CN116218889A - A kind of mRNA vaccine expressing feline parvovirus VP2 protein and its preparation method - Google Patents

Abstract

The invention discloses an mRNA vaccine for expressing cat parvovirus VP2 protein and a preparation method thereof, and relates to the field of nucleic acid vaccines. The invention successfully prepares an mRNA vaccine for expressing the cat parvovirus protein, constructs an mRNA vector plasmid containing the cat parvovirus VP2 protein, is used for preparing capped modified mRNA, is combined with a liposome encapsulation scheme to prepare the mRNA vaccine, and is applied to the prevention of cat panleukopenia. The method has application value in cat leukopenia research and vaccine creation, and can greatly promote the transformation application of the vaccine.

Description

A kind of mRNA vaccine expressing feline parvovirus VP2 protein and its preparation method

technical field

The invention relates to the field of nucleic acid vaccines, in particular to an mRNA vaccine expressing feline parvovirus VP2 protein, including the preparation of the feline parvovirus VP2 protein mRNA vaccine.

Background technique

Feline panleukopenia (FP) is an acute, highly contagious disease of cats caused by feline parvovirus (FPV). Typical clinical symptoms of FPV infection are biphasic fever, vomiting, diarrhea, dehydration, circulatory disturbance, severe leukopenia and hemorrhagic enteritis. It mainly infects kittens under 1 year old. Usually, the fatality rate for cats under 1 year old is 50% to 60%, and the mortality rate for kittens under 5 months of age is as high as 80% to 90%. If the female cat is infected with the virus during pregnancy, it will cause symptoms such as stillbirth and miscarriage. The virus was first identified by Verge in the 1990s, and it was found in Canada, the United States, Japan and many other countries. The virus was isolated in my country for the first time in 1985, and Zhang Zhenxing, Li Gang and others reported it in Hebei, Henan, Feline parvovirus cases have occurred in Zhejiang and other places. The disease is widespread in the world and is considered to be one of the important infectious diseases of cats. FPV has a wide range of hosts. In addition to infecting domestic cats, it can also infect other cats (such as tigers and leopards), mustelids (such as mink) and bears (such as raccoons). It seriously endangers the life and health of pet cats and wild cats. FPV belongs to the family Parvoviridae, the genus Parvovirus, and is a non-enveloped single-stranded positive-strand DNA virus capable of self-replication. FPV virus particles are about 20-30nm, and are equiaxed and symmetrical icosahedrons. The FPV genome is about 5Kb long, including a long coding region and two unique palindromic structures at both ends. The palindromic sequence base pairs to form a hairpin duplex. Structural proteins NS (NS1 and NS2) and viral structural proteins VP (VP1 and VP2).

VP2 protein accounts for about 90% of all structural proteins, and VP1 protein accounts for 10% of all structural proteins. Therefore, VP2 protein is the main antigenic protein encoded by FPV and plays an important role in the process of immune response, recognition of receptors and infected tissues . The tertiary structure of the VP2 protein is mainly formed by the five ring structures of Loop1-Loop5 surrounding the three-fold unit fibril, shoulder and top formed by eight antiparallel anti-folded sheets, which is related to the hemagglutination characteristics and pathogenicity of the virus Sex is closely related. Among them, Loop3 is the shoulder part of the fibrils that make up the three-fold protein unit of the virus. It does not directly participate in the recognition of the transferrin receptor by the virus capsid protein, but improves the infection efficiency of the virus in the three-dimensional space. There are multiple antigen recognition sites on the VP2 protein, which can stimulate the body to produce neutralizing antibodies and play an immune protective role. The virus-like particles composed of VP2 protein can be used to prepare candidate vaccines for various viruses, which play a decisive role in the replication of the virus and the range of infected hosts, and can well induce humoral and cellular immunity.

