Porcine rotavirus RNA vaccine and preparation method thereof

Abstract

The invention discloses a porcine rotavirus RNA vaccine and a preparation method thereof. According to the invention, amino acid sequences and nucleotide sequences of G9 type and G4 type VP4 proteins of porcine rotaviruses are optimized, and optimized translation starting elements are used, so that optimized RNA molecules with nucleotide sequences shown as SEQ ID NO.18 and SEQ ID NO.24 are finally prepared. The optimized RNA has higher immunogenicity in a mouse body. Furthermore, the porcine rotavirus RNA vaccine provided by the invention comprises a vaccine vector and the optimized RNA molecule. The porcine rotavirus G9-G4 type bivalent RNA vaccine disclosed by the invention is superior to a porcine rotavirus inactivated vaccine in the aspect of immune protection efficacy in a pig body.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to a porcine rotavirus RNA vaccine and a preparation method thereof, and in particular comprises a porcine rotavirus G9-G4 type bivalent RNA vaccine and a preparation method thereof. Background Art

[0002] Porcine rotavirus (RV) is a non-enveloped double-stranded RNA virus belonging to the genus Rotavirus of the family Reoviridae. It can cause diseases in piglets with vomiting, anorexia, dehydration, diarrhea and acid-base imbalance as the main symptoms. The incidence rate of piglets less than 4 weeks old can reach more than 80%, and the mortality rate can reach 7-20%. The rotavirus gene encodes 11 proteins, namely inner capsid protein (VP1, VP2, VP3 and VP6), outer capsid protein (VP7 and VP4) and non-structural protein (NSP1, NSP2, NS34, NS35 and NS28). Rotavirus has different serogroups and serotypes. According to VP6, it is divided into 7 serogroups (types) (AG) and two subgroups (Ⅰ and Ⅱ). Subgroup A rotavirus is the main pathogen that causes gastrointestinal diseases in humans and animals. Different P types are divided based on the differences in the VP4 gene sequence, and different G types are divided based on the differences in the VP7 gene sequence. Currently, there are 27 G types and 37 P types, and the cross-reactivity between different types is poor.

[0003] Porcine rotavirus infection causes viral diarrhea in piglets, which causes illness or even death in piglets, causing huge economic losses to the pig farming industry. Currently, there is no effective drug to treat diseases caused by porcine rotavirus infection, so it is particularly important to prevent porcine rotavirus infection, and vaccination is an effective means of prevention and control. Problems with existing vaccine technology:

[0004] (1) At present, the most widely used vaccine in clinical practice in China is the triple live vaccine for porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus (G5 type) (weakened Chinese strain + attenuated CV777 strain + NX strain), which uses porcine rotavirus G5 type. However, G9 type has replaced G5 as the main prevalent strain in my country. VP7 gene sequencing and BLAST sequence comparison of diseased samples nationwide showed that among the 9 porcine rotavirus types in my country, the positive rate of G9 type has reached 56.55%, followed by the positive rate of G5 type with a positive rate of 14.48. In addition, the positive rate of G4 type has reached 6.21% and is on an upward trend (doi:10.3390 / v16030453). Existing vaccines cannot cross-protect against prevalent strains, so it is necessary to develop new multivalent vaccines against prevalent strains in order to protect against multiple types of porcine rotavirus.

[0005] (2) The VP4 protein contains virus-neutralizing B cell epitopes and is considered to be one of the main targets for the development of new vaccines. However, correctly expressing the VP4 antigen and maintaining its immunogenicity is a challenge in the industry. The full-length VP4 protein is expressed at a low level in cells or exists in the form of inclusion bodies (doi: 10.1016 / j.vaccine.2018.03.011). Truncated VP4 can effectively improve expression, but it will destroy immunogenicity to a certain extent. The complete VP4 protein contains the VP5 domain and the VP8 domain. For example, Padilla-Noriega L et al. expressed VP5 and VP8 proteins respectively, but the immunogenicity of these two proteins was significantly weaker than that of the full-length VP4 protein (doi: 10.1128 / jcm.30.6.1392-1397). Wen et al. found that the truncated VP8 protein (aa65-223) could be expressed soluble and in high yield, inducing high-titer neutralizing antibodies in guinea pigs, but the immune response induced by this protein in mice was weak (doi:10.1016 / j.vaccine.2012.07.078); the CD4+T cell epitope P2 was further introduced into the protein, but the positive rate of neutralizing antibodies induced in vivo was still low (doi:10.1016 / j.vaccine.2014.06.060). YijianLi et al. constructed a truncated VP4 protein (aa26-476), which contains the VP8 domain and the STALK domain of VP5. After being expressed in a soluble form, the protein can form a stable trimer structure, which can induce high levels of neutralizing antibodies in mice and achieve protection against the virus. The effect is significantly better than the single VP8 domain, the stalk domain of VP5 and the inactivated vaccine (doi:10.1016 / j.vaccine.2018.03.011); however, further studies have shown that the immune response and protective effect of this protein in the animal pig are significantly weaker than the existing inactivated vaccine technology (doi:10.1128 / jvi.00212-24). Summary of the invention

