An mRNA vaccine expressing feline calicivirus vp1 protein and a preparation method thereof

Abstract

The application discloses an mRNA vaccine for expressing feline calicivirus VP1 protein and a preparation method thereof, and relates to the field of nucleic acid vaccines.The application prepares an mRNA vaccine for expressing feline calicivirus VP1 protein, constructs an mRNA vector plasmid containing feline calicivirus VP1 protein, and is used for preparing capped modified mRNA, and combines a liposome wrapping scheme to prepare the mRNA vaccine, and is applied to the immunization of feline calicivirus.The method has application value in the research and vaccine creation of feline calicivirus, and can greatly promote the transformation and application of the vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nucleic acid vaccines, in particular to an mRNA vaccine expressing feline calicivirus VP1 protein, the construction of the mRNA synthetic plasmid, the synthesis of the mRNA, and the preparation method of the mRNA vaccine. BACKGROUND

[0002] Feline calicivirus disease (FCVD) is a disease caused by feline calicivirus (FCV) with upper respiratory tract infection as the main symptom. The disease mainly attacks 7-84-day-old kittens, often occurs in 56-84-day-old kittens, and kittens under 1 year old are most susceptible. The younger the kitten, the higher the mortality rate after infection. The incubation period is 2-3 days, and the natural course is 7-10 days. The main clinical symptoms are oral ulcer, pneumonia, chronic gastritis, arthritis, and lameness. Cats show depression, increased eye and nasal discharge, sometimes salivation and keratitis. Since FCV was first isolated and identified by Fastier in 1957, FCV has been found in Europe, America, and Asia, and has shown worldwide distribution. In recent years, virulent strains caused by the high variability of FCV can cause severe, acute, and fatal systemic diseases (VSD), which pose a serious threat to the health of cats and felines. It is listed as one of the three viral infectious diseases of cats. FCV belongs to the Calicivirudae family of Vesivirus and is a icosahedral spherical single-stranded positive-sense RNA virus without a capsid membrane, with a diameter of 35-39 nm and a genome size of about 7.8 kb. The genome encodes three open reading frames (ORFs). ORF1 encodes non-structural proteins, including 2C helicase, 3C cysteine protease, and 3D RNA-dependent RNA polymerase; ORF2 encodes the major capsid protein VP1; and ORF3 encodes a small structural protein, VP2.

[0003] The VP1 protein is a structural protein and plays a key role in the formation of virus particles, antigenic determination and virus-host cell interaction. The VP1 is further divided into six regions A-F based on the sequence conservation analysis. The A region is located at the amino terminal end of the VP1 and is cut off in the process of forming the capsid protein, between 1aa-125aa. The B region contains potential myristoylated glycine and ATP / GTP binding sites, between 126aa-397aa. The c region is a short hypervariable sequence, between about 398aa-401aa. The D region is highly conserved, between 402aa-426aa. The E region is about 427-524aa and is further divided into two hypervariable regions (HRV) of 5' end (33aa) and 3' end (34aa) by 28 conserved bases (conE), which may determine that the FCV has only one serotype. The E region is also the region for producing neutralizing monoclonal antibodies (MAb) and contains multiple antigenic epitopes. The F region is located at the carboxy terminal end of the highly conserved capsid protein and is the connection site of non-neutralizing MAb, between 525aa-668aa.

[0004] Compared with the traditional vaccine, the mRNA vaccine as a new form of vaccine has simple production process, fast development speed, no need for cell culture and low cost. Compared with the DNA vaccine, the mRNA vaccine does not need to enter the nucleus and has no risk of integration into the host genome, and the half-life can be adjusted by modification. The mRNA vaccine can provide comprehensive stimulation of adaptive and innate immunity, i.e. in situ antigen expression and danger signal transmission; can induce a "balanced" immune response, including humoral and cellular effectors and immune memory. SUMMARY

[0005] The present application aims at the deficiencies existing in the existing cat calicivirus vaccine products and provides a preparation method of an mRNA vaccine for preventing or treating cat respiratory disease and application thereof.

