MRNA vaccine for preventing and / or treating pseudorabies and application thereof

Abstract

The invention discloses an mRNA (messenger ribonucleic acid) vaccine for preventing and / or treating pseudorabies and application of the mRNA vaccine. Codon optimization is performed on a gene sequence of a pseudorabies virus gD protein antigen to obtain an optimized gene sequence of the pseudorabies virus gD protein antigen; the pseudorabies virus truncated gD protein antigen is obtained by optimizing the sequence of the pseudorabies virus gD protein antigen, the gene sequence of the truncated gD protein antigen is subjected to codon optimization, and experiments show that the optimized pseudorabies virus truncated gD protein antigen has higher expression quantity; the sequence-optimized pseudorabies virus truncated gD protein is further prepared into an mRNA vaccine, and after the mRNA vaccine is used for immunizing mice and piglets, it is found that the mRNA vaccine of the sgD protein can play an immune protection role on the mice and the piglets, and the mRNA vaccine is obviously superior to commercially available pseudorabies inactivated vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus vaccines, and relates to an mRNA vaccine and an application thereof, and specifically to an mRNA vaccine for preventing and / or treating pseudorabies and an application thereof. Background Art

[0002] Pseudorabies virus (PRV) is a herpesviridae virus that mainly infects pigs and can cause an acute, febrile, highly contagious disease, namely pseudorabies. This disease not only affects the health of pigs, but also causes economic losses. The clinical symptoms of pseudorabies virus infection may vary depending on the host species. In pigs, infection with PRV is usually manifested by fever, loss of appetite, difficulty breathing, neurological symptoms (such as convulsions, ataxia), and high mortality. In dogs, infection with PRV may cause symptoms such as fever, vomiting, diarrhea, and in severe cases may be fatal.

[0003] In pseudorabies virus, glycoprotein D (gD) plays an important role. gD is one of the key factors for the virus to enter the host cell. It mediates the adsorption and membrane fusion process of the virus by binding to specific receptors on the surface of the host cell. This binding is an essential step in the early stage of viral infection because it triggers subsequent viral endocytosis and membrane fusion, allowing the viral genome to enter the interior of the host cell. Studies have shown that gD protein can not only induce the host to produce specific antibodies, but also stimulate the production of neutralizing antibodies, which are essential for protecting the host from pseudorabies virus. Therefore, gD protein is an important target in vaccine design and antiviral drug development. In vaccine development, gD protein can be used as a vaccine component to induce the host to produce a specific immune response against PRV. Studies have shown that gD protein expressed by mammalian cells has good immunogenicity and can stimulate the production of high levels of anti-pseudorabies virus-specific antibodies and neutralizing antibodies in mice and piglets.

[0004] At present, the technology related to mRNA vaccines is developing rapidly. Compared with traditional vaccines, mRNA vaccines can induce immune responses more quickly, provide faster and more lasting immune protection, and are safer. In addition, mRNA vaccines have high flexibility and can quickly adjust and design different antigen protein coding sequences according to the diversity and variation of the virus. This flexibility enables mRNA vaccines to quickly respond to the challenges of new virus strains or mutant strains, ensuring the effectiveness and adaptability of the vaccine. Since mRNA vaccines do not require processes such as live virus cultivation and inactivation treatment, the production process is simpler and more cost-effective. At the same time, the production scale and degree of automation are high, which is conducive to large-scale production and supply.

[0005] However, there is currently no mRNA vaccine for pseudorabies virus. If an mRNA vaccine based on gD protein with high expression and immunogenicity can be developed and designed, it will provide an effective immune strategy for the control and prevention of pseudorabies. Summary of the invention

[0006] The object of the present invention is to provide an mRNA vaccine for preventing and / or treating pseudorabies and its application, by optimizing the antigen sequence of pseudorabies G protein and preparing it into an mRNA vaccine, which can effectively immunize and protect the administered host from infection by rabies virus and has good safety.

[0007] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0008] In one aspect, the present invention provides a polynucleotide comprising a first nucleotide sequence encoding a pseudorabies virus gD protein or an immunogenic fragment thereof, wherein the amino acid sequence of the pseudorabies virus gD protein or an immunogenic fragment thereof has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:1.

[0009] In one or more embodiments of the present invention, the sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:2 or SEQ ID NO:3.

[0010] In one or more embodiments of the present invention, the immunological fragment is a truncated gD protein of pseudorabies virus, and the amino acid sequence of the truncated gD protein of pseudorabies virus (sgD protein) has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:4.

