Cat infectious peritonitis mRNA vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of mRNA vaccines, and discloses a feline infectious peritonitis mRNA vaccine as well as a preparation method and application thereof. The feline infectious peritonitis mRNA vaccine comprises mRNA molecules; the mRNA molecule comprises an FIBV-S protein coding sequence as shown in SEQ ID NO: 6. According to the invention, the mRNA sequence for coding the natural FIBV-S protein is modified, so that the FIBV-S protein obtained by translation can be stably maintained at the prefusion conformation and has higher immunogenicity, thereby endowing the mRNA vaccine with a better immune effect and being beneficial to the prevention of feline infectious peritonitis.

Description

Technical Field

[0001] This invention relates to the field of mRNA vaccine technology, and in particular to a feline infectious peritonitis mRNA vaccine, its preparation method, and its application. Background Technology

[0002] Feline infectious peritonitis virus (FIPV) can infect multiple systems throughout a cat's body, causing highly fatal feline infectious peritonitis (FIP). Cats can be infected and develop the disease at any age, and it can also cause mild to severe eye and neurological disorders. FIPV-induced FIP was first reported by Holzworth J in the United States in 1963. Its pathophysiology is not fully understood, but it is generally believed that FIPV mutates during the replication process of FCoV. Accurate clinical diagnosis is difficult, and there is currently no safe, effective, and standardized treatment. Therefore, developing a vaccine to prevent FIP ​​is of great importance. However, there are currently few vaccines specifically for preventing FIP on the market. Vaccinated cats are prone to antibody-dependent enhancement (ADE) when challenged with high doses of heterologous viral strains. Therefore, to date, there is no sufficiently safe and effective vaccine to prevent FIPV infection.

[0003] The basic mechanism of mRNA vaccine technology is based on the use of genetically engineered vectors to transfer antigens encoded by nucleic acid molecules into the host's target cells, thereby inducing the host cells to produce antibodies. When pathogens carrying the antigens invade again, the host's immune system can rapidly trigger humoral and cellular immune responses, thus preventing disease. mRNA vaccines have a high effective protection rate, and because the translation site is in the cytoplasm of animals, they have high safety. Furthermore, compared with traditional recombinant protein vaccines, inactivated vaccines, and attenuated vaccines, mRNA vaccines are simpler to prepare and purify, require fewer doses, and are easier to popularize, thus showing good application prospects.

[0004] The trimeric S protein on the surface of FIPV plays a crucial role in mediating host cell invasion, thus the S protein is considered a primary antigen in vaccine design. For example, patent CN114015702A produced an mRNA vaccine by adding at least one of a 5'-cap structure, a 3'-terminal polyadenylated nucleotide sequence, a 5'-UTR, and a 3'-UTR to the mRNA sequence encoding the FIPV-S protein. However, the FIPV-S protein is highly unstable and undergoes significant conformational changes during receptor binding, transitioning from an active to an inactive state. The pre-fusion conformation of the FIPV-S protein exhibits better immunogenicity, while the post-fusion conformation shows poorer immunogenicity. Therefore, if an mRNA vaccine is prepared by simply adding a 5'-cap structure, a 3'-terminal polyadenylated nucleotide sequence, a 5'-UTR, and a 3'-UTR to the mRNA sequence encoding the FIPV-S protein, the S protein translated from the mRNA vaccine in the host is unstable, resulting in poor immunization efficacy. Summary of the Invention

[0005] To address the technical problem of poor immunization efficacy of existing FIPV-mRNA vaccines, this invention provides a feline infectious peritonitis (FIPV) mRNA vaccine, its preparation method, and its application. The FIPV-S protein translated from the mRNA vaccine of this invention can be stably maintained in its pre-fusion conformation, thereby resulting in a better immunization effect of the mRNA vaccine.

[0006] The specific technical solutions of the present invention are: In a first aspect, the present invention provides a feline infectious peritonitis mRNA vaccine comprising an mRNA molecule; said mRNA molecule comprising the FIPV-S protein coding sequence as shown in SEQ ID NO:6.

