A bivalent swine foot-and-mouth disease mRNA vaccine and its preparation method and application

Abstract

The present application discloses a bivalent swine foot-and-mouth disease mRNA vaccine and its preparation method and application, and belongs to the field of vaccine production technology in the field of biotechnology. The technical problem to be solved by the present application is: how to effectively prevent swine foot-and-mouth disease virus. To this end, the present application provides a bivalent swine foot-and-mouth disease mRNA vaccine, the bivalent swine foot-and-mouth disease mRNA vaccine contains mRNA molecule 1 and mRNA molecule 2, the nucleotide sequence of mRNA molecule 1 is SEQ ID No.5; the nucleotide sequence of mRNA molecule 2 is SEQ ID No.6. The present application selects the key VP1 protein of foot-and-mouth disease as the mRNA antigen, and utilizes the principle that GAG protein interacts with GP41 protein CTD to assemble into VLPS, and designs GAG fusion O-type VP1 and A-type VP1 fusion GP41 to interact to form a bivalent VLPS mRNA vaccine.

Description

Technical Field

[0001] The present application relates to vaccine production technology in the field of biotechnology, and specifically to a bivalent swine foot-and-mouth disease mRNA vaccine and a preparation method and application thereof. Background Art

[0002] Animal vaccines play a vital role in the prevention and control of animal diseases, preventing millions of cases of animal diseases each year and significantly reducing animal deaths and property losses. Through widespread vaccination, the incidence of common animal diseases such as influenza, foot-and-mouth disease, and swine fever has dropped significantly worldwide, achieving effective prevention of most diseases. Traditional vaccine approaches, such as live attenuated vaccines, inactivated pathogen vaccines, and subunit vaccines, provide long-lasting and effective protection against a variety of high-risk diseases. However, despite remarkable achievements, vaccine development still faces great challenges for some highly variable infectious pathogens, especially those that are good at evading adaptive immune responses. For most emerging virus vaccines, the main challenge is not the effectiveness of traditional methods, but how to develop and deploy them rapidly in a short period of time. Therefore, when responding to rapid and large-scale outbreaks, traditional vaccine approaches may not be sufficient to meet the needs of rapid prevention and control.

[0003] Nucleic acid therapy has shown great promise as a potential alternative to traditional vaccine approaches. As early as 1990, studies reported the successful production of reporter gene proteins in mice through in vitro transcription (IVT) mRNA technology. Subsequent studies in 1992 further demonstrated that injection of vasopressin-encoding mRNA into the rat hypothalamus could induce physiological responses. However, despite these promising early research results, challenges such as mRNA instability, high innate immunogenicity, and inefficient in vivo delivery did not trigger large-scale investment in the development of mRNA therapies at the time. Instead, the field was more inclined to explore DNA- and protein-based treatments.

[0004] In the past few years, mRNA vaccines have emerged in the vaccine field and have become a highly promising tool in traditional vaccines and protein replacement therapies. Compared with inactivated viruses, attenuated live viruses and DNA vaccines, mRNA vaccines have shown several significant advantages. First, in terms of safety, mRNA, as a non-infectious, non-integrating platform, eliminates the risk of infection or gene insertion mutation. In addition, mRNA is degraded through natural cellular processes, its half-life can be adjusted by a variety of modifications and delivery methods, and its immunogenicity can also be downregulated by technical means, thereby ensuring higher safety. Secondly, in terms of efficacy, mRNA has higher stability and translation efficiency after modification. With the assistance of carrier molecules, mRNA can be effectively delivered to the body and rapidly expressed in the cytoplasm. As the smallest genetic carrier, mRNA avoids anti-carrier immunity and can be repeatedly administered, providing more possibilities for vaccine application. Finally, the production of mRNA vaccines has the potential for rapid, low-cost and scalability, mainly due to the high yield of in vitro transcription reactions. The recent successful development of the new crown vaccine has fully demonstrated the huge potential of mRNA vaccines in the development of infectious disease vaccines and provided new ideas for solving many problems in vaccine development.

[0005] Gag protein, as the precursor structural protein of HIV-1 virus, can form matrix protein (MA), capsid protein (CA), nucleocapsid protein (NC) and p6 after protease hydrolysis. Among them, matrix protein is embedded in the lipid bilayer of the viral envelope, while capsid protein, nucleocapsid protein and viral RNA together form the viral core. Regardless of the presence of other viral components, Gag protein can self-assemble to form virus-like particles (VLPs). Env protein is composed of a trimer composed of heterodimeric monomers, each monomer includes receptor binding protein gp120 (surface glycoprotein, SU) and transmembrane fusion protein GP41 (transmembrane protein, TM), and the two are connected by non-covalent interactions. The CTD part of Env protein interacts with the matrix domain (MA) of Gag protein, while GP41 is anchored to the viral envelope through its transmembrane region, and the C-terminal cytoplasmic domain is located in the viral cavity, together forming virus-like particles.

[0006] FMDV genetic engineering subunit vaccine mainly relies on the expression system to express VP1 protein. Kupper et al. (1981) successfully cloned the FMDV VP1 gene and inserted it into the downstream of the prokaryotic expression vector PL promoter to achieve prokaryotic expression of VP1 protein. Through indirect ELISA and radioimmunoassay, it was confirmed that the expression product was antigenic, which provided theoretical support for the development of FMDV genetic engineering subunit vaccine. Further studies have shown that the use of type A FMDV VP1 protein expressed by Escherichia coli to immunize pigs and cattle can effectively induce the production of neutralizing antibodies and resist the attack of FMDV virulent. Summary of the invention

[0007] The technical problem to be solved by this application is: how to effectively prevent swine foot-and-mouth disease virus.

