MRNA (messenger ribonucleic acid) and application

Abstract

The invention aims to provide mRNA (messenger Ribonucleic Acid) and application. According to the invention, on the basis of gene sequences of VP60 protein receptor binding regions of Chinese native isolated epidemic rabbit hemorrhagic disease virus type 1 and type 2 strains, candidate mRNA (messenger ribonucleic acid) for expressing RHDV1 and RHDV2 VP60 target antigens is constructed by using a messenger RNA (messenger ribonucleic acid) technology. The third-generation rabbit plague vaccine is prepared from the mRNA, carries the sequence information of the antigen protein, can be effectively translated into the antigen protein in cytoplasm, and activates cellular immune response and humoral immune response at the same time. Experiments of the vaccine for immunizing young rabbits show that the vaccine can induce generation of high-level antibodies aiming at RHDV1 and RHDV2, has good protectiveness on epidemic isolated strains of RHDV1 and RHDV2, and has great 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) is an acute, highly lethal infectious disease of rabbits. RHD is commonly known as rabbit plague, rabbit hemorrhagic pneumonia, and rabbit hemorrhagic disease. Its pathogen is rabbit hemorrhagic disease virus (RHDV). According to the different VP60 antigen structures, it can be divided into RHDV1 and RHDV2. RHDV1 was first reported and detected in China in 1984, and then quickly spread globally. RHDV2 first appeared and was reported in France in 2010. Until April 2020, RHDV2 first broke out in Sichuan Province, my country, and caused serious economic losses to many rabbit production areas in my country. RHDV is a single-stranded positive-stranded non-enveloped RNA virus. The genome has two open reading frames, ORF1 and ORF2. ORF1 encodes 7 non-structural proteins and the main structural capsid protein VP60. Each VP60 monomer consists of a shell domain (S) and a protruding domain (P). The P domain is subdivided into P1 and P2, with the P2 domain being the most exposed region of the capsid. This region shows the greatest genetic and antigenic variation due to the selective pressure generated by host antibody recognition. ORF2 encodes VP10, a minor structural protein.

[0003] There is no specific medicine for RHD on the market, and prevention is still the main treatment for this disease. There are many problems to be overcome in the research of RHDV, one of which is that cells that can be used for in vitro culture of RHDV have not yet been developed, which seriously hinders the research on RHDV and the development of its vaccine. The rabbit plague vaccines currently circulating on the market include rabbit plague tissue inactivated vaccine and rabbit plague baculovirus vector inactivated vaccine. Rabbit plague tissue inactivated vaccine is prepared by grinding the liver and spleen of rabbits infected by artificial inoculation of the virus and inactivating them with chemical reagents such as formaldehyde. The application of this vaccine provides key support for the prevention and control of rabbit plague in clinical practice. At the same time, the biosafety issues of tissue inactivated vaccines, pollutant residues and animal welfare and many other drawbacks cannot be ignored. VP60 is the main capsid protein and antigen protein of RHDV. Rabbit plague baculovirus vector inactivated vaccine is a genetically engineered subunit vaccine prepared by introducing the VP60 gene into a baculovirus vector, expressing the VP60 protein by 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 purpose of the present invention is to provide an mRNA and its application, to construct a candidate mRNA expressing the target antigen of RHDV1 and RHDV2 VP60 by using messenger RNA (mRNA) technology, and to apply it to the preparation of rabbit plague vaccine to significantly improve the immune protection of rabbit hemorrhagic disease virus. In order to achieve this technical purpose, the technical solution adopted by the present invention is: In a first aspect, the present invention provides an mRNA, wherein the mRNA sequence is: T in the sequence shown in SEQ ID NO.2 or SEQ ID NO.5 is changed to U.

[0005] 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, 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.

[0006] In a second aspect, the present invention provides a method for preparing the mRNA according to the first aspect, comprising the following steps: 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 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.

[0007] 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, polyadenylic acid are constructed on pTnTR vector.

[0008] 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, a PEGylated phospholipid mixture with the mRNA described in the first aspect, namely, RHDV1-mRNA lipid nanoparticles or RHDV2-mRNA lipid nanoparticles.

[0009] 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, The molar ratio of the ionizable cationic liposome, the neutral phospholipid, the sterol lipid and the PEGylated phospholipid mixture is 48-52:8-12:36-40:1-3, preferably 50:10:38.5:1.5.