[0006] The technical scheme of the present application is as follows:

[0007] A recombinant mRNA synthesis plasmid comprises a plasmid backbone sequence and a gene for encoding a cat calicivirus VP1 protein. The gene sequence of the cat calicivirus VP1 protein is shown in SEQ ID NO. 1, and the gene for the cat calicivirus VP1 protein lacks the 4th-372th nucleotide sequence at the 5' end.

[0008] The cat calicivirus (FCV) belongs to the Calicivirudae virus and mainly harms 7-84-day-old kittens, causing upper respiratory tract infection as the main symptom.

[0009] The VP1 protein of FCV has most of the neutralizing antibody epitopes and is the main antibody immunization point. Since the VP1 protein structure is divided into six regions A-F, and the A region is located at the amino-terminal end of VP1 and is cut off during the formation of the capsid protein, the nucleotide sequence of 4-372 of the VP1 protein gene is deleted and modified.

[0010] The plasmid backbone sequence comprises a T7 promoter sequence, a 5' UTR region, a 3' UTR region, and a 3' terminal PolyA tail. The recombinant mRNA synthesis plasmid can be constructed in any cloning vector, and a Bsa I restriction endonuclease site is reserved at the end of the PolyA tail for plasmid linearization preparation.

[0011] Preferably, the restriction endonuclease site is BspQ I, Bsa I, or Mlu I.

[0012] The alphavirus non-structural protein gene coding region is a non-structural protein 1-4 gene coding region, and the cat calicivirus VP1 protein gene sequence is codon-optimized. Preferably, the alphavirus is Venezuelan equine encephalitis virus (VEE). The VEE replicase gene nsp 1-4 region comprises a VEE virus replicase gene sequence, and the expressed VEE virus replicase can synthesize mRNA in vivo to function as a replicase.

[0013] The application also provides the use of the recombinant mRNA synthesis plasmid in the preparation of a vaccine for preventing cat respiratory disease caused by cat calicivirus.

[0014] The application also provides an mRNA for expressing a cat calicivirus VP1 protein, comprising a 5' UTR region, a 3' UTR region, and a 3' terminal Poly A tail, and a cat calicivirus VP1 protein mutant-encoding mRNA. The amino acid sequence of the cat calicivirus VP1 protein mutant is shown in SEQ ID NO. 2.

[0015] The mRNA also comprises a Venezuelan equine encephalitis virus replicase 1-4 coding region.

[0016] Preferably, the mRNA also comprises a 5' cap structure, and the 5' cap structure is a 7-methylguanosine cap structure.

[0017] The mRNA sequence for expressing the VP1 protein is 1913 bp in size (the sequence is shown in SEQ ID NO. 3), and the replicative mRNA sequence for expressing the VP1 protein is 9426 bp in size (the sequence is shown in SEQ ID NO. 4).

[0018] The application also provides an mRNA vaccine expressing feline calicivirus VP1 protein, comprising the mRNA expressing feline calicivirus VP1 protein.

[0019] At present, mRNA vaccines mainly exist in two sequence structures, traditional non-replicating mRNA sequences and self-amplifying (replicating) mRNA vaccine sequences. The self-amplifying mRNA sequence contains a replicase gene, which can amplify the mRNA in the cell, thereby producing more antigens with less mRNA dose. The non-replicating mRNA vaccine structure is simple, cannot self-replicate in the human body, and needs mature optimization process to induce effective immune response at a lower dose.

[0020] The application also provides a preparation method of the mRNA vaccine, wherein the mRNA expressing feline calicivirus VP1 protein is mixed with cationic lipids, distearoyl phosphatidylcholine, polyethylene glycol lipids and cholesterol to prepare lipid nanoparticles, and the replicating mRNA vaccine is obtained after microfluidic device or mixed dialysis.