[0008] In order to solve the above technical problems, the present application provides a bivalent swine foot-and-mouth disease mRNA vaccine, wherein the bivalent swine foot-and-mouth disease mRNA vaccine contains mRNA molecule 1 and mRNA molecule 2;

[0009] The mRNA molecule 1 encodes a protein 1 comprising an antigen protein 1, and the antigen protein 1 may be any of the following proteins:

[0010] a1) the amino acid sequence of the protein having the amino acid sequence of positions 513-725 of SEQ ID No. 2;

[0011] a2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues with the amino acid sequence shown in a1) that has more than 80% identity with the amino acid sequence shown in a1) and has the same antigenicity as O-type foot-and-mouth disease VP1 protein;

[0012] a3) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2);

[0013] The mRNA molecule 2 encodes a protein 2 comprising an antigen protein 2, and the antigen protein 2 may be any of the following proteins:

[0014] a4) the amino acid sequence of the protein having the amino acid sequence of positions 11 to 222 of SEQ ID No. 4;

[0015] a5) A protein obtained by substitution and / or deletion and / or addition of amino acid residues with the amino acid sequence shown in a4) that has more than 80% identity with the amino acid sequence shown in a1) and has the same antigenicity as type A foot-and-mouth disease VP1 protein;

[0016] a6) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a4) or a5).

[0017] Furthermore, in the bivalent swine foot-and-mouth disease mRNA vaccine, the protein 1 may be any of the following proteins;

[0018] a7) a protein whose amino acid sequence is SEQ ID No. 2;

[0019] a8) A protein obtained by substitution and / or deletion and / or addition of amino acid residues with the amino acid sequence shown in a7) that has more than 80% identity with the amino acid sequence shown in a7) and has the same antigenicity as O-type foot-and-mouth disease VP1 protein;

[0020] a9) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of a7) or a8);

[0021] The protein 2 may be any of the following proteins;

[0022] a10) a protein whose amino acid sequence is SEQ ID No. 4;

[0023] a11) A protein obtained by substitution and / or deletion and / or addition of amino acid residues with the amino acid sequence shown in a10) that has more than 80% identity with the amino acid sequence shown in a10) and has the same antigenicity as type A foot-and-mouth disease VP1 protein;

[0024] a12) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of a10) or a11);

[0025] In the present application, SEQ ID No. 2 consists of 734 amino acid residues.

[0026] In the present application, SEQ ID No. 4 consists of 410 amino acid residues.

[0027] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0028] The linkage described in a3), a6), a9) and / or a12) may be via a peptide bond.

[0029] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0030] In one embodiment of the present application, the protein 1 is a fusion protein FMDV O CHA31 2010GAG VP1. The amino acid sequence of the fusion protein FMDV O CHA31 2010GAG VP1 is SEQ ID No. 2. Among them, SEQ ID No. 2 1-500 is the amino acid sequence of HIVGAG core protein, SEQ ID No. 2 501-512 is the amino acid sequence of the connecting peptide, 513-725 is the amino acid sequence of O-type foot-and-mouth disease VP1 protein, and SEQ ID No. 2 726-734 is the amino acid sequence of the immune-enhancing αvβ3 / β5integrin protein.

[0031] In one embodiment of the present application, the protein 2 is a fusion protein FMDV AWH09VP1 HIV Gp41, and the amino acid sequence of the fusion protein FMDV AWH09VP1 HIV Gp41 is SEQ ID No. 4. Among them, positions 1-10 of SEQ ID No. 4 are the amino acid sequence of the immune-enhancing αvβ3 / β5integrin protein, and positions 11-222 of SEQ ID No. 4 are the amino acid sequence of the A-type foot-and-mouth disease VP1 protein, positions 223-234 are the amino acid sequence of the connecting peptide, and positions 235-410 are the amino acid sequence of the HIVGP41 protein.

[0032] Furthermore, in the bivalent swine foot-and-mouth disease mRNA vaccine, the mRNA molecule 1 comprises, from the 5' end to the 3' end, a 5' cap structure, a 5' UTR, a nucleic acid encoding αvβ3 / β5integrin, a nucleic acid encoding O-type foot-and-mouth disease VP1, a nucleic acid encoding a connecting peptide, a nucleic acid encoding HIVGAG, a 3' UTR and a 3' polyadenylic acid tail;

[0033] The mRNA molecule 2 comprises, from 5' to 3' end, a 5' cap structure, a 5' UTR, a nucleic acid encoding αvβ3 / β5integrin, a nucleic acid encoding type A foot-and-mouth disease VP1, a nucleic acid encoding a connecting peptide, a nucleic acid encoding HIVGP41, a 3' UTR and a 3' polyadenylic acid tail.

[0034] Furthermore, the αvβ3 / β5integrin may be a protein having an amino acid sequence of positions 726-734 of SEQ ID No. 2 and / or positions 1-10 of SEQ ID No. 4.

[0035] The nucleotide sequence of the nucleic acid encoding αvβ3 / β5integrin (integrin αvβ3 protein and / or integrin αvβ5 protein) can specifically be an RNA molecule whose nucleotide sequence is shown at positions 2286-2315 of SEQ ID No.5 and / or an RNA molecule whose nucleotide sequence is shown at positions 111-140 of SEQ ID No.6.

[0036] Furthermore, the O-type foot-and-mouth disease VP1 may be a protein whose amino acid sequence is positions 513-725 of SEQ ID No.2.

[0037] The nucleic acid encoding O-type foot-and-mouth disease VP1 may specifically be an RNA molecule whose nucleotide sequence is shown at positions 1647-2285 of SEQ ID No.5.

[0038] Furthermore, the connecting peptide may be a protein having an amino acid sequence of positions 501-512 of SEQ ID No. 2 and / or a protein having positions 223-234 of SEQ ID No. 4.

[0039] The nucleic acid encoding the connecting peptide may specifically be an RNA molecule whose nucleotide sequence is shown at positions 1611-1646 of SEQ ID No.5 and / or an RNA molecule whose nucleotide sequence is shown at positions 777-812 of SEQ ID No.6.

[0040] Furthermore, the HIVGAG may be a protein having an amino acid sequence of positions 1 to 500 of SEQ ID No.2.

[0041] The nucleic acid encoding HIVGAG may specifically be an RNA molecule whose nucleotide sequence is shown at positions 111-1610 of SEQ ID No.5.

[0042] Furthermore, the type A foot-and-mouth disease VP1 may be a protein whose amino acid sequence is positions 11-222 of SEQ ID No.4.