[0010] 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 100 mM sodium citrate solution, and the ionizable cationic liposomes, neutral phospholipids, sterol lipids, and PEGylated phospholipids mixture is 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, and the aqueous phase and the oil phase are mixed with a volume ratio of 2 to 5:1.

[0011] In a fourth aspect, the present invention provides the use of the mRNA lipid nanoparticles described in the third aspect in the preparation of a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague.

[0012] 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 described in claim 5 or 6.

[0013] 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.

[0014] The vaccine according to claim 9 is characterized in that the delivery system of the vaccine is a cationic or polycationic compound, preferably LNP.

[0015] The present invention is based on the gene sequence of the VP60 protein receptor binding region of the rabbit hemorrhagic disease virus type 1 and type 2 strains isolated and prevalent in China, and uses messenger RNA (mRNA) technology to construct candidate mRNAs expressing the RHDV1 and RHDV2 VP60 target antigens, and uses them to prepare rabbit plague vaccines, i.e., third-generation vaccines, which carry the sequence information of antigenic proteins and can be effectively translated into antigenic proteins in the cytoplasm, while activating cellular and humoral immune responses. Experiments using the vaccine to immunize rabbits have shown that it can induce the production of high-level antibodies against RHDV1 and RHDV2, and has good protection against the prevalent isolated strains of RHDV1 and RHDV2. The safety and immune efficacy of the vaccine show great advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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: 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; Figure 2 Western Blot test results in the protein expression verification experiment of BHK-21 cells transfected with mRNA; Figure 3 The IFA test results of the protein expression verification experiment of BHK-21 cells transfected with mRNA, where Figure A corresponds to RHDV1-VP60P2, Figure B corresponds to RHDV2-VP60P2, and Figure C corresponds to the empty vector pTnTR; Figure 4 The electron microscopy analysis results after LNP-mRNA packaging, where Figure A corresponds to RHDV1-VP60P2 and Figure B corresponds to RHDV2-VP60P2; Figure 5 The particle size distribution diagram after LNP-mRNA packaging, where Figure A corresponds to RHDV1-VP60P2 and Figure B corresponds to RHDV2-VP60P2; Figure 6 The antibody-time curves of rabbits immunized with bivalent mRNA vaccine of rabbit hemorrhagic disease virus type 1 and type 2 and commercially available vaccine, respectively. Graph A corresponds to the RHDV1 antibody level, and Graph B corresponds to the RHDV2 antibody level. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0018] 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.

[0019] Specifically, the mRNA contains the VP60 protein receptor binding domain encoding RHDV1 and RHDV2. The VP60 protein is the most important viral surface membrane protein for rabbit plague virus to invade target cells. Therefore, after the mRNA encoding the corresponding antigen is delivered to the animal body, it can produce the VP60 protein receptor binding domain of rabbit plague virus to trigger an effective protective immune response as an antigen. The VP60 protein consists of two parts, the S region and the P region. When the virus invades the host cell, the P2 region is the receptor binding domain that binds to the cell receptor and is the main determinant of the virus's attachment to the target cell. Therefore, the P2 region has good antigenicity.

[0020] 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.

[0021] The coding region of the mRNA encodes the full-length sequence of the receptor binding region of the RHDV1 VP60 protein, which is derived from the JX / CHA / 97 strain (GenBank: DQ205345). The full-length sequence of the receptor binding region of the RHDV1 VP60 protein is shown in sequence SEQ ID NO.1. The coding region of the mRNA encodes the full-length sequence of the receptor binding region of the RHDV2 VP60 protein, which is derived from the SCMS-2020 strain (GenBank: OQ570964). The full-length sequence of the receptor binding region of the RHDV2 VP60 protein is shown in sequence SEQ ID NO.4.

[0022] 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.

[0023] The present invention also provides a method for preparing the mRNA, comprising the following steps: Step (1): optimizing and modifying the sequence between the transcription promoter and the transcription end point on the plasmid; Step (2): extract the plasmid and ensure that it is not contaminated by protein, and select a suitable restriction endonuclease BspQ I to perform a single enzyme digestion on the extracted plasmid; Step (3): purifying the plasmid after enzyme digestion by phenol or chloroform extraction and ethanol precipitation to obtain purified template DNA; 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; 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; Step (6): Use the linearized plasmid as a template, add 2 μL 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 balance is Nuclease-free Water. Mix well and react at 37°C for 3 h to perform in vitro transcription and capping of mRNA; Step (7): Add 1 μL DNase I, mix well, and react at 37°C for 15 min to degrade the template DNA; Step (8): Purify mRNA using lithium chloride precipitation method. Add 1 / 2 volume of LiCl precipitation solution to the in vitro transcription reaction solution, mix well and let stand 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, let stand at room temperature until the organic solvent evaporates, and add RNase free water to re-dissolve.