[0006] The object of the present invention is to provide a porcine rotavirus RNA vaccine and a method for preparing the same. First, by optimizing the porcine rotavirus G9 and G4 VP4 proteins and their coding regions, an optimized circular RNA molecule is provided for the optimized VP4 protein to obtain P4-6 circular RNA and P4-9 circular RNA molecules, thereby alleviating the technical problem of low immunogenicity caused by low activity and poor translation efficiency of RNA molecules constructed in the prior art. The vaccine vector is further mixed with the above-mentioned optimized circular RNA to obtain a porcine rotavirus RNA vaccine; the method is simple and easy to operate, can achieve large-scale production, and effectively reduce production costs. The porcine rotavirus RNA vaccine comprising P4-6 circular RNA and / or P4-9 circular RNA molecules of the present invention, especially the porcine rotavirus G9-G4 type bivalent RNA vaccine, solves the problem that the existing commercial vaccines are not ideal for protecting the epidemic strains, realizes cross protection against the epidemic strains, and the effect is better than the G9-G4 type bivalent inactivated vaccine prepared by the existing inactivated vaccine technology.

[0007] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0008] <First aspect>

[0009] The invention provides an optimized VP4 protein of porcine rotavirus, wherein the optimized VP4 protein is one of VP4 protein aa26-776 / 225th P mutation to A / 226th P mutation to A and VP4 protein aa26-776 / 225th P mutation to Q / 226th P mutation to Q.

[0010] The above-mentioned VP4 protein aa26-776 / P mutation at position 225 to A / P mutation at position 226 to A refers to a truncated sequence of aa26-776 in which the amino acid at position 225 on the VP4 protein mutates from P to A, and the amino acid at position 226 mutates from P to A; VP4 protein aa26-776 / P mutation at position 225 to Q / P mutation at position 226 to Q refers to a truncated sequence of aa26-776 in which the amino acid at position 225 on the VP4 protein mutates from P to Q, and the amino acid at position 226 mutates from P to Q.

[0011] As an embodiment of the present invention, the amino acid sequence of the optimized VP4 protein is one of SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.22.

[0012] The full-length amino acid sequence of the wild-type VP4 protein of the G9 strain is SEQ ID NO.4, the amino acid sequence of the truncated portion of the VP4 protein aa26-476 is SEQ ID NO.8, the amino acid sequence of the VP4 protein aa26-776 / the 225th P mutation to A / the 226th P mutation to A is SEQ ID NO.9, and the amino acid sequence of the VP4 protein aa26-776 / the 225th P mutation to Q / the 226th P mutation to Q is SEQ ID NO.10;

[0013] The full-length amino acid sequence of the wild-type VP4 protein of the G4 strain is SEQ ID NO.19, and the amino acid sequence of the VP4 protein aa26-776 / P mutation at position 225 to Q mutation at position 226 to Q is SEQ ID NO.22.

[0014] <Second Aspect>

[0015] The present invention provides a nucleotide sequence encoding the VP4 protein described in any one of the above items, and the nucleotide sequence encoding the optimized VP4 protein is one of SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.23.

[0016] The nucleotide sequence encoding the full length of the VP4 protein of the G9 strain is shown in SEQ ID NO.3, and the sequence after codon optimization is shown in SEQ ID NO.11; the nucleotide sequence encoding aa26-476 of the VP4 protein is shown in SEQ ID NO.12; the nucleotide sequence encoding aa26-776 of the VP4 protein / mutation of P at position 225 to A / mutation of P at position 226 to A is shown in SEQ ID NO.13, and the nucleotide sequence encoding aa26-776 of the VP4 protein / mutation of P at position 225 to Q / mutation of P at position 226 to Q is shown in SEQ ID NO.14;

[0017] The nucleotide sequence encoding the full length of the VP4 protein of the G4 strain is shown in SEQ ID NO.20, and the nucleotide sequence encoding the VP4 protein aa26-776 / P mutation at position 225 to Q mutation at position 226 to Q is shown in SEQ ID NO.23.

[0018] <Third Aspect>

[0019] The present invention provides an application of the VP4 protein or nucleotide sequence described above in preparing a recombinant protein antigen or in preparing a genetically engineered vaccine.

[0020] As an embodiment of the present invention, the genetic engineering vaccine includes a nucleic acid vaccine, which includes a DNA vaccine, a linear RNA vaccine, a self-replicating RNA vaccine, and a circular RNA vaccine.