[0021] The specific steps are as follows:

[0022] (1) linearizing the recombinant mRNA synthesis plasmid T7-VP1 using a restriction enzyme;

[0023] (2) performing in vitro transcription reaction on the linearized T7-VP1 plasmid using a T7 promoter sequence to obtain VP1-mRNA;

[0024] (3) performing Cap capping modification reaction on the obtained VP1-mRNA using vaccinia virus capping enzyme and 2'-O-methyltransferase by enzyme method to obtain cap-VP1-mRNA;

[0025] (4) mixing the cap-VP1-mRNA after capping modification with cationic lipids (SM-102), distearoyl phosphatidylcholine (DSPC), cholesterol and polyethylene glycol lipids (DMG-PEG2000) in a certain proportion to obtain LNP-VP1-mRNA vaccine after microfluidic device or mixed dialysis.

[0026] The application also provides the application of the mRNA vaccine of feline calicivirus VP1 protein in preventing feline calicivirus infection. Specific antibodies against feline calicivirus VP1 protein exist in the animals immunized with the LNP-VP1-mRNA vaccine, which has the effect of preventing feline calicivirus infection.

[0027] The application has the following beneficial effects:

[0028] The application prepares an mRNA vaccine expressing feline calicivirus VP1 protein, constructs an mRNA vector plasmid containing feline calicivirus VP1 protein, and is used for preparing mRNA, and an mRNA vaccine is prepared by combining a liposome wrapping scheme, and is applied to the immunization of feline calicivirus. The method has application value in feline calicivirus research and vaccine development, and can greatly promote the transformation and application of vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A schematic diagram of a recombinant mRNA synthesis plasmid T7-VP1 for expressing feline calicivirus VP1 gene.

[0030] Figure 2 A schematic diagram of a recombinant replicative mRNA synthesis plasmid VEE-VP1 for expressing feline calicivirus VP1 gene.

[0031] Figure 3 It is an agarose gel electrophoresis verification result graph of T7-VP1 mRNA synthesis plasmid enzyme cutting linearization; wherein, M: DNA Maker, 1: T7-FCV-VP1 plasmid, 2: T7-FCV-VP1 plasmid enzyme cutting linearization.

[0032] Figure 4 It is an agarose gel electrophoresis verification result graph of replicative mRNA synthesis plasmid VEE-VP1 enzyme cutting linearization; wherein, M: DNA Maker, 1: VEE-VP1 plasmid, 2: VEE-FCV-VP1 plasmid enzyme cutting linearization.

[0033] Figure 5 It is an agarose gel electrophoresis verification result graph of feline calicivirus VP1 protein cap-VP1-mRNA prepared after cap modification; wherein, M: DNA Maker, 1: T7-FCV-VP1 plasmid, 2: cap-VP1-mRNA.

[0034] Figure 6 It is an agarose gel electrophoresis verification result graph of feline calicivirus VP1 protein replicative cap-VEE-VP1-mRNA prepared after cap modification; wherein, M: DNA Maker, 1: VEE-VP1 plasmid, 2: cap-VEE-VP1-mRNA.

[0035] Figure 7 It is an indirect immunofluorescence detection result graph of feline calicivirus VP1 protein expressed by cell transfection of cap-VP1-mRNA.

[0036] Figure 8 It is an indirect immunofluorescence detection result graph of feline calicivirus VP1 protein expressed by cell transfection of cap-VEE-VP1-mRNA.

[0037] Figure 9 Figure for the results of determining the level of VP1 protein specific antibody in serum after immunization with LNP-VP1-mRNA vaccine by enzyme-linked immunosorbent assay.

[0038] Figure 10 Figure for the results of determining the level of VP1 protein specific antibody in serum after immunization with replicative LNP-VEE-VP1-mRNA vaccine by enzyme-linked immunosorbent assay. DETAILED DESCRIPTION

[0039] The technical solutions of the present application will be further described in detail below through specific embodiments, but the present application will not be limited in the scope of the described embodiments.

[0040] The mRNA vaccine developed by the present application for feline calicivirus mainly adopts two ways of (1) preparing capped modified mRNA expressing feline calicivirus VP1 protein and replicative mRNA; (2) preparing two new types of LNP-VP1-mRNA and LNP-VEE-VP1-mRNA vaccines.