[0043] The nucleic acid encoding type A foot-and-mouth disease VP1 may specifically be an RNA molecule whose nucleotide sequence is shown in positions 141-776 of SEQ ID No.4.

[0044] Furthermore, the HIVGP41 may be a protein having an amino acid sequence of positions 235-410 of SEQ ID No.4.

[0045] The nucleic acid encoding HIV GP41 may specifically be an RNA molecule whose nucleotide sequence is shown at positions 813-1343 of SEQ ID No.4.

[0046] Furthermore, the connecting peptide may be a GS connecting peptide.

[0047] Further, the amino acid sequence of the connecting peptide may be (GGGS)n, where n may be a natural number greater than or equal to 1 and less than or equal to 4, such as n is a natural number greater than or equal to 1 and less than or equal to 3. In some embodiments of the present application, n is 3.

[0048] Further, the amino acid sequence of the connecting peptide may be (GGS)n, where n may be a natural number greater than or equal to 1 and less than or equal to 4. In some embodiments of the present application, n is 4.

[0049] Furthermore, in the bivalent swine foot-and-mouth disease mRNA vaccine,

[0050] The nucleotide sequence of the mRNA molecule 1 is SEQ ID No. 5;

[0051] The nucleotide sequence of the mRNA molecule 2 is SEQ ID No.6.

[0052] In the present application, the 5' end of SEQ ID No. 5 and SEQ ID No. 6 has a cap structure Cap1 (m7G (5') ppp (5') (2'OMeA) pG).

[0053] In some embodiments of the present application, positions 1-110 of SEQ ID No.5 are the mRNA sequence of 5'-UTR, positions 111-1610 are the mRNA sequence of GAG, positions 1611-1646 are the mRNA sequence of the connecting peptide, positions 1647-2285 are the mRNA sequence of FMDVO type VP1, positions 2286-2315 are the mRNA sequence of αvβ3 / β5integrin, positions 2316-2613 are the mRNA sequence of 3'-UTR, and positions 2614-2723 are the mRNA sequence of poly(A).

[0054] In some embodiments of the present application, positions 1-110 of SEQ ID No.6 are the mRNA sequence of 5'-UTR, positions 111-140 are the mRNA of αvβ3 / β5integrin gene, positions 141-776 are the mRNA sequence of FMDV type A VP1, positions 777-812 are the mRNA sequence of GS connecting peptide, positions 813-1343 are the mRNA sequence of GP41, positions 1344-1641 are the mRNA sequence of 3'-UTR, and positions 1642-1751 are the mRNA sequence of poly(A).

[0055] Furthermore, the active ingredients of the bivalent swine foot-and-mouth disease mRNA vaccine may be mRNA molecule 1 and mRNA molecule 2.

[0056] Furthermore, in the bivalent swine foot-and-mouth disease mRNA vaccine, the bivalent swine foot-and-mouth disease mRNA vaccine is formed by encapsulating the mRNA molecule 1 and the mRNA molecule 2 together in liposome nanoparticles.

[0057] Furthermore, in the bivalent swine foot-and-mouth disease mRNA vaccine, the mass ratio of the mRNA molecule 1 and the mRNA molecule 2 is 1:1.

[0058] In the present application, four lipids including cationic lipid material D-Lin-MC3-DMA, cholesterol (CHOLESTEROL), auxiliary lipid (DSPC) and PEG lipid (PEG2000-DMG) were selected as the basic components of LNP.

[0059] In some embodiments of the present application, the four lipids in LNP are assembled according to the following molar ratio (D-Lin-MC3-DMA: CHOLESTEROL: DSPC: PEG2000-DMG = 50:38.5:10:1.5).

[0060] The present application also provides biological materials related to the above-mentioned bivalent swine foot-and-mouth disease mRNA vaccine, wherein the biological material is selected from any one of the following:

[0061] B1) a DNA molecule encoding the above mRNA molecule 1 and / or mRNA molecule 2;

[0062] B2) an expression cassette containing the DNA molecule described in B1);

[0063] B3) a recombinant vector containing the DNA molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

[0064] B4) a recombinant microorganism containing the DNA molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0065] B5) a transgenic plant cell line containing the DNA molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2) or a transgenic animal cell line containing the recombinant vector described in B3);

[0066] B6) transgenic plant tissue containing the DNA molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2) or transgenic animal tissue containing the recombinant vector described in B3);

[0067] B7) A transgenic plant organ containing the DNA molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2) or a transgenic animal organ containing the recombinant vector described in B3).

[0068] Furthermore, in the above-mentioned biological material, the DNA molecule described in B1) may be a DNA molecule having a nucleotide sequence of positions 916-3645 of SEQ ID No. 1 and / or a DNA molecule having a nucleotide sequence of positions 917-2673 of SEQ ID No. 3.

[0069] Furthermore, in the above-mentioned biological material, the recombinant vector described in B3) may be a DNA molecule having a nucleotide sequence of SEQ ID No.1 and / or a DNA molecule having a nucleotide sequence of SEQ ID No.3.

[0070] In some embodiments of the present application, positions 898-916 of SEQ ID No.1 are the nucleotide sequence of the T7 promoter, positions 917-1026 are the nucleotide sequence of the mRNA 5'UTR, positions 1027-2526 are the nucleotide sequence of the HIVGAG encoding gene, positions 2527-2562 are the nucleotide sequence of the GS connecting peptide encoding gene, positions 2563-3201 are the nucleotide sequence of the O type foot-and-mouth disease VP1 encoding gene, positions 3202-3231 are the nucleotide sequence of the αvβ3 / β5integrin encoding gene, positions 3232-3529 are the nucleotide sequence of the 3'UTR, positions 3530-3639 of SEQ ID No.1 are the nucleotide sequence of the poly(A) gene, and positions 3640-3645 of SEQ ID No.1 are linearization sites.