[0021] <Fourth Aspect>

[0022] The present invention provides a circular RNA molecule, wherein the vector DNA for preparing the circular RNA comprises the following elements in sequence from 5' to 3': an intron fragment II, a translation initiation element truncated fragment II, an ORF, an insertion element, a translation initiation element truncated fragment I, and an intron fragment I;

[0023] The ORF is a coding sequence corresponding to the optimized VP4 protein of porcine rotavirus G9 strain, specifically any one of ORF-3 or ORF-4;

[0024] The amino acid sequence of ORF-3 is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.13;

[0025] The amino acid sequence of ORF-4 is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.14;

[0026] Alternatively, the ORF is a coding element corresponding to the optimized VP4 protein of porcine rotavirus G4 strain, denoted as ORF-6, the amino acid sequence of ORF-6 is shown in SEQ ID NO.22, and the nucleotide sequence is shown in SEQ ID NO.23.

[0027] As an embodiment of the present invention, the nucleotide sequence of the intron fragment II is shown as SEQ ID NO.1, the nucleotide sequence of the insertion element is shown as SEQ ID NO.5, and the nucleotide sequence of the intron fragment I is shown as SEQ ID NO.7.

[0028] As one embodiment of the present invention, the nucleotide sequence of the translation initiation element truncated fragment II is shown as SEQ ID NO.2, and the nucleotide sequence of the translation initiation element truncated fragment I is shown as SEQ ID NO.6;

[0029] Alternatively, the nucleotide sequence of the translation initiation element truncated fragment II is shown as SEQ ID NO.15, and the nucleotide sequence of the translation initiation element truncated fragment I is shown as SEQ ID NO.16.

[0030] As an embodiment of the present invention, the nucleotide sequence of the circular RNA molecule is shown in SEQ ID NO.18, which is recorded as P4-6 circular RNA molecule;

[0031] Alternatively, the nucleotide sequence of the circular RNA molecule is as shown in SEQ ID NO.24, recorded as P4-9 circular RNA molecule.

[0032] <Fifth Aspect>

[0033] The present invention provides a composition comprising any one of the circular RNA molecules described above, and a pharmaceutically acceptable carrier.

[0034] <Sixth Aspect>

[0035] The present invention provides a porcine rotavirus G9-G4 type bivalent RNA vaccine, which is prepared by mixing the P4-6 circular RNA molecule and the P4-9 circular RNA molecule as described above with lipids.

[0036] As an embodiment of the present invention, the G9-G4 type bivalent RNA vaccine is prepared by a method comprising the following steps:

[0037] S1, dissolving P4-6 circular RNA molecules and P4-9 circular RNA molecules in a citric acid buffered saline solution at a mass ratio of 15:1-1:15 to prepare a solution I;

[0038] S2, mixing and dissolving CMAX4, cholesterol, DSPC and PEG-DMG in ethanol to obtain solution II;

[0039] S3. Solution I obtained in S1 and solution II obtained in S2 are mixed by using a microfluidic device, diluted with Tris-HCl solution, and tangential flow filtration is used to remove ethanol in the solution and concentrate it, and a bivalent RNA vaccine is prepared by sterile filtration.

[0040] As an embodiment of the present invention, the molar ratios of CMAX4, cholesterol, DSPC and PEG-DMG are 30% to 60%, 30% to 50%, 5% to 20% and 0.5% to 2.0%, respectively.

[0041] In some implementation examples, the molar ratio of CMAX4, cholesterol, DSPC, and PEG-DMG is 50:38.5:10:1.5.

[0042] As an embodiment of the present invention, the N / P ratio of solution II to solution I is 4:1-12:1. Specifically, it can be 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1. In some implementation examples, the preferred N / P ratio of solution II to solution I is 7:1.

[0043] As an embodiment of the present invention, the pH of the citric acid buffered saline solution in step S1 is 2.0-6.0, and the concentration is 5mM-50mM. In some implementation examples, the pH of the citric acid buffered saline solution in step S1 is 4.0, and the concentration is 10mM.

[0044] As an embodiment of the present invention, the Tris-HCl solution in step S3 contains 2-15% sucrose by volume, and the solution pH is 7.0-9.0. In some implementation examples, the Tris-HCl solution in step S3 contains 8% sucrose by volume, and the solution pH is 7.4.

[0045] As an embodiment of the present invention, after removing ethanol in step S3, the solution is concentrated to an RNA concentration of 25-200 μg / mL. In some implementation examples, after removing ethanol in step S3, the solution is concentrated to an RNA concentration of 100 μg / mL.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1) The present invention optimizes the amino acids and nucleotides of the VP4 proteins of porcine rotavirus G9 and G4, and adds a novel translation initiation element to prepare an optimized mRNA sequence with nucleotide sequences such as SEQ ID NO.18 and SEQ ID NO.NO.24. Compared with the RNA designed using the public technology (SEQ ID NO.17 and SEQ ID NO.21), the optimized RNA has higher translation efficiency in mice and stronger immunogenicity;

[0048] 2) The present invention optimizes the vaccine preparation process parameters to obtain a more efficient production process. The method is simple and easy to operate, and is easy to scale up production, which can effectively reduce production costs;