[0007] This invention modifies the mRNA sequence encoding the natural FIPV-S protein, using the mRNA sequence shown in SEQ ID NO:6. This addresses the problem of unstable immunogenicity caused by the conformational instability of the FIPV-S protein. The FIPV-S protein obtained after translating the mRNA sequence shown in SEQ ID NO:6 can stably maintain its pre-fusion conformation and has high immunogenicity, thereby giving the mRNA vaccine a better immune effect and benefiting the prevention of feline infectious peritonitis.

[0008] Preferably, the mRNA molecule further includes at least one of a 5' cap sequence, a 5' UTR, a Kozak sequence, a 3' UTR sequence, and a PolyA tail sequence.

[0009] Preferably, the full sequence of the mRNA molecule is shown in SEQ ID NO:7.

[0010] Preferably, the feline infectious peritonitis mRNA vaccine further includes at least one of a pharmaceutically acceptable carrier, excipient, and dispersion medium.

[0011] Furthermore, the carrier is lipid nanoparticles.

[0012] In a second aspect, the present invention provides a DNA for preparing the feline infectious peritonitis mRNA vaccine, comprising the FIPV-S protein-coding sequence as shown in SEQ ID NO:3.

[0013] Preferably, the DNA further includes at least one of the following: the T7 promoter sequence shown in SEQ ID NO:1, the 5'UTR sequence shown in SEQ ID NO:2, the Kozak sequence, the 3'UTR sequence shown in SEQ ID NO:4, and the PolyA tail sequence shown in SEQ ID NO:5; wherein the Kozak sequence is GCCACC.

[0014] Thirdly, the present invention provides a recombinant plasmid, wherein the recombinant plasmid is a cloning vector or expression vector carrying the DNA.

[0015] Fourthly, the present invention provides a method for preparing the feline infectious peritonitis mRNA vaccine, comprising the following steps: constructing a recombinant plasmid containing a DNA sequence encoding the mRNA molecule, linearizing the plasmid, performing in vitro transcription and 5' capping to obtain the mRNA molecule, and then preparing the mRNA molecule into an mRNA vaccine.

[0016] Fifthly, the present invention provides the use of the feline infectious peritonitis mRNA vaccine or the DNA or the recombinant plasmid in the preparation of a medicament for the prevention of feline infectious peritonitis.

[0017] Compared with the prior art, the present invention has the following advantages: This invention, through specific modification of the mRNA sequence encoding the natural FIPV-S protein, enables the translated FIPV-S protein to have a stable pre-fusion conformation and high immunogenicity, thereby giving the mRNA vaccine a better immune effect and benefiting the prevention of feline infectious peritonitis. Attached Figure Description

[0018] Figure 1 The results show the antibody titer of the FIPV-mRNA vaccine. Figure 1 (A) is the antibody titer curve measured 14 days after the initial immunization; Figure 1 (B) is the IgG antibody titer measured 14 days after the initial immunization; Figure 1 (C) Antibody titer curve measured 14 days after booster immunization; Figure 1 (D) is the IgG antibody titer measured 14 days after booster immunization.

[0019] Figure 2 This is a map of recombinant plasmids used to prepare FIPV-mRNA vaccines.

[0020] Figure 3 This is a schematic diagram of the core components in the recombinant plasmid used to prepare the FIPV-mRNA vaccine.

[0021] Figure 4 This is a graph showing the results of PAGE analysis for mRNA denaturation.

[0022] Figure 5 This is a graph showing the detection results of mRNA expression.

[0023] Figure 6 This is a negative-stained electron microscope image of the FIPV-mRNA vaccine. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments.

[0025] General Implementation Examples First, the present invention relates to a feline infectious peritonitis mRNA vaccine comprising an mRNA molecule; said mRNA molecule comprising the FIPV-S protein coding sequence as shown in SEQ ID NO:6.

[0026] In some specific embodiments, the mRNA molecule further includes at least one of a 5' cap sequence, a 5' UTR, a Kozak sequence, a 3' UTR sequence, and a PolyA tail sequence.

[0027] In some specific embodiments, the full sequence of the mRNA molecule is shown in SEQ ID NO:7.

