Anti-pseudorabies virus circular RNA vaccine and preparation method thereof

Abstract

The invention discloses an anti-pseudorabies virus circular RNA vaccine and a preparation method thereof. The pseudorabies virus gD truncated mutant protein comprises a circular RNA (Ribonucleic Acid) molecule for coding pseudorabies virus gD truncated mutant protein and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the pseudorabies virus gD truncated mutant protein is shown as SEQ ID No.12 or a degenerate sequence thereof, and the nucleotide sequence of the pseudorabies virus gD truncated mutant protein is shown as SEQ ID No.13 or a degenerate sequence thereof; the nucleotide sequence of the circular RNA molecule is as shown in SEQ ID No. 16. At present, no RNA vaccine for resisting pseudorabies virus infection exists, the vaccine is prepared from the circular RNA for coding the pseudorabies virus gD truncated and mutant protein and the lipid nanoparticles, and the vaccine has the advantages of simple production process, safety, high efficiency, low cost and the like; a high-level neutralizing antibody can be generated and CD8 + T cell immune response can be successfully activated by injecting a low-dose circular RNA vaccine (80mu g / ml). The vaccine has good safety and effectiveness, protects animals from attacking toxic substances, and is safe to piglets. The vaccine is stable after being stored at-20 DEG C for 6 months, and a basis is provided for large-scale vaccine preparation and clinical application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a circular RNA vaccine for resisting pseudorabies virus infection and a preparation method thereof. Background Art

[0002] Pseudorabies in pigs is an acute, virulent infectious disease caused by pseudorabies virus (PRV) that seriously harms the pig breeding industry. The main clinical symptoms are pruritus, high fever, reproductive disorders and encephalomyelitis. The virus can lurk in the trigeminal ganglion or sacral ganglion to cause latent infection, making sick pigs carry the virus for a long time and difficult to eradicate. In my country, at present, attenuated live vaccines are mainly used to prevent and control PRV. However, due to the stronger pathogenicity of the newly emerged strains, the traditional vaccine has poor protection against the new PRV. In recent years, the number of pseudorabies-positive pig farms in my country has increased significantly. The prevalence of new pseudorabies viruses has brought great challenges to the prevention and control of the disease in my country (doi:10.1007 / s11250-018-1555-1). In addition, my country's prevention and control goal for pseudorabies is "purification and elimination". At this time, vaccination with infectious live vaccines will no longer be applicable. In view of the huge economic losses caused by the new outbreak of pseudorabies to my country's pig industry and the need to purify and eliminate the pseudorabies virus, the development of a new vaccine against PRV is of great significance for the prevention and control and purification of the disease.

[0003] PRV belongs to the family Herpesviridae. It is a member of the subfamily and is a double-stranded DNA virus with a genome size of about 150kb, at least 75 open reading frames, and is predicted to encode nearly 100 proteins. It is oval or round in shape, with a diameter of about 150 to 300nm. It consists of a four-layer structure, from the inside to the outside, which is the virus core containing the viral genome, the capsid that encapsulates the viral DNA, the membrane composed of functional proteins, and the capsule rich in PRV glycoprotein. At present, the new vaccines for pseudorabies mainly include inactivated vaccines and recombinant subunit vaccines. The inactivated vaccine is mainly made of strains through amplification, inactivation, emulsification and other processes, which plays a certain role in the prevention of pseudorabies disease. However, the traditional inactivated vaccine has a short protection period for animals and poor protection against mutant virus strains. In addition, the inactivated vaccine has high requirements for the production environment and production personnel during the production and preparation process. There is a risk of virus strain leakage and incomplete inactivation during the production and preparation process, so the production cost is relatively high. Another vaccine technology route is the recombinant subunit vaccine for pseudorabies, which is still in the research and development stage. Currently, the envelope glycoproteins (gB, gC, gD, gH, and gL proteins) on PRV virus particles are usually used as targets for subunit vaccine design, but as of now, no pseudorabies subunit vaccine has been approved worldwide. The development of new vaccines for pseudorabies virus still needs breakthroughs.

[0004] mRNA vaccines are vaccines that directly introduce mRNA containing antigenic proteins into the body, so that it is directly translated in the body to complete the expression of the target protein, thereby causing a specific immune response in the body. They have played an important role in the prevention of major respiratory infectious diseases in humans. At present, the mRNA used in clinical or preclinical applications is mainly linear mRNA. The structure of linear mRNA includes 5' cap structure (5'Cap), 3' polyadenosine tail (PolyA tail), 5' untranslational sequence (5'untranslational region, 5'UTR), 3' untranslational sequence (3'untranslational region, 3'UTR) and open reading frame (Open reading frame, ORF). The 5' cap structure is a basic feature of eukaryotic mRNA, which is obtained by adding N7-methylguanosine to the 5' end of mRNA. The 5' cap structure and 3' polyadenosine tail are necessary components of linear RNA, which can effectively prevent mRNA degradation and promote the translation of mRNA into protein. The 5' cap structure promotes mRNA translation by binding to the translation initiation complex eIF4E, and can effectively prevent mRNA degradation and reduce the immunogenicity of mRNA. The main function of the 3' polyadenosine tail is to bind to PolyA binding protein (PABP), which interacts with eIF4G and eIF4E, mediates the formation of mRNA into a ring, promotes the translation process, and prevents mRNA degradation.

[0005] Circular RNA (circRNAs) is a common type of RNA in eukaryotes. Currently, the most efficient technology for constructing circular RNA in vitro is the self-cleavage circular system design technology (patent CN 114574483 A). The design principle of this technology is to connect the exon E1 and E2 sequences end to end through molecular cloning to form a continuous circular plasmid. The intron is cut and broken by restriction endonuclease to obtain a linear plasmid. Then, in vitro transcription is performed through the T7 promoter upstream of the inverted 3' intron to obtain a linear RNA containing a 3' intron-E2-E1-5' intron structure. Similar to the natural class I intron system, the specific conserved sequence of the splicing site of exon E1 is broken by the nucleophilic attack of the free guanosine 3' hydroxyl, and exon E1 produces a naked 3' hydroxyl, while guanosine binds to the broken 5' intron. Afterwards, the exposed 3'hydroxyl group of exon E1 attacks the conserved sequence between the 3'intron and exon E2, excises the 3'intron, and exons E2 and E1 undergo a circularization reaction to obtain a circular E1-E2 RNA. Compared with linear mRNA, circular mRNA molecules often have a longer half-life and better stability, and have important application prospects in protein expression and clinical treatment. In addition, the preparation process of circular RNA designed based on the self-cleaving circularization system is simpler and has lower process costs. After transcription, only circularization is performed, and there is no need to use expensive capping enzymes and tailing enzymes to modify additional 5' cap structures and 3' polyadenosine tail structures, nor is there a need for additional circularization processes using T4 RNA ligase as in traditional circular RNA. In addition, it can avoid the introduction of additional exon sequences in circular RNA, thereby improving the sequence accuracy of circular RNA molecules.

[0006] As of now, no new pseudorabies virus vaccine, including RNA vaccine, has entered clinical use worldwide, and breakthroughs in related fields are yet to be made. Summary of the invention

[0007] The purpose of the present invention is to address the many problems existing in the existing pseudorabies live vaccines and inactivated vaccines in clinical use and the recombinant subunit vaccines under research, such as the live pseudorabies virus vaccine of porcine cannot effectively prevent and control newly emerging strains and is not suitable for virus purification and elimination, the inactivated vaccine has a short protection period for animals, poor protection against mutant virus strains, and high production costs, the recombinant subprotein vaccine has unstable efficacy, low production efficiency, and difficulties in the purification process, and the reported pseudorabies virus linear RNA vaccine cannot activate CD8+ cell immune response and has insufficient immune protection efficacy (doi: 10.1016 / j.vetmic.2020.108886). A circular RNA vaccine against pseudorabies virus and a preparation method thereof are provided, which can produce neutralizing antibodies, successfully activate CD8+T cell immune response, and effectively prevent pseudorabies virus infection, and has many advantages such as safety, high efficiency, and low cost. The pseudorabies virus circular RNA vaccine comprises a coding circular RNA molecule and a pharmaceutically acceptable carrier, wherein the circular RNA molecule comprises optimized pseudorabies virus gD protein optimized nucleotides and an optimized backbone.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a circular RNA vaccine against pseudorabies virus, wherein the circular RNA vaccine comprises a circular RNA molecule encoding a truncated and mutant protein of pseudorabies virus envelope protein gD and a pharmaceutically acceptable carrier.

[0010] As an embodiment of the present invention, the amino acid sequence of the truncated and mutant protein of the pseudorabies virus envelope protein gD is shown in SEQ ID No. 12 or its degenerate sequence.