[0049] 3) The porcine rotavirus G9-G4 type bivalent RNA vaccine prepared by the present invention has better immune protection efficacy in pigs than that of the inactivated porcine rotavirus vaccine; and can achieve cross-protection against prevalent strains; it will provide a technical basis for the clinical prevention and control of porcine rotavirus disease and the elimination of the virus in pig herds, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] 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:

[0051] Figure 1 The figure is a result of capillary gel electrophoresis (CGE) of each RNA prepared in Example 2;

[0052] Figure 2 This is a graph showing the results of measuring the neutralizing antibody titers produced by each RNA in mice in Example 4;

[0053] Figure 3 This is a graph showing the results of measuring the neutralizing antibody titers produced by each RNA in mice in Example 5;

[0054] Figure 4 This is a graph showing the results of measuring the neutralizing antibody titers against the G9 virus produced by the RNA bivalent vaccine and the inactivated vaccine in pigs in Example 7;

[0055] Figure 5 This is a graph showing the results of measuring the neutralizing antibody titers against G4 virus produced by the RNA bivalent vaccine and the inactivated vaccine in pigs in Example 7;

[0056] Figure 6 This is a graph showing the results of the specific lymphocyte proliferation activity stimulated by viral VP4 antigen in Example 7. DETAILED DESCRIPTION

[0057] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0058] 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".

[0059] 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.

[0060] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0061] 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.

[0062] 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".

[0063] 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).

[0064] 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 RNA".

[0065] 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 RNA".

[0066] In the present invention, the term "linear RNA" refers to RNA with translation function including a 5' cap structure (5' Cap), a 3' polyadenosine tail (PolyA tail), a 5' untranslational sequence (5' untranslational region, 5' UTR), a 3' untranslational sequence (3' untranslational region, 3' UTR) and an open reading frame (Open reading frame, ORF) and the like, which can also be called "linear RNA".

[0067] 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.

[0068] 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, the translation initiation element includes an IRES sequence, an m6A modification sequence, or a rolling circle translation initiation sequence, etc.

[0069] 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 RNA start codon is properly oriented in the ribosome for translation. The IRES sequence is usually located in the 5'UTR of the RNA (just upstream of the start codon). IRES functionally replaces the need for various protein factors that interact with the eukaryotic translation machinery.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Example 1. Design and optimization of RNA molecular sequence of porcine rotavirus vaccine

[0080] The porcine rotavirus vaccine disclosed in the present invention comprises an optimized circular RNA molecule, which encodes an optimized porcine rotavirus VP4 protein. The key elements of the vector DNA transcribed into the circular RNA molecule along the direction from 5' to 3' are intron fragment II, translation initiation element truncated fragment II (IRES fragment II), ORF, insertion element, translation initiation element truncated fragment I (IRES fragment I), and intron fragment I. Linear RNA is obtained by transcription from the vector DNA, and the linear RNA forms circular RNA according to the following mechanism in the presence of GTP and MgCl2: ribozyme recognizes the connection position of IRES fragment I and intron fragment I to first generate a break, releasing intron fragment I; then ribozyme recognizes the connection position of IRES fragment II and intron fragment II to generate a break, releasing intron fragment II; the 3' end of IRES fragment I is connected to the 5' end of IRES fragment II to form a circular molecule.

[0081] The scheme for optimizing the circular RNA molecule sequence of the present invention is shown in Table 1, and is specifically as follows:

[0082] The construction of P4-1 circular RNA mainly refers to patent CN114574483B. The vector DNA for preparing the circular RNA includes the following elements in sequence from 5' to 3': intron fragment II, translation initiation element truncated fragment II, ORF, insertion element, translation initiation element truncated fragment I, intron fragment I; wherein the translation initiation element is a Caprine kobuvirus IRES sequence in which the 80th G is mutated to T, the 81st A is mutated to C, and the 82nd G is mutated to T to obtain a mutant sequence (numbered CKV in this application), specifically, intron fragment II (numbered A-II in this application, the nucleotide sequence is SEQ ID NO.1), translation initiation element truncated fragment II (numbered CKV-II in this application, the nucleotide sequence is SEQ ID NO.2), ORF (the nucleotide sequence of porcine rotavirus G9 type VP4 protein, referred to as "natural sequence" in Table 1, the nucleotide sequence is SEQ ID NO.3, and the amino acid sequence is SEQ ID NO.4), insertion element (numbered A-polyAC in this application, the nucleotide sequence is SEQ ID NO.5), translation initiation element truncation fragment I (numbered CKV-I in this application, the nucleotide sequence is SEQ ID NO.6), intron fragment I (numbered AI in this application, the nucleotide sequence is SEQ ID NO.7).

