mRNA and its application

Abstract

The present invention aims to provide mRNA and its application. Based on the gene sequence of the VP60 protein receptor binding region of rabbit hemorrhagic disease virus type 1 and type 2 strains isolated and prevalent in China, candidate mRNA expressing the VP60 target antigen of RHDV1 and RHDV2 was constructed using messenger RNA (mRNA) technology. This mRNA was then used to prepare a third-generation rabbit plague vaccine, which carries the sequence information of the antigenic protein and can be effectively translated into the antigenic protein in the cytoplasm, stimulating both cellular and humoral immune responses. Experiments using this vaccine to immunize rabbits have shown that it can induce high levels of antibodies against RHDV1 and RHDV2, and provides good protection against prevalent isolates of RHDV1 and RHDV2. The vaccine exhibits significant advantages in safety and immune efficacy.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to mRNA and its application. Background Art

[0002] Rabbit hemorrhagic disease (RHD), commonly known as rabbit plague, rabbit hemorrhagic pneumonia, or rabbit hemorrhagic disease, is an acute, highly lethal infectious disease of rabbits. It is caused by the rabbit hemorrhagic disease virus (RHDV). Based on the structure of the VP60 antigen, RHDV is divided into RHDV1 and RHDV2. RHDV is a single-stranded, positive-sense, non-enveloped RNA virus. Its genome contains two open reading frames, ORF1 and ORF2. ORF1 encodes seven nonstructural proteins and the major structural capsid protein, VP60. Each VP60 monomer consists of a capsid domain (S) and a protruding domain (P). The P domain is subdivided into P1 and P2, with the P2 domain located at the outermost region of the capsid. This region exhibits the greatest genetic and antigenic variation due to selective pressure generated by host antibody recognition. ORF2 encodes VP10, a minor structural protein.

[0003] Currently, there is no specific treatment for RHD on the market, and prevention remains the primary approach to the disease. RHDV research faces numerous challenges, including the lack of cells suitable for in vitro culture of RHDV, which significantly hinders research and vaccine development. Currently available vaccines for rabbit plague include inactivated tissue-based vaccines and baculovirus-vectored vaccines. The inactivated tissue-based vaccine is prepared by grinding the liver and spleen of infected rabbits and inactivating them with chemicals such as formaldehyde. Its use provides crucial support for the clinical prevention and control of rabbit plague. However, the biosafety, residual contaminants, and animal welfare issues of inactivated tissue-based vaccines should not be overlooked. VP60 is the primary capsid protein and antigenic protein of RHDV. The inactivated baculovirus-vectored vaccine is a genetically engineered subunit vaccine produced by introducing the VP60 gene into a baculovirus vector, expressing the VP60 protein in insect cells, and then purifying the target antigen. Recombinant subunit vaccines are more safe and have long-lasting humoral immunity, but the activation of cellular immunity requires the screening of excellent adjuvants to assist. Therefore, the inactivated rabbit plague baculovirus vector vaccine has high requirements for adjuvants, which also increases the complexity and difficulty of vaccine updates. Summary of the Invention

[0004] The present invention aims to provide an mRNA and its application. Using messenger RNA (mRNA) technology, candidate mRNAs expressing the RHDV1 and RHDV2 VP60 target antigens are constructed and applied to the preparation of rabbit plague vaccines to significantly enhance the immune protection against rabbit hemorrhagic disease virus. To achieve this technical objective, the present invention employs the following technical solutions:

[0005] In a first aspect, the present invention provides an mRNA, wherein the mRNA sequence is the sequence shown in SEQ ID NO. 2 or SEQ ID NO. 5, wherein T is changed to U. .

[0006] Preferably, the mRNA is obtained by codon-optimizing the VP60 protein receptor binding region of rabbit plague virus type 1 or the VP60 protein receptor binding region of rabbit plague virus type 2, and the original sequence of the VP60 protein receptor binding region of rabbit plague virus type 1 is shown as SEQ ID NO.1, and the original sequence of the VP60 protein receptor binding region of rabbit plague virus type 2 is shown as SEQ ID NO.4.

[0007] In a second aspect, the present invention provides a method for preparing the mRNA according to the first aspect, comprising the following steps:

[0008] Step 1, constructing a recombinant plasmid and modifying the sequence between the transcription promoter and the transcription terminator on the recombinant plasmid;

[0009] Step 2, extracting the recombinant plasmid, selecting the restriction endonuclease BspQI to perform single enzyme digestion on the extracted recombinant plasmid and purifying it to obtain template DNA;

[0010] Step 3: Using the template DNA to perform in vitro transcription and capping of mRNA, then removing the template DNA and purifying the mRNA to obtain the mRNA.