[0011] In one or more embodiments of the present invention, the sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:5 or SEQ ID NO:6.

[0012] In one or more embodiments of the present invention, the 5' end of the first nucleotide is further connected to a second nucleotide sequence encoding a signal peptide;

[0013] Preferably, the signal peptide is tPA, IL-2, IL-6 or a neuropeptide, and the neuropeptide is preferably VIP, CGRP or NPY;

[0014] Further preferably, the 5' end of the second nucleotide sequence is connected to a 5'UTR sequence, and the nucleotide sequence of the 5'UTR sequence is preferably as shown in SEQ ID NO: 9, or

[0015] The 3' end of the first nucleotide is also connected to a 3'UTR sequence, and the end of the 3'UTR sequence is also connected to a PolyA sequence, and the nucleotide sequence of the 3'UTR sequence is preferably as shown in SEQ ID NO: 10;

[0016] Also preferably, a T7 promoter is further provided at the 5' end of the 5'UTR sequence, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 7. The polynucleotide is preferably provided with a FLAG tag.

[0017] In one or more embodiments of the present invention, the polynucleotide is DNA or RNA;

[0018] Preferably, when the polynucleotide is RNA, the 5' end of the 5'UTR sequence of the RNA is further provided with a 5' capping structure, and the 5' capping structure is preferably Cap1 (methylation of the ribose of the adjacent nucleotide of m7GpppN), Cap2 (methylation of the ribose of the second nucleotide downstream of m7GpppN), Cap3 (methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (methylation of the ribose of the fourth nucleotide downstream of m7GpppN) or Cap 101.

[0019] On the other hand, the present invention also provides a vector constructed from the above-mentioned polynucleotide.

[0020] In another aspect, the present invention also provides a cell transformed by the above-mentioned vector.

[0021] In yet another aspect, the present invention further provides a composition for preventing and / or treating pseudorabies, comprising the above-mentioned polynucleotide or mRNA transcribed from the above-mentioned vector.

[0022] In one or more embodiments of the present invention, the composition is an mRNA vaccine, which includes mRNA selected from those expressing pseudorabies virus gD protein and / or immunogenic fragments thereof.

[0023] In one or more embodiments of the present invention, the sequence of the mRNA expressing the pseudorabies virus gD protein or an immunogenic fragment thereof has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO: 3 or 6.

[0024] In one or more embodiments of the present invention, the composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is preferably a liposome, and the mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles of the liposome, and the particle size of the liposome nanoparticles is preferably 50 to 200 nm;

[0025] Preferably, the liposome nanoparticles are selected from at least one of cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol and DMG-2000;

[0026] More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts of cationic lipids, 5-15 parts of distearoylphosphatidylcholine (DSPC), 24-52 parts of cholesterol and 1-2 parts of DMG-2000;

[0027] Further preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts of cationic lipids, 10 parts of distearoylphosphatidylcholine (DSPC), 38.5 parts of cholesterol and 1.5 parts of DMG-2000.

[0028] In yet another aspect, the present invention further provides a use of the above-mentioned polynucleotide, vector, or composition in the preparation of a drug for preventing and / or treating pseudorabies.

[0029] In another aspect, the present invention also provides a method for using the above-mentioned polynucleotide, vector, composition or mRNA vaccine to prevent and / or treat pseudorabies, comprising administering an appropriate amount of the polynucleotide, vector or composition to a host in need thereof, wherein the host may be a human or an animal such as a cat, dog, pig, or the like.

[0030] In particular, administration may be orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

[0031] Compared with the prior art, the mRNA vaccine for preventing and / or treating pseudorabies of the present invention and its application obtain the optimized gene sequence of pseudorabies virus gD protein antigen by codon optimization of the gene sequence of pseudorabies virus gD protein antigen; the pseudorabies virus truncated gD protein antigen is obtained by sequence optimization of pseudorabies virus gD protein antigen, and the gene sequence of the truncated gD protein antigen is codon optimized. Experiments show that the expression level of the optimized pseudorabies virus truncated gD protein antigen is higher; the sequence-optimized pseudorabies virus truncated gD protein is further prepared into an mRNA vaccine, and after using it for immunization of mice and piglets, it is found that the mRNA vaccine of the sgD protein can play an immune protective role in mice and piglets, and is significantly better than the commercially available pseudorabies inactivated vaccine, and is relatively safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is an agarose gel electrophoresis diagram of the transcribed mRNA in Example 2 of the present invention, wherein Marker is RNAmarker6000, 1 is the mRNA of the sgD protein, and 2 is the mRNA of the gD protein;