[0083] Circular RNAs P4-2, P4-3, P4-4, and P4-5 are obtained by replacing the ORF nucleotide sequence of circular RNA P4-1 with the ORF-1, ORF-2, ORF-3, and ORF-4 nucleotide sequences in Table 1, respectively. Among them, ORF-1 encodes the full-length VP4 protein (amino acid sequence is SEQ ID NO.4, nucleotide sequence is SEQ ID NO.11), ORF-2 encodes the optimized VP4 protein-2 (aa26-476) (amino acid sequence is SEQ ID NO.8, nucleotide sequence is SEQ ID NO.12), ORF-3 encodes the optimized VP4 protein-3 (aa26-776, P at position 225 mutated to A, P at position 226 mutated to A) (amino acid sequence is SEQ ID NO.9, nucleotide sequence is SEQ ID NO.13), ORF-4 encodes the optimized VP4 protein-4 (aa26-776, P at position 225 mutated to Q, P at position 226 mutated to Q) (amino acid sequence is SEQ ID NO.10, nucleotide sequence is SEQ ID NO.14), and the nucleotide sequences of ORF-1, ORF-2, ORF-3, and ORF-4 are obtained by adjusting the commonly used codon preferences during expression in pigs, increasing the GC content of the sequences, and other optimizations.

[0084] The P4-6 circular RNA is based on the P4-5 circular RNA sequence, with the translation initiation elements CKV-II and CKV-I replaced by translation initiation elements X-II (nucleotide sequence is SEQ ID NO.15) and XI (nucleotide sequence is SEQ ID NO.16), respectively. The translation initiation element IRES fragment II (X-II) sequence and the translation initiation element IRES fragment I (XI) sequence were discovered accidentally and can improve the expression efficiency of porcine rotavirus VP4 protein.

[0085] Table 1 RNA sequence optimization (G9 type)

[0086]

[0087] Example 2. Preparation of porcine rotavirus vaccine RNA molecules

[0088] The DNA vector for transcribing RNA was synthesized commercially, and the RNA molecules for preparing the vaccine were obtained through the following steps: bacterial strain preparation, plasmid extraction, plasmid restriction and purification, in vitro transcription, transcription product precipitation by LiCl solution (final concentration 2.5M), RNA formation of circular RNA in the presence of GTP (final concentration 20mM) and MgCl2 (final concentration 100mM), circular RNA purification by chromatographic column SEC-1000 (SEPAX) in 150mM phosphate buffer mobile phase, RNA replacement into nuclease-free water by TFF Capsule (LV Centramate, PALL) and concentration to ~2mg / ml, wherein the membrane area of ​​TFF capsule is 0.02m 2 , the flow rate is 120mL / min, and the specific method refers to the circular RNA preparation patent CN117305328A. Capillary gel electrophoresis (CGE) analysis showed that the purity of RNA molecules P4-1, P4-2, P4-3, P4-4, P4-5, P4-6, and P4-7 were 92.3%, 90.4%, 91.5%, 94.2%, 91.1%, 95.2%, and 93.5%, respectively, all greater than 85%, meeting the requirements. The results are shown in Figure 1 .

[0089] Example 3: Preliminary preparation of test vaccine

[0090] The circular RNA molecules P4-1, P4-2, P4-3, P4-4, P4-5, P4-6, and P4-7 prepared above were respectively prepared into LNP-encapsulated RNA vaccines using commercial LNPs, and the corresponding numbers were P4-1, P4-2, P4-3, P4-4, P4-5, P4-6, and P4-7 vaccines. The specific preparation method is referred to the literature (doi:10.3390 / pharmaceutics15020477). The circular RNA was diluted to a final concentration of 200 μg / mL with citric acid-sodium citrate buffer, and LNP (Mianna (Shanghai) Instrument Technology Co., Ltd.) and RNA were mixed in a volume ratio of 1:3 using a microfluidic device. The buffer was replaced with a Tris-HCl solution (containing 8% sucrose (w / v)) at pH 7.4 using tangential flow technology and concentrated to an RNA concentration of about 100 μg / mL, and the test vaccine was obtained by filtration and sterilization. After testing, the physical properties of the vaccines in each test group met the requirements (Table 2).

[0091] Table 2 Physical properties of commercial LNP-encapsulated RNA vaccines

[0092] serial number Particle size / nm Encapsulation rate / % Surface potential Polydispersity coefficient P4-1 96 90 -4.73 0.097 P4-2 85 88 -1.23 0.050 P4-3 89 91 -2.56 0.064 P4-4 110 89 -2.24 0.063 P4-5 95 86 -3.36 0.086 P4-6 102 89 -1.74 0.091 P4-7 98 90 -2.25 0.085

[0093] Example 4: Mouse experiment and neutralizing antibody titer determination

[0094] BABL / c female mice aged 6 to 8 weeks were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. and randomly divided into 7 groups, with 4 mice in each group. The RNA vaccines prepared in Example 3 were used for intramuscular immunization of mice at a dose of 100 μl / mouse. At the same time, mice vaccinated with LNP were used as negative controls. The mice were boosted 21 days after immunization, and serum was collected on the 14th day after the second immunization for virus neutralizing antibody detection. The neutralizing antibody titer for porcine rotavirus G9 strain was determined according to the method of patent CN114854697B.