[0071] In some embodiments of the present application, positions 898-916 of SEQ ID No.3 are the nucleotide sequence of the T7 promoter, positions 917-1026 are the nucleotide sequence of the mRNA 5'UTR, positions 1027-1056 are the nucleotide sequence of the αvβ3 / β5integrin gene, positions 1057-1692 are the nucleotide sequence of the A-type foot-and-mouth disease VP1 encoding gene, positions 1693-1728 are the nucleotide sequence of the GS connecting peptide encoding gene, positions 1729-2259 are the nucleotide sequence of the HIV GP41 encoding gene, positions 2260-2557 are the nucleotide sequence of the 3'UTR, positions 2558-2667 of SEQ ID No.3 are the nucleotide sequence of the poly(A) gene, and positions 2668-2673 of SEQ ID No.3 are linearization sites.

[0072] In one embodiment of the present application, the recombinant vector described in B3) may be the recombinant expression plasmid FMDV O CHA312010GAG and / or the recombinant expression plasmid FMDV AWH09VP1 HIV Gp41.

[0073] This application also provides any of the following substances:

[0074] C1) mRNA molecule, wherein the mRNA molecule is the mRNA molecule 1 and / or mRNA molecule 2 described above;

[0075] C2) liposome nanoparticles loaded with the mRNA molecule described in C1);

[0076] C3) a monovalent swine foot-and-mouth disease mRNA vaccine whose active ingredient is the mRNA molecule 1 or mRNA molecule 2 according to any one of claims 1 to 6;

[0077] C4) an mRNA molecule composition consisting of mRNA molecule 1 and mRNA molecule 2;

[0078] C5) Liposome nanoparticles loaded with the mRNA molecule composition described in C4).

[0079] This application also provides any of the following applications:

[0080] D1) Use of the substances described in C3), C4) and / or C5) above in the preparation of the above-mentioned bivalent swine foot-and-mouth disease mRNA vaccine;

[0081] D2) Use of the above substances in the preparation of products for preventing swine foot-and-mouth disease virus infection;

[0082] D3) Use of the above substances in the preparation of products for neutralizing swine foot-and-mouth disease virus;

[0083] D4) Use of the above substances in the preparation of products for specific binding to VP1 protein of swine foot-and-mouth disease virus;

[0084] D5) Use of the above substances in the preparation of antibodies against swine foot-and-mouth disease virus;

[0085] Furthermore, the antibody is a neutralizing antibody; and / or

[0086] Furthermore, the swine foot-and-mouth disease virus is foot-and-mouth disease serotype O and / or foot-and-mouth disease serotype A.

[0087] Furthermore, the antibody is a neutralizing antibody; and / or

[0088] Furthermore, the swine foot-and-mouth disease virus is foot-and-mouth disease serotype O and / or foot-and-mouth disease serotype A.

[0089] Compared with the prior art, the beneficial technical effects achieved by this application are as follows:

[0090] In this application, the VP1 proteins of O-type and A-type foot-and-mouth disease were selected as mRNA antigens, aiming to develop an innovative vaccine strategy. Based on the interaction principle between GAG protein and GP41 protein CTD (cytotoxic T lymphocyte domain), we designed a bivalent VLPS (virus-like particle) mRNA vaccine. The core of this vaccine lies in the interaction between GAG fusion O-type VP1 and A-type VP1 fusion GP41, thereby forming an efficient antigen display platform.

[0091] To achieve this goal, when designing chimeric VP1 mRNA, the coding sequence of type A VP1 protein was fused to the N-terminus of the CTD sequence of the GP41 protein of HIV-1. At the same time, the coding sequence of type O VP1 protein was fused to the C-terminus of the HIVGAG protein. In this way, two mRNAs were successfully constructed: one encoding a fusion protein of type A VP1 protein fused to the GP41 of HIV-1, and the other encoding a fusion protein of IVGAG fused to type O VP1 protein. The two mRNAs were encapsulated and immunized in a 1:1 ratio.

[0092] This strategy not only makes full use of the natural assembly mechanism between GAG and GP41, but also achieves comprehensive immune defense against O and A types of foot-and-mouth disease virus by fusing different types of VP1 proteins. The bivalent VLPS mRNA vaccine will provide effective protection for the prevention and control of O and A types of foot-and-mouth disease virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is the physical map of the recombinant expression plasmid FMDV O CHA31 2010GAG VP1.

[0094] Figure 2 This is the physical map of the recombinant expression plasmid FMDV AWH09VP1 HIV Gp41.

[0095] Figure 3 The figure shows the electrophoresis pattern of plasmid transcription products and plasmid.

[0096] Figure 4 This is the electrophoresis pattern of mRNA after capping and purification.

[0097] Figure 5 The results are from Western Blot.

[0098] Figure 6 Immunization process for pigs.

[0099] Figure 7 This is the mRNA encapsulation test report.

[0100] Figure 8 The raw data of particle size and PDI detection of the final product (after dialysis and ultrafiltration concentration). DETAILED DESCRIPTION

[0101] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0102] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0103] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0104] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0105] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0106] BHK cells are products of Beijing Yishengke Biotechnology Co., Ltd.

[0107] Example 1. Obtaining mRNA vector encoding O / AVP1 protein fusion gene

[0108] 1.1. Synthesis of GAG-O-VP1 target gene

[0109] In order to improve the high immunogenicity of wild-type vp1, the O-type foot-and-mouth disease VP1 gene was fused to the 3' end of the GAG ​​functional region gene to obtain the GAG-O-VP1 coding gene.

[0110] Qingke Biotechnology was commissioned to synthesize a recombinant vector containing the GAG-O-VP1 encoding gene, and the nucleotide sequence of the recombinant vector was SEQ ID No. 1. The GAG-O-VP1 encoding gene was a DNA molecule with a nucleotide sequence of positions 898-3645 of SEQ ID No. 1. Among them, positions 898-916 of SEQ ID No. 1 are the nucleotide sequence of T7 promoter, positions 917-1026 are the nucleotide sequence of mRNA 5'UTR, positions 1027-2526 are the nucleotide sequence of HIVGAG encoding gene, positions 2527-2562 are the nucleotide sequence of GS connecting peptide encoding gene, positions 2563-3201 are the nucleotide sequence of O-type foot-and-mouth disease VP1 encoding gene, positions 3202-3231 are the nucleotide sequence of αvβ3 / β5integrin encoding gene, positions 3232-3529 are the nucleotide sequence of 3'UTR, positions 3530-3639 of SEQ ID No. 1 are the nucleotide sequence of poly (A) gene, and positions 3640-3645 of SEQ ID No. 1 are linearization sites. The recombinant expression plasmid obtained by replacing the sequence between hindIII and XbaI of the pcda3.1(+) vector with the DNA molecule shown in position 898-3645 of SEQ ID No.1 and keeping the other nucleotide sequences of the pcda3.1(+) vector unchanged was named as the recombinant expression plasmid FMDV O CHA31 2010GAG VP1. The nucleotide sequence of the recombinant expression plasmid FMDV O CHA31 2010GAG VP1 is SEQ ID No.1. The physical map of the recombinant expression plasmid FMDV O CHA31 2010GAG VP1 is shown in Figure 1 shown.