[0028] In some specific embodiments, the feline infectious peritonitis mRNA vaccine further includes at least one of a pharmaceutically acceptable carrier, excipient, and dispersion medium. Optionally or preferably, the carrier is a lipid nanoparticle.

[0029] Second, the present invention relates to a DNA for preparing the feline infectious peritonitis mRNA vaccine, comprising the FIPV-S protein coding sequence as shown in SEQ ID NO:3.

[0030] In some specific embodiments, the DNA further includes at least one of the following: the T7 promoter sequence shown in SEQ ID NO:1, the 5'UTR sequence shown in SEQ ID NO:2, the Kozak sequence, the 3'UTR sequence shown in SEQ ID NO:4, and the PolyA tail sequence shown in SEQ ID NO:5; wherein the Kozak sequence is GCCACC.

[0031] Third, the present invention relates to a recombinant plasmid, wherein the recombinant plasmid is a cloning vector or expression vector carrying the DNA.

[0032] Fourth, the present invention relates to a method for preparing a feline infectious peritonitis mRNA vaccine, comprising the following steps: constructing a recombinant plasmid containing a DNA sequence encoding the mRNA molecule, linearizing the plasmid, performing in vitro transcription and capping the 5' end to obtain the mRNA molecule, and then preparing the mRNA molecule into an mRNA vaccine.

[0033] Fifth, the present invention relates to the use of the feline infectious peritonitis mRNA vaccine or the DNA or recombinant plasmid in the preparation of a drug for the prevention of feline infectious peritonitis. Specific Implementation The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0035] The definitions of the technical terms used in this invention are as follows: (1) Untranslated Region (UTR): This refers to any segment located at either end of the coding sequence of the mRNA strand. If it is located at the 5' end, it is called the 5' untranslated region (or "leader sequence"), and if it is located at the 3' end, it is called the 3' untranslated region (or "tail sequence"). The untranslated region sequence is not a codon and cannot be translated into amino acids, but it can control the degradation and transcription efficiency of the mRNA product through RNA-binding proteins. Therefore, every mRNA vaccine needs to consider how to design the UTR components of the product to improve the stability and transcription efficiency of the mRNA vaccine in the target cells.

[0036] (2) PolyA: Most eukaryotic mRNAs have a PolyA tail at the 3' end, consisting of 100-200 adenosine nucleotides polymerized together. This tail can prevent exonuclease degradation and also serves as a marker of the nuclear pore transport system, involved in the transport of mature mRNA through nuclear pores to the cytoplasm. The length of the PolyA tail is also a very important optimization indicator when designing the sequence of mRNA vaccine products.

[0037] (3) Kozak sequence: The eukaryotic translation initiation sequence is often referred to as the "Kozak" common sequence. The Kozak sequence is a nucleic acid sequence located between the 5'-UTR of eukaryotic mRNA and the start codon AUG, usually ACCACCAUGG, GCCAUGAUGG, etc., where AUG represents the translation initiation site of the methionine codon at position +1. The Kozak sequence on the mRNA molecule is recognized by ribosomes as a translation initiation site, and it can bind to translation initiation factors to mediate the translation initiation of mRNA containing a 5' cap structure. The Kozak sequence can be used to enhance the translation efficiency of eukaryotic genes. Kozak sequences different from those of this invention can be selected and replaced or deleted using conventional techniques. For example, the Kozak sequence can be replaced with GCC, ACC, GCCACC, or GCCANN, where N refers to A, T / U, C, or G.

[0038] (4) 5' Cap: In eukaryotes, the 5' end of messenger RNA (mRNA) is a dinucleotide terminus formed by modification. It is formed after mRNA transcription by pyrophosphorylation of a methylated guanine nucleotide (m7G) to the 5' nucleotide of the mRNA. The 5' cap structure plays an important role in many biological functions of mRNA, including mRNA splicing, stabilization, transport, and ribosome recruitment. Due to the important biological functions of the 5' cap structure, it is frequently used in mRNA vaccine development.

[0039] (5) Antibody titer: used to measure the minimum concentration (i.e., maximum dilution) required for a certain antibody to recognize a specific epitope.