[0011] Preferably, the recombinant plasmid is constructed as follows: from 5' to 3' end, T7 promoter, 5'UTR, Kozak sequence, original sequence of RHDV1-VP60 receptor binding region or original sequence of RHDV2-VP60 receptor binding region, 3'UTR, polyadenylation are constructed into pTnTR vector.

[0012] In a third aspect, the present invention provides an mRNA lipid nanoparticle, which is prepared by mixing an ionizable cationic liposome, a neutral phospholipid, a sterol lipid, and a PEGylated phospholipid mixture with the mRNA described in the first aspect, namely, RHDV1-mRNA lipid nanoparticles or RHDV2-mRNA lipid nanoparticles.

[0013] Preferably, the ionizable cationic liposome is selected from at least one of SM-102, L319, and ALC-0159; the neutral phospholipid is selected from DSPC; the sterol lipid is selected from cholesterol; the PEGylated phospholipid mixture is selected from DMG-PEG 2000; and / or,

[0014] The molar ratio of the mixture of ionizable cationic liposomes, neutral phospholipids, sterol lipids and PEGylated phospholipids is 48-52:8-12:36-40:1-3, preferably 50:10:38.5:1.5.

[0015] Preferably, the mixture of ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipids is mixed with the mRNA described in the first aspect, and the mRNA is prepared by preparing an aqueous phase with a volume ratio of 0.5 to 1.5:1 with a 100mM sodium citrate solution. At the same time, the ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipid mixture are prepared with anhydrous ethanol as a solvent to prepare an oil phase with a concentration of 5.99 to 6.49 mol / L, 1 to 1.5 mol / L, 4.49 to 4.99 mol / L, and 0.12 to 0.37 mol / L, respectively. The aqueous phase and the oil phase are mixed at a volume ratio of 2 to 5:1.

[0016] In a fourth aspect, the present invention provides use of the mRNA lipid nanoparticles described in the third aspect in preparing a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague.

[0017] In a fifth aspect, the present invention provides a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague, characterized in that it comprises the mRNA lipid nanoparticles according to claim 5 or 6.

[0018] The vaccine according to claim 8 is characterized in that the vaccine is a bivalent vaccine, comprising RHDV1-mRNA lipid nanoparticles and RHDV2-mRNA lipid nanoparticles, and the mass ratio of RHDV1-mRNA lipid nanoparticles to RHDV2-mRNA lipid nanoparticles is 1~2:1~2.

[0019] The vaccine according to claim 9 is characterized in that the delivery system of the vaccine is a cationic or polycationic compound, preferably LNP.

[0020] Based on the gene sequence of the VP60 protein receptor binding region of rabbit hemorrhagic disease virus type 1 and type 2 strains isolated and prevalent in China, this invention utilizes messenger RNA (mRNA) technology to construct candidate mRNAs expressing the RHDV1 and RHDV2 VP60 target antigens. This mRNA is then used to prepare a rabbit plague vaccine, a third-generation vaccine. This vaccine carries the sequence information of the antigenic protein and can be effectively translated into the antigenic protein within the cytoplasm, activating both cellular and humoral immune responses. Experiments using this vaccine to immunize rabbits have shown that it can induce high levels of antibodies against RHDV1 and RHDV2, providing good protection against prevalent isolates of RHDV1 and RHDV2. The vaccine demonstrates significant advantages in both safety and immune efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 The plasmid maps of RHDV1-VP60P2 (Figure A) and RHDV2-VP60P2 (Figure B) constructed using the pTnTR vector in the embodiments of the present invention;

[0023] Figure 2 Western Blot analysis results for protein expression validation of mRNA transfected into BHK-21 cells;

[0024] Figure 3 These are the IFA test results for protein expression verification of BHK-21 cells transfected with mRNA. Panel A corresponds to RHDV1-VP60P2, Panel B corresponds to RHDV2-VP60P2, and Panel C corresponds to the empty vector pTnTR.