[0111] The recombinant expression plasmid FMDV O CHA31 2010GAG VP1 can be transcribed to obtain an mRNA molecule whose nucleotide sequence is SEQ ID No.5.

[0112] 1.2. Synthesis of FMDV AWH09VP1 HIV Gp41 target gene

[0113] Qingke Biotechnology was commissioned to synthesize a recombinant vector containing the FMDV AWH09VP1 HIV Gp41 encoding gene, and the nucleotide sequence of the recombinant vector is SEQ ID No. 3. The nucleotide sequence of the FMDV AWH09VP1 HIV Gp41 encoding gene is shown in SEQ ID No. 3, positions 898-2673. Among them, positions 898-916 of SEQ ID No.3 are the nucleotide sequence of T7 promoter, positions 917-1026 are the nucleotide sequence of mRNA 5'UTR, positions 1027-1056 are the nucleotide sequence of αvβ3 / β5integrin gene, positions 1057-1692 are the nucleotide sequence of A-type foot-and-mouth disease VP1 encoding gene, positions 1693-1728 are the nucleotide sequence of GS connecting peptide encoding gene, positions 1729-2259 are the nucleotide sequence of HIV GP41 encoding gene, positions 2260-2557 are the nucleotide sequence of 3'UTR, positions 2558-2667 of SEQ ID No.3 are the nucleotide sequence of poly (A) gene, and positions 2668-2673 of SEQ ID No.3 are linearization sites. The DNA molecule shown in position 917-2673 of SEQ ID No.3 was used to replace the sequence between hindIII and XbaI of the pcda3.1(+) vector, and the other nucleotide sequences of the pcda3.1(+) vector were kept unchanged to obtain the recombinant expression plasmid named as the recombinant expression plasmid FMDV AWH09VP1 HIV Gp41. The nucleotide sequence of the recombinant expression plasmid FMDVAWH09VP1 HIV Gp41 is SEQ ID No.3. The physical map of the recombinant expression plasmid FMDV AWH09VP1 HIV Gp41 is shown in Figure 2 shown.

[0114] The recombinant expression plasmid FMDV AWH09VP1 HIV Gp41 can be transcribed to obtain the mRNA molecule whose nucleotide sequence is SEQ ID No.6.

[0115] Example 2. Obtaining mRNA LNP encoding O / AVP1 protein fusion gene

[0116] The correctly sequenced recombinant plasmids FMDV O CHA31 2010GAG VP1 and FMDV AWH09VP1 HIV Gp41 were transformed into DH5a, and the recombinant strains were named DH5a / FMDV O CHA31 2010GAG VP1 strain and DH5a / FMDV AWH09VP1 HIV Gp41 strain, respectively.

[0117] 2.1. Bacteria expansion

[0118] DH5a / FMDV O CHA31 2010GAG VP1 and DH5a / FMDV AWH09VP1 HIV Gp41 strains were inoculated into LB medium containing 100 μg / mL penicillin sodium and cultured at 37°C and 220 rpm for 12 hours. The plasmids were extracted using Tiangen endotoxin-free kit for later use.

[0119] 2.2. Vector linearization

[0120] Plasmids with T7 promoter can be used as transcription templates. The linearization and purity of the plasmid affect the transcription yield and RNA integrity. Circular plasmids will transcribe RNA products of different lengths due to the lack of effective termination. In order to obtain specific RNA, the vector must be linearized.

[0121] The concentration and purity of the recombinant plasmid FMDV O CHA31 2010GAG VP1 and the recombinant expression plasmid FMDVAWH09VP1HIV Gp41 extracted in large quantities were determined using an ultra-micro spectrophotometer. The enzyme digestion system was: 2 μL of ECORV, 10 μL of 10×Buffer, 50 μg of recombinant plasmid, and ddH2O was added to 100 μL. The reaction conditions were: 37°C overnight.

[0122] 2.3 In vitro transcription reaction

[0123] T7 High Yield RNA Transcription kit (Nearshore Protein, catalog number E131) was used for in vitro transcription according to the product instructions. The synthesized RNA was analyzed by electrophoresis and purified, and can be used for downstream experiments. The electrophoresis results are shown in Figure 3 . Figure 3 MK in the middle is DL5000, lane 1 is the RNA transcribed from the recombinant plasmid FMDV O CHA31 2010GAG VP1, lane 2 is the RNA transcribed from FMDV AWH09VP1HIV Gp411, lane 3 is the recombinant plasmid FMDV O CHA31 2010GAGVP1, and lane 4 is the recombinant plasmid FMDV AWH09VP1 HIV Gp411.

[0124] 2.4 Product Purification

[0125] 1) Add 30 μL RNase Free Water and 30 μL LithiumChloride Precipitation Solution to 20 μL of transcription product in the previous step.

[0126] 2) After mixing, place at -20℃ for at least 30 minutes.

[0127] 3) Centrifuge at 12000 pm for 15 min, remove the supernatant and collect the precipitate.

[0128] 4) Wash three times with pre-cooled 70% ethanol.

[0129] 5) Detection after reconstitution with RNase Free Water.

[0130] 2.5. mRNA capping reaction

[0131] The kit cap1 Capping System (nearshore protein, Cat. No.: M082) was selected and operated according to the product instructions. The reaction system is shown in Table 1. Follow the steps below:

[0132] 1) Dilute an appropriate amount of RNA to 100uL with RNase-free water;

[0133] 2) Heat the RNA at 65°C for 5 minutes and then place on ice for 5 minutes;

[0134] 3) Add the components in Table 3 in sequence and react at 37°C for 30 min.