Compared with traditional vaccines, mRNA vaccines, as a new form of vaccine, have the advantages of simple production process, fast development speed, no need for cell culture, and low cost. Compared with DNA vaccines, mRNA vaccines do not need to enter the nucleus, there is no risk of integration into the host genome, and the half-life can be adjusted through modification. mRNA vaccines can provide integrated stimulation of adaptive and innate immunity, namely in situ antigen expression and danger signal transmission; and can induce "balanced" immune responses, including humoral and cellular effectors and immune memory.

Contents of the invention

Aiming at the deficiencies in existing pet vaccine products, the invention provides a method for preparing an mRNA vaccine expressing feline parvovirus VP2 protein for preventing or treating feline panleukopenia and its application.

Technical scheme of the present invention:

A recombinant mRNA synthesis plasmid, comprising a plasmid backbone sequence and a gene encoding feline parvovirus VP2 protein; the gene sequence encoding feline parvovirus VP2 protein is shown in SEQ ID NO.1.

Among them, feline parvovirus (Feline panleukopenia virus, FPV) belongs to the parvoviridae virus (Panleukopenia), mainly infecting young cats under 1 year old, causing biphasic fever, vomiting, diarrhea, dehydration, circulatory disturbance, severe leukopenia and hemorrhagic enteritis.

Among them, the VP2 protein of FPV has most of the neutralizing antibody epitopes and is the main point of antibody immunity. There are multiple antigen recognition sites on the VP2 protein, which can stimulate the body to produce neutralizing antibodies and play an immune protective role.

The plasmid backbone sequence includes a T7 promoter sequence, a 5' UTR region, a 3' UTR region and a 3' terminal PolyA tail. It can be constructed in any cloning vector, and a restriction endonuclease site is reserved at the end of the PolyA tail for plasmid linearization preparation. Preferably, the restriction endonuclease site is BspQ I, Bsa I or Mlu I.

Venezuelan equine encephalitis virus replicase 1-4 coding region; gene sequence encoding feline calicivirus VP2 protein codon-optimized. The VEE replicase gene nsp 1-4 region contains the VEE virus replicase gene sequence, and the expressed VEE virus replicase can synthesize mRNA in vivo and play the function of replicating mRNA.

The present invention also provides the use of the recombinant mRNA synthesis plasmid in preparing a vaccine for preventing feline panleukopenia caused by feline parvovirus infection.

The present invention also provides an mRNA expressing feline parvovirus VP2 protein, including a 5'UTR region, a 3'UTR region and a 3' terminal PolyA tail, an mRNA encoding a feline calicivirus VP2 protein; the feline calicivirus VP2 The amino acid sequence of the protein is shown in SEQ ID NO.2.

Also included is the mRNA for the replicase 1-4 coding region of Venezuelan equine encephalitis virus.

Preferably, a 5' cap structure is also included, and the 5' cap structure is a 7-methylguanosine cap structure.

Among them, the size of the gene mRNA sequence expressing VP2 protein is 2018bp (sequence shown in SEQ ID NO.3), and the gene replication type mRNA sequence size expressing VP2 protein is 9528bp (sequence shown in SEQ ID NO.4).

The present invention also provides an mRNA vaccine expressing feline parvovirus VP2 protein, comprising the mRNA expressing feline parvovirus VP2 protein.

Currently, mRNA vaccines mainly exist in two sequence structures, the traditional non-replicating mRNA sequence and the self-amplifying (replicating) mRNA vaccine sequence. Self-amplifying mRNA sequences contain replicase genes that amplify the mRNA within the cell, thereby producing more antigen with less mRNA dosage. The non-replicating mRNA vaccine has a simple structure and cannot replicate itself in the human body. It requires a mature optimization process to induce an effective immune response at a lower dose.

The present invention also provides the preparation method of the mRNA vaccine, which is prepared by mixing the mRNA expressing feline parvovirus VP2 protein with cationic lipid, distearoylphosphatidylcholine, polyethylene glycol lipid and cholesterol Lipid nanoparticle, the replicative mRNA vaccine is obtained after passing through a microfluidic device or hybrid dialysis.