[0040] (6) Neutralizing antibody: is an antibody that has protective power to prevent cells from being damaged by a certain antigen or infectious agent.

[0041] Example 1: Preparation of feline infectious peritonitis mRNA vaccine 1.1 Construction of recombinant plasmids Build as Figure 2The recombinant plasmid shown comprises, from upstream to downstream, a T7 promoter, a 5'UTR sequence, a Kozak sequence, a coding sequence, a 3'UTR sequence, and a PolyA tail. The DNA sequences of each component are as follows: the DNA sequence of the T7 promoter is shown in SEQ ID NO:1; the DNA sequence of the 5'UTR sequence is shown in SEQ ID NO:2; the Kozak sequence is GCCACC; the coding sequence is the DNA sequence encoding the FIPV-S protein, as shown in SEQ ID NO:3; the 3'UTR sequence is shown in SEQ ID NO:4; and the DNA sequence of the PolyA tail is shown in SEQ ID NO:5.

[0042] 1.2 Plasmid template linearization plasmid preparation: The recombinant plasmid constructed in 1.1 was transformed into Escherichia coli and cultured in LB liquid medium; 15-25 mL of overnight culture was taken and the plasmid was extracted using a small-volume extraction kit, with a elution concentration of approximately 300-400 μg / μL and OD260 / 280, 260 / 230≈2.

[0043] Plasmid linearization: Take 30 μg of plasmid, approximately 100 μL. Digest the plasmid with 6 μL of BsaI (nearshore protein) (1 μL of plasmid is digested with 1 μL of 10 U / μL BsaI), and digest the mixture in a metal bath at 37°C for 1 h.

[0044] Enzyme removal: The enzyme was treated with proteinase K (5 mg / mL, 100×) at a working concentration of 50 μg / mL, 1.2 μL, and digested at 37 °C for 1 h; then diluted to 200 μL with DNase / RNase-free ultrapure water to obtain an aqueous phase containing linearized template.

[0045] Protein removal and DNA purification: Prepare a Tris-saturated phenol, chloroform, and isoamyl alcohol mixture at a volume ratio of 25:24:1 (pH ≥ 7.8). Add an equal volume (200 μL) to the aqueous phase containing the linearized template, mix thoroughly, and let stand for 1 min until the mixture shows milky white bubbles. Centrifuge the mixture at 1000 rpm for 10 min at room temperature. The upper layer is a clear aqueous layer containing nucleic acids, the lower layer is a yellow phenol layer, and the middle layer is a white protein layer. Transfer the upper aqueous phase to a new RNase-free EP tube (1.5 mL), being careful not to aspirate the middle layer. Extract twice with an equal volume of chloroform / isoamyl alcohol mixture (chloroform to isoamyl alcohol volume ratio 24:1), centrifuge at 1000 rpm for 10 min at room temperature, and collect the upper aqueous phase into a new RNase-free EP tube.

[0046] DNA purification: Add 150 μL of isopropanol (1:1 ratio) to precipitate the DNA, mix well by pipetting, and incubate at -20°C for 1 hour. Centrifuge at 15,000 rpm for 15 minutes at 4°C, then discard the supernatant. A small amount of pale yellow, transparent, glassy precipitate can be observed. Wash twice with 300 μL of pre-chilled 70% ethanol, centrifuge at 15,000 rpm for 15 minutes at 4°C, discard the supernatant, and dry in a clean bench for 15 minutes. Dissolve in 20 μL of DNase / RNase-free ultrapure water, and measure OD260 / 280 = 1.8–2.0 to obtain the linearized template solution, which should be stored at -20°C.