[0135] Table 1 Reaction system of mRNA capping reaction

[0136] Components Volume (μL) Denatured RNA 67 10×Capping action Buffer 10 GTP(10mM) 10 SAM(20mM) 2.5 Recombinant RNase Inhibitor(40U / μL) 2.5 mRNA Cap 2'-O-Methyltransferase(100U / μL) 4 Vaccinia Capping Enzyme(10U / μL) 4 RNase Free Water Up to 100

[0137] 2.6. Cap-mRNA purification is the same as 2.4.

[0138] The RNA capping product obtained by transcription of the recombinant plasmid FMDV O CHA31 2010GAG VP1 was named mRNA molecule 1, and the RNA capping product obtained by transcription of the recombinant plasmid FMDV AWH09VP1 HIV Gp411 was named mRNA molecule 2. The product purification results are shown in Figure 4 . Figure 4 The results show that after capping and purification, obvious mRNA bands can be seen on the DNA agarose gel. Lanes 1 and 2 are mRNA molecule 1; Lane 3 is mRNA molecule 2. After capping, the mRNA can be used for subsequent encapsulation experiments.

[0139] The nucleotide sequence of mRNA molecule 1 is SEQ ID No.5; the 5' end of SEQ ID No.5 carries the mRNA cap structure Cap1 (m7G(5')ppp(5')(2'OMeA)pG). Positions 1-110 of SEQ ID No.5 are the mRNA sequence of 5'-UTR, positions 111-1610 are the mRNA sequence of GAG, positions 1611-1646 are the mRNA sequence of the connecting peptide, positions 1647-2285 are the mRNA sequence of FMDVO type VP1, positions 2286-2315 are the mRNA sequence of αvβ3 / β5integrin, positions 2316-2613 are the mRNA sequence of 3'-UTR, and positions 2614-2723 are the mRNA sequence of poly(A).

[0140] mRNA molecule 1 can express fusion protein FMDV O CHA31 2010GAG VP1, and the amino acid sequence of fusion protein FMDV O CHA312010GAG VP1 is SEQ ID No. 2. Among them, SEQ ID No. 2 1-500 is the amino acid sequence of HIVGAG core protein, SEQ ID No. 2 501-512 is the amino acid sequence of connecting peptide, 513-725 is the amino acid sequence of O-type foot-and-mouth disease VP1 protein, and SEQ ID No. 2 726-734 is the amino acid sequence of immune enhancement αvβ3 / β5integrin protein.

[0141] The nucleotide sequence of mRNA molecule 2 is SEQ ID No. 6. The 5' end of SEQ ID No. 6 carries the cap structure Cap1 (m7G (5') ppp (5') (2'OMeA) pG) of mRNA. Positions 1-110 of SEQ ID No. 6 are the mRNA sequence of 5'-UTR, positions 111-140 are the mRNA of αvβ3 / β5integrin gene, positions 141-776 are the mRNA sequence of FMDV A type VP1, positions 777-812 are the mRNA sequence of GS connecting peptide, positions 813-1343 are the mRNA sequence of GP41, positions 1344-1641 are the mRNA sequence of 3'-UTR, and positions 1642-1751 are the mRNA sequence of poly (A).

[0142] mRNA molecule 2 can express fusion protein FMDV AWH09VP1 HIV Gp41, and the amino acid sequence of fusion protein FMDV AWH09VP1HIV Gp41 is SEQ ID No. 4. Among them, SEQ ID No. 4, 1-10 are the amino acid sequence of immune enhancement αvβ3 / β5integrin protein, SEQ ID No. 2, 11-222 are the amino acid sequence of type A foot-and-mouth disease VP1 protein, 223-234 are the amino acid sequence of the connecting peptide, and 235-410 are the amino acid sequence of HIVGP41 protein.

[0143] 2.7. Cap-mRNA in vitro cell transfection assay

[0144] Lipofectamine 2000 was used for mRNA cell transfection experiment. The specific operation steps are as follows:

[0145] BHK cells were cultured at 1×10 5 The number of cells / well was inoculated into a 6-well plate, and DMEM medium containing 10% high-quality fetal bovine serum was selected for culture. The cells were cultured in a 37°C incubator containing 5% CO2 until the cell confluence was close to 80%; the serum-free DMEM medium was replaced, and 500 μL was added to each well;

[0146] Step 1: One day before transfection, seed the cells at an appropriate cell density on a 6-well culture plate; during transfection, the cells should reach 90-95% confluence;

[0147] Step 2, mix solution 1 and solution 2 and place at room temperature for 20 minutes;

[0148] Solution 1: 240 μL DMEM serum-free medium) + 10 μL Lipofectamine TM 3000 (Thermo Fisher Scientific, catalog number L3000015) / well, incubate for 5 min (total volume per well 250 μL);

[0149] Solution 2: 225 μL serum-free medium (DMEM serum-free medium + 25 μL (4 μg) mRNA / well (total volume per well 250 μL);

[0150] Step 3: Rinse the cells in the 6-well plate in step 1 twice with serum-free medium, and then add 1 mL of serum-free medium;

[0151] Step 4: Add the mixture of solution 1 and solution 2 dropwise into the wells, shake the culture plate and gently mix; incubate at 37°C, 5% CO2 for 6 hours;

[0152] Step 5: Replace the culture medium with DMEM containing 10% fetal bovine serum and culture at 37°C and 5% CO2. After 24 hours, take the supernatant for immunoblotting.

[0153] 2.8. Western Blot

[0154] After transfection of BHK cells with FMDV O CHA31 2010GAG VP1 and FMDV AWH09VP1 HIV Gp41 mRNA for 12 hours, 40 μL of the cell mixture was taken, and 1 / 5 volume of 5×SDS loading solution was added. After mixing, the mixture was boiled in boiling water for 10 minutes, and 20 μg of the sample was loaded and subjected to 10% SDS-PAGE gel electrophoresis (polyacrylamide gel electrophoresis). The specific operation steps are as follows:

[0155] 1) Electrophoresis: First, 120V for 30min to allow the protein samples to enter the separation gel uniformly, then adjust the voltage to 120V and end the electrophoresis when the band approaches the bottom of the gel block;

[0156] 2) Transfer: The proteins in the gel were transferred to a 0.45 μm nitrocellulose membrane (NC membrane) using a wet method, with the parameters set at 120 V for 90 min.