[0025] Figure 4 The electron microscopy analysis results after LNP-mRNA packaging are shown, where Figure A corresponds to RHDV1-VP60P2 and Figure B corresponds to RHDV2-VP60P2;

[0026] Figure 5 Figure 2 is the particle size distribution diagram after LNP-mRNA packaging, where Figure A corresponds to RHDV1-VP60P2 and Figure B corresponds to RHDV2-VP60P2;

[0027] Figure 6 These are the antibody-time curves of rabbits immunized with the bivalent mRNA vaccine for rabbit hemorrhagic disease virus type 1 and type 2 and the commercially available vaccine, respectively. Graph A corresponds to the RHDV1 antibody level, and Graph B corresponds to the RHDV2 antibody level. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] An embodiment of the present invention provides an mRNA, which contains a nucleotide sequence encoding a VP60 protein receptor binding region of rabbit plague virus type 1 or a VP60 protein receptor binding region of rabbit plague virus type 2 that causes rabbit viral hemorrhagic disease, wherein the nucleotide sequence has a nucleotide sequence as shown in SEQ ID NO.2 or SEQ ID NO.5, and T therein is changed to U.

[0030] Specifically, the mRNA contains the VP60 protein receptor binding domain encoding RHDV1 and RHDV2. VP60 is the primary viral surface membrane protein used by rabbit plague viruses to invade target cells. Therefore, when mRNA encoding the corresponding antigen is delivered into an animal, it can produce the VP60 protein receptor binding domain of rabbit plague viruses, acting as an antigen to trigger an effective protective immune response. The VP60 protein consists of two parts: the S region and the P region. When the virus invades host cells, the P2 region is the receptor binding domain that binds to cell receptors and is the primary determinant of viral attachment to target cells. Therefore, the P2 region has strong antigenicity.

[0031] The nucleotide sequence of the VP60 protein receptor binding region encoded by the mRNA coding region is derived from the serotype 1 and serotype 2 RHDV strains prevalent in China.

[0032] The coding region of the mRNA encodes the full-length sequence of the RHDV1 VP60 protein receptor binding region, which is derived from the JX / CHA / 97 strain (GenBank: DQ205345). The full-length sequence of the RHDV1 VP60 protein receptor binding region is shown in SEQ ID NO.1. The coding region of the mRNA encodes the full-length sequence of the RHDV2 VP60 protein receptor binding region, which is derived from the SCMS-2020 strain (GenBank: OQ570964). The full-length sequence of the RHDV2 VP60 protein receptor binding region is shown in SEQ ID NO.4.

[0033] Furthermore, the nucleotide sequence of the VP60 protein receptor binding region encoded by the mRNA coding region can also be derived from other serotype 1 RHDV strains: GenBank: AJ302016, JN165233, HM623309; serotype 2 RHDV strains: GenBank: PP387654, MT724773, PP387652.

[0034] The present invention also provides a method for preparing the mRNA, comprising the following steps:

[0035] Step (1): optimizing and modifying the sequence between the transcription promoter and the transcription end point on the plasmid;

[0036] Step (2): extract the plasmid and ensure that it is not contaminated by proteins, and select the appropriate restriction endonuclease BspQ I to perform single enzyme digestion on the extracted plasmid;

[0037] Step (3): Purify the enzyme-digested plasmid using phenol or chloroform extraction and ethanol precipitation to obtain purified template DNA;

[0038] Step (4): Take 2 μL of template DNA and dilute it to 200 μL with TE buffer. Then use UV spectrophotometry to determine the concentration and purity of the template DNA.

[0039] Step (5): Use agarose gel electrophoresis to determine whether the linearization reaction of the plasmid DNA is complete. The band on the electrophoresis graph should be single;

[0040] Step (6): Use the linearized plasmid as a template and add 2 μL each of CTP / GTP / ATP / N1-Me-Pseudo UTP (100 mM each), 2 μL 10× Reaction Buffer, 2 μL Enzyme Mix, 2 μL GAG (100 mM), and the remainder Nuclease-free Water. Mix well and incubate at 37°C for 3 h to perform in vitro transcription and capping of the mRNA.

[0041] Step (7): Add 1 μL DNase I, mix well, and react at 37°C for 15 min to degrade the template DNA;

[0042] Step (8): Purify the mRNA using lithium chloride precipitation. Add 1 / 2 volume of LiCl precipitation solution to the in vitro transcription reaction solution, mix well, and let it stand at -20°C for 8 hours. Then, centrifuge at 15,000 rpm for 15 minutes to collect the precipitate. Wash the collected precipitate with pre-cooled 70% ethanol, let it stand at room temperature until the organic solvent evaporates, and add RNase-free water to re-dissolve it.

[0043] Step (9): Determine the concentration and purity of the purified mRNA using ultraviolet spectrophotometry.