[0095] The results are as follows Figure 2 As shown, the neutralizing antibody titer of the experimental vaccine P4-5 is significantly higher than that of P4-1 to P4-4. The optimized ORF-4 provided by the present invention can significantly improve the immunogenicity of RNA vaccines in mice. The experimental vaccine P4-6 is much higher than P4-5, indicating that when other elements remain unchanged, the translation initiation element truncation fragment II (X-II) and the translation initiation element truncation fragment I (XI) can more efficiently express the optimized VP4 protein-4 than the disclosed translation initiation element truncation fragment II (CKV-II) and translation initiation element truncation fragment I (CKV-I). In addition, the experimental vaccine P4-6 is better than P4-7, indicating that even the use of a new translation initiation element cannot improve the expression efficiency and immunogenicity of the natural VP4 protein, further proving that the present invention is effective in optimizing the VP4 protein sequence and nucleotide sequence.

[0096] The circular RNA P4-1 of the present invention is a circular RNA designed using public technology and encoding the VP4 protein of porcine rotavirus G9 type, and the nucleotide sequence is SEQ ID NO. 17. The circular RNA P4-6 is the RNA with a sequence of SEQ ID NO. 18 provided by the present invention, and the RNA encodes the optimized VP4 protein-4 of porcine rotavirus G9 type (amino acid sequence is SEQ ID NO. 10, nucleotide sequence is SEQ ID NO. 14).

[0097] Example 5: Optimization of G4-type VP4 protein

[0098] The amino acid sequence homology of G4 and G9 VP4 proteins is more than 85%, and the two proteins are very conservative at key amino acid sites, so it is believed that the two proteins have similar structural characteristics and expression characteristics. Therefore, the optimization of G9 VP4 protein is also applicable to G4 VP4 protein. This study further verified this.

[0099] The RNA molecule P4-8 was designed by the existing public method, and the RNA expressed the natural protein of VP4 of porcine rotavirus G4 type (amino acid sequence is SEQ ID NO.19, nucleotide sequence is SEQ ID NO.20), and its nucleotide sequence is SEQ ID NO.21. The RNA molecule P4-9 was designed with reference to the structure of RNA molecule P4-6 of the present application, and the RNA expressed the optimized VP4-5 protein of porcine rotavirus G4 type (G4 type VP4 protein aa26-776, P mutated to Q at position 225, P mutated to Q at position 226, amino acid sequence is SEQ ID NO.22, nucleotide sequence is SEQ ID NO.23), and its nucleotide sequence is SEQ ID NO.24. The design method is shown in Table 3. The specific method is as follows: the nucleotide sequence of the natural ORF in the P4-1 RNA molecule is replaced with the ORF-5 nucleotide sequence (SEQ ID NO.20) to obtain the P4-8 circular RNA molecule, and the ORF-4 nucleotide sequence in the P4-6 RNA molecule is replaced with the ORF-6 nucleotide sequence (SEQ ID NO.23) to obtain the P4-9 circular RNA.

[0100] Table 3 RNA sequence optimization (G4 type)

[0101]

[0102] RNA molecules P4-8 and P4-9 were prepared according to the method of Example 2 (their capillary electrophoresis purities were 93% and 91%, respectively, see Figure 1), and the two RNA molecules were prepared into test vaccines according to the method of Example 3, and mice were immunized according to the method of Example 4. Serum was collected on the 14th day after the second immunization for virus neutralizing antibody detection. The neutralizing antibody titer for porcine rotavirus G4 strain was determined according to the method of patent CN114854697B. The results are shown in Figure 3 As shown, the antibodies produced by the P4-9 RNA vaccine were significantly increased, indicating that the molecule has higher immunogenicity in mice.

[0103] Table 4 Physical properties of commercial LNP-encapsulated RNA vaccines

[0104] serial number Particle size / nm Encapsulation rate / % Surface potential Polydispersity coefficient P4-8 101 92 -3.14 0.062 P4-9 98 93 -2.28 0.038

[0105] Example 6. Preparation method of bivalent porcine rotavirus RNA vaccine

[0106] After the RNA molecules were initially selected, the preparation method of the bivalent RNA vaccine was further studied. The two qualified circular RNA molecules were dissolved in an equal mass ratio in a 10 mM citric acid / sodium citrate buffered saline solution at pH 4.0, with a final concentration of 200 μg / mL of RNA molecules to prepare Solution I.