The specific steps are:

(1) Linearize the recombinant mRNA synthesis plasmid T7-VP2 using restriction endonucleases;

(2) performing an in vitro transcription reaction on the linearized T7-VP2 plasmid using the T7 promoter sequence, and purifying to obtain VP2-mRNA;

(3) Using vaccinia virus capping enzyme and 2'-O-methyltransferase to obtain VP2-mRNA, carry out Cap capping modification reaction by enzymatic method, and purify to obtain cap-VP2-mRNA;

(4) capped modified cap-VP2-mRNA and cationic lipid (SM-102), distearoyl phosphatidylcholine (DSPC), cholesterol, polyethylene glycol lipid (DMG-PEG2000) passed in proportion LNP-VP2-mRNA vaccine was obtained after microfluidic device or hybrid dialysis.

The invention also provides the mRNA vaccine of the feline parvovirus VP2 protein as an application in preventing feline parvovirus infection. Animals immunized with the LNP-VP2-mRNA vaccine have specific antibodies against feline parvovirus VP2 protein, which can prevent feline parvovirus infection.

Beneficial effects of the present invention:

The present invention successfully prepared an mRNA vaccine expressing feline parvovirus protein, constructed an mRNA carrier plasmid containing feline parvovirus VP2 protein, and used it to prepare cap-VP2-mRNA, combined with liposome encapsulation scheme to prepare LNP-VP2-mRNA vaccine , and applied to the prevention of cat panleukopenia. This method has application value in the research of feline panleukopenia and vaccine creation, and can vigorously promote the transformation and application of vaccines.

Description of drawings

Fig. 1 is a schematic diagram of recombinant mRNA synthesis plasmid T7-VP2 expressing feline parvovirus VP2 gene.

Fig. 2 is a schematic diagram of the recombinant mRNA synthesis plasmid VEE-VP2 expressing the feline parvovirus VP2 gene.

Figure 3 is the result of agarose gel electrophoresis verification of the linearization of mRNA synthesis plasmid T7-VP2 enzyme digestion; among them, M: DNA Maker, 1: T7-FPV-VP2 plasmid, 2: T7-FPV-VP2 plasmid enzyme digestion linearization change.

Figure 4 is the agarose gel electrophoresis verification result of the linearization of the replication-type mRNA synthesis plasmid VEE-VP2 enzyme digestion; wherein, M: DNA Maker, 1: VEE-VP2 plasmid, 2: linearization of the enzyme digestion of VEE-VP2 plasmid.

Figure 5 is a graph showing the results of agarose gel electrophoresis verification of feline parvovirus VP2 protein cap-VP2-mRNA prepared after capping modification; wherein, M: DNA Maker, 1: T7-FPV-VP2 plasmid, 2: cap-VP2 -mRNA.

Figure 6 is the result of agarose gel electrophoresis verification of feline parvovirus VP2 protein replication type cap-VEE-VP2-mRNA prepared after capping modification; wherein, M: DNA Maker, 1: VEE-VP2 plasmid, 2: cap -VEE-VP2-mRNA.

Fig. 7 is a graph showing the results of indirect immunofluorescence detection of cells transfected with cap-VP2-mRNA to express feline parvovirus VP2 protein.

Fig. 8 is a diagram showing the detection results of feline parvovirus VP2 protein expressed in cells transfected with cap-VEE-VP2-mRNA by indirect immunofluorescence method.

Fig. 9 is a graph showing the results of measuring the level of VP2 protein-specific antibody in serum after immunization with LNP-VP2-mRNA vaccine by enzyme-linked immunosorbent assay.

Fig. 10 is a graph showing the results of measuring the level of VP2 protein-specific antibody in serum after immunizing the replicative LNP-VEE-VP2-mRNA vaccine by enzyme-linked immunosorbent assay.