[0044] Based on the above, the present invention also prepares the mRNA into a bivalent mRNA vaccine for rabbit hemorrhagic disease virus for preventing rabbit plague. The vaccine is prepared by mixing two mRNA nanoparticles. The preparation process of the two mRNA nanoparticles specifically includes the following steps:

[0045] Step 1: The mixture of ionizable cationic liposomes, neutral phospholipids, sterol lipids, PEGylated phospholipids and purified mRNA aqueous solution was mixed through a nano-microfluidic device (Mian Na INano L + ) were mixed to prepare LNP-mRNA particles (mRNA lipid nanoparticles) of uniform size. Ultrafiltration was used to purify and recover the mRNA lipid nanoparticles. Two types of mRNA nanoparticles were obtained: RHDV1-mRNA and RHDV2-mRNA.

[0046] The mRNA vaccine contains two mRNA lipid nanoparticles: RHDV1-mRNA and RHDV2-mRNA, wherein the mass ratio of the lipid nanoparticles of RHDV1-mRNA and RHDV2-mRNA is 1~2:1~2, preferably 1:1.

[0047] This mRNA is primarily used to prepare a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague. The vaccine also contains a 5'-cap structure, a 3'-terminal polyadenylation sequence, a 5'-UTR, and a 3'-UTR. The vaccine is effective in preventing diseases caused by rabbit plague virus infection, particularly rabbit viral hemorrhagic disease.

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0049] Example 1: Construction of an antigen expression vector containing mRNA encoding the receptor binding region of the VP60 protein of rabbit plague virus type 1 that causes rabbit viral hemorrhagic disease

[0050] (1) The following gene sequences were synthesized by Yaohai Biotechnology Co., Ltd.: T7 promoter, 5'UTR, kozak sequence, RHDV1-VP60 receptor binding sequence, 3'UTR, and polyadenylation were constructed into the pTnTR vector. The synthesized plasmid was named pTnTR-RHDV1-VP60P2. Figure 1 As shown in A;

[0051] Original sequence of the RHDV1-VP60P2 receptor binding region (SEQ ID NO.1)

[0052] TGGTCAAGCCCTCGGTTTGCCGACATTGACCATCGAAGAGGCAGTGCAAGTTATTCTGGGAACAGCTCCACCAACGTGCTCCAGTTTTGGTACGCTAATGCTGGGTCTGCAATTGACAACCCTATCTCCCAGGTTGCACCGGACGGCTTTCCTGACATGTCATTCGTGCCCTTTAACAGCCCCAACATTCCGACCGCGGGGTGGGTCGGGTTTGGTGGTATTTGGAACAGTAACAACGGTGCCCCCGCTGCCACGACTGTGCAGGCCTATGAGTTAGGTTTTGCCACTGGGGCACCAAACAACCTCCAGCCCACCACCAACACTTCAGGTGCACAGACTGTCGCTAAGTCCATTTATGCCGTGGTGACCGGCACAAACCAAAACCCAACCGGACTGTTTGTGATGGCCTCGGGTGTTATCTCCACCCCAAACGCCAGCGCCGTCACATACACGCCCCAACCAGATAGAATTGTGACTACACCTGGCACTCCTGCCGCTGCACCTGTGGGTAAGAACACACCCATCATGTTCGCGTCTGTTGTCAGGCGCACCGGTGACGTCAACGCCGCAGCTGGGTCAACCAACGGGACCCAG;

[0053] Codon-optimized sequence of the RHDV1-VP60 P2 receptor-binding region (SEQ ID NO.2)

[0054] TGGTCCAGCCCCCGGTTCGCCGACATCGACCACCGCAGGGGCTCCGCTTCCTACTCCGGCAACAGCTCCACCAACGTGCTGCAATTCTGGTACGCCAACGCCGGCTCCGCCATCGACAACCCCATCAGCCAGGTGGCCCCCGACGGCTTCCCCGATATGAGCTTCGTGCCCTTCAACAGCCCCAACATCCCCACCGCCGGCTGGGTGGGATTCGGCGGAATCTGGAACTCCAACAACGGCGCCCCCGCCGCCACCACAGTGCAGGCTTACGAGCTGGGCTTCGCCACCGGCGCCCCTAACAACCTGCAACCCACCACCAACACCAGCGGCGCCCAGACCGTGGCCAAGAGCATCTACGCCGTGGTGACCGGCACCAACCAGAACCCCACCGGCCTGTTCGTGATGGCCAGCGGCGTGATCAGCACCCCCAACGCCAGCGCCGTGACCTACACCCCCCAGCCTGACCGCATCGTGACCACCCCCGGCACCCCTGCTGCCGCTCCTGTGGGAAAGAACACCCCCATCATGTTCGCCAGCGTGGTGCGCCGCACCGGCGATGTGAACGCCGCTGCTGGCAGCACCAACGGCACCCAG;