[0107] 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) and cholesterol (5-cholesten-3β-ol), DSPC (distearoylphosphatidylcholine) and PEG-DMG (polyethylene glycol-dimyristol) are dissolved in ethanol to obtain solution II. The molar ratio of CMAX4 is 30% to 60%, specifically 30% or 50% or 60%, the molar ratio of cholesterol is 30% to 50%, specifically 30% or 32.5% or 38.5% or 50%, the molar ratio of DSPC is 5% to 20%, specifically 5% or 10% or 20%, and the molar ratio of PEG-DMG is 0.5% to 2.0%, specifically 0.5% or 1.5% or 2.0%.

[0108] Solution I and solution II were mixed in different proportions (the N / P ratio of solution II to solution I was 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1) by using a microfluidic device, and the flow rate during mixing was 20 ml / min. The mixed solution was immediately diluted 4 times with Tris-HCl solution (pH 7.4, containing 8% sucrose (w / v)), and tangential flow filtration (TFF) was used to remove the ethanol component in the solution and concentrate it to an RNA concentration of 50 μg / mL, and the RNA vaccine was obtained by sterile filtration.

[0109] When the N / P ratio of solution II and solution I is 7:1, and the molar ratio of CMAX4 / cholesterol / DSPC / PEG-DMG is 50:38.5:10:1.5, the molecular polydispersity coefficient obtained at this time is the lowest (0.026), the encapsulation efficiency is the highest (94%), the surface potential is electronegative, the average particle size is 98nm, and the production process meets the requirements. The obtained vaccine is the porcine rotavirus bivalent RNA vaccine of the present invention.

[0110] Example 7: Target Animal Experiment

[0111] The porcine rotavirus bivalent RNA vaccine prepared by the screening formula of Example 6 was used for the immunization test of the target animal: newborn piglets. Newborn piglets (both PoRV antigen and antibody were negative) were randomly divided into 4 groups, 5 piglets / group, and immunized with 50μg / pig of porcine rotavirus bivalent RNA vaccine, 80μg / pig of porcine rotavirus bivalent RNA vaccine, 1ml of porcine rotavirus G9 and G4 bivalent inactivated vaccine, and sterile saline, respectively, by intramuscular injection in the neck, and booster immunization was performed once 14 days after the first immunization. On the 7th day after the second immunization, blood was collected and placed in 5mM EDTA, and PBMC cells were quickly separated with Lymphoprep lymphocyte separation solution (TBD Company, Tianjin), and resuspended with RPMI culture medium containing 15% FBS (Sigma Company). Cells (2×10 6cells / ml, 100μl), the experimental group was stimulated with 10μg G9 virus VP4 protein or G4 virus VP4 protein polypeptide pool, the negative control group was not stimulated with antigen, and the positive control group was stimulated with ConA, wherein the VP4 protein polypeptide pool was synthesized and provided by Shenlian Bio, consisting of 15mers with 11 overlapping amino acids, and the polypeptide purity was more than 85%. Incubate at 37°C for 72 hours, add 50μl MTT for 3 hours, add 100μl DMSO, and test the OD570nm value. Compared with the control group, the proportion of lymphocytes proliferated by antigen-specific stimulation was calculated. Serum was collected 10 days after the second immunization to determine the neutralizing antibody titer. The results are expressed as mean ± SD values. The significant difference was analyzed by student t test, ns means no significant difference, * means p<0.05, ** means p<0.01, *** means p<0.005.

[0112] The preparation method of the G9-G4 bivalent inactivated vaccine of porcine rotavirus is referred to the prior patent CN114854697B, which is as follows: the suspension PK-15 cells are cultured for 72 hours, and the cell density reaches 6.0-6.5×10 6 When the cell viability is above 95%, porcine rotavirus G9 or G4 is inoculated with a cell density of 2.0×10 6 / ml of suspended PK-15 cells, and add trypsin at a final concentration of 6μg / ml, culture for 24 hours to harvest the virus liquid. After centrifugation, the supernatant of the virus liquid was clarified by filtering with a 0.45μm filter membrane, and the clarified liquid was harvested after washing with PBS, and then purified by chromatography through Q Focurose 6FF. 0.5mol / L BEI solution was added to the purified antigen to make its final concentration 0.005mol / L, mixed evenly and inactivated at 37℃ for 24 hours, and then filtered and sterilized 1.0mol / L Na2S2O3 solution was added to the final concentration of 0.005mol / L to terminate the inactivation. The two inactivated porcine rotavirus liquids were diluted with PBS to the vaccine concentration (10 8.0 TCID50 / ml), then mix evenly at a volume ratio of 1:1, preheat to 32°C as the water phase, preheat ISA201VG adjuvant to 32°C as the oil phase, slowly add the water phase to ISA201 VG adjuvant at a volume ratio of water phase: oil phase = 1:1, emulsify at 100-120r / min for 60 minutes, quantitatively package, cover and seal, and store at 2-8°C.