Detailed ways

The present invention is aimed at the mRNA vaccine of feline panleukopenia development, mainly adopts (1) prepares the mRNA and replicative mRNA of capped modified expression feline parvovirus VP2 protein; (2) prepares novel LNP-VP2-mRNA vaccine and LNP- VEE-VP2-mRNA vaccine in both ways.

Example 1 Constructing the recombinant mRNA synthesis plasmid of feline parvovirus VP2 gene and the recombinant replication type mRNA synthesis plasmid

Feline parvovirus-positive cat nasal swab samples were collected from a cat with feline panleukopenia admitted to a pet hospital in Guangzhou, Guangdong Province, China, in October 2021. To extract feline parvovirus samples, viral genomic DNA was extracted by a DNA extraction kit and analyzed by PCR using primers FPV-VP2-F (5'-ATGAGTGATGGAGCAGTTCA-3') and FPV-VP2-R (5'-TTAATATAATTTTCTAGGTG-3') The amplified VP2 gene sequence is shown in SEQ ID NO.1.

According to the T7 promoter sequence, 5'UTR region, feline parvovirus VP2 protein (amino acid sequence shown in SEQ ID NO.2) gene, 3'UTR region and 3' terminal Poly(A) tail sequence, it was constructed in the pUC cloning vector, Gene synthesis of feline parvovirus VP2 protein gene recombinant mRNA synthesis plasmid T7-FPV-VP2, map shown in Figure 1. There is a BsaI restriction endonuclease site at the end of the Poly(A) tail, which is used for plasmid linearization preparation.

According to the sequence of T7 promoter sequence, 5'UTR region, VEE replicase gene nsp 1-4 region, codon-optimized feline parvovirus VP2 protein gene, 3'UTR region and 3' terminal Poly(A) tail, it was constructed in VEE clone Vector, to construct the replication-type mRNA plasmid VEE-VP2, the map is shown in Figure 2, and the sequence is shown in SEQ ID NO.9. There is a Bsa I restriction endonuclease site at the end of the Poly(A) tail, which is used for plasmid linearization preparation.

Plasmid T7-FPV-VP2 or plasmid VEE-VP2 was stored in Escherichia coli T1 competent strains, a single clone was picked at random, and inoculated into 200mL LB liquid medium containing ampicillin resistance, cultured on a shaker at 37°C, according to Omega Plasmid DNA Mass Kit Instructions Extract the cloned T7-FPV-VP2 plasmid or plasmid VEE-VP2, measure the concentration and store it for use.

Example 2 Preparation of FPV-VP2-mRNA and replication type VEE-VP2-mRNA by in vitro transcription

As shown in Figure 1, a Bsa I restriction endonuclease site is reserved at the Poly(A) tail end of the recombinant mRNA synthesis plasmid T7-FPV-VP2 of the feline parvovirus VP2 protein gene, and the restriction endonuclease Bsa I is used to Carrier plasmid carries out restriction endonuclease linearization reaction; As shown in Figure 2, the Poly (A) tail end of the poly(A) tail end of the recombination replication type mRNA synthesis plasmid T7-VP2 of feline parvovirus VP2 protein gene retains a Mlu I restriction endonuclease site, The vector plasmid was digested and linearized with the restriction endonuclease Mlu I. The two enzyme digestion linearization reactions follow the steps below:

(1) The T7-FPV-VP2 plasmid and T7-VP2 plasmid constructed according to Example 1 were linearized by Bsa I enzyme or Mlu I enzyme.

a) First, take 25 μg of T7-FPV-VP2 plasmid and digest it with Bsa I for 2 hours at 37°C. The enzyme digestion reaction system prepared is as follows:

Take 25 μg of the VEE-VP2 plasmid and digest it with MluI for 2 hours at 37°C. The enzyme digestion reaction system is prepared as follows:

b) Add 5 μL of 10% SDS solution after enzyme digestion, so that the final concentration of SDS is 0.5%;

c) Add 0.5 μL of 20 mg/μL proteinase K so that the final concentration of protease is 50-100 μg/mL;

d) Incubate at 37°C for 1 hour, then place on ice, add 200 μL of nuclease-free water, 300 μL of Phenol/CHCl3/IAA mixture, vortex and let stand for 5 minutes;

e) Centrifuge at room temperature for 10 minutes at 12,000 rpm, absorb the supernatant, add 750 μL of absolute ethanol, and let stand in a -20°C refrigerator for 30 minutes;

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

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

h) Add 20 μL of nuclease-free water to dissolve the DNA, take 1 μL and dilute it 10 times, measure the linearized DNA concentration, and store at -20°C;

i) Verify the linearization of plasmid T7-FPV-VP2 and plasmid T7-VP2 by agarose gel electrophoresis.

As shown in Figure 3, the size of the T7-FPV-VP2 plasmid agarose gel electrophoresis band is smaller than the T7-FPV-VP2 digested linearized DNA fragment, which proves that the T7-FPV-VP2 plasmid has been linearized.

As shown in Figure 4, the size of the VEE-VP2 plasmid agarose gel electrophoresis band is smaller than the VEE-VP2 digested linearized DNA fragment, which proves that the VEE-VP2 plasmid has been linearized.

(2) Using the linearized DNA in step (1) as a template, VP2-mRNA and VEE-VP2-mRNA were obtained by in vitro transcription.

The mRNA synthesis plasmid T7-FPV-VP2 and the T7 promoter upstream of the target gene of VEE-VP2 were used to prepare and express the mRNA of the target gene feline parvovirus VP2 protein through in vitro transcription reaction.

a) First take 1-2 μg of linearized T7-FPV-VP2 plasmid or VEE-VP2 plasmid, and use T7 RNA polymerase to synthesize mRNA at 37°C, and prepare the in vitro transcription reaction system as follows:

b) Incubate the in vitro transcription system at 37°C for 4 hours, then add 1 μL DNase and incubate at 37°C for 15 minutes to digest the DNA template;

c) Extract VP2-mRNA (sequence shown in SEQ ID NO.3) or VEE-VP2-mRNA (sequence shown in SEQ ID NO.4) by Trizol method or affinity chromatography, take 1 μ L and dilute 10 times The mRNA concentration was measured and stored at -80°C.

Example 3 mRNA capping modification reaction to prepare cap-VP2-mRNA and replicative cap-VEE-VP2-mRNA

A 7-methylguanosine cap is added to the 5' end of mRNA using vaccinia virus capping enzyme and related components. It makes mRNA more stable and facilitates transport and translation.

a) The VP2-mRNA or VEE-VP2-mRNA obtained by in vitro transcription was capped with vaccinia virus capping enzyme and Cap2′-O-methyltransferase at 37°C. The prepared capping modification reaction system was as follows:

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

c) Extract cap-VP2-mRNA or cap-VEE-VP2-mRNA by Trizol method or affinity chromatography, take 1 μL and dilute it 10 times, measure the mRNA concentration, and store at -80°C;

d) Verifying the capped modified feline parvovirus VP2 protein cap-VP2-mRNA or cap-VEE-VP2-mRNA by agarose gel electrophoresis.

As shown in Figure 5, the agarose gel electrophoresis shows the band size of the mRNA, and the T7-FPV-VP2 plasmid is used as a control, which proves that the feline parvovirus VP2 protein cap-VP2-mRNA is effectively synthesized.

As shown in Figure 6, agarose gel electrophoresis shows the band size of mRNA, and the VEE-VP2 plasmid is used as a control, which proves that feline parvovirus VP2 protein replication type cap-VEE-VP2-mRNA is effectively synthesized.