[0011] As an embodiment of the present invention, the optimized nucleotide sequence of the truncated and mutant protein of the pseudorabies virus envelope protein gD is shown in SEQ ID No. 13 or its degenerate sequence.

[0012] As one embodiment of the present invention, the circular RNA is obtained by transcription and circularization of a vector comprising the following elements: an intron fragment as shown in SEQ ID NO.8, a translation initiation element truncated fragment as shown in SEQ ID NO.10, a protein coding element as shown in SEQ ID NO.13, an insertion element as shown in SEQ ID NO.5, a translation initiation element truncated fragment as shown in SEQ ID NO.11 and an intron fragment as shown in SEQ ID NO.9.

[0013] As one embodiment of the present invention, the nucleotide sequence of the circular RNA molecule (PPV2-gD2135) is as SEQ ID No.16.

[0014] As an embodiment of the present invention, the content of the circular RNA molecule (PPV2-gD2135) in the circular RNA vaccine against pseudorabies virus is 30-200 μg / ml, including 30-60 μg / ml, 60-90 μg / ml, 90-120 μg / ml, 120-150 μg / ml, 150-180 μg / ml, 180-200 μg / ml, preferably 60-100 μg / ml.

[0015] As one embodiment of the present invention, the pharmaceutically acceptable carrier comprises lipid nanoparticles.

[0016] As an embodiment of the present invention, the ratio of lipid nanoparticles to circular RNA molecules in the anti-pseudorabies virus circular RNA vaccine is 1:1-1:3 (V / V).

[0017] As an embodiment of the present invention, the raw material composition of the lipid nanoparticles includes: 45% to 55% cationic lipids, 35% to 44% cholesterol, 3% to 10% neutral lipids and 0.8% to 1.8% PEG-modified lipids.

[0018] As one embodiment of the present invention, the cationic lipid is CMAX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoate) or SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecanyloxy)hexyl]amino]-octanoate.

[0019] As an embodiment of the present invention, the cholesterol is 5-cholesten-3β-ol.

[0020] As an embodiment of the present invention, the neutral lipid is DSPC (distearoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine).

[0021] As an embodiment of the present invention, the PEG-modified lipid is PEG-DMG (polyethylene glycol-dimyristyl glyceride), PEG-DSG (bis-distearoyl-rac-glycerol-polyethylene glycol) or PEG-DPG (polyethylene glycol diphosphoglycerate).

[0022] As an embodiment of the present invention, the preparation method of the lipid nanoparticles comprises: dissolving 45% to 55% cationic lipids, 35% to 44% cholesterol, 3% to 10% neutral lipids, and 0.8% to 1.8% PEG-modified lipids in ethanol.

[0023] As an embodiment of the present invention, the cationic lipid is CMAX4; the cholesterol is 5-cholesten-3β-ol; the neutral lipid is DSPC; the PEG-modified lipid is PEG-DMG; the molar ratio of CMAX4:5-cholesten-3β-ol:DSPC:PEG-DMG is 45.8:43.4:9.3:1.5.

[0024] The present invention also provides a method for preparing a circular RNA vaccine against pseudorabies virus, the method comprising the following steps:

[0025] S1, synthesizing and inserting the gD truncated plus mutant protein gene and looping element in the pseudorabies virus into a cloning vector to obtain a recombinant vector containing the gD truncated plus mutant protein gene and looping element in the pseudorabies virus;

[0026] S2, transforming or transducing a host with the recombinant vector containing the truncated gD plus mutant protein gene and the looping element in the pseudorabies virus obtained in step S1 to obtain a recombinant containing the recombinant vector;

[0027] S3, culturing the recombinant obtained in step S2 to prepare a plasmid containing the gD truncated plus mutant protein gene and the circular element in the pseudorabies virus;

[0028] S4, using the plasmid containing the pseudorabies virus gD truncated plus mutant protein gene and the circular element obtained in step S3 to prepare a circular mRNA encoding the pseudorabies virus gD truncated plus mutant protein;

[0029] S5. Add the circular mRNA encoding the truncated and mutant protein of pseudorabies virus gD obtained in step 4 to a pharmaceutically acceptable carrier to obtain the anti-pseudorabies virus circular RNA vaccine.

[0030] The present invention also provides use of a circular RNA vaccine against pseudorabies virus in preparing a drug for preventing and / or treating pseudorabies virus infection.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1) There is currently no pseudorabies virus RNA vaccine. The present invention uses a circular RNA molecule encoding a gD truncated and mutant protein optimized nucleotide sequence as an antigen to prepare a circular RNA vaccine against pseudorabies infection, which has many advantages such as safety, high efficiency, and low cost.

[0033] 2) In the present invention, when a low-dose circular RNA vaccine (RNA content of 80 μg / ml) against pseudorabies infection is injected, significantly increased neutralizing antibodies and cellular immune responses can be produced in the second week after the secondary immunization, and the vaccine is highly effective.

[0034] 3) Compared with linear RNA vaccines and protein vaccines, circular RNA vaccines against pseudorabies infection produce higher levels of neutralizing antibodies in pigs.

[0035] 4) Compared with linear RNA vaccines and protein vaccines that cannot activate CD8+T cell immune responses, circular RNA vaccines against pseudorabies infection successfully activated CD8+T cell immune responses, further improving the protective effect of the vaccine.

[0036] 5) In the present invention, the circular RNA vaccine against pseudorabies infection is safe and effective, protects animals from developing the disease after being infected with the virus, and is safe for piglets.

[0037] 6) The circular RNA vaccine against pseudorabies infection prepared by the optimized process of the present invention is stable when stored at -20°C for 6 months, providing a basis for large-scale vaccine preparation and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0039] Figure 1 This is the proportion of CD4+CD8+T lymphocytes in PBMC cells of pigs after immunization with each group of vaccines (14 days after the second immunization). DETAILED DESCRIPTION

[0040] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0041] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0042] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0043] In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event occurs and instances where it does not occur.

[0044] In the present invention, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about".

[0045] In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0046] In the present invention, the term "circular RNA" refers to an RNA molecule in a closed circular shape. In the present invention, the circular RNA used has protein translation activity and can also be called "circular mRNA".

[0047] In the present invention, the term "linear RNA" refers to an RNA precursor that can be circularized to form a circular RNA, which is generally transcribed from a linear DNA molecule and can also be called "linear mRNA".

[0048] In the present invention, the term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide may be included in a suitable vector, and the vector may be used to transform into a suitable host cell. The polynucleotide is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc. In the present invention, the recombinant nucleic acid molecule comprises a coding element encoding a target polypeptide, and a translation initiation element connected upstream of the coding element.

[0049] In the present invention, the term "translation initiation element" refers to any sequence that can recruit ribosomes and initiate the translation process of RNA molecules. Exemplary translation initiation elements include IRES sequences, m 6 A modification sequence, or the initiation sequence of rolling circle translation, etc.

[0050] In the present invention, the term "IRES" (Internal ribosome entry site, IRES) is also called internal ribosome entry site, "internal ribosome entry site" (IRES) belongs to the translation control sequence, which is usually located at the 5' end of the gene of interest and enables the translation of RNA in a cap-independent manner. The transcribed IRES can directly bind to the ribosomal subunit so that the mRNA start codon is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the mRNA (just upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0051] In the present invention, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0052] In the present invention, the term "wild type" is synonymous with "naturally occurring", and refers to an object that can be found in nature and has not been modified by humans.

[0053] In the present invention, the terms "variant" and "mutant" are used interchangeably and refer to nucleotides or amino acids that contain changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a "wild-type" polynucleotide or polypeptide. Substitution refers to replacing a nucleotide / amino acid occupying a position with a different nucleotide / amino acid. Deletion refers to the removal of a nucleotide / amino acid occupying a position. Insertion refers to the addition of a nucleotide / amino acid adjacent to and immediately following the nucleotide / amino acid occupying the position.

[0054] In the present invention, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). The sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0055] In the present invention, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0056] In the present invention, "treatment" means: after suffering from a disease, the subject is exposed to (e.g., administered) the RNA molecule described in the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0057] In the present invention, "prevention" means that before contracting a disease, by exposing a subject to the RNA molecule described in the present invention, the probability of contracting a disease and / or the symptoms after contracting a disease are reduced compared to when the subject is not exposed, and it does not mean that the disease must be completely suppressed.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include inevitable systematic errors.

[0059] <circular RNA>

[0060] The present invention provides a circular RNA, wherein the circular RNA comprises a coding element, wherein the coding element encodes a truncated and mutant protein of pseudorabies virus gD; the amino acid sequence of the truncated and mutant protein of pseudorabies virus gD is shown in SEQ ID No. 12 or its degenerate sequence; the optimized nucleotide sequence of the truncated and mutant protein of pseudorabies virus gD is shown in SEQ ID No. 13 or its degenerate sequence; the nucleotide sequence of the circular RNA molecule (numbered PPV2-gD2135) is shown in SEQ ID No. 16.