[0055] Amino acid sequence of the RHDV1-VP60 P2 receptor-binding region (SEQ ID NO.3)

[0056] WSSPRFADIDHRRGSASYSGNSSTNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNAAAGSTNGTQ;

[0057] (2) The following gene sequences were synthesized by Yaohai Biotechnology Co., Ltd.: T7 promoter, 5'UTR, kozak sequence, RHDV2-VP60 receptor binding sequence, 3'UTR, and polyadenylation were constructed into the pTnTR vector. The synthesized plasmid was named pTnTR-RHDV2-VP60P2. Figure 1 As shown in B;

[0058] Original sequence of the RHDV2-VP60P2 receptor binding region (SEQ ID NO.4)

[0059] ;

[0060] RHDV2-VP60P2 receptor binding region codon optimized sequence (SEQ ID NO.5)

[0061] TGGTCCAGCCCTAGATTCGCCGCCATCGACCACGACAGGGGAAACGCTTCCTTCCCCGGAAGCAGCTCCTCCAACGTGCTGGAACTGTGGTACGCCTCCGCCGGATCTGCTGCTGATAACCCTATCTCCCAGATCGCCCCCGACGGATTCCCTGATATGAGCTTCGTGCCCTTCTCCGGCACCACCATCCCTACAGCTGGCTGGGTGGGATTCGGCGGAATCTGGAACTCCTCCAACGGCGCTCCCTACGTGACCACAATGCAGGCTTACGAGCTGGGCTTCGCCACCGGAGTGCCTTCCAATCCTAAGCCCACCACCACCACCAGCGGCGCTCAGATCGTGGCTAAGTCCATCTACGGCGTGGCCAACGGCATCAACCAGACCACAGCCGGACTGTTCGTGATGGCCAGCGGAGTGATCTCCACCCCTAACAGCAGCGCCACCACCTACACCCCTCAGCCTAACAGGATCGTGAACGCCCCCGGAACCCCTGCTGCTGCTCCTATCGGAAAGAACACCCCCATCATGTTCGCCAGCGTGGTGAGGAGGACCGGAGATATTAACGCCGAGGCCGGCAGCACCAACGGAACCCAG;

[0062] Amino acid sequence of the RHDV2-VP60 P2 receptor-binding region (SEQ ID NO.6)

[0063] WSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPKPTTTTSGAQIVAKSIYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQ;

[0064] Example 2: Preparation and detection of mRNA

[0065] In vitro synthesis method of mRNA sequence, comprising the following steps:

[0066] (1) Extraction of plasmid: The designed sequence is synthesized into a plasmid template by a gene synthesis company. The transformation competent cell seed solution frozen at -80°C is streaked on an LB plate. After culturing overnight at 37°C, a single colony is picked and inoculated into LB medium and expanded at 37°C. After the culture is completed, the plasmid is extracted using a commercial plasmid extraction kit. After the plasmid solution is diluted 10 times with TE Buffer, the concentration and purity of the obtained plasmid solution are determined by ultraviolet spectrophotometry. The purity is judged by the absorbance value (A260 / A280) of the plasmid solution at 260nm and 280nm. When the absorbance value is in the range of 1.8-2.0, it indicates that the purity of the extracted plasmid solution is good. If it is lower than 1.8, there may be protein contamination;

[0067] Preparation of template DNA: The plasmids obtained in Example 1 (pTnTR-RHDV1-VP60P2; pTnTR-RHDV2-VP60P2) were digested with BspQI restriction endonuclease from New England Biolabs to linearize the plasmids. The linearized DNA was then purified using phenol / chloroform extraction and ethanol precipitation. The template DNA was diluted 10-fold with TE Buffer, and the concentration and purity of the obtained template DNA were determined by UV spectrophotometry. Agarose gel electrophoresis was used to determine whether the linearization reaction of the plasmid DNA was complete. If linearization is incomplete, the unlinearized plasmid exists as a supercoiled ring, which migrates faster than the linearized plasmid. Therefore, if there are extra bands below the correct position on the electrophoretogram, it indicates that the plasmid has not been completely linearized.