Example 4 Detection of feline parvovirus VP2 protein expressed by transfected cap-VP2-mRNA and replicative cap-VEE-VP2-mRNA by immunofluorescence experiment

Transfection of cap-VP2-mRNA or cap-VEE-VP2-mRNA in cells can express feline parvovirus VP2 protein, and the translation of VP2-mRNA or VEE-VP2-mRNA can be detected effectively by using the specific antibody of feline parvovirus VP2 protein , follow the steps below:

a) Lay adherent cells such as BHK-21 on a 48-well plate, culture in a 5% CO ₂ incubator at 37°C, and when the cell density reaches about 70%, the obtained cap-VP2-mRNA or cap-VEE-VP2 -mRNA for transfection;

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-VP2-mRNA or cap-VEE-VP2- mRNA, gently tap to mix, incubate for 10min;

c) Wash the cells once with Opti-MEM medium, add the incubated mixture, use the specific rabbit polyclonal antibody of feline parvovirus VP2 protein after 24 hours, and detect the expression of VP2 protein by indirect immunofluorescence;

The specific rabbit polyclonal antibody of VP2 protein is derived from the antibody serum prepared by immunizing New Zealand white rabbits by using VP2 protein polypeptide segment CQPDGGQPAVRNERA and QTDENQAADGDPRYC synthetic peptide coupling.

As shown in Figure 7, the results show that the BHK-21 cells transfected with cap-VP2-mRNA can clearly observe the green fluorescent signal under the fluorescence microscope, indicating that cap-VP2-mRNA can effectively express feline parvovirus VP2 protein.

As shown in Figure 8, the results show that the BHK-21 cells transfected with cap-VEE-VP2-mRNA can clearly observe the green fluorescent signal under the fluorescence microscope, indicating that cap-VEE-VP2-mRNA can effectively express feline parvovirus VP2 protein.

Example 5 Preparation of lipid nanoparticle mRNA vaccine LNP-VP2-mRNA and lipid nanoparticle replicating mRNA vaccine LNP-VEE-VP2-mRNA

mRNA is a negatively charged biomacromolecule, which is difficult to pass through the negatively charged cell membrane by passive transport. Lipid nanoparticles (LNP) can be used to deliver RNA, which is an effective drug delivery method for mRNA vaccines.

The preparation of lipid nanoparticle mRNA vaccine LNP-VP2-mRNA or lipid nanoparticle replicating mRNA vaccine LNP-VEE-VP2-mRNA is performed according to the following steps:

a) Dissolve SM-102, DSPC, DMG-PEG2000 and cholesterol in 30 μL of absolute ethanol with a molar ratio of 50:10:38.5:1.5 and a total mass of 200 μg;

b) Under the condition of vortexing, quickly inject the ethanol solution into 90 μL of 20 mM sodium acetate buffer containing 5 μg of cap-VP2-mRNA or replicating cap-VEE-VP2-mRNA prepared in Example 3, and stir vigorously for 20 s , and then left to stand for 10 minutes to obtain nanoparticles;

c) Dialyze the prepared ethanol-sodium acetate mixed solution containing nanoparticles in 10 mM PBS solution for 2 to 4 hours to remove ethanol, and obtain the final product LNP-VP2-mRNA or replica LNP-VEE- VP2-mRNA.

Example 6 Feline parvovirus protein mRNA vaccine LNP-VP2-mRNA or replication type mRNA vaccine LNP-VEE-VP2-mRNA immunization experiment

The cap-VP2-mRNA or replication-type cap-VEE-VP2-mRNA prepared by Example 3 can effectively express feline parvovirus VP2 protein, and in combination with the preparation method of lipid nanoparticle mRNA vaccine in Example 5, prepare feline parvovirus protein For LNP-VP2-mRNA vaccine or replica LNP-VEE-VP2-mRNA vaccine, the vaccine effect is detected by immunoassay.