[0041] Example 1 Construction of recombinant mRNA and replicative mRNA synthesis plasmid of feline calicivirus VP1 gene

[0042] The cat calicivirus positive cat nasal swab sample was collected from a pet hospital in Guangzhou, Guangdong Province, China in October 2021 for cats with pneumonia and keratoconjunctivitis. To extract the feline calicivirus sample, the cDNA was obtained by reverse transcription after Trizol method to extract RNA, and the primer FCV-VP1-F (5'-ATGTGCTCAACCTGCGCTAA-3') and FCV-VP1-R (5'-TCATAACTTAGTCATGGGAC-3') were used to amplify the VP1 gene sequence as shown in SEQ ID NO. 1.

[0043] According to the sequence of T7 promoter, 5'UTR region, feline calicivirus VP1 protein gene (amino acid sequence as shown in SEQ ID NO. 2) after deleting the 4-372th nucleotide sequence, 3'UTR region and 3' terminal Poly(A) tail, the recombinant mRNA synthesis plasmid T7-FCV-VP1 of feline calicivirus VP1 protein gene was constructed in pUC cloning vector, and the map is as shown in Figure 1 The end of the Poly(A) tail has a Bsa I restriction endonuclease site, which is used for plasmid linearization preparation.

[0044] According to the T7 promoter sequence, 5' UTR region, VEE replicase gene nsp 1-4 region, cat coronavirus VP1 protein gene (the VP1 gene sequence is optimized for mammalian protein expression codon sequence) after deleting the 4-372th nucleotide sequence, 3' UTR region and 3' terminal Poly(A) tail, the VEE cloning vector is constructed in sequence, and a replicative mRNA plasmid VEE-VP1 is constructed, as shown in the map of Figure 2 The Poly(A) tail end has an Mlu I restriction endonuclease site, which is used for plasmid linearization preparation.

[0045] The plasmid T7-FCV-VP1 or the plasmid VEE-VP1 is stored in an E. coli T1 competent strain, a single clone is randomly picked, and 200 mL of LB liquid medium containing ampicillin resistance is inoculated, and cultured at 37°C on a shaking table. The cloned T7-FCV-VP1 plasmid or the plasmid VEE-VP1 is extracted according to the Omega plasmid DNA large kit instructions, the concentration is determined, and then stored for use.

[0046] Example 2 In vitro transcription preparation of VP1-mRNA and replicative VEE-VP1-mRNA

[0047] As shown in Figure 1 , the Poly(A) tail end of the cat coronavirus VP1 protein gene recombinant mRNA synthesis plasmid T7-FCV-VP1 retains a Bsa I restriction endonuclease site, and the vector plasmid is subjected to enzyme cutting linearization reaction using the restriction endonuclease Bsa I; as shown in Figure 2 , the Poly(A) tail end of the cat coronavirus VP1 protein gene recombinant replicative mRNA synthesis plasmid VEE-VP1 retains an Mlu I restriction endonuclease site, and the vector plasmid is subjected to enzyme cutting linearization reaction using the restriction endonuclease Mlu I. The two enzyme cutting linearization reactions are operated according to the following steps:

[0048] (1) The T7-FCV-VP1 plasmid and the VEE-VP1 plasmid constructed according to Example 1 are linearized by Bsa I enzyme or Mlu I enzyme.

[0049] a) First, 25 μg of the T7-FCV-VP1 plasmid is cut with Bsa I enzyme at 37°C for 2 h, and the enzyme cutting reaction system is prepared as follows:

[0050]

[0051] Take 25 μg of the VEE-VP1 plasmid, cut with Mlu I enzyme at 37°C for 2 h, and prepare the enzyme cutting reaction system as follows:

[0052]

[0053] b) After enzyme digestion, add 5 μL of 10% SDS solution to make the final SDS concentration 0.5%;

[0054] c) Add 0.5 μL of 20 mg / μL proteinase K to make the final proteinase concentration 50-100 μg / mL;

[0055] d) Incubate at 37℃ for 1 h, then place on ice, add 200 μL of nuclease-free water and 300 μL of Phenol / CHCl3 / IAA mixture, vortex and let stand for 5 min;

[0056] e) Centrifuge at 12000 rpm at room temperature for 10 min, aspirate the supernatant, add 750 μL of anhydrous ethanol, and let stand in a -20℃ refrigerator for 30 min;

[0057] f) Centrifuge at 12000 rpm at 4°C for 15 min, and discard the supernatant;

[0058] g) Add 1 mL of 75% ethanol, centrifuge at 12000 rpm at 4°C for 5 min, repeat this step twice, discard the supernatant, and air dry the centrifuge tube.