[0068] In vitro transcription of mRNA: Use Yisheng Bio's T7 High Yield RNA Synthesis Kit for Co-transcription for in vitro transcription. A 20 μL in vitro transcription reaction system includes 1 μg template, 2 μL each of 100 mM CTP / GTP / ATP / N1-Me-Pseudo UTP, 2 μL 10× Reaction Buffer, 2 μL Enzyme Mix, 2 μL GAG (100 mM), and the balance is Nuclease-free Water. Mix well and incubate at 37°C for 3 h for in vitro transcription and capping of the mRNA.

[0069] Removal of template DNA: After the reaction is complete, add 1 μL of DNase I (RNase-free) to each tube and incubate at 37°C for 15 min.

[0070] mRNA purification: Add 1 / 2 volume of LiCl precipitation solution (7.5 M) to the in vitro transcription reaction solution, mix well, and incubate at -20°C for 8 h. Then, centrifuge at 15,000 rpm for 15 min to collect the precipitate. Wash the collected precipitate with pre-cooled 70% ethanol, incubate at room temperature until the organic solvent evaporates, and reconstitute it with nuclease-free water. Determine the purity and concentration by UV spectrophotometry. Measure the RNA concentration using a Nanodrop instrument and store the RNA at -80°C.

[0071] The nucleotide sequences of the two mRNAs are shown below:

[0072] mRNA (RHDV1-VP60P2): T in the sequence shown in SEQ ID NO. 2 is changed to U;

[0073] ;

[0074] mRNA (RHDV2-VP60P2): T in the sequence shown in SEQ ID NO. 5 is changed to U;

[0075] .

[0076] Example 3: RHDV1-VP60P2, RHDV2-VP60P2 protein expression verification test

[0077] The purified in vitro transcribed RNA was transfected into BHK-21 cells using ThermoFisher's Lipofectamine 3000 reagent to verify expression. BHK-21 cells were evenly plated in a six-well plate, and 2 μg of mRNA was transfected into each well. 24 h after transfection, commercial RIPA cell lysis buffer supplemented with protease inhibitors (PMSF) was added to fully lyse the cells. After centrifugation, the supernatant was collected to determine the protein concentration. 5× SDS loading buffer was added, and the cells were transferred to a membrane after reducing SDS-PAGE electrophoresis. Western blot results were obtained using SantaCruz's anti-His tag antibody. The results are shown in Figure 2. Figure 2As shown, compared with the empty vector control group (pTnTR), protein bands with expected sizes (expected protein size is 21 kDa) appeared in the experimental groups transfected with RHDV1-VP60P2 mRNA and RHDV2-VP60P2 mRNA.

[0078] IFA detection: The purified in vitro transcribed RNA was transfected into BHK-21 cells using ThermoFisher's Lipofectamine 3000 reagent to verify expression. BHK-21 cells were evenly plated in a 24-well plate, and 1 μg of mRNA was transfected into each well. 24 hours after transfection, the original culture medium in the culture plate was discarded and the cells were fixed with formaldehyde. Santa Cruz's anti-His tag antibody and FITC-labeled goat anti-mouse secondary antibody were used for incubation in sequence. The expression of the target antigen protein in the transfected well cells was observed using a fluorescence microscope. The results are as follows: Figure 3 As shown, compared with the empty vector control group (pTnTR), the experimental groups transfected with RHDV1-VP60P2 mRNA and RHDV2-VP60P2 mRNA both produced specific yellow-green fluorescence.

[0079] Example 4: Preparation and Detection of mRNA Lipid Nanoparticles

[0080] The mRNA lipid nanoparticles were prepared as follows: the obtained mRNA was mixed with 100 mM sodium citrate (pH 4.0) at a volume ratio of 1:1 to prepare the aqueous phase. Simultaneously, SM-102:DSPC:Cholesterol:DMG-PEG 2000 were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to prepare the lipid phase (the concentrations of SM-102, DSPC, Cholesterol, and DMG-PEG 2000 after dissolution were 6.25 mol / L, 1.25 mol / L, 4.8 mol / L, and 0.186 mol / L, respectively).

[0081] The prepared aqueous phase and lipid phase were loaded into syringes of appropriate volumes in a volume ratio of 3:1, and the two phases were passed through a nano-microfluidic device (Mian Na INano L + ), to create a LNP-mRNA stock solution. Ultrafiltration was used to purify and recover the mRNA lipid nanoparticles. Two types of mRNA nanoparticles were obtained: RHDV1-mRNA and RHDV2-mRNA.

[0082] The two mRNA lipid nanoparticle samples prepared above were stained with 1% phosphotungstic acid solution and observed under a transmission electron microscope at an accelerating voltage of 80 kV using an HT7820 (Hitachi, Japan). The results were as follows: Figure 4 As shown in Figures A and B, lipid nanoparticles have an average diameter of approximately 100 nm.