[0047] 1.3 mRNA in vitro transcription To construct the reaction system, add 4.5 μL of DNase / RNase-free ultrapure water, 2 μL of ATP, 2 μL of CTP, 2 μL of GTP, 2 μL of 1-N-Met-UTP, and 2 μL of CleanCap AG (3'OMe), and mix by pipetting. Add 4 μL of 5×Reaction Buffer and mix by pipetting. Add 2 μL of the linearized template solution prepared in step 1.2 (530 ng / μL, approximately 1.06 μg in total) and mix by pipetting. Add 1.5 μL of Enzyme. Mix thoroughly by pipetting; after thorough mixing, centrifuge at low speed and incubate at 37℃ for 2–4 h; then add 2 μL of DNase I, mix by pipetting / vortexing, centrifuge at low speed, and incubate at 37℃ for 30 min to allow the reaction to proceed; finally, add 30 μL of DNase / RNase-free water and 30 μL of LiCl to terminate the reaction, the solution will become visibly turbid, and incubate at -20℃ for 30 min; then centrifuge at 12000 rpm for 15 min at 4℃, discard the supernatant, wash three times with 200 μL of pre-cooled 70% ethanol, and centrifuge at 15000 rpm for 10 min at 4℃. Open the lid and dry in a clean bench for 15 min; dissolve in DNase / RNase-free water, and measure OD260 / 280 = 1.8–2.0 to obtain the mRNA solution, which should be stored at -20℃.

[0048] 1.4 Preparation of mRNA-LNP (lipid nanoparticles loaded with mRNA) mRNA aqueous phase preparation: Dilute the mRNA solution prepared in step 1.3 to 0.15 μg / μL with 100 mM sodium citrate (pH 4.0) to prepare an mRNA aqueous phase with a total volume of approximately 667 μL (26 μL mRNA solution + 320.5 μL 100 mM sodium citrate + 320.5 μL DNase / RNase-free water, mix by pipetting), and incubate at 4 °C.

[0049] Lipid phase preparation: Take 36.36 μL ALC-0315, 24.60 μL DSPC, 55.89 μL Chole and 3.14 μL ALC-0159, then add anhydrous ethanol to make up to a total volume of 222 μL, mix well, and obtain the Lipid phase.

[0050] Mixing: Set the flow rate of the Lipid channel (left channel) to 3 mL / min and the flow rate of the mRNA channel (right channel) to 9 mL / min. Mix 0.2 mL of the Lipid phase and 0.6 mL of the mRNA aqueous phase to obtain the mRNA / Lipid mixture. Initial waste solution: 0.2 mL; final waste solution: 0.05 mL.

[0051] Calculate the final concentration: The volume of the mixture is 550 mL. If the encapsulation rate is 90%, the final concentration of mRNA is 0.15 × 600 × 0.9 / 800 = 0.10125 μg / mL, which is approximately 100 ng / μL.

[0052] Alcohol removal: Transfer the mRNA / Lipid mixture into a 10 kDa ultrafiltration tube (MiliPore) and centrifuge at 3000 rpm and 4°C to 500 mL; then add 6 times the amount of 1×PBS (approximately 2.5–3 mL) for dilution, and continue centrifuging to 500 μL, with a concentration of approximately 100 ng / μL.

[0053] After filtration through a 0.22 μm filter membrane, the FIPV-mRNA vaccine (mRNA-LNP dispersion) was obtained and can be directly administered to animals.

[0054] In FIPV-mRNA vaccines, the core components of the mRNA molecule include... Figure 3 As shown, from upstream to downstream, the sequence includes a 5' cap (5'm7G), a 5' UTR, a Kozak sequence, an mRNA sequence encoding the FIPV-S protein (as shown in SEQ ID NO:6; since "t" is used to represent thymine T in DNA and uracil U in RNA according to the WIPOST.26 standard, the specific sequence of SEQ ID NO:6 is the same as that of SEQ ID NO:3), a 3' UTR, and a PolyA tail. The sequence of the entire mRNA molecule is shown in SEQ ID NO:7.

[0055] Example 2: mRNA quality assessment 2.1 Identification of mRNA denaturation by PAGE Prepare formaldehyde denaturing gel: Total volume 25mL, specifically 0.25g agarose, add 18.34mL water to an Erlenmeyer flask, heat to boiling, and cool slightly; add 4.16mL 40% formaldehyde, 2.5mL 10×MOPS buffer, and 2.5μL YeaRed in sequence, and pour into a mold.