[0059] h) Add 20 μL of nuclease-free water to dissolve the DNA. Take 1 μL, dilute it 10 times, and measure the concentration of linearized DNA. Store at -20℃.

[0060] i) The linearization of plasmid TT-FCV-VP1 and plasmid VEE-VP1 by enzyme digestion was verified by agarose gel electrophoresis.

[0061] like Figure 3 As shown, the size of the T7-FCV-VP1 plasmid band on agarose gel electrophoresis is smaller than that of the T7-FCV-VP1 enzyme-digested linearized DNA fragment, proving that the T7-FCV-VP1 plasmid linearization was completed;

[0062] like Figure 4 As shown, the agarose gel electrophoresis band of the VEE-VP1 plasmid is smaller than the linearized DNA fragment of VEE-VP1, proving that the linearization of the VEE-VP1 plasmid is complete.

[0063] (2) Using the linearized DNA from step (1) as a template, VP1-mRNA and VEE-VP1-mRNA were obtained by in vitro transcription.

[0064] The T7 promoter upstream of the target gene T7-FCV-VP1 and VEE-VP1 plasmids were synthesized using mRNA. The mRNA of the target gene feline calicivirus VP1 protein was prepared and expressed by in vitro transcription reaction.

[0065] a) First, 1-2 μg of linearized T7-FCV-VP1 plasmid or VEE-VP1 plasmid was used to synthesize mRNA with T7 RNA polymerase at 37°C, and the prepared in vitro transcription reaction system was as follows:

[0066]

[0067]

[0068] b) The in vitro transcription system was incubated at 37°C for 4 h, and then 1 μL of DNase was added and incubated at 37°C for 15 min to digest the DNA template;

[0069] c) VP1-mRNA (the sequence is shown in SEQ ID NO. 3) or VEE-VP1-mRNA (the sequence is shown in SEQ ID NO. 4) was extracted by the Trizol method or affinity chromatography, 1 μL of which was diluted 10 times to determine the mRNA concentration, and stored at -80°C.

[0070] Example 3 Preparation of cap-VP1-mRNA and replicative cap-VEE-VP1-mRNA by mRNA capping modification reaction

[0071] The 7-methylguanosine cap structure is added to the 5' end of mRNA by using vaccinia virus capping enzyme and related components, so that the mRNA is more stable, and is conducive to transport and translation.

[0072] a) The VP1-mRNA or VEE-VP1-mRNA obtained by in vitro transcription was subjected to capping modification reaction with vaccinia virus capping enzyme and Cap 2'-O-methyltransferase at 37°C, and the prepared capping modification reaction system was as follows:

[0073]

[0074] b) The capping modification reaction system was incubated at 37°C for 60 min;

[0075] c) cap-VP1-mRNA or cap-VEE-VP1-mRNA was extracted by the Trizol method or affinity chromatography, 1 μL of which was diluted 10 times to determine the mRNA concentration, and stored at -80°C;

[0076] d) The cat calicivirus VP1 protein cap-VP1-mRNA or cap-VEE-VP1-mRNA after capping modification was verified by agarose gel electrophoresis.

[0077] As Figure 5As shown, the agarose gel electrophoresis shows the band size of mRNA, and the T7-FCV-VP1 plasmid is used as a control to prove that the feline calicivirus VP1 protein cap-VP1-mRNA is effectively synthesized.