[0157] 3) Blocking: Place the NC membrane in 5% skim milk and block for 2 h;

[0158] 4) Washing: After blocking, wash 3 times with TBST to remove the skim milk residue on the membrane surface;

[0159] 5) Primary antibody incubation: 5% BSA was used to prepare a 1:2000 dilution of the primary antibody, which was a mouse His antibody (Beijing Quanshijin Biotechnology Co., Ltd. Anti-His Mouse Monoclonal Antibody (catalog number: HT501-01), incubate at room temperature for 2 hours or at 4°C overnight;

[0160] 6) Washing: Wash with TBST 3 times, 5 minutes each time;

[0161] 7) Secondary antibody incubation: Use TBST to prepare a 1:2000 dilution of secondary antibody (HRP-labeled goat anti-mouse IgG, Beijing Solebao Biological Product No.: SA36), goat anti-mouse IgG antibody, and incubate at room temperature with shaking for 120 minutes;

[0162] 8) Washing: Wash with TBST 3 times, 10 min each time;

[0163] 9) Development: Add ECL developer solution to the membrane and stop color development after 1 minute.

[0164] Results Figure 5 , Figure 5 MK in the middle is full-form gold Proteinruler II12-120KD, lane 1 is cell control; lane 2 is fusion protein FMDV AWH09VP1 HIV Gp41; lane 3 is fusion protein FMDV O CHA31 2010GAG VP1.

[0165] 2.9 Encapsulation

[0166] mRNA molecule 1 and mRNA molecule 2 were mixed at a molar ratio of 1:1 and then encapsulated. The encapsulated samples were tested and the results were as follows: mRNA-LNP encapsulation efficiency was 97.5%, mRNA concentration was 62.82ng / μL, and encapsulation volume was 6mL ( Figure 7 ). The random sampling test results showed that the mRNA-LNP particle size was 80.05-80.32nm, PDI: 0.085-0.113, indicating that the mRNA-LNP particle size and uniformity were both qualified ( Figure 8 ).

[0167] The encapsulation was entrusted to Shanghai Pengzan Biotechnology Co., Ltd. The specific process is as follows:

[0168] 1). Prepare the compound lipid-ethanol solution:

[0169] Cationic lipid material D-Lin-MC3-DMA, cholesterol (CHOLESTEROL), auxiliary lipid (DSPC) and PEG lipid (PEG2000-DMG) were selected as components of LNP. A lipid mixture was prepared according to the molar ratio of D-Lin-MC3-DMA: CHOLESTEROL: DSPC: PEG2000-DMG = 50:38.5:10:1.5, and the lipid mixture was dissolved in ethanol to prepare a 12mM (about 7.5mg / mL) composite lipid-ethanol solution.

[0170] 2). Prepare citric acid buffer

[0171] Use ultrapure water to prepare 100mM citric acid monohydrate (molecular weight: 210.14, weigh 1.05g) and sodium citrate dihydrate (molecular weight: 294.10, weigh 1.47g) solutions, 50mL each. Take 33.0mL citric acid solution and 17.0mL sodium citrate solution and mix them, and adjust to pH = 4 with NaOH. After mixing 1:1, use ultrapure water to make up to 100mL, add DEPC with a final concentration of 0.1% and let stand for 30 minutes. Autoclave to remove DEPC to obtain 50mM citric acid buffer (included in the encapsulation kit). Citric acid buffer and phospholipids prepared with ethanol need to be filtered with 0.22μm MCE filter membrane (citric acid buffer) and PTFE filter membrane (phospholipids prepared with ethanol) respectively to ensure that the final product does not contain tiny solid particles. Dissolve the mRNA mixture in citric acid buffer to prepare mRNA-sodium citrate buffer with an RNA concentration of 128.52ng / μL.

[0172] 3) Microfluidic mixing

[0173] The mRNA-citric acid buffer solution / compound lipid-ethanol solution were mixed in an intelligent LNP synthesizer at a flow rate ratio of mRNA-citric acid buffer solution / compound lipid-ethanol solution flow rate ratio (FRR) = 3 to obtain an encapsulation complex of lipid nanoparticles and mRNA (mRNA-LNP).

[0174] 4) Encapsulation result detection

[0175] ① Use Qubit 4.0 Invitrogen to determine the LNP encapsulation efficiency and utilization rate.

[0176] ② Detection of particle size and PDI: The particle size and uniformity of mRNA-LNP products can be measured using a dynamic light scattering instrument.

[0177] Example 3: Animal Experiment

[0178] 3.1 Experimental Materials

[0179] (1) The experimental pigs were selected from 5 three-way piglets (three-way pigs) weighing about 10 kg from a non-immune healthy pig herd (purchased from a pig farm in Fangshan), and the experiment was carried out under strict isolation conditions in the company's animal room.

[0180] (2) mRNA vaccine immunization and kit testing

[0181] The encapsulated mixed mRNA vaccine was randomly selected and administered to the test pigs by intramuscular injection. The dosage was 10 μg / head. Five pigs were immunized. The day of administration was recorded as the first day of the first immunization. The second immunization (second immunization) was performed 14 days after the first immunization (the 15th day of the first immunization). Blood was collected on the 7th, 14th, 21st and 28th days after the first immunization to detect serum antibody titers. The foot-and-mouth disease O type antibody liquid phase blocking ELISA detection kit and the foot-and-mouth disease A type antibody liquid phase blocking ELISA detection kit (Lanzhou Animal Research Biotechnology Co., Ltd.) were used to detect antibodies according to the product instructions. The immunization process was as follows Figure 6 .