[0056] RNA pretreatment: Mix 1–2 μL of the mRNA solution prepared in step 1.3 (for loading > 5 μg), 0.5 μL of 10×MOPS, 1 μL of formaldehyde (40%), and 2.5 μL of 2×loading dye (Invitrogen) to prepare a total loading system of 5 μL. Incubate the prepared loading system at 65 °C for 10 min, then place it on ice for 5 min. Add the loading system to the wells of the prepared denaturing gel and electrophoresis at 100 V for 30 min (the buffer in the electrophoresis tank is a mixture of 800 mL 1×MOPS buffer, 80 mL 10×MOPS, and 720 mL DEPC water).

[0057] Electrophoresis results as follows Figure 4 As shown. Figure 4 The results show that the size of the electrophoretic bands matches the size of the FIPV S protein coding sequence, indicating that the preparation of the mRNA molecule in Example 1 was successful.

[0058] 2.2 Detection of mRNA expression (1) Preheat OPTI-MEM medium; prepare 8 EP tubes, add 100mL of medium to each tube, label them, and divide 4 EP tubes into two groups, namely the mRNA group and the lipo group; (2) Add 2 μg of the mRNA solution prepared in step 1.3 to the mRNA group, add 4 μL of transfection reagent to the lipo group, and mix well; add the mixture of the mRNA group to the lipo group, mix well, and let stand for 20 min; (3) Add all the solution from each tube to a 12-well plate containing HEK293T cells and shake evenly to spread the cells. After 4-6 hours, change the medium (remove all the medium from the pump head and add 1 mL of fresh DMEM medium) or add medium (remove half of the medium and add half of the medium). (4) Harvest cells 24 h after transfection. Remove the culture medium from the 12-well plate, wash once with PBS and discard; add 200 μL of lysis buffer (prepared by mixing 25 mM HEPES, 150 mM NaCl solution at pH 7.4, 1 mM DTT and 1% Triton X-100 with added protease inhibitors) to each well; scrape the cells and transfer them to 1.5 mL EP tubes; place on ice for 15 min, centrifuge at 13000 rpm for 15 min, and collect the supernatant; (5) Take 15 μL of supernatant and add 5 μL of 4× loading buffer, boil at 100℃ for 5 min; add the obtained sample to the gel well of SDS-PAGE, electrophoresis at 90V for 30 min, and electrophoresis at 180V for 40 min. (6) Activate the PVDF membrane with methanol, rinse with ddH2O, and balance the wet transfer buffer; pre-balance the filter paper with the wet transfer buffer; soak the transfer clamp and cotton in the wet transfer buffer, assemble the membrane according to the following steps: anode (red) double-layer filter paper - PVDF membrane - adhesive - (black) double-layer filter paper, and remove air bubbles; add ice box and ice pack, and transfer the membrane at 300mA for 180min; (7) Cut the PVDF membrane according to the loading channels; block with 5% skim milk (made by mixing 20 mL TBST and 1 g skim milk) for 1 h; wash once with TBST, shake for 5 min, and repeat 3 times; dilute the FIPV antibody at 1:5000, add 2.4 μL of diluted antibody to 12 mL TBST, and incubate for 1 h; recover the antibody, wash once with TBST, shake for 5 min, and repeat 3 times; dilute the secondary antibody from the corresponding source at 1:5000, incubate for 1 h, wash once with TBST, shake for 5 min, and repeat 3 times; take 0.5 mL TBST, 0.25 mL luminescent solution, and 0.25 mL chromogenic solution and mix them. The colorimetric mixture is drawn onto the packaging bag, and air bubbles are removed. The PVDF film is immersed in the mixture with the PVDF film facing down and reacted in the dark for 3 minutes. Water is sprayed on both sides of the film in the dark clamp, and the film is wiped clean with absorbent paper. The film is placed with the front side facing up, and the air bubbles are removed. The film is developed in a darkroom. Three X-ray films are taken, one corner is creased, and the films are pressed for 2 minutes / 5 minutes. The films are then developed by machine.

[0059] Development results as follows Figure 5 As shown.