To evaluate the immune efficacy of LNP-VP2-mRNA vaccine, follow the steps below:

a) 12 6-week-old female BALB/c strain mice were randomly divided into 3 groups (negative control group, LNP-VP2-mRNA vaccine (or LNP-VEE-VP2-mRNA vaccine) group and Miaosanduo Vaccine group), BALB/c mice injected LNP-VP2-mRNA vaccine (or LNP-VEE-VP2-mRNA vaccine) or Miaosanduo inactivated vaccine with 5 μg/intramuscular route on the 0th day and the 30th day;

b) On the 7th day after the second immunization, blood was collected through the orbital route, and the collected serum samples were collected;

c) Determination of VP2 protein-specific antibody level in serum after immunization with LNP-VP2-mRNA vaccine (replicative LNP-VEE-VP2-mRNA vaccine) by double-antibody one-step sandwich enzyme-linked immunosorbent assay: pre-coated feline parvovirus antigen Serum samples and HRP-labeled detection antibodies were added to the coated microwells in sequence, incubated and washed thoroughly. Use the substrate TMB to develop the color, and convert it into the final yellow under the action of 1M sulfuric acid, and measure the absorbance (OD value) at a wavelength of 450nm with a microplate reader

As shown in FIG. 9 , the level of antibody specific to feline parvovirus protein in the sample was determined. Compared with the Miaosanduo control group, the immunized LNP-VP2-mRNA vaccine group produced different levels of feline parvovirus-specific antibodies, and the relative level of antibodies in the Miaosanduo control group was higher. Protein mRNA vaccine can effectively activate humoral immunity to produce feline parvovirus-specific antibodies.

As shown in FIG. 10 , the level of antibody specific to feline parvovirus protein in the sample was determined. The over-replicated LNP-VEE-VP2-mRNA vaccine group produced different levels of feline parvovirus-specific antibodies compared with the Miaosanduo control group, and the relative level of antibodies in the Miaosanduo control group was relatively high. The replicative mRNA vaccine of feline parvovirus VP2 protein can effectively activate humoral immunity to produce feline parvovirus-specific antibodies.

Claims (10)

1. A recombinant mRNA synthesis plasmid comprising a plasmid backbone sequence, a gene encoding a feline parvovirus VP2 protein; the gene sequence of the encoding cat parvovirus VP2 protein is shown as SEQ ID NO. 1.

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 PolyA tail.

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

4. The recombinant mRNA synthesis plasmid of claim 1, further comprising a venezuelan equine encephalitis virus replicase 1-4 coding region; the gene sequence of VP2 protein of the encoding cat calicivirus is subjected to codon optimization.

5. Use of the recombinant mRNA synthesis plasmid of claims 1-4 in the preparation of a vaccine for preventing feline panleukopenia caused by feline parvovirus infection.

6. An mRNA for expressing the feline parvovirus VP2 protein, comprising a 5' utr region, a 3' utr region, and a 3' terminal poly a tail, the mRNA encoding the feline calicivirus VP2 protein; the amino acid sequence of the feline calicivirus VP2 protein is shown as SEQ ID NO. 2.

7. The mRNA of claim 6, wherein the mRNA expresses the feline parvovirus VP2 protein, further comprising the mRNA encoding the venezuelan equine encephalitis virus replicase 1-4 coding region.

8. The mRNA of claim 6, further comprising a 5 'cap structure, wherein the 5' cap structure is a 7-methylguanosine cap structure.

9. An mRNA vaccine for expressing the VP2 protein of feline parvovirus comprising an mRNA for expressing the VP2 protein of feline parvovirus according to any one of claims 6 to 8.

10. The method for preparing an mRNA vaccine according to claim 9, wherein the mRNA expressing the feline parvovirus VP2 protein according to any one of claims 6 to 8 is mixed with cationic lipid, distearoyl phosphatidylcholine, polyethylene glycol lipid and cholesterol to prepare lipid nanoparticles, and the mRNA vaccine is obtained.

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