[0034] Figure 2 This is a Western Blot diagram of protein expression verification of the transcribed mRNA in Example 3 of the present invention, wherein Marker is a protein marker, 1 is the mRNA of the gD protein, and 2 is the mRNA of the sgD protein;

[0035] Figure 3 This is a particle size distribution diagram of the mRNA vaccine of the sgD protein packaged in Example 4 of the present invention;

[0036] Figure 4 This is a flow chart of the animal experiment in Example 5 of the present invention;

[0037] Figure 5 The antibody statistics of mice immunized with the mRNA vaccine of the sgD protein in Example 5 of the present invention are shown in Figure 5, wherein A is a statistical diagram of serum IgG antibodies, and B is a statistical diagram of serum neutralizing antibodies, wherein # indicates a comparison with the blank control group, and * indicates a comparison with the positive control group, ##: P<0.01; *: P<0.05; **: P<0.01;

[0038] Figure 6 This is a statistical result diagram of the survival rate of mice immunized with the mRNA vaccine of the sgD protein in Example 5 of the present invention after virus attack;

[0039] Figure 7 This is a pathological section of mice immunized with the mRNA vaccine of the sgD protein in Example 5 of the present invention after being challenged with the virus;

[0040] Figure 8The antibody statistics of pigs immunized with the mRNA vaccine of the sgD protein in Example 6 of the present invention are shown in Figure 6, where A is a statistical chart of serum IgG antibodies, and B is a statistical chart of serum neutralizing antibodies, where # indicates a comparison with the blank control group, and * indicates a comparison with the positive control group, ##: P<0.01; *: P<0.05; **: P<0.01;

[0041] Fig. 9 This is a statistical result diagram of the survival rate of pigs immunized with the mRNA vaccine of the sgD protein in Example 6 of the present invention after being challenged with a virus;

[0042] Fig.10 This is a pathological section of pigs immunized with the mRNA vaccine of sgD protein in Example 6 of the present invention after challenge. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, the “reagents” or “materials” herein can be commercially obtained from conventional channels.

[0045] Unless otherwise specified, "sgD" or "sgD protein" herein refers to "pseudorabies virus truncated gD protein".

[0046] Unless otherwise specified, "PRV-gD mRNA" herein refers to "mRNA of gD protein".

[0047] Unless otherwise specified, "PRV-sgD mRNA" herein refers to "mRNA of sgD protein".

[0048] Unless otherwise specified, “PRV-sgD LNP-mRNA” in this article refers to “mRNA vaccine of sgD protein”.

[0049] Unless otherwise specified, "sgD mRNA" and "sgD mRNA vaccine" in this article refer to "mRNA vaccine of sgD protein".

[0050] Example 1. Acquisition of antigen expression vector of pseudorabies virus mRNA vaccine

[0051] In order to improve the translation efficiency of the target mRNA vaccine, an attempt was made to optimize the gene sequence of the rabies virus gD protein antigen (amino acid sequence is SEQ ID NO: 1) by codon optimization to obtain the optimized gene sequence of the pseudorabies virus gD protein antigen, which is shown in SEQ ID NO: 2.

[0052] In addition, the sequence of the pseudorabies virus gD protein antigen was optimized to obtain a pseudorabies virus truncated gD protein antigen, whose amino acid sequence is shown in SEQ ID NO:4; in order to further improve the translation efficiency of the obtained mRNA vaccine, an attempt was made to further codon optimize the gene sequence of the optimized pseudorabies virus truncated gD protein antigen to obtain the optimized gene sequence of the pseudorabies virus truncated gD protein antigen, and the gene sequence (first nucleotide sequence) is shown in SEQ ID NO:5.

[0053] The designed sequence is then synthesized and the antigen expression vector of the mRNA vaccine is constructed, specifically: the following gene sequences are synthesized by Shanghai Shenggong Biological Company: from 5' to 3', respectively, T7 promoter (SEQ ID NO: 7), 5'UTR (SEQ ID NO: 9), kozak sequence (GCCACC), tPA signal peptide sequence (SEQ ID NO: 8), gD nucleotide sequence (SEQ ID NO: 2) or sgD nucleotide sequence (SEQ ID NO: 5), 3'UTR (SEQ ID NO: 10), polyA (SEQ ID NO: 11). FLAG tags are added after gD protein and sgD protein to verify protein expression. The target gene sequence is constructed into a cloning vector by using genetic engineering technology, and transformed into Escherichia coli competent cells for mass culture of bacteria and mass extraction of plasmids.