[0182] 3.3 Determination of serum antibody titer

[0183] For the 4 virus antigen control wells, discard the highest and lowest D 450nm values, calculate the average D 450nm value of the remaining 2 wells, and divide it by 2, which is the 50% control value. This value is the critical value, which represents the control D 450nm value that blocks 50% of the reaction. The wells with D450nm values ​​of the tested serum greater than the critical value are negative wells, and the wells with D450nm values ​​less than or equal to the critical value are positive wells. If the critical value is the same as the D450nm value of the positive well with the highest dilution multiple, the highest dilution multiple of the positive well of the tested serum is used as the antibody titer of the serum; if the critical value is between the D 450nm values ​​of two dilution wells, the antibody titer is the middle value of the antilogarithm of the dilution multiples of the adjacent positive wells and negative wells. For example, if it is between 1:64 (antilogarithm is 1.8) and 1:128 (antilogarithm is 2.1), the antibody titer of the serum is judged to be 1:90 (antilogarithm is 1.95).

[0184] For specific determination, see the antibody titer calculation comparison table in Table 2. Antibody titers greater than or equal to 1:128 are judged as positive for foot-and-mouth disease O / A type antibodies; 1:64 to 1:128, it is judged as suspicious; less than 1:64, it is judged as negative. Suspicious serum samples can be tested again. If the antibody titer is greater than or equal to 1:128, it is judged as positive, and less than 1:128 is judged as negative.

[0185] Table 2 Antibody titer calculation comparison table

[0186]

[0187] 3.4 Determination of the relationship between antibody results and protection rate

[0188] Relationship between ELISA antibody titer and protection of immunized animals against virus infection

[0189] Cattle and sheep:

[0190] Antibody titer ≥1:128, more than 99% protection;

[0191] Antibody titer ≤1:16, no protection;

[0192] The antibody titer was between 1:22-1:90, with 50% protection.

[0193] pig:

[0194] Antibody titer ≥1:64, more than 99% protection;

[0195] Antibody titer <1:4, no protection;

[0196] The antibody titer was 1:4-1:45, with 50% protection.

[0197] The test instrument is Bio-Rad Imark enzyme-linked immunosorbent assay analyzer.

[0198] 3.5 Analysis of the results of the foot-and-mouth disease efficacy experiment

[0199] Foot-and-mouth disease is listed as a legally reportable severe infectious disease by the World Organization for Animal Health. At present, developing countries mainly adopt the prevention and control strategy of vaccine immunization, and the inactivated foot-and-mouth disease vaccine widely used for vaccine immunization has the disadvantages of immunity duration and worry about incomplete inactivation. Therefore, we used the MRNA expression technology platform, the HIVGAG protein self-assembly VLPS characteristics, and the GP41 can be displayed on VLPS to produce high immunogenicity, and successfully constructed a bivalent OA VP1vlpsmRNA vaccine. Pigs, one of the natural hosts of foot-and-mouth disease, were used as experimental animals for immunization experiments. The experimental animals in the vaccine group began to produce a large number of antibodies 14 days after the first immunization, and a higher level of antibodies began to be produced on the 7th day of the second immunization. At the same time, this result also confirmed that the VP1vlpsmRNA vaccine can also induce immunity in animals. This shows that the vaccine has the prospect of being used for foot-and-mouth disease immunization and can quickly enable susceptible animals to obtain immune protection. On the 28th day after immunization, the antibody level continued to increase, and there was no downward trend, which may also be due to the high dose of immunization. This has opened up a new path for the development of new foot-and-mouth disease vaccines and improved conventional foot-and-mouth disease vaccines. The specific results are shown in Table 3.

[0200] Table 3. Antibody titer of swine foot-and-mouth disease serum in mRNA immunoassay

[0201]

[0202] Table 4 Partial sequences in this application

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. A bivalent swine foot-and-mouth disease mRNA vaccine, characterized in that: The bivalent swine foot-and-mouth disease mRNA vaccine contains mRNA molecule 1 and mRNA molecule 2; The mRNA molecule 1 encodes protein 1, and the protein 1 is a protein whose amino acid sequence is SEQ ID No. 2; The mRNA molecule 2 encodes protein 2, and the protein 2 is a protein whose amino acid sequence is SEQ ID No.

4.

2. The bivalent swine foot-and-mouth disease mRNA vaccine according to claim 1, characterized in that The nucleotide sequence of the mRNA molecule 1 is SEQ ID No. 5; The nucleotide sequence of the mRNA molecule 2 is SEQ ID No.

6.

3. The bivalent swine foot-and-mouth disease mRNA vaccine according to claim 1 or 2, characterized in that: The bivalent swine foot-and-mouth disease mRNA vaccine is a substance formed by encapsulating the mRNA molecule 1 and the mRNA molecule 2 in liposome nanoparticles.

4. The bivalent swine foot-and-mouth disease mRNA vaccine according to claim 3, characterized in that: In the bivalent swine foot-and-mouth disease mRNA vaccine, the mass ratio of the mRNA molecule 1 to the mRNA molecule 2 is 1:

1.

5. Biomaterial, characterized in that The biological material is selected from any one of the following: B1) a DNA molecule encoding the mRNA molecule 1 and the mRNA molecule 2 according to any one of claims 1 to 4; B2) an expression cassette containing the DNA molecule described in B1); B3) a recombinant vector containing the DNA molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the DNA molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

6. The biomaterial according to claim 5, characterized in that B1) The DNA molecule described is a DNA molecule having a nucleotide sequence of 917-3645 positions of SEQ ID No.1 and a DNA molecule having a nucleotide sequence of 917-2673 positions of SEQ ID No.

3.

7. Any of the following substances: C1) mRNA molecules, wherein the mRNA molecules are the mRNA molecule 1 and the mRNA molecule 2 according to any one of claims 1 to 4; C2) liposome nanoparticles loaded with the mRNA molecule described in C1); C3) an mRNA molecule composition consisting of the mRNA molecule 1 and the mRNA molecule 2 according to any one of claims 1 to 4; C4) Liposome nanoparticles loaded with the mRNA molecule composition described in C3).

8. Use of the substance described in C3) or C4) in claim 7 in the preparation of the bivalent swine foot-and-mouth disease mRNA vaccine described in any one of claims 1-4.

Images