[0083] Take 10 μL of LNP-mRNA liquid and add it to 2 mL of PBS solution. Use a pipette to gently pipette to mix and then add it to the cuvette. Place the cuvette in the nanoanalyzer (Omni NanoBrook) for detection. The detection results of the two mRNAs are as follows: Figure 5 As shown in Figures A and B, the average particle size of the prepared RHDV1 mRNA lipid nanoparticles was 93.65 nm, and the PDI was less than 0.146; the average particle size of the RHDV2 mRNA lipid nanoparticles was 100.06 nm, and the PDI was less than 0.108.

[0084] Example 5: Safety Experiment of Bivalent mRNA Vaccine

[0085] A safety study of vaccination was conducted in SPF New Zealand pups. Ten pups, five in each group, were vaccinated intramuscularly with a high-dose bivalent mRNA vaccine (RHDV1-VP60P2:RHDV2-VP60P2) at a mass ratio of 1:1, at 60 μg / pup. The other group received PBS as a control. Two weeks after the first vaccination, a second vaccination was administered using the same dose and route. The rabbits' health was observed and recorded daily. The results are shown in Table 1.

[0086] Table 1 Safety test results of bivalent mRNA vaccine

[0087]

[0088] The results in Table 1 show that all the rabbits in the PBS group and the bivalent mRNA vaccine group were in good health, with normal body temperature and no inflammatory reactions such as redness, swelling, heat, and pain at the injection site, indicating that the bivalent mRNA vaccine is safe.

[0089] Example 6: Immunogenicity experiment of bivalent mRNA vaccine

[0090] Seventy one-month-old SPF New Zealand pups (double negative for RHDV antigen and antibody) were selected. Twenty rabbits were placed in the mRNA vaccine group and received intramuscular injections of 30 μg / rabbit of a bivalent rabbit viral hemorrhagic disease mRNA vaccine (RHDV1-VP60P2 to RHDV2-VP60P2 mass ratio of 1:1). Ten rabbits were placed in the PBS blank control group, and the remaining 40 rabbits were untreated and fed normally. The mRNA vaccine group received a second vaccination 14 days after the first vaccination, using the same dose as the first vaccination. Simultaneously, 40 untreated rabbits were randomly divided into two groups: one group received a subcutaneous injection of 1 ml / rabbit of the bivalent inactivated rabbit viral hemorrhagic disease virus vaccine (1 ml / rabbit), and the other group received a subcutaneous injection of 1 ml / rabbit of the inactivated rabbit viral hemorrhagic disease baculovirus vector vaccine (1 ml / rabbit). Both the bivalent inactivated rabbit viral hemorrhagic disease vaccine and the bivalent inactivated rabbit viral hemorrhagic disease baculovirus vector vaccine were commercially available and administered only once according to the manufacturer's instructions. Blood was collected and serum was separated before immunization, 2 weeks after the first immunization, and 2 weeks after the booster immunization. The antibody titer in the serum of the immunized rabbits was analyzed by hemagglutination test. The results are as follows: Figure 6 shown.

[0091] The results showed that all three vaccines activated high levels of specific antibodies 14 days after immunization, with HI titers significantly higher than those before immunization and the PBS blank group. Hemagglutination antibody titers continued to rise 14 days after the second immunization of the mRNA vaccine, indicating that the prepared mRNA vaccine can induce high levels of humoral immunity.

[0092] Example 7: mRNA vaccine challenge experiment

[0093] The experimental rabbits of Example 6 were challenged with the bivalent tissue-inactivated rabbit viral hemorrhagic disease vaccine group and the bivalent baculovirus vector inactivated vaccine group 14 days after the first vaccination, and the bivalent mRNA vaccine group 14 days after the second vaccination. The challenge method was to subcutaneously inject RHDV1 strain (10000 LD 50 ), and another 10 rabbits were injected subcutaneously with RHDV2 strain (10000LD 50 After the challenge, the rabbits were observed for 10 days, the incidence and mortality of the rabbits were recorded, and the challenge protection rate was calculated. The results of the challenge experiment are shown in Table 2.

[0094] Table 2 Results of the challenge experiment

[0095]

[0096] The data in Table 2 demonstrate that immunization of experimental rabbits with the bivalent RHDV mRNA vaccine produced high antibody titers, significantly higher than those in the PBS control group. Furthermore, the vaccine demonstrated a high protection rate against challenge with the prevalent RHDV strain, demonstrating excellent protection against challenge with the prevalent RHDV strain. This data lays the foundation for the development of a new, safe, and highly effective RHDV vaccine.