[0054] The sequences of the above designed and synthesized sequences and the sequences of the antigenic protein are as follows:

[0055] The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO:9:

[0056] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0057] The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO: 10:

[0058] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCC CTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0059] The sequence of polyadenylic acid (polyA) is shown in SEQ ID NO: 11, which contains 104 bases A:

[0060] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0061] The DNA sequence of the T7 promoter is shown in SEQ ID NO:7:

[0062] TAATACGACTCACTATAGG

[0063] The DNA sequence of the signal peptide tPA sequence is shown in SEQ ID NO:8:

[0064] ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC

[0065] The gD antigen DNA sequence of PRV is shown in SEQ ID NO: 2:

[0066]

[0067] The amino acid sequence of the gD antigen of PRV is shown in SEQ ID NO: 1:

[0068] MLLAALLAALVARTTLGADVDAVPAPTFPPPAYPYTESWQLTLTTVPSPFVGPADVYHTRPLEDPCGVVALISDPQVDRLLNEAVAHRRPTYRAHVAWYRIADGCAHLLYFIEY X(A / T) DCDPRQIFGRCRRRTTPMW WTPSADYMFPTEDELGLLMVAPGRFNEGQYRRLVSVDGVNILTDFMVALPEGQECPFARVDQHRTYKFGACWSDDSFKRGVDVMRFLTPFYQQPPHREVVNYWYRKNGRTLPRAYAAATPYAIDPARPSAGSPRPRPRPRPRPRPKPEPAPATPAPPGRLPEPATRDHAAGGRPTPRPPRPETPHRPFAPPAVVPSGWPQPAEPFPPRTTAAPGVSRHRSVIVGTGTAMGALLVGVCVYIFFRLRGAKGYRLLGGPADADELKAQPGPDYKDDDDK (the italic bold underlined part indicates that the position X can be A or T). In this embodiment, X is A.

[0069] The gD antigen mRNA sequence of PRV is shown in SEQ ID NO:3:

[0070]

[0071] The DNA sequence of the sgD antigen of PRV is shown in SEQ ID NO:5:

[0072]

[0073] The amino acid sequence of the sgD antigen of PRV is shown in SEQ ID NO: 4:

[0074] MLLAALLAALVARTTLGADVDAVPAPTFPPPAYPYTESWQLTLTTVPSPFVGPADVYHTRPLEDPCGVVALISDPQVDRLLNEAVAHRRPTYRAHVAWYRIADGCAHLLYFIEYADCDPRQIFGRCRRRTTPMWWTPSADYMFPTEDELGLLMVAPGRFNEGQYRRLVSVDGVNILTDFMVALPEGQECPFARVDQHRTYKFGACWSDDSFKRGVDVMRFLTPFYQQPPHREVVNYWYRKNGRTLPRAYAAATPYAIDPARPSAGSPRPRPRPRPRPRPKPEPAPATPAPPGRLPEPATRDHAAGGRPTPRPPRPETPHRPFAPPAVVPSGWPQPAEPFPPRTTAAPGVSRHRSVDYKDDDDK

[0075] The mRNA sequence of the sgD antigen of PRV is shown in SEQ ID NO: 6:

[0076]

[0077] Example 2. mRNA transcription verification test

[0078] The plasmids pUC57-gD and pUC57-sgD obtained in Example 1 were digested with Bsa I restriction endonuclease from Novagen Biotech and reacted at 37° C. for 1 hour to linearize them. The DNA template was recovered using a DNA recovery kit from TransGen. The linearization system is shown in Table 1.

[0079] Table 1 Plasmid linearization reaction system

[0080]

[0081] The mRNA was synthesized in vitro using the T7 transcription kit of Yisheng Biotechnology, and the mRNA cap structure was added using Cap101 of Cangzhou Weixin Biotechnology. The reaction was carried out at 37°C for 3 hours. The specific reaction conditions are shown in Table 2.

[0082] Table 2 In vitro transcription reaction system

[0083]

[0084] After the reaction, add 1.5 times the volume of lithium chloride solution to the system and let it stand at -20°C for 1 hour to precipitate mRNA. Then, centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, and redissolve the mRNA with DEPC-treated water. The concentration of RNA was measured using the Nanodrop device and stored at -80°C. Prepare 1% agarose gel and perform electrophoresis to evaluate the quality of in vitro synthesized RNA. The results are shown in Figure 1 shown.

