation method according to claim 17, characterized in that, The molar ratio of D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG is 50:10:37.5:2.

5.

20. The preparation method according to claim 19, wherein, The preparation method includes mixing D-Lin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol and PEGylated lipid PEG-DMG in an organic solvent according to the molar ratio to obtain lipid nanoparticles, then mixing with the construct, and then obtaining the mRNA vaccine after dialysis, concentration and sterile filtration in sequence.

21. The preparation method according to claim 20, characterized in that, The organic solvent is selected from ethanol, isopropanol or methanol.

22. The preparation method according to claim 21, wherein, The organic solvent is ethanol.

23. The preparation method according to claim 20, characterized in that, The lipid nanoparticles and the construct are mixed at a volume flow rate of 1:(1 - 10).

24. The preparation method according to claim 23, wherein The lipid nanoparticles and the construct are mixed at a volume flow rate of 1:

3.

25. The mRNA vaccine prepared by the preparation method described in claim 15, wherein, The mRNA vaccine has a particle size of 90 - 100 nm, a Zeta potential of -20 - -7.5 mV, and an encapsulation efficiency of more than 90%.

Images