[0061] In some embodiments, the circular RNA described in the present invention is obtained by transcription and circularization of a vector DNA comprising the following elements (in the direction from 5' to 3'): intron fragment II (numbered B-II, nucleotide sequence such as SEQ ID NO.8), translation initiation element truncated fragment II (numbered PPV-II, nucleotide sequence such as SEQ ID NO.10), protein coding element (numbered gD2135, nucleotide sequence such as SEQ ID NO.13), insertion element (numbered polyAC, nucleotide sequence such as SEQ ID NO.5), translation initiation element truncated fragment I (numbered PPV-I, nucleotide sequence such as SEQ ID NO.11), and intron fragment I (numbered BI, nucleotide sequence such as SEQ ID NO.9).

[0062] In other embodiments, the circular RNA of the present invention is prepared by a clean PIE system, under which conditions, the circular RNA comprises elements arranged in the order shown in (c) or (d):

[0063] (c) translation initiation elements and coding elements;

[0064] (d) Translation initiation elements, coding elements and insertion elements.

[0065] In some preferred embodiments, the circular RNA of the present invention is prepared by a clean PIE system, and the circular RNA comprises the elements arranged in the order shown in (d) above. The clean PIE system for preparing circular RNA can refer to the contents shown in patents CN202210200112.6 and CN202210200186.X, and the entire contents of the above patents are incorporated into this application by reference.

[0066] In some embodiments, the translation initiation element described herein can be any type of element that can initiate translation of a coding element in a circular RNA.

[0067] In some specific embodiments, the translation initiation element of the present invention comprises one or more of the following sequences with translation initiation activity: IRES sequence, 5'UTR sequence, Kozak sequence, m 6 A modified (N(6) methyladenosine modification) sequence, complementary sequence of ribosomal 18S rRNA.

[0068] In some preferred embodiments, the translation initiation element of the present invention comprises an IRES sequence. Exemplary, the IRES sequence includes but is not limited to an IRES sequence derived from Echovirus, Human poliovirus, Human Enterovirus, Coxsackievirus, Human rhinovirus, Canine picornavirus, Turdivirus 3, Hepatovirus, Passerivirus, Picornaviridae, Tremovirus A, Feline kobuvirus, Murine kobuvirus, Kobuvirus sewage Kathmandu, Ferret kobuvirus, Marmot kobuvirus, Humanparechovirus, Chicken picornavirus, Falcon picornavirus, Feline picornavirus, French Guiana picornavirus, etc.

[0069] In some embodiments, the insertion element described in the present invention can be used to regulate the transcription of recombinant nucleic acid molecules, to regulate the translation of circular RNA, to achieve specific expression of circular RNA in different tissues, or to purify circular RNA, etc.

[0070] In some specific embodiments, the insertion element of the present invention comprises one or more of the following sequences: untranslated region sequence, polyN sequence, aptamer sequence, ribosomal switch sequence, and sequence binding to transcriptional regulatory factors; wherein, in the polyN sequence, N is selected from at least one of A, T, G, and C. In some preferred embodiments, the insertion element of the present invention comprises a polyAC sequence; preferably, the polyAC sequence comprises a sequence as shown in SEQ ID NO.9.

[0071] In some more preferred embodiments, the circular RNA described in the present invention has a sequence as shown in any one of SEQ ID NOs. 14 to 16.

[0072] <Recombinant Nucleic Acid Molecule>

[0073] The present invention provides a recombinant nucleic acid molecule, which forms the above-mentioned linear RNA after transcription.

[0074] In some embodiments, the recombinant nucleic acid molecule of the present invention may contain a regulatory sequence in addition to the elements contained in the linear RNA described above. Exemplarily, the regulatory sequence is a T7 promoter.

[0075] <Nucleic acid lipid nanoparticle composition>

[0076] The present invention provides a nucleic acid lipid nanoparticle composition, which comprises lipid nanoparticles and the above-mentioned circular RNA.

[0077] By encapsulating the above circular RNA into lipid nanoparticles, the body's uptake efficiency of circular RNA can be improved, while the storage stability of circular RNA is improved, providing assistance for the development and application of pseudorabies vaccine.

[0078] <Medical Use>

[0079] The circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition provided by the present invention can be used to prevent and / or treat pseudorabies.

[0080] The circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition provided by the present invention can be used to prepare a drug for preventing and / or treating pseudorabies.

[0081] The present invention provides a method for preventing and / or treating pseudorabies, comprising administering an effective amount of the above-mentioned circular RNA, linear RNA, recombinant nucleic acid molecule and / or nucleic acid lipid nanoparticle composition to a subject; preferably, the administration is performed by injection.

[0082] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0083] In the examples, if the specific conditions are not specified, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0084] Example 1: Gene design and synthesis

[0085] Plasmid vector DNA encoding different RNA molecules was designed for the pseudorabies virus gD protein (see Table 1). The vector DNA contained the following elements arranged in the following order from 5' to 3': intron fragment II, translation initiation element truncated fragment-II, protein coding element, insertion element, translation initiation element truncated fragment-I, intron fragment I.

[0086] Table 1 Vector DNA design and construction

[0087]

[0088] Among them, intron fragment II (numbered A-II), translation initiation element truncated fragment II (numbered EVE-II), insertion element (polyAC), translation initiation element truncated fragment I (numbered EVE-I), and intron fragment I (numbered AI) were designed with reference to patent CN114574483 A, and intron fragment II (numbered B-II), intron fragment I (numbered BI), translation initiation element truncated fragment II (numbered PPV-II), translation initiation element truncated fragment I (numbered PPV-I), and optimized nucleotide sequences of pseudorabies virus JS-2012 strain gD truncated plus mutant protein (N-terminal truncated 27 amino acids and containing mutations V154Y and Q187D) were obtained through preliminary screening. The specific information is as follows:

[0089] The nucleotide sequence of intron fragment II (numbered A-II) is shown in SEQ ID NO.1;

[0090] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as EVE-II) is shown in SEQ ID NO.2;

[0091] The protein coding element comprises: a nucleotide sequence (as shown in SEQ ID NO.4) encoding the wild-type gD protein of pseudorabies virus JS-2012 strain (numbered gD1103) (amino acid sequence as shown in SEQ ID NO.3), and a stop codon (as shown in (TAA)x(TAG)y(TGA)z, wherein x+y+z≥1, x, y, z are independently integers greater than or equal to 0, and the stop codon is preferably TGA);

[0092] The nucleotide sequence of the insertion element (polyAC) is shown in SEQ ID NO.5;

[0093] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as EVE-I) is shown in SEQ ID NO.6;

[0094] The nucleotide sequence of intron fragment I (numbered as AI) is shown in SEQ ID NO.7.

[0095] The nucleotide sequence of intron fragment II (numbered B-II) is shown in SEQ ID NO.8;

[0096] The nucleotide sequence of intron fragment I (numbered BI) is shown in SEQ ID NO.9;

[0097] The nucleotide sequence of the translation initiation element truncation fragment II (numbered as PPV-II) is shown in SEQ ID NO.10;

[0098] The nucleotide sequence of the translation initiation element truncation fragment I (numbered as PPV-I) is shown in SEQ ID NO.11;

[0099] The protein coding element includes: an optimized nucleotide sequence (as shown in SEQ ID NO.13) encoding a truncated and mutant protein of gD of pseudorabies virus JS-2012 strain (27 amino acids truncated at the N-terminus and including mutations V154Y and Q187D, numbered gD2135, and the amino acid sequence is shown in SEQ ID NO.12), and a stop codon (as shown in (TAA)x(TAG)y(TGA)z, wherein x+y+z≥1, x, y, z are independently integers greater than or equal to 0, and the preferred stop codon is TGA); the above gene was commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis and cloning. The obtained gene fragment was connected to the BsaI restriction enzyme site of the pUC57 vector and verified to be correct by gene sequencing.

[0100] Example 2: Construction of recombinant bacteria

[0101] 2.1 Construction of original seeds. The above-mentioned vector DNA molecules pEVE-gD1103, pPPV-gD2135, and pPPV2-gD2135 were transformed into E. coli DH5α competent cells, respectively, and spread on LB medium plates containing corresponding antibiotics (100 μg / ml ampicillin), and cultured at 37°C. When the colonies on the plates were clearly visible, single colonies were picked and placed in 3 ml liquid LB medium containing corresponding antibiotics (100 μg / ml ampicillin), cultured at 37°C, and 1 ml of bacterial solution was added to a final concentration of 8% glycerol, and stored at -80°C to obtain recombinant engineered bacteria (numbered b-pEVE-gD1103, b-pPPV-gD2135, and b-pPPV2-gD2135, respectively) as original seed banks.