[0085] from Figure 1 It can be seen that the gD mRNA and sgD mRNA synthesized in vitro have clear and single bands, and their sizes are consistent with expectations.

[0086] Example 3. Protein expression verification test

[0087] HEK-293T cells (purchased from ADCC) were transfected with TransGen's PEI reagent to verify the expression of gD mRNA and sgD mRNA purified in Example 2. 293T cells were evenly spread in a 24-well plate, and 0.8 μg of mRNA was transfected into each well. 48 hours after transfection, the cells were collected for SDS-PAGE electrophoresis, and the cells were blocked overnight after transfer. TransGen Biotech's anti-FLAG tag protein monoclonal antibody and TransGen's HRP-labeled goat anti-rabbit IgG antibody were used for incubation. After incubation at room temperature, ECL color development was performed, and finally the Western Blot results were obtained. The results are as shown in the figure. Figure 2 shown.

[0088] from Figure 2 It can be seen that compared with the blank control, protein bands with expected sizes and specific bands appeared after transfection of PRV-gD mRNA and PRV-sgD mRNA, indicating that mRNA can be expressed in cells. In addition, the protein content expressed by PRV-sgD mRNA is significantly higher than that of PRV-gD mRNA, indicating that the expression level of the optimized sgD protein sequence is higher, so we will use it for subsequent animal experiments.

[0089] Example 4. Preparation of mRNA vaccine of sgD protein

[0090] First, the alcohol phase was prepared. The lipids were dissolved in anhydrous ethanol in a ratio of cationic lipid (SM102): distearoylphosphatidylcholine (DSPC): cholesterol: DMG-2000 = 50:10:38.5:1.5 (mass ratio). Secondly, the aqueous phase was prepared. A citric acid buffer (50mM) with a pH of 4.0 was selected as the solute for dissolving mRNA; the mRNA was packaged using a microfluidic device with an alcohol phase: aqueous phase (volume ratio of 1:3). RNase-free PBS buffer was used for dilution, and a 30kDa ultrafiltration tube was used for concentration and liquid exchange. On this basis, an equal volume of PBS solution with a sucrose concentration of 20% was added, the mRNA concentration was adjusted to 100μg / mL, the sucrose concentration was 10%, and it was filtered using a 0.22μm filter membrane to finally obtain PRV-sgD LNP-mRNA, which was stored at -20℃ after aliquoting.

[0091] Then, the dynamic light detection particle size distribution experiment (see Trivalent mRNA Vaccine against SARS-CoV-2 and Variants with Effective Immunization, Wang J, et al.) showed that the prepared PRV-sgD LNP-mRNA (mRNA vaccine of sgD protein) was as follows Figure 3 As shown, from Figure 3 It can be seen that the prepared PRV-sgD LNP-mRNA particles are uniform and about 110 nm.

[0092] Example 5. Mouse immune evaluation experiment of sgD protein mRNA vaccine

[0093] In order to study the protective effect of the mRNA vaccine of sgD protein prepared in Example 4 against pseudorabies virus attack, 20 8-week-old female SPF Balb / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly divided into 4 groups, with 5 mice in each group. Figure 4 The first group was vaccinated with 20 μL of pseudorabies live vaccine (Keweijing, Keqian Biotechnology) as the positive control group, the second group was vaccinated with sgD protein mRNA vaccine (10 μg), and the third group was vaccinated with 0.1 ml PBS as the blank control group. Two weeks after the second immunization, serum samples were separated for titer detection, and the PRVHeN1 strain (2×10 4 The fourth group was a negative control group, which was only injected with the vaccine and not challenged with the virus (specific antibody results are not shown). 14 days after the challenge, the mice were euthanized, and brain, lung and other tissue samples were taken for hematoxylin and eosin (H&E) staining.

[0094] The antibody test was performed using the ELISA kit from Shanghai ELISA Biotechnology. The results were as follows: Figure 5 As shown in A. Figure 5 As can be seen from A, the titer of specific anti-gD antibodies in the mouse serum two weeks after the second immunization with the vaccine was significantly higher than that in the blank control group (##: P<0.01), and the titer of the sgD protein mRNA vaccine group was significantly higher than that in the positive control group (*: P<0.05), indicating that the mRNA vaccine can induce humoral immunity in mice.

[0095] Neutralizing antibody detection was performed using a pseudovirus experiment (see