[0024] Step (9): Determine the concentration and purity of the purified mRNA using ultraviolet spectrophotometry.

[0025] Based on the above content, the embodiment of the present invention also prepares the mRNA into a bivalent mRNA vaccine of 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: Step 1: Ionizable cationic liposomes, neutral phospholipids, sterol lipids, PEGylated phospholipids and purified mRNA aqueous solution were mixed through a nano-microfluidic device (Miana INano L +) were mixed to prepare LNP-mRNA particles (mRNA lipid nanoparticles) of uniform size. The mRNA lipid nanoparticles were purified and recovered using an ultrafiltration tube. Two types of mRNA nanoparticles were obtained: RHDV1-mRNA and RHDV2-mRNA.

[0026] 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.

[0027] The mRNA is mainly used to prepare a rabbit hemorrhagic disease virus vaccine for preventing rabbit plague. The vaccine also has a 5'-cap structure, a 3'-terminal polyadenylic acid sequence, a 5'-UTR and a 3'-UTR. The vaccine can effectively prevent diseases caused by rabbit plague virus infection, wherein the disease caused by rabbit plague virus infection is rabbit viral hemorrhagic disease.

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0029] 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 (1) The following gene sequence was synthesized by Yaohai Biotechnology Co., Ltd.: from 5' to 3' end, T7 promoter, 5'UTR, kozak sequence, RHDV1-VP60 receptor binding sequence, 3'UTR, polyadenylation sequence were constructed on the pTnTR vector. The synthetic plasmid was named pTnTR-RHDV1-VP60P2. Figure 1 As shown in A; Original sequence of RHDV1-VP60P2 receptor binding region (SEQ ID NO.1) TGGTCAAGCCCTCGGTTTGCCGACATTGACCATCGAAGAGGCAGTGCAAGTTATTCTGGGAACAGCTCCACCAACGTGCTCCAGTTTTGGTACGCTAATGCTGGGTCTGCAATTGACAACCCTATCTCCCAGGTTGCACCGGACGGCTTTCCTGACATGTCATTCGTGCCCTTTAACAGCCCCAACATTCCGACCGCGGGGTGGGTCGGGTTTGGTGGTATTTGGAACAGTAACAACGGTGCCCCCGCTGCCACGACTGTGCAGGCCTATGAGTTAGGTTTTGCCACTGGGGCACCAAACAACCTCCAGCCCACCACCAACACTTCAGGTGCACAGACTGTCGCTAAGTCCATTTATGCCGTGGTGACCGGCACAAACCAAAACCCAACCGGACTGTTTGTGATGGCCTCGGGTGTTATCTCCACCCCAAACGCCAGCGCCGTCACATACACGCCCCAACCAGATAGAATTGTGACTACACCTGGCACTCCTGCCGCTGCACCTGTGGGTAAGAACACACCCATCATGTTCGCGTCTGTTGTCAGGCGCACCGGTGACGTCAACGCCGCAGCTGGGTCAACCAACGGGACCCAG; Codon-optimized sequence of the RHDV1-VP60 P2 receptor-binding region (SEQ ID NO.2) ; Amino acid sequence of the RHDV1-VP60P2 receptor binding region (SEQ ID NO.3) WSSPRFADIDHRRGSASYSGNSSTNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNAAAGSTNGTQ; (2) The following gene sequences were synthesized by Yaohai Biotechnology Co., Ltd.: from 5' to 3' end, they were T7 promoter, 5'UTR, kozak sequence, RHDV2-VP60 receptor binding sequence, 3'UTR, and polyadenylation sequence, which were constructed on the pTnTR vector. The synthesized plasmid was named pTnTR-RHDV2-VP60P2. Figure 1as shown in B; Original sequence of the RHDV2-VP60 P2 receptor-binding region (SEQ ID NO.4) TGGTCAAGCCCGCGGTTTGCCGCCATTGACCACGACAGAGGCAACGCAAGTTTCCCTGGAAGCAGTTCGTCAAACGTGCTTGAGCTTTGGTATGCTAGTGCCGGGTCTGCAGCTGACAACCCCATCTCCCAAATTGCTCCAGATGGTTTCCCTGACATGTCATTTGTACCCTTCAGCGGTACCACCATCCCTACCGCAGGGTGGGTCGGGTTCGGTGGGATCTGGAACAGCAGTAATGGTGCCCCCTACGTCACGACCATGCAGGCTTATGAGTTGGGTTTTGCCACTGGAGTACCGAGCAACCCCAAACCCACCACCACCACTTCAGGGGCTCAGATTGTTGCCAAGTCCATCTATGGCGTTGCAAACGGCATAAACCAGACAACAGCCGGGTTGTTTGTGATGGCATCTGGTGTCATATCCACTCCAAACAGCAGTGCCACTACGTACACACCTCAGCCAAACAGGATTGTTAACGCACCTGGCACCCCTGCTGCCGCCCCTATTGGCAAGAACACACCCATCATGTTCGCGTCTGTTGTTAGGCGCACCGGCGACATCAACGCTGAGGCCGGTTCAACTAACGGAACCCAG; Codon-optimized sequence of the RHDV2-VP60 P2 receptor-binding region (SEQ ID NO.5) TGGTCCAGCCCTAGATTCGCCGCCATCGACCACGACAGGGGAAACGCTTCCTTCCCCGGAAGCAGCTCCTCCAACGTGCTGGAACTGTGGTACGCCTCCGCCGGATCTGCTGCTGATAACCCTATCTCCCAGATCGCCCCCGACGGATTCCCTGATATGAGCTTCGTGCCCTTCTCCGGCACCACCATCCCTACAGCTGGCTGGGTGGGATTCGGCGGAATCTGGAACTCCTCCAACGGCGCTCCCTACGTGACCACAATGCAGGCTTACGAGCTGGGCTTCGCCACCGGAGTGCCTTCCAATCCTAAGCCCACCACCACCACCAGCGGCGCTCAGATCGTGGCTAAGTCCATCTACGGCGTGGCCAACGGCATCAACCAGACCACAGCCGGACTGTTCGTGATGGCCAGCGGAGTGATCTCCACCCCTAACAGCAGCGCCACCACCTACACCCCTCAGCCTAACAGGATCGTGAACGCCCCCGGAACCCCTGCTGCTGCTCCTATCGGAAAGAACACCCCCATCATGTTCGCCAGCGTGGTGAGGAGGACCGGAGATATTAACGCCGAGGCCGGCAGCACCAACGGAACCCAG; Amino acid sequence of the RHDV2-VP60 P2 receptor binding region (SEQ ID NO.6) WSSPRFAAIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTIPTAGWVGFGGIWNSSNGAPYVTTMQAYELGFATGVPSNPKPTTTTSGAQIVAKSIYGVANGINQTTAGLFVMASGVISTPNSSATTYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQ; Example 2: Preparation and detection of mRNA In vitro synthesis method of mRNA sequence, comprising the following steps: (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 taken and 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; 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 next step is to purify the linearized DNA, using phenol / chloroform extraction and ethanol precipitation to purify the digested plasmid. After the template DNA was diluted 10 times with TE Buffer, the concentration and purity of the template DNA were determined by ultraviolet spectrophotometry. Agarose gel electrophoresis was used to determine whether the linearization reaction of the plasmid DNA was complete. When the linearization was incomplete, the unlinearized plasmid existed in the form of a supercoiled ring, and its swimming rate was faster than that of the linearized plasmid. Therefore, if there were extra bands below the correct position on the electrophoresis graph, it indicated that the linearization of the plasmid was not complete; mRNA in vitro transcription: Use T7 High Yield RNA Synthesis Kit for Co-transcription from Yisheng Biotechnology for in vitro transcription. 20μL in vitro transcription reaction system: 1 μg template, 2 μL each of CTP / GTP / ATP / N1-Me-Pseudo UTP (100mmM each), 2 μL 10×Reaction Buffer, 2 μL EnzymeMix, 2 μL GAG (100mM), and the remainder is Nuclease-free Water. After mixing, react at 37℃ for 3 hours to perform in vitro transcription and capping of mRNA.

[0030] Removal of template DNA: After the reaction is completed, add 1 μL DNase I (RNase free) to each tube and incubate at 37°C for 15 min.

[0031] mRNA purification: Add 1 / 2 volume of LiCl precipitation solution (7.5M) to the in vitro transcription reaction solution, mix well and let stand at -20℃ for 8 h, then centrifuge at 15000 rpm for 15 min to collect the precipitate; wash and collect the precipitate with pre-cooled 70% ethanol, let stand at room temperature until the organic solvent evaporates, add Nuclease-free Water to re-dissolve, and determine the purity and concentration by UV spectrophotometry; measure the RNA concentration using a Nano drop instrument, and store the RNA at -80℃.

[0032] The nucleotide sequences of the two mRNAs are shown below: mRNA (RHDV1-VP60P2): T in the sequence shown in SEQ ID NO.2 is changed to U; ; mRNA (RHDV2-VP60P2): T in the sequence shown in SEQ ID NO.5 is changed to U; .

[0033] Example 3: RHDV1-VP60P2, RHDV2-VP60P2 protein expression verification test 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 inhibitor (PMSF) was added to fully lyse the cells, and the supernatant was taken after centrifugation to determine the protein concentration. 5×SDS loading buffer was added, and the cells were transferred to the membrane after reducing SDS-PAGE electrophoresis, and the Western blot results were obtained using SantaCruz's anti-His tag antibody. The results are shown in 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.

[0034] 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 h after transfection, the original culture medium in the culture plate was discarded and the cells were fixed with formaldehyde. Anti-His tag antibody and FITC-labeled goat anti-mouse secondary antibody from Santa Cruz were used for incubation in sequence. The expression of the target antigen protein in the cells of the transfected wells was observed using a fluorescence microscope. The results are shown in Figure 2. 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.

[0035] Example 4: Preparation and detection of mRNA lipid nanoparticles The preparation process of mRNA lipid nanoparticles is as follows: the obtained mRNA is mixed with 100mM sodium citrate (pH adjusted to 4.0) at a volume ratio of 1:1 to prepare an aqueous phase for use. At the same time, it is dissolved in anhydrous ethanol at a molar ratio of SM-102: DSPC: Cholesterol: DMG-PEG 2000 = 50: 10: 38.5: 1.5 as a lipid phase for use (the concentrations of SM-102, DSPC, Cholesterol, and DMG-PEG 2000 after dissolution are 6.25 mol / L, 1.25 mol / L, 4.8 mol / L, and 0.186 mol / L, respectively).

[0036] 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 (Miana INano L + ), LNP-mRNA stock solution was prepared. The mRNA lipid nanoparticles were purified and recovered using an ultrafiltration tube. Two types of mRNA nanoparticles were obtained: RHDV1-mRNA and RHDV2-mRNA.

[0037] 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 under HT7820 (Hitachi, Japan). The results were as follows: Figure 4As shown in Figures A and B, lipid nanoparticles have an average diameter of approximately 100 nm.

[0038] Take 10 μL of LNP-mRNA liquid and add it to 2 mL of PBS solution. Use a pipette to gently blow and mix, then add it to the cuvette. Place the cuvette in a 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.

[0039] Example 5: Safety experiment of bivalent mRNA vaccine SPF New Zealand rabbits were selected for the safety experiment of vaccine immunization. A total of 10 rabbits, 5 in each group, one group was intramuscularly inoculated with a high-dose bivalent mRNA vaccine, in which the mass ratio of RHDV1-VP60P2 and RHDV2-VP60P2 was 1:1, 60μg / head; the other group was injected with PBS as a control. Two weeks after the first vaccination, the second vaccination was carried out according to the same dose and route. The health status of the rabbits was observed and recorded daily. The results are shown in Table 1.

[0040] Table 1 Safety test results of bivalent mRNA vaccine

[0041] 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.

[0042] Example 6: Immunogenicity experiment of bivalent mRNA vaccine 70 one-month-old SPF New Zealand rabbits (double negative for RHDV antigen and antibody) were selected, including 20 in the mRNA vaccine group, which were intramuscularly injected with 30 ug / rabbit viral hemorrhagic disease bivalent mRNA vaccine (the mass ratio of RHDV1-VP60P2 and RHDV2-VP60P2 was 1:1), 10 in the PBS blank control group, and the remaining 40 rabbits were not treated and continued to be fed normally. The mRNA vaccine group was vaccinated for the second time 14 days after the first vaccination, and the vaccination dose was the same as the first vaccination. At the same time, 40 untreated rabbits were randomly divided into two groups, one group was subcutaneously injected with 1 ml / rabbit viral hemorrhagic disease bivalent tissue inactivated virus vaccine (20 rabbits), and the other group was subcutaneously injected with 1 ml / rabbit viral hemorrhagic disease baculovirus vector inactivated vaccine (20 rabbits). Both the rabbit viral hemorrhagic disease bivalent tissue inactivated virus vaccine and the rabbit viral hemorrhagic disease bivalent baculovirus vector inactivated vaccine are commercially available vaccines, and they were only immunized once according to the instructions for use. 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.

[0043] The results showed that all three vaccines activated high specific antibody levels 14 days after immunization, and the HI titer was significantly higher than that before immunization and the PBS blank group. The hemagglutination antibody titer continued to increase 14 days after the second immunization of the mRNA vaccine, indicating that the prepared mRNA vaccine can induce a higher level of humoral immunity.

[0044] Example 7: mRNA vaccine challenge experiment The experimental rabbits of Example 6 were challenged with RHDV1 strain (10000 LD) by subcutaneous injection of 10 rabbits in each group. 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.

[0045] Table 2 Results of the virus attack experiment

[0046] The data in Table 2 show that the rabbits were immunized with the bivalent mRNA vaccine of rabbit hemorrhagic disease virus, and high titer antibodies were produced in the rabbits, which was significantly different from the PBS control group, and the protection rate against the virus was high, which produced a good protection effect against the attack of the epidemic strain of RHDV. This laid the foundation for the development of a new safe and efficient vaccine for RHDV.

[0047] In summary, this study was based on the gene sequence of the VP60 protein receptor binding region of the rabbit hemorrhagic disease virus type 1 and type 2 strains isolated and prevalent in China, and codon optimization was used to improve translation efficiency. T7 promoter, 5'UTR, and "cap" structure were added to the front of the RNA sequence encoding the VP60 protein receptor binding domain, and 3'UTR and polyadenylic acid tail (poly A) were added to the rear end of the RNA sequence encoding the VP60 protein receptor binding domain to improve the translation efficiency and stability of the mRNA vaccine, and to construct a candidate mRNA vaccine expressing the RHDV1 and RHDV2 VP60 target antigen. The expression of the constructed candidate mRNA vaccine in BHK-21 cells was verified by in vitro cell transfection experiments and Western Blot experiments.

[0048] In addition, in terms of immune effect, the plasmids that have been verified for expression are used to prepare mRNA through in vitro transcription. After lipid nanoparticle packaging, quality analysis such as electron microscopy and particle size detection is performed to prepare bivalent mRNA vaccines for rabbit hemorrhagic disease virus type 1 and type 2. Immunizing rabbits with the constructed candidate bivalent mRNA vaccine can induce them to produce high levels of antibodies against RHDV1 and RHDV2, and it has good protection against the epidemic isolates of RHDV1 and RHDV2.

[0049] Although the embodiments of the present invention have been shown and described, it is understood that a person of ordinary skill in the art may make various changes, modifications, substitutions and variations 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 attached claims and their equivalents. The above-described embodiments only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that a person of ordinary skill in the art may make several variations and improvements without departing from the concept of the present invention, and these all belong to the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims.

Claims

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

2. The mRNA according to claim 1, characterized in that 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 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, polyadenylic acid are constructed on pTnTR vector.

5. An mRNA lipid nanoparticle, characterized in that: The ionizable cationic liposome, neutral phospholipid, sterol lipid, PEGylated phospholipid mixture and the mRNA according to claim 1 or 2 are mixed to obtain RHDV1-mRNA lipid nanoparticles or RHDV2-mRNA lipid nanoparticles.

6. The mRNA lipid nanoparticle according to claim 5, characterized in that 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 ionizable cationic liposome, the neutral phospholipid, the sterol lipid and the PEGylated phospholipid mixture is 48-52:8-12:36-40:1-3, preferably 50:10:38.5:1.

5.

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 described in 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 the RHDV1-mRNA lipid nanoparticles to the 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, preferably LNP.

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