[0102] 2.2 Identification of original seeds.

[0103] (1) Morphological and biochemical characteristics: This engineered bacterium is a Gram-negative short rod. It can ferment and decompose glucose to produce acid and gas. The indole test and methyl red test are both positive; the Voges-Proskauer test (VP test) and citrate test are both negative.

[0104] (2) Culture characteristics: It grows on LB solid medium to form round, shiny, smooth, milky white colonies with neat edges.

[0105] (3) Pure test: The test is carried out according to the pure test method in the appendix of the "Pharmacopoeia of the People's Republic of China", and the results are pure.

[0106] Example 3: Fermentation of recombinant bacteria

[0107] The b-pEVE-gD1103, b-pPPV-gD2135, and b-pPPV2-gD2135 strains were inoculated into 500 mL of LB medium containing the corresponding antibiotics (100 μg / ml ampicillin), and cultured at 37°C with shaking until the OD600 value was about 1.2 to 1.5. The seed solution was inoculated into a 5L fermentation tank at a 10% inoculation amount for fermentation culture. When the OD600 value of the bacteria reached about 80 to 100, the bacteria were collected by centrifugation with a wet weight of about 1000 g.

[0108] Example 4 Plasmid DNA preparation and testing

[0109] 4.1 Bacterial lysis

[0110] (1) Take 200 g of the E. coli fermented cells harvested in Example 3, add 1.6 L of Solution I stored at 4° C. in a mass volume ratio of 1:8 between the cells and Solution I (50 mM Tris-HL, 10 mM EDTA, pH 8.0), and stir until dissolved.

[0111] (2) After dissolution, add 1.6 L of solution II (0.2 M NaOH, 1.0% SDS), stir slowly, and then let stand to react for 10 min.

[0112] (3) After the reaction is completed, add 1.6 L of solution III (3 M KAc, pH 5.5, pre-cooled at 4°C), shake well, and let it stand for reaction for 30 min.

[0113] (4) After the reaction is completed, add 1.6 L of 2M CaCl2 solution, shake gently, and let it stand for 1 h. The cleavage solution is divided into two layers, the upper layer is solid and the lower layer is liquid. The liquid is discharged with a peristaltic pump and filtered through a filter element with a pore size of 1.0 μm to remove impurities.

[0114] 4.2 Ultrafiltration concentration

[0115] The lysate was filtered through a Quikstand ultrafiltration system using a 300KD hollow fiber ultrafiltration membrane (GE Healthcare Life Science, USA, membrane area 850 cm 2 , membrane fiber tube diameter 1mm) filtration, membrane negative pressure (TMP) is 1psi, operation time is 1.5h, and concentration multiple is 5-10 times volume.

[0116] 4.3 Ion exchange chromatography

[0117] Plasmid was purified by AKTA purifier TM 100 purification system, Capto TM DEAE (GE Healthcare Life Science, USA) was used to purify the plasmid. Before purification, 5 column volumes were equilibrated with equilibration solution (0.5M KAc, pH 5.5). After loading the sample, the column was cleaned with cleaning solution (0.55M NaCl, 50Mm Tris-HCL, 10mm EDTA, pH 8.0); then the column was gradient eluted with eluent (0.65M NaCl, 50Mm Tris-HCl, 10Mm EDTA, pH 8.0) solution, and the eluted sample was collected; then it was cleaned and regenerated with CIP (2M NaCl, 0.5M NaOH), and equilibrated with equilibration solution.

[0118] 4.4 Gel filtration chromatography

[0119] First, Capto TMCore 700 (GE Healthcare Life Science, USA) column was equilibrated with PBS (NaCl 8.5 g / L, Na2HPO4·12H2O 2.9 g / L, KH2PO4 0.25 g / L, pH 7.4) for 3 column volumes, and the sample collected by ion exchange column purification was loaded onto Capto TM core 700, after the sample loading is completed, use PBS to elute and collect the eluted sample.

[0120] 4.5 Determination of plasmid DNA concentration and yield

[0121] For plasmid DNA (pPPV2-gD2135), according to the "Technical Guidelines for Preclinical Research of DNA Vaccines for Prevention", the plasmid DNA concentration was determined by spectrophotometry, the bacterial endotoxin residue was detected by the horseshoe crab reagent gel method, the RNA residue was detected by agarose gel electrophoresis, and the protein residue was tested by BCA method to confirm the process.

[0122] Through optimized culture medium and culture conditions, the Escherichia coli DH5α strain was expanded to obtain bacterial cells. The lysis method was adopted to remove large molecular RNA and protein impurities by adding calcium chloride to obtain a crude plasmid DNA extract sample. The hollow fiber system ultrafiltration concentrated lysate can further remove RNA and protein impurities, and the yield can reach more than 95%.

[0123] The yield of plasmid DNA produced at each stage of this process and the preliminary quality inspection results are shown in Table 2. Ion exchange medium and gel filtration purification were used to further remove impurities such as RNA and endotoxin to obtain plasmid DNA. The ion exchange yield was 69%, the gel filtration yield was 93%, and the total yield was 50.37%. The production process met the requirements.

[0124] Table 2 Plasmid DNA yield (pPPV2-gD2135)

[0125]

[0126] 4.6 Plasmid testing

[0127] The plasmids (pEVE-gD1103, pPPV-gD2135, pPPV2-gD2135) were tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0128] (1) Identification: The plasmid was sequenced and analyzed, and the sequence was consistent with the target sequence.

[0129] (2) Concentration analysis: The concentration of the plasmid was measured by UV spectrophotometer. The results are shown in Table 3.

[0130] (3) Purity analysis: A260 / A280, supercoil content, and impurity residues were analyzed according to the methods specified by regulations. A260 / A280 was between 1.8 and 2.0, supercoil content was not less than 90%, host RNA residue was less than 100 pg / ml, host DNA was less than 1 ng / ml, host protein residue was less than 100 pg / ml, and antibiotic residual activity was negative.

[0131] (4) Safety: Endotoxin test was performed according to the method specified by the regulations, and the endotoxin content was less than 10EU / mg.

[0132] (5) Others: Analyze appearance and pH according to the methods specified by regulations. The appearance inspection should be free of foreign matter and the pH value should be between 6.5 and 7.5.

[0133] Table 3 DNA plasmid yield and purity

[0134]

[0135]

[0136] Example 5: Preparation and testing of circular RNA

[0137] 5.1 CircRNA Preparation

[0138] (1) Plasmid digestion and purification: The plasmid was digested by restriction endonuclease Bsa I (nearshore protein) / Bsa I (Takara), and the digestion system was as follows: 10× buffer: 2 ml, plasmid: 100 mg, Bsa I: 3 ml, water was added to 20 ml, and digestion was carried out at 37°C overnight. After digestion, Capto Q ImpRes (Source 30Q) (Cytiva) column was used for chromatography purification to obtain linearized plasmid.

[0139] (2) In vitro transcription: The transcription system is configured as shown in Table 1 below:

[0140] Table 4

[0141] Reagents volume 10× Reaction Buffer 100ml ATP 100ml CTP 100ml UTP 100ml GTP 100ml Linearized plasmid 30ml T7 RNA Polymerase 100ml RNase inhibitor (40U / μl) 100ml Nuclease free water Total 1000ml

[0142] Reaction conditions: Constant temperature shaking reaction at 20-40°C / 220rpm for 1-4h, then add 100ml of deoxyribonuclease I (DNaseI) (1000U / ml) to the transcription system, and constant temperature shaking reaction at 37°C / 220rpm for 15min.

[0143] It is difficult to obtain stable and complete RNA molecules using conventional in vitro transcription reaction systems for long linear RNA. In this study, the following conditions were optimized one by one for plasmid pPPV2-gD2135 to obtain the optimal transcription conditions:

[0144] 1) Optimization of linearized plasmid template concentration: The final concentrations of the plasmid template in the system were set to 25 mg, 50 mg, 75 mg, and 100 mg respectively.

[0145] 2) Optimization of nucleotide concentration: set the final concentrations of GTP / ATP / CTP / UTP in the system to 8mM / 8mM / 8mM / 8mM, 32mM / 8mM / 8mM / 8mM, 16mM / 32mM / 8mM / 8mM, and 16mM / 16mM / 8mM / 8mM, respectively.

[0146] 3) Optimization of T7 RNA polymerase (RNA Polymerase) concentration: The concentrations of T7 RNA polymerase (RNA Polymerase) in the system were set to 750KU, 900KU, 1000kU, and 1250kU respectively.

[0147] 4) Optimization of enzymatic reaction temperature: The enzymatic reaction temperature was set to 20°C, 30°C, 37°C, and 40°C, respectively.

[0148] 5) Optimization of enzymatic reaction duration: The enzymatic reaction duration was set to 1 h, 2 h, 3 h, and 4 h respectively.

[0149] The RNA content was determined by UV spectrophotometry, and the integrity of the RNA was analyzed by capillary electrophoresis.

[0150] The above conditions have a great influence on the results of RNA in vitro transcription. Without optimization of the conditions, the RNA content obtained by in vitro transcription of plasmid pPPV2-gD2135 is only 0.4 g / 1000 ml reaction, which contains a lot of incomplete RNA.

[0151] The optimal in vitro transcription conditions were obtained through experimental screening: the concentration of linearized plasmid template was 75mg, the final concentration of GTP / ATP / CTP / UTP was 16mM / 32mM / 8mM / 8mM, the final concentration of T7 RNA polymerase was 1000KU, the enzymatic reaction temperature was 37°C, and the enzymatic reaction time was 3h. Under these conditions, the RNA content obtained by in vitro transcription of plasmid pPPV2-gD2135 can be increased to 3.2g / 1000ml reaction, and the production process meets the requirements.

[0152] (3) Precipitation of mRNA of the transcription product: Add LiCl solution to the above transcription product and place it at -20°C for overnight precipitation; centrifuge the overnight treatment solution to obtain mRNA precipitate, dry it at room temperature, and resuspend the precipitate in enzyme-free water to obtain a linear mRNA aqueous solution.

[0153] (4) mRNA cyclization and concentration: The cyclization system and conditions are shown in Table 5 below:

[0154] Table 5

[0155]

[0156] The above solutions are mixed thoroughly and heated at 48-60°C for 8-20 minutes. The circularized mRNA product is concentrated by ultrafiltration tube centrifugation, and the filtrate is discarded; and the ultrafiltration liquid is transferred to a new centrifuge tube.

[0157] It is difficult to obtain stable and complete circular RNA molecules using conventional reaction systems. In this study, the following conditions were optimized one by one for plasmid pPPV2-gD2135 to obtain the optimal circularization conditions:

[0158] 1) Optimization of linear mRNA concentration: The final concentrations of linear mRNA in the system were set to 3 g, 3.5 g, and 4 g, respectively.

[0159] 2) Optimization of magnesium ion concentration: set the final magnesium ion concentration in the system to 5mM, 8mM, 10mM, 12mM, and 15mM, respectively.

[0160] 3) Optimization of cyclization reaction temperature: the reaction temperatures were set to 48°C, 52°C, 55°C, 58°C, and 62°C, respectively.

[0161] 4) Optimization of cyclization reaction time: set the reaction time to 8 min, 10 min, 12 min, 15 min, and 20 min respectively.

[0162] The above conditions have a great influence on the RNA cyclization results, and complete circular RNA cannot be prepared without optimization. The optimal cyclization conditions were obtained through experimental screening: the linear mRNA concentration was 3.5g, the magnesium ion concentration was 10mM, the cyclization reaction temperature was 52℃, and the cyclization reaction time was 12min. Under these conditions, the content of circular RNA obtained by in vitro transcription and cyclization of plasmid pPPV2-gD2135 can reach 3.2g / 1000ml reaction, and the production process meets the requirements.

[0163] (5) Circular mRNA HPLC purification: A certain amount of circular mRNA was taken and purified by Capto TMCore 700 (GE Healthcare Life Science, USA) column was used for separation and the main peak components were collected.

[0164] (6) Concentration of purified circular mRNA: The circular mRNA tube was concentrated to the desired concentration using a TFF capsule (LV Centramate, PALL). The membrane area of ​​the TFF capsule was 0.02 m 2 , flow rate is 120mL / min, and the ultrafiltered liquid is transferred to a new sample bottle.

[0165] Through the above process, a circular mRNA with the following sequence structure was successfully prepared:

[0166] The circular mRNA prepared from the DNA plasmid pEVE-gD1103 was named EVE-gD1103, and the sequence is shown in SEQ ID NO.14.

[0167] The circular mRNA prepared from DNA plasmid pPPV-gD2135 was named PPV-gD2135, and its sequence is shown in SEQ ID NO.15.

[0168] The circular mRNA prepared from DNA plasmid pPPV2-gD2135 was named PPV2-gD2135, and its sequence is shown in SEQ ID NO.16.

[0169] 5.2 mRNA testing

[0170] Circular mRNA was tested according to Analytical Procedures for mRNA Vaccine Quality 2rd USP, and all indicators met the requirements.

[0171] (1) Identification: Sequence the mRNA and the sequence is consistent with the target sequence.

[0172] (2) Concentration analysis: The concentration of mRNA was measured by UV spectrophotometer. The results are shown in Table 6.

[0173] (3) Integrity analysis: The integrity of mRNA was determined by capillary gel electrophoresis (CGE), which was no less than 80%. The results are shown in Table 6.

[0174] (4) Purity analysis: The impurity residues were analyzed according to the prescribed method. The dsRNA content was less than 0.5%, the DNA template residue was less than 100 pg / mg RNA, the free nucleotide residue was less than 100 pg / mg RNA, and the T7 RNA polymerase residue was less than 50 ppm.

[0175] (5) Efficacy analysis: The cells were transfected according to the prescribed method, and the target protein expression was detected to meet the requirements. 48 hours after transfection, the target protein expression level of the PPV2-gD2135 group reached 97.6 ng / ml.

[0176] (6) Safety: Endotoxin was analyzed according to the prescribed method and the endotoxin content was less than 0.1 EU / ml.

[0177] (7) Others: Analyze the appearance and pH value according to the prescribed methods. The appearance inspection should be free of foreign matter and the pH value should be between 6.5 and 7.5.

[0178] Compared with the PPV-gD2135 group, the RNA yield in the PPV2-gD2135 group increased by 2.4 times, indicating that intron optimization in the non-coding region was effective and necessary, and successfully improved the yield and purity of circular RNA molecules.

[0179] Therefore, the circular RNA molecules (numbered PPV2-gD2135), plasmid DNA molecules (numbered pPPV2-gD2135) and Escherichia coli strains containing the plasmid DNA molecules (numbered b-pPPV2-gD2135) corresponding to the PPV2-gD2135 group are preferred.

[0180] Table 6 CircRNA Molecular Yield and Purity

[0181]

[0182] Example 6: Comparison of the immune effects of various circular RNA molecules

[0183] 6.1 Commercial LNP Preparation

[0184] The circular RNA molecules (numbered EVE-gD1103, PPV2-gD2135) obtained in Example 5 and the control circular RNA molecule (numbered HM-gD2135) were prepared into LNP-encapsulated RNA using commercial LNPs to prepare for further animal experiments.

[0185] The control circular RNA is an optimized nucleotide sequence (as shown in SEQ ID NO.13) encoding the truncated and mutant protein of gD of pseudorabies virus JS-2012 strain (27 amino acids are truncated at the N-terminus and contain mutations V154Y and Q187D, and the amino acid sequence is as shown in SEQ ID NO.12) prepared by a commercial circular RNA synthesis kit (Biyuntian Biotechnology).

[0186] (1) Test method

[0187] The commercial LNP-mRNA preparation reference is 10.1016 / j.cell.2022.03.044, and the specific process is as follows:

[0188] a) Dilute the circular RNA to a final concentration of 170 μg / mL using PNI formulation buffer (Precision NanoSystems, Cat. No. NWW0043).

[0189] b) On a NanoAssembler Ignite (Precision NanoSystems), commercial LNP (Precision NanoSystems) and circular RNA were mixed at a volume ratio of 1:3 using Ignite NxGenCartridge (Precision NanoSystems, Catalog No. NIT0002).

[0190] c) using tangential flow technology to replace the buffer environment with PBS at pH 7.4 to remove ethanol, and prepare LNP-mRNA, which are numbered as c-LNP / EVE-gD1103, c-LNP / PPV2-gD2135, and c-LNP / HM-gD2135, respectively.

[0191] d) Using Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM The particle size, PDI and surface potential of LNP particles were measured by dynamic light scattering method on a Zeta potential laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern).

[0192] (2) Test results

[0193] The mRNA-encapsulated LNPs prepared in this step passed the inspection: the particle size was small and uniformly distributed, the encapsulation effect was good, and the encapsulation rate was greater than 90%; the surface potential was electronegative, avoiding the cytotoxicity caused by the positive charge on the surface of the nanoparticles, and the biosafety was good.

[0194] 6.2 Mouse immunization and neutralizing antibody assay

[0195] (1) Experimental methods

[0196] Female BABL / c mice aged 6 to 8 weeks were randomly divided into 4 groups, 10 mice in each group, and the three circular mRNAs (numbered c-LNP / EVE-gD1103, c-LNP / PPV2-gD2135, c-LNP / HM-gD2135) encapsulated by the commercial LNP prepared in this example were immunized with mice at a dose of 30 μg / mouse. The mice inoculated with commercial LNP were negative controls, and booster immunization was performed once on the 21st day after the initial immunization. Blood was collected 14 days after the second immunization to separate serum for virus neutralizing antibody detection. The detection method was referenced to CN_112430625_B, and the neutralizing antibody titer of each group was expressed as mean ± SD value.

[0197] (2) Experimental results

[0198] The results are shown in Table 7. Compared with the circular RNA molecule containing the wild-type gD protein nucleotide sequence and the disclosed circular RNA backbone, the optimization of gD protein and nucleotides and the circular RNA molecular backbone successfully significantly increased the neutralizing antibody titer in mice: the neutralizing antibody titer of the c-LNP / PPV2-gD2135 group was 26.1 times that of the c-LNP / EVE-gD1103 group, and relevant optimization is necessary.

[0199] Furthermore, the circular RNA molecular skeleton provided by this study is superior to the commercial circular RNA molecular skeleton: the neutralizing antibody titer of the c-LNP / PPV2-gD2135 group is 1.9 times that of the c-LNP / HM-gD2135 group.

[0200] Table 7 Neutralizing antibody titers induced in mice

[0201] Group Mean SD value c-LNP / EVE-gD1103 13.9 5.2 c-LNP / PPV2-gD2135 362.3 103.7 c-LNP / HM-gD2135 187.6 86.4 LNP none none

[0202] Example 7: LNP-mRNA production process research

[0203] This section studies the production process of LNP-encapsulated mRNA.

[0204] 7.1 Preparation of LNP-mRNA (1) Preparation of solution I: Dissolve cationic lipids, cholesterol, neutral lipids, and PEG-modified lipids in ethanol to obtain solution I. (2) Preparation of solution II: Dissolve the preferred circular RNA (No. PPV2-gD2135) of Example 6 in 10 mM citric acid buffered saline solution at pH 4.0.

[0205] (3) Using microfluidics technology to quickly mix the two phases and using tangential flow technology to replace the buffer environment with PBS at pH 7.4 to remove ethanol, LNP-mRNA was prepared. In this section, the liposome preparation and vaccine preparation processes were optimized, specifically involving liposome components and the ratio of each component, and the ratio of LNP to mRNA, as follows:

[0206] 3.1) Screening of liposome components and ratio conditions: Solution I was prepared according to different components and different ratios, including cationic lipids (CMAX4 (4-[(3-{[3-({3-[bis(3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino]propyl}(methyl)amino)propyl](3-{4-[(2-butyloctanoyl)oxy]butoxy}-3-oxypropyl)amino}propoxy)oxy]butyl 2-octanoate) or SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate)) was set to 45% to 55%, cholesterol (5-cholesten-3β-ol) was set to 35% to 44%, and neutral lipid (DSPC (distearylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine)) was set to 3% to 10%, PEG-modified lipids (PEG-DMG (polyethylene glycol-dimyristyl glyceride) or PEG-DSG (bis-distearoyl-rac-glycerol-polyethylene glycol) or PEG-DPG (diphosphoglycerate polyethylene glycol))) were set to 0.8% to 1.8%, wherein the cationic lipid CMAX4 was purchased from Suzhou Coremade Biopharmaceutical Technology Co., Ltd., and the remaining lipids were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., and LNP-encapsulated circular RAN molecules were prepared with a LNP / circular RNA (No. PPV2-gD2135) volume ratio of 1:1, and the particle size and encapsulation efficiency of each group were compared.

[0207] The results showed that the composition and ratio of liposomes had a great influence on the results: for example, the combination of CMAX4 / cholesterol / DOPE / PEG-DPG at a ratio of 54.5:34.5:9.2:1.8 was ineffective, with an encapsulation efficiency of less than 10%.

[0208] Through experiments, the preferred liposome components were obtained to be CMAX4 / cholesterol / DSPC / PEG-DMG, and the preferred ratio of each component was 45.8:43.4:9.3:1.5. At this time, the molecular particle size was the smallest (96nm) and the encapsulation rate reached 93.6%, and the production process met the requirements.

[0209] 3.2) Optimization of LNP / mRNA ratio: LNP and circular RNA (numbered PPV2-gD2135) were mixed in different ratios (volume ratio of solution I:II 1:1, 1:2, 1:3, 1:4, 1:5, 1:6), and the optimal LNP / mRNA ratio was verified by gel retardation test. The results showed that when the volume ratio of liposome nanomaterial (LNP) and circular mRNA molecule (numbered PPV2-gD2135) was greater than 1:3, the migration of PPV2-gD2135 could be completely blocked. Therefore, the volume ratio of LNP / circular RNA molecule was set to 1:3.

[0210] (4) The circular RNA molecule (numbered PPV2-gD2135) is encapsulated with LNP according to the above preferred conditions and sterile filtered to obtain an RNA vaccine, numbered LNP / PPV2-gD2135.

[0211] (5) Using Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM The encapsulation efficiency was determined by R11490, and the particle size, polydispersity index (PDI) and surface potential of the LNP particles were measured by dynamic light scattering on a Zeta potential-laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern).

[0212] 7.2 Experimental Results

[0213] The results are shown in Table 8. The indicators of LNP-encapsulated mRNA were qualified: the particle size was less than 90nm, the PDI was less than 0.1, the encapsulation rate was greater than 90%, and the surface potential was electronegative. The overall particle size was small and uniformly distributed. The surface electronegativity avoided the cytotoxicity caused by the positive charge on the surface of the nanoparticles, and the biosafety was good, providing a feasibility basis for the large-scale preparation of vaccines.

[0214] Table 8 LNP-mRNA sample characterization

[0215] serial number Particle size (nm) PDI Surface potential (mV) Encapsulation rate (%) LNP / PPV2-gD2135 67 0.08 -5.4 94.6

[0216] Example 8 Evaluation of the immune effect of LNP-encapsulated RNA

[0217] For the vaccine prepared with the preferred LNP in Example 7, the immune effect was evaluated and the immune dose was determined

[0218] 8.1 Mouse immunization and neutralizing antibody assay

[0219] (1) Experimental methods

[0220] Female C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 3 groups, 10 mice in each group, and the LNP / PPV2-gD2135 prepared with the preferred LNP in this example and the c-LNP / PPV2-gD2135 prepared with the commercial LNP in Example 6 were used for immunization of mice, with an inoculation dose of 30 μg / mouse. The mice inoculated with the commercial LNP were used as negative controls, and booster immunization was performed once on the 21st day after the initial immunization. Blood was collected 14 days after the second immunization to separate serum for virus neutralizing antibody detection. The detection method was referred to CN_112430625_B, and the neutralizing antibody titer of each group was expressed as mean ± SD value.

[0221] (2) Experimental results

[0222] The results are shown in Table 9. The LNP-encapsulated RNA vaccine prepared by the optimal process was superior to the commercial LNP-encapsulated LNP vaccine, and successfully further improved the neutralizing antibody titer in mice: the neutralizing antibody titer of the LNP / PPV2-gD2135 group was 1.5 times that of the c-LNP / PPV2-gD2135 group, providing a feasibility basis for further research.

[0223] Table 9 Neutralizing antibody titers induced in mice

[0224] Group Mean SD value LNP / PPV2-gD2135 519.7 25.2 c-LNP / PPV2-gD2135 358.9 87.6 LNP none none

[0225] 8.2 Pig Immunization and Vaccine Immunization Dosage Determination

[0226] (1) Test method

[0227] The screening criteria for experimental pigs are shown in Table 10 below, and a total of 28 piglets were screened.

[0228] Table 10 Screening criteria for experimental pigs

[0229]

[0230]

[0231] The experimental animals were divided into 4 groups, with 7 animals in each group.

[0232] Experimental group 1: LNP / PPV2-gD2135 prepared in Example 7 was inoculated into pigs (neck muscle), with an immunization dose of 60 μg / head, numbered PPV2-gD2135-60 μg.

[0233] Experimental Group 2: Pigs (neck muscle) were inoculated with LNP / PPV2-gD2135 prepared in Example 7, with an immunization dose of 80 μg / head, numbered PPV2-gD2135-80 μg.

[0234] Experimental Group 3: Pigs (neck muscle) were inoculated with LNP / PPV2-gD2135 prepared in Example 7, with an immunization dose of 100 μg / head, numbered PPV2-gD2135-100 μg.

[0235] Control group 1: immunized with PBS only, with an immunization dose of 1 ml / head, numbered PBS group.

[0236] The animals were boosted with a booster immunization on the 28th day after the first immunization. Serum was collected 21 days after the second immunization to prepare serum for neutralizing antibody detection. The detection method was based on CN_112430625_B, and the neutralizing antibody titer of each group was expressed as mean ± SD value.

[0237] Furthermore, 14 days after the second immunization, blood from the preferred group was collected to prepare serum, and the IFN-γ and IL-4 levels in the serum of each group were detected using the MILLIPLEX MAPPorcine Cytokine and Chemokine Magnetic Bead Panel-Immunology Multiplex Assay Kit (MERCK, Catalog No. PCYTMAG-23K-09) to preliminarily evaluate the cellular immune response. The specific operation was carried out according to the kit instructions.

[0238] (2) Test results

[0239] The results of neutralizing antibodies in pigs are shown in Table 11. Using low-dose immunization (80 μg), the PPV2-gD2135 group can effectively induce pigs to produce high levels of neutralizing antibody titers, indicating that the vaccine is highly effective.

[0240] The levels of various cytokines in pig blood are shown in Table 12. Compared with the PBS group, the PPV2-gD2135 group can effectively increase the levels of IFN-γ and IL-4 in pig serum, suggesting that the cellular immune response is effectively activated, further improving the protective effect of the vaccine, and providing a basis for further research.

[0241] Table 11 Neutralizing antibody titers induced in pigs

[0242]

[0243]

[0244] Table 12 IFN-γ and IL-4 content in pig serum (mean ± SD value) (pg / ml)

[0245] Group IFN-γ IL-4 PPV2-gD2135-60μg 6122.9±1259.8 807.7±531.4 PPV2-gD2135-80μg 15457.4±6412.5 1670.6±902.5 PPV2-gD2135-100 μg 18093.4±9550.1 1937.0±927.5 PBS 176.6±82.3 80.7±23.8

[0246] Example 9 Preparation of control recombinant subunit vaccine

[0247] This example prepares a recombinant subunit vaccine of the gD protein of the pseudorabies virus of pigs, providing a basis for comparing the vaccine effect. First, the gD protein of the PRV JS-2012 strain was prepared by recombinant expression in eukaryotic cells according to conventional methods. The specific method refers to patent CN_110327461_B, the expression vector is pCI-GS, the expression host is the CHO-S cell line, wherein the amino acid sequence encoding the wild-type gD protein is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0248] The expressed protein was tested by SDS-PAGE. The specific operation is as follows: take 40μl of the target protein solution, add 10μl of 5× loading buffer, boil in water for 5 minutes, centrifuge at 12000r / min for 1 minute, take the supernatant for SDS-PAGE gel (12% concentration gel) electrophoresis, take the gel after electrophoresis, stain and decolorize to observe the target band. Using pCI-GS as a vector and CHO-S cell line as a host, the pseudorabies virus gD protein was successfully prepared, and its molecular weight was consistent with the expected molecular weight after SDS-PAGE gel electrophoresis analysis.

[0249] The control gD subunit vaccine was further prepared with reference to the method of patent CN_115838440_A. The specific method is as follows: the purified gD protein was quantified using a BCA protein quantification kit, then filtered and sterilized using a 0.22 μm filter, the protein was diluted to 100 μg / mL using sterile PBS, and the vaccine (50 μg / mL) was fully mixed and emulsified with ISA 201VG adjuvant (Seppic) at a volume ratio of 1:1, and stored at 4°C after aliquoting, which was recorded as the control gD protein vaccine group.

[0250] Example 10 Preparation of control linear RNA vaccine

[0251] This embodiment prepares a linear RNA vaccine to provide a basis for comparing vaccine effects. The linear RNA of the wild-type protein of pseudorabies virus gD is prepared as a reference (doi: 10.1016 / j.vetmic.2020.108886.): The DNA vector is designed to be numbered plinear-gD1103, and the coding region of the linear RNA contains the wild-type protein of gD of PRV JS-2012 strain (amino acid sequence see SEQ ID NO.3, nucleotide sequence see SEQ ID NO.4), and the DNA vector is commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis and cloning, and the resulting gene fragment is connected to the pUC57 vector. A linear RNA molecule (numbered linear-gD1103) was further prepared. The linear RNA molecule linear-gD1103 synthesized in vitro was quality analyzed using an Agilent 2100 bioanalyzer, and the band was consistent with the target band, and the purity met the requirements.

[0252] The linear mRNA prepared above was prepared into a lipid nanoparticle / RNA composition according to the preferred method of Example 7, recorded as LNP / linear-gD1103, and passed the inspection: the LNP particle size encapsulated mRNA was small and uniformly distributed, the encapsulation effect was good, and the encapsulation rate was greater than 90%; the surface potential was electronegative, and the biosafety was good.

[0253] Example 11: Study on pig immunity and vaccine effectiveness

[0254] 11.1 Experimental Animals.

[0255] The screening criteria for experimental pigs were the same as in Example 8, and a total of 28 piglets were screened.

[0256] 11.2 Immunity experiments.

[0257] The experimental animals were divided into 4 groups, with 7 animals in each group.

[0258] Experimental group 1: LNP / PPV2-gD2135 prepared in Example 7 was inoculated into pigs (neck muscle), with an immunization dose of 80 μg / head, numbered LNP / PPV2-gD2135.

[0259] Experimental group 2: LNP / linear-gD1103 prepared in Example 10 was inoculated into pigs (neck muscle), with an immunization dose of 80 μg / head, numbered LNP / linear-gD1103.

[0260] Experimental Group 3: The pseudorabies virus gD subunit protein vaccine prepared in Example 9 was inoculated into pigs (neck muscle), with an immunization dose of 50 ug / head, numbered as gD protein vaccine group.

[0261] Control group: immunized with PBS only, with an immunization dose of 1 ml / head, numbered PBS group.

[0262] A booster vaccination was given on the 28th day after the initial vaccination.

[0263] 11.3 Neutralizing Antibody Detection

[0264] Serum was collected 21 days after the second immunization to prepare serum for neutralizing antibody detection. The detection method refers to CN_112430625_B, and the neutralizing antibody titer of each group is expressed as mean ± SD value. The results are shown in Table 13. LNP / linear-gD1103 and gD protein vaccines can produce neutralizing antibodies to a certain extent, but the neutralizing antibody titer of LNP / PPV2-gD2135 prepared by the optimized process is significantly better than that of LNP / linear-gD1103 and gD protein vaccines, indicating that process optimization is very necessary for the development of pseudorabies virus circular RNA vaccine.

[0265] Table 13 Neutralizing antibody titers induced in pigs

[0266] Group Mean SD value LNP / PPV2-gD2135 614.1 79.4 LNP / linear-gD1103 23.9 8.7 gD protein vaccine 40.2 6.9 PBS none none

[0267] 11.4 Cellular Immune Response Detection

[0268] The cellular immune response stimulated by each vaccine group in the pigs was further detected. Blood PBMC cells were collected from each group immunized in this example 14 days after the second immunization, and the corresponding lymphocyte subtypes were detected by flow cytometry. The specific operation is as follows:

[0269] (1) 14 days after the second immunization, anticoagulated blood was collected from each group and single cell suspensions were prepared;

[0270] (2) Cell density adjustment and density counting: After filtering the above single cell suspension through a 100 μm cell sieve, take 18 μL and add it to a 200 μL EP tube, add 2 μL of trypan blue and mix well, then take 10 μL and add it to a counting plate for cell counting, and dilute it with an appropriate amount of PBS solution until the density of the single cell suspension is 1×10 6 / mL; observe the number of dead cells and living cells under a microscope. Antibody incubation can be carried out only after the cell density and cell viability of the single cell suspension meet the requirements.

[0271] (3) Antibody incubation:

[0272] (4) Preparation of single-stained tubes: Take 0.5 mL of the single-cell suspension of each group into three 1.5 mL EP tubes, and add 1 μL of fluorescein isothiocyanate (FITC)-labeled mouse anti-swine CD3 antibody (BD Company), 1 μL of phycoerythrin PE-labeled mouse anti-swine CD4 antibody (BD Company), and 1 μL of allophycocyanin fluorescein APC-labeled mouse anti-swine CD8 antibody (BD Company) in sequence. Incubate at 4°C in the dark for 30 min, then centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend with 0.5 mL PBS, and place on ice. Add 2.5 μL of 7-aminoactinomycin D (eBioscience Company) to each tube before loading.

[0273] (5) Preparation of sample tubes: Take 1 mL of the single cell suspension from each group into a 1.5 mL EP tube and add 2 μL of FITC-labeled mouse anti-swine CD3 antibody (BD Company), 5 μL of PE-labeled mouse anti-swine CD4 antibody (BD Company), and 5 μL of APC-labeled mouse anti-swine CD8 antibody (BD Company). Incubate at 4°C in the dark for 30 min, then centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend with 1 mL of PBS, and place on ice. Add 5 μL of 7-aminoactinomycin D (eBioscience Company) to each tube before loading.

[0274] (6) Sorting on the flow cytometer: Sorting was performed on the flow cytometer (BD Aria SORP, BD). The above tubes were filtered into the flow tubes with a 100 μm cell sieve and marked. First, the blank tube was used to determine the location of lymphocytes, and then the single-stained tube was used to determine whether the antibody labeling was good. If the labeling was good (i.e., the antibody fluorescence signal value was 10 3The samples were placed in the sample tubes, and CD4-CD8- double negative cells, CD4+CD8+ double positive cells, CD4+CD8- single positive cells, and CD4-CD8+ single positive cells were circled and sorted. Unpaired t-test was used for statistical significance analysis, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0275] The results are as follows Figure 1 As shown, the LNP / linear-gD1103 group and the gD protein vaccine group induced an increase in CD4+ cells in pigs, but failed to stimulate CD8+ cells; while the ratio of the two T cells in the LNP / PPV2-gD2135 group prepared by the optimized process was significantly higher than that in the other groups, indicating that this group successfully activated the cellular immune response, especially the CD8+ cellular immune response, further improving the protective effect of the vaccine.

[0276] 11.5 Challenge protection test

[0277] In this example, the LNP / PPV2-gD2135 group and the PBS group were challenged with 1 ml of PRV Fa virus solution (10 6 PFU) and observed for 7 days. The results are shown in Table 14. All 7 pigs in the control group became ill, and all were observed to have emaciation and obvious dyspnea. Among them, 5 pigs lay on the ground, and had typical neurological symptoms such as paddling on the ground and extreme irritability. No obvious symptoms were observed in the vaccine group, indicating that the vaccine is effective in protecting against the virus.

[0278] Table 14 Attack protection effect

[0279]

[0280] Note: "+" indicates death or neurological symptoms or respiratory symptoms. Neurological symptoms include typical neurological symptoms such as falling to the ground and paddling, extreme irritability, etc. Respiratory system symptoms include dyspnea, etc. "-" indicates no neurological symptoms or respiratory symptoms, and no death.

[0281] Example 12 Safety test of newborn piglets

[0282] This experiment further studied the safety of the porcine pseudorabies virus circular RNA / lipid nanoparticle composition in pigs.

[0283] 12.1 Test method

[0284] Sixteen 0-day-old pseudorabies-negative piglets (negative for both gB and gE antibodies) were selected;

[0285] Group 1 is the vaccination group, in which 8 piglets were vaccinated with LNP / PPV2-gD2135 prepared in Example 7 (neck muscle), with an immunization dose of 80 μg / pig. ;

[0286] Group 1 was the control group, and 8 piglets were inoculated with sterile PBS at a dose of 1 ml / pig.

[0287] Afterwards, the clinical symptoms of each animal were continuously observed and recorded, body temperature was monitored, and weaning weight was recorded.

[0288] 12.2 Test results

[0289] The results are shown in Table 15. The vaccine is safe. No abnormal body temperature, other obvious side effects or abnormal clinical symptoms were observed within 14 days after vaccination. The weight of the LNP / PPV2-gD2135 group was not significantly different from that of the control group when weaned, indicating that the vaccine provided by the present invention has good safety in clinical application.

[0290] Table 15 Body temperature and weight of piglets after vaccination (mean ± SD value)

[0291]

[0292] Example 13 Vaccine Stability Study

[0293] The stability of the vaccine was further evaluated as follows:

[0294] The LNP / PPV2-gD2135 prepared in Example 7 was aseptically dispensed into vials (5 ml / vial) after filtration and sterilization, and stored at -20°C. Samples were taken for testing at 0 day, 1 month, 2 months, 3 months, and 6 months:

[0295] Using Quant-iT TM RiboGreen TM RNA Assay Kit (Invitrogen TM R11490) to determine the mRNA content and encapsulation efficiency. The particle size of LNP particles was measured using a Zeta potential-laser particle size analyzer Malvern Zetasizer Nano-ZEN 3600 (Malvern). The mRNA integrity was determined by capillary gel electrophoresis (CGE). pH and appearance inspection were performed.

[0296] The test results are shown in Table 16. The vaccine is stable when stored at -20°C for 6 months, with no significant changes in encapsulation efficiency, particle size, mRNA content, mRNA integrity, pH, and appearance, providing a basis for large-scale vaccine preparation and clinical application.

[0297] Table 16 Vaccine Storage Stability

[0298] Day 0 1 month 2 months 3 months 6 months Encapsulation rate / % 94.3 93.7 94.9 94.1 93.5 Particle size / nm 66.5 67.4 66.7 66.4 67.2 mRNA content / mg / ml 0.51 0.50 0.51 0.51 0.51 mRNA integrity / % 90.4 90.7 90.1 90.2 90.7 pH 7.11 7.14 7.11 7.12 7.12 Appearance No foreign matter found No foreign matter found No foreign matter found No foreign matter found No foreign matter found

[0299] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A circular RNA vaccine against pseudorabies virus, comprising a circular RNA molecule encoding a truncated plus mutant protein of pseudorabies virus envelope protein gD and a pharmaceutically acceptable carrier.

2. The anti-pseudorabies virus circular RNA vaccine according to claim 1, characterized in that: The amino acid sequence of the truncated and mutant protein of the pseudorabies virus envelope protein gD is shown in SEQ ID No. 12 or its degenerate sequence.

3. The anti-pseudorabies virus circular RNA vaccine according to claim 1, characterized in that: The nucleotide sequence of the truncated and mutant protein of the pseudorabies virus envelope protein gD is shown in SEQ ID No. 13 or its degenerate sequence.

4. The circular RNA vaccine against pseudorabies virus according to claim 1, characterized in that: The circular RNA is obtained by transcription and circularization of a vector comprising the following elements: an intron fragment as shown in SEQ ID NO.8, a translation initiation element truncated fragment as shown in SEQ ID NO.10, a protein coding element as shown in SEQ ID NO.13, an insertion element as shown in SEQ ID NO.5, a translation initiation element truncated fragment as shown in SEQ ID NO.11 and an intron fragment as shown in SEQ ID NO.

9.

5. The anti-pseudorabies virus circular RNA vaccine according to claim 1, characterized in that: The nucleotide sequence of the circular RNA molecule is as shown in SEQ ID No.

16.

6. The circular RNA vaccine against pseudorabies virus according to claim 1, characterized in that: The content of circular RNA molecules in the circular RNA vaccine is 30-200 μg / ml.

7. The circular RNA vaccine against pseudorabies virus according to claim 1, characterized in that: The pharmaceutically acceptable carrier includes lipid nanoparticles; the volume ratio of lipid nanoparticles to circular RNA molecules in the circular RNA vaccine is 1:1-1:

3.

8. The anti-pseudorabies virus circular RNA vaccine according to claim 1, characterized in that: The raw material composition of the lipid nanoparticles includes: 45% to 55% of cationic lipids, 35% to 44% of cholesterol, 3% to 10% of neutral lipids and 0.8% to 1.8% of PEG-modified lipids; the cationic lipids are CMAX4 or SM-102; the cholesterol is 5-cholesten-3β-ol; the neutral lipids are DSPC or DOPE; the PEG-modified lipids are PEG-DMG, PEG-DSG or PEG-DPG.

9. The circular RNA vaccine against pseudorabies virus according to claim 8, characterized in that: The cationic lipid is CMAX4; the cholesterol is 5-cholestene-3β-ol; the neutral lipid is DSPC; the PEG-modified lipid is PEG-DMG; the molar ratio of CMAX4:5-cholestene-3β-ol:DSPC:PEG-DMG is 45.8:43.4:9.3:1.

5.

10. A method for preparing a circular RNA vaccine against pseudorabies virus according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: S1, synthesizing and inserting the gD truncated plus mutant protein gene and looping element in the pseudorabies virus into a cloning vector to obtain a recombinant vector containing the gD truncated plus mutant protein gene and looping element in the pseudorabies virus; S2, transforming or transducing a host with the recombinant vector containing the truncated gD plus mutant protein gene and the looping element in the pseudorabies virus obtained in step S1 to obtain a recombinant containing the recombinant vector; S3, culturing the recombinant obtained in step S2 to prepare a plasmid containing the gD truncated plus mutant protein gene and the circular element in the pseudorabies virus; S4, using the plasmid containing the pseudorabies virus gD truncated plus mutant protein gene and the circular element obtained in step S3 to prepare a circular mRNA encoding the pseudorabies virus gD truncated plus mutant protein; S5. Add the circular mRNA encoding the truncated and mutant protein of pseudorabies virus gD obtained in step 4 to a pharmaceutically acceptable carrier to obtain the anti-pseudorabies virus circular RNA vaccine.

11. Use of the anti-pseudorabies virus circular RNA vaccine according to any one of claims 1 to 9 in the preparation of a medicament for preventing and / or treating pseudorabies virus infection.

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