[0078] As shown, the agarose gel electrophoresis shows the band size of mRNA, and the VEE-VP1 plasmid is used as a control to prove that the feline calicivirus VP1 protein cap-VEE-VP1-mRNA is effectively synthesized. Figure 6

[0079] Example 4 Immunofluorescence experiment to detect the expression of feline calicivirus VP1 protein by transfection of cap-VP1-mRNA and cap-VEE-VP1-mRNA

[0080] The transfection of cap-VP1-mRNA or cap-VEE-VP1-mRNA in cells can express feline calicivirus VP1 protein, and the specific antibody of feline calicivirus VP1 protein can detect the translation of VP1-mRNA or VEE-VP1-mRNA effectively, and the operation is as follows:

[0081] a) In a 48-well plate, the wall cells such as BHK-21 are plated and cultured in a 5% CO2 incubator at 37°C, and when the cell density reaches about 70%, the obtained cap-VP1-mRNA or cap-VEE-VP1-mRNA is transfected;

[0082] b) Take a clean EP tube, add 300 μL of Opti-MEM medium and 4 μL of DMRIE-C transfection reagent, vortex to mix, incubate at room temperature for 30 min, add 2 μg of cap-VP1-mRNA or cap-VEE-VP1-mRNA, and mix gently, incubate for 10 min;

[0083] c) Wash the cells with Opti-MEM medium once, add the incubated mixture, and after 24 h, use the specific rabbit polyclonal antibody of feline calicivirus VP1 protein to detect the expression of VP1 protein by indirect immunofluorescence;

[0084] The specific rabbit polyclonal antibody of VP1 protein is obtained from the antibody serum prepared by coupling the peptides synthesized by using the VP1 protein polypeptide segments STLPETGARGGNHPC and TATLDGDNNNKINPC to immunize a New Zealand white rabbit.

[0085] As shown, the results show that the BHK-21 cells transfected with cap-VP1-mRNA can be observed under a fluorescence microscope to have a green fluorescent signal, indicating that cap-VP1-mRNA can effectively express feline calicivirus VP1 protein. Figure 7 ​​

[0086] As shown in Figure 8 The results show that the BHK-21 cells transfected with cap-VEE-VP1-mRNA can be observed under fluorescence microscope with green fluorescence signal, indicating that the cap-VEE-VP1-mRNA can effectively express the feline calicivirus VP1 protein.

[0087] Example 5 Preparation of lipid nanoparticle mRNA vaccine LNP-VP1-mRNA and lipid nanoparticle replicon mRNA vaccine LNP-VEE-VP1-mRNA

[0088] The mRNA is a negatively charged biological macromolecule, which is difficult to pass through the negatively charged cell membrane by passive transport. The lipid nanoparticle (LNP) can be used to deliver RNA, which is an effective drug delivery method for mRNA vaccine.

[0089] The preparation of the lipid nanoparticle mRNA vaccine LNP-VP1-mRNA or the lipid nanoparticle replicon mRNA vaccine LNP-VEE-VP1-mRNA is performed according to the following steps:

[0090] a) SM-102, DSPC, DMG-PEG2000 and cholesterol are dissolved in 30 μL of anhydrous ethanol according to the formula of a molar ratio of 50:10:38.5:1.5, and the total mass is 200 μg;

[0091] b) The ethanol solution is quickly injected into 90 μL of 20 mM sodium acetate buffer containing 5 μg of cap-VP1-mRNA or cap-VEE-VP1-mRNA prepared in Example 3 under the condition of vortex, and stirred vigorously for 20 s, and then left for 10 minutes to prepare the nanoparticles;

[0092] c) The prepared ethanol-sodium acetate mixed solution containing nanoparticles is dialyzed in 10 mM PBS solution for 2-4 hours to remove ethanol, and the final product LNP-VP1-mRNA or replicon LNP-VEE-VP1-mRNA is obtained after ultrafiltration concentration.

[0093] Example 6 Immunization experiment of feline calicivirus protein mRNA vaccine LNP-VP1-mRNA and replicon mRNA vaccine LNP-VEE-VP1-mRNA

[0094] The cap-VP1-mRNA or replicon cap-VEE-VP1-mRNA prepared in Example 3 can effectively express the feline calicivirus VP1 protein, and the LNP-VP1-mRNA vaccine or replicon LNP-VEE-VP1-mRNA vaccine of feline calicivirus protein is prepared by combining the preparation method of the lipid nanoparticle mRNA vaccine in Example 5. The vaccine effect is detected by immunization experiment.

[0095] The immune effect of LNP-VP1-mRNA vaccine or replicative LNP-VEE-VP1-mRNA vaccine was evaluated according to the following steps:

[0096] a) 12 six-week-old female BALB / c mice were equally divided into three groups (negative control group, LNP-VP1-mRNA vaccine group (or replicative LNP-VEE-VP1-mRNA vaccine) and Miao Sanduo vaccine group), and the BALB / c mice were injected with 5 μg per mouse of LNP-VP1-mRNA vaccine (or replicative LNP-VEE-VP1-mRNA vaccine) or existing Miao Sanduo inactivated vaccine by intramuscular route at day 0 and day 30;

[0097] b) On day 7 after the second immunization, blood was collected by the orbital route, and the collected serum samples were collected;

[0098] c) The level of VP1 protein-specific antibodies in the serum after immunization with LNP-VP1-mRNA vaccine or replicative LNP-VEE-VP1-mRNA vaccine was determined by double-antibody sandwich enzyme-linked immunosorbent assay: cat calicivirus antigen-coated micro-wells were pre-coated, and serum samples, HRP-labeled detection antibodies were added in turn, incubated and thoroughly washed. Color development was performed with substrate TMB, and the final yellow color was converted under the action of 1M sulfuric acid, and the absorbance (OD value) was determined at 450 nm wavelength by an enzyme-labeled instrument.

[0099] As shown in Figure 9 , the level of cat calicivirus-specific antibodies in the sample was determined. The LNP-VP1-mRNA vaccine group and the Miao Sanduo control group produced different levels of cat calicivirus-specific antibodies, respectively, and the relative level of antibodies in the Miao Sanduo control group was higher, so that immunization with mRNA vaccine expressing cat calicivirus VP1 protein can effectively activate humoral immunity to produce cat calicivirus-specific antibodies.

[0100] As shown in Figure 10 , the level of cat calicivirus-specific antibodies in the sample was determined. The LNP-VEE-VP1-mRNA vaccine group and the Miao Sanduo control group produced different levels of cat calicivirus-specific antibodies, respectively, and the relative level of antibodies in the LNP-VEE-VP1-mRNA vaccine group was higher, so that immunization with replicative mRNA vaccine expressing cat calicivirus VP1 protein can effectively activate humoral immunity to produce cat calicivirus-specific antibodies.

Claims

1. A recombinant mRNA synthesis plasmid, characterized in that, The plasmid backbone sequence comprises a T7 promoter sequence, a 5' UTR region, a 3' UTR region and a 3' terminal Poly A tail.

2. The recombinant mRNA synthesis plasmid of claim 1, wherein, The plasmid backbone sequence comprises a T7 promoter sequence, a 5' UTR region, a 3' UTR region and a 3' terminal Poly A tail. The sequence of the recombinant mRNA synthesis plasmid is shown as SEQ ID NO.

9.

3. The recombinant mRNA synthesis plasmid of claim 2, wherein, The 3' terminal Poly A tail comprises a restriction enzyme site for linearization preparation of the plasmid.

4. Use of the recombinant mRNA synthesis plasmid of claims 1-3 in the preparation of a vaccine for preventing feline respiratory disease caused by feline calicivirus.

5. An mRNA expressing a mutant of feline calicivirus VP1 protein, characterized in that, The mRNA sequence expressing the feline calicivirus VP1 protein mutant is shown as SEQ ID NO.

3.

6. The mRNA expressing a mutant of feline calicivirus VP1 protein according to claim 5, characterized in that, Further comprising a 5' cap structure, which is a 7-methylguanosine cap structure.

7. An mRNA vaccine expressing a mutant of feline calicivirus VP1 protein, characterized in that, The mRNA expressing the feline calicivirus VP1 protein mutant of claim 5 or 6.

8. The preparation method of the mRNA vaccine according to claim 7, characterized in that, The mRNA expressing the feline calicivirus VP1 protein mutant of claim 5 or 6 is mixed with a cationic lipid, disstearylphosphatidylcholine, polyethylene glycol lipid and cholesterol to prepare a lipid nanoparticle, thereby obtaining the mRNA vaccine.

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