[0060] Example 3: Quality assessment of FIPV-mRNA vaccine (observation using negative staining electron microscopy) Glow discharge was performed with a discharge time of 60 s and a current of 15 mA for 10 s. The hydrophilicized carrier mesh was held face up with self-locking tweezers, and 2.5 μL of the FIPV-mRNA vaccine (mRNA-LNP dispersion) prepared in Example 1 was added. The mixture was allowed to stand for 1 min for adsorption. Excess sample was blotted away with filter paper. 5 μL of uranium acetate was added and allowed to stand for 10 s. Excess staining was blotted away with filter paper. After drying, uranium acetate was added once more, and finally 5 μL of uranium acetate was added and allowed to stand for 1 min for thorough staining. After blotting away excess staining with filter paper, the mixture was air-dried at room temperature for at least 15 min before being used for negative staining electron microscopy observation. The results are as follows: Figure 6 As shown. Figure 6 The electron microscope revealed that the small, round particles had a clear overall outline, consistent with the morphology of lipid nanoparticles.

[0061] Example 4: Evaluation of the Immunization Efficacy of FIPV-mRNA Vaccine (Antibody Titer Detection) The experimental group design was as follows: Mice in the experimental group were injected with 7 μg of the FIPV-mRNA vaccine (mRNA-LNP dispersion) prepared in Example 1 at the time of primary immunization. Two weeks after the primary immunization, mice in the experimental group were boosted with the same dose. Control group mice were injected with the same dose of PBS at both the primary and booster immunization times. To detect the level of feline infectious peritonitis (FIP) antibodies in mouse serum, purified FIPV S protein was used as the coating antigen. The IgG antibodies in the serum of immunized mice were detected by ELISA. The results at 14 days after the primary immunization are shown below. Figure 1 (A) and Figure 1 As shown in (B), the test results were obtained 14 days after the booster immunization. Figure 1 (C) and Figure 1 As shown in (D).

[0062] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0063] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A feline infectious peritonitis mRNA vaccine, characterized in that, Includes mRNA molecules; said mRNA molecules include the FIPV-S protein-coding sequence as shown in SEQ ID NO:

6.

2. The feline infectious peritonitis mRNA vaccine according to claim 1, characterized in that, The mRNA molecule also includes at least one of the following: a 5' cap sequence, a 5' UTR, a Kozak sequence, a 3' UTR sequence, and a PolyA tail sequence.

3. The feline infectious peritonitis mRNA vaccine according to claim 2, characterized in that, The full sequence of the mRNA molecule is shown in SEQ ID NO:

7.

4. The feline infectious peritonitis mRNA vaccine according to claim 1 or 3, characterized in that, It also includes at least one of pharmaceutically acceptable carriers, excipients, and dispersion media.

5. The feline infectious peritonitis mRNA vaccine according to claim 4, characterized in that, The carrier is lipid nanoparticles.

6. A DNA for preparing a feline infectious peritonitis mRNA vaccine according to any one of claims 1 to 5, characterized in that, Including the FIPV-S protein coding sequence shown in SEQ ID NO:

3.

7. The DNA according to claim 6, characterized in that, It also includes at least one of the following: the T7 promoter sequence shown in SEQ ID NO:1, the 5'UTR sequence shown in SEQ ID NO:2, the Kozak sequence, the 3'UTR sequence shown in SEQ ID NO:4, and the PolyA tail sequence shown in SEQ ID NO:5; wherein the Kozak sequence is GCCACC.

8. A recombinant plasmid, characterized in that, The recombinant plasmid is a cloning vector or expression vector carrying the DNA of claim 6 or 7.

9. A method for preparing a feline infectious peritonitis mRNA vaccine according to claim 3, characterized in that, Includes the following steps: A recombinant plasmid containing a DNA sequence encoding the mRNA molecule was constructed. After plasmid linearization, in vitro transcription was performed and the 5' end was capped to obtain the mRNA molecule. The mRNA molecule was then used to make an mRNA vaccine.

10. The use of the feline infectious peritonitis mRNA vaccine according to any one of claims 1 to 5, or the DNA according to claim 6 or 7, or the recombinant plasmid according to claim 8, in the preparation of a medicament for the prevention of feline infectious peritonitis.

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