[0097] In summary, this study used the gene sequences of the VP60 receptor-binding domain of rabbit hemorrhagic disease virus type 1 and type 2 strains isolated and prevalent in China as the basis for codon optimization to enhance translation efficiency. A T7 promoter, 5' untranslated region (UTR), and a "cap" construct were added to the RNA sequence encoding the VP60 receptor-binding domain. A 3' untranslated region (UTR) and a polyadenylation tail (poly A) were added to the end of the RNA sequence encoding the VP60 receptor-binding domain to enhance translation efficiency and stability. A candidate mRNA vaccine expressing the VP60 target antigen of RHDV1 and RHDV2 was constructed. Expression of the candidate mRNA vaccine in BHK-21 cells was verified by in vitro cell transfection and Western blot analysis.

[0098] Furthermore, the expression plasmid was verified for immune efficacy by in vitro transcription to produce mRNA. After packaging into lipid nanoparticles and conducting quality analysis, including electron microscopy and particle size measurement, a bivalent mRNA vaccine for rabbit hemorrhagic disease virus types 1 and 2 was prepared. Immunization of the constructed candidate bivalent mRNA vaccine with rabbits induced the production of high levels of antibodies against RHDV1 and RHDV2, and demonstrated good protection against prevalent isolates of both RHDV1 and RHDV2.

[0099] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for those skilled in the art, several variations and improvements may be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An mRNA, characterized in that The mRNA sequence is the sequence shown in SEQ ID NO.2 or SEQ ID NO.5, wherein T is changed to U.

2. The mRNA according to claim 1, wherein The mRNA is obtained by codon-optimizing the VP60 protein receptor binding region of rabbit plague virus type 1 or the VP60 protein receptor binding region of rabbit plague virus type 2. The original sequence of the VP60 protein receptor binding region of rabbit plague virus type 1 is shown in SEQ ID NO.1, and the original sequence of the VP60 protein receptor binding region of rabbit plague virus type 2 is shown in SEQ ID NO.

4.

3. The method for preparing mRNA according to claim 1 or 2, characterized in that The following steps are involved: Step 1, constructing a recombinant plasmid and modifying the sequence between the transcription promoter and the transcription terminator on the recombinant plasmid; Step 2, extracting the recombinant plasmid, selecting the restriction endonuclease BspQI to perform single enzyme digestion on the extracted recombinant plasmid and purifying it to obtain template DNA; Step 3: Using the template DNA to perform in vitro transcription and capping of mRNA, then removing the template DNA and purifying the mRNA to obtain the mRNA.

4. The preparation method according to claim 3, characterized in that The recombinant plasmid is constructed as follows: from 5' to 3' end, T7 promoter, 5'UTR, Kozak sequence, original sequence of RHDV1-VP60 receptor binding region or original sequence of RHDV2-VP60 receptor binding region, 3'UTR, polyadenylation are constructed on pTnTR vector.

5. An mRNA lipid nanoparticle, characterized in that The RHDV1-mRNA lipid nanoparticles or RHDV2-mRNA lipid nanoparticles are prepared by mixing a mixture of ionizable cationic liposomes, neutral phospholipids, sterol lipids and PEGylated phospholipids with the mRNA according to claim 1 or 2.

6. The mRNA lipid nanoparticle according to claim 5, wherein The ionizable cationic liposome is selected from at least one of SM-102, L319, and ALC-0159; the neutral phospholipid is selected from DSPC; the sterol lipid is selected from cholesterol; the PEGylated phospholipid mixture is selected from DMG-PEG 2000; and / or, The molar ratio of the mixture of ionizable cationic liposomes, neutral phospholipids, sterol lipids and PEGylated phospholipids is 48-52:8-12:36-40:1-3.

7. Use of the mRNA lipid nanoparticles according to claim 5 or 6 in the preparation of a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague.

8. A rabbit hemorrhagic disease virus vaccine for preventing rabbit plague, characterized in that: Comprising the mRNA lipid nanoparticles according to claim 5 or 6.

9. The vaccine according to claim 8, characterized in that The vaccine is a bivalent vaccine, comprising RHDV1-mRNA lipid nanoparticles and RHDV2-mRNA lipid nanoparticles, and the mass ratio of RHDV1-mRNA lipid nanoparticles to RHDV2-mRNA lipid nanoparticles is 1-2:1-2.

10. The vaccine according to claim 9, characterized in that The delivery system of the vaccine is a cationic or polycationic compound.

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