[0113] from Figure 4 and Figure 5 It can be seen that the porcine rotavirus bivalent RNA vaccine 80 μg group prepared in Example 6 can increase the neutralizing antibody in the serum of newborn piglets and significantly increase the proliferation activity of PBMC cells after specific stimulation with the virus VP4 antigen ( Figure 6 ), which are higher than the inactivated vaccine, indicating that the porcine rotavirus bivalent RNA vaccine provided by the present invention has a higher immune protection effect on piglets. For diarrhea viruses, the virus neutralizing antibody titer is positively correlated with the protective effect, and the viral antigen-specific lymphocyte proliferation activity is stronger after immunization, indicating the activation of a stronger cellular immune response, which are the key mechanisms for the RNA vaccine of the present invention to provide immune protection.

[0114] 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. An optimized VP4 protein of porcine rotavirus, characterized in that: The optimized VP4 protein is one of VP4 protein aa26-776 / P mutation at position 225 to A / P mutation at position 226 to A, and VP4 protein aa26-776 / P mutation at position 225 to Q / P mutation at position 226 to Q.

2. The optimized VP4 protein according to claim 1, characterized in that The amino acid sequence of the optimized VP4 protein is one of SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.

22.

3. A nucleotide sequence encoding the VP4 protein according to claim 1 or 2, characterized in that: The nucleotide sequence encoding the optimized VP4 protein is one of SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.

23.

4. Use of the VP4 protein sequence according to claim 1 or 2 or the nucleotide sequence according to claim 3 in preparing a recombinant protein antigen or a genetically engineered vaccine; The genetically engineered vaccines include nucleic acid vaccines; the nucleic acid vaccines include DNA vaccines, linear RNA vaccines, self-replicating RNA vaccines and circular RNA vaccines.

5. A circular RNA molecule, characterized in that The vector DNA for preparing the circular RNA includes the following elements in order from 5' to 3': intron fragment II, translation initiation element truncated fragment II, ORF, insertion element, translation initiation element truncated fragment I, intron fragment I; The ORF is a coding sequence corresponding to the optimized VP4 protein of porcine rotavirus G9 strain, specifically ORF-3 or ORF-4; The amino acid sequence of ORF-3 is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.13; The amino acid sequence of ORF-4 is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.14; Alternatively, the ORF is a coding element corresponding to the optimized VP4 protein of porcine rotavirus G4 strain, denoted as ORF-6, the amino acid sequence of ORF-6 is shown in SEQ ID NO.22, and the nucleotide sequence is shown in SEQ ID NO.

23.

6. The circular RNA molecule according to claim 5, characterized in that The nucleotide sequence of the translation initiation element truncated fragment II is shown in SEQ ID NO.2, and the nucleotide sequence of the translation initiation element truncated fragment I is shown in SEQ ID NO.6; Alternatively, the nucleotide sequence of the translation initiation element truncated fragment II is shown as SEQ ID NO.15, and the nucleotide sequence of the translation initiation element truncated fragment I is shown as SEQ ID NO.

16.

7. The circular RNA molecule according to claim 5, characterized in that The nucleotide sequence of the intron fragment II is shown as SEQ ID NO.1, the nucleotide sequence of the insertion element is shown as SEQ ID NO.5, and the nucleotide sequence of the intron fragment I is shown as SEQ ID NO.

7.

8. The circular RNA molecule according to claim 5, characterized in that The nucleotide sequence of the circular RNA molecule is shown in SEQ ID NO.18, and is recorded as P4-6 circular RNA molecule; Alternatively, the nucleotide sequence of the circular RNA molecule is as shown in SEQ ID NO.24, recorded as P4-9 circular RNA molecule.

9. A composition, characterized in that Comprising the circular RNA molecule according to any one of claims 5 to 8, and a pharmaceutically acceptable carrier.

10. A porcine rotavirus G9-G4 type bivalent RNA vaccine, characterized in that: The G9-G4 type bivalent RNA vaccine is prepared by mixing the P4-6 circular RNA molecule and the P4-9 circular RNA molecule in the circular RNA molecule as described in claim 8 with lipids.

11. The G9-G4 type bivalent RNA vaccine according to claim 10, characterized in that: The G9-G4 type bivalent RNA vaccine is prepared by a method comprising the following steps: S1, dissolving P4-6 circular RNA molecules and P4-9 circular RNA molecules in a citric acid buffered saline solution at a mass ratio of 15:1-1:15 to prepare a solution I; S2, mixing and dissolving CMAX4, cholesterol, DSPC and PEG-DMG in ethanol to obtain solution II; S3. Solution I obtained in S1 and solution II obtained in S2 are mixed by using a microfluidic device, diluted with Tris-HCl solution, and tangential flow filtration is used to remove ethanol in the solution and concentrate it, and a bivalent RNA vaccine is prepared by sterile filtration.

12. The G9-G4 type bivalent RNA vaccine according to claim 11, characterized in that: The molar ratios of CMAX4, cholesterol, DSPC and PEG-DMG are 30% to 60%, 30% to 50%, 5% to 20% and 0.5% to 2.0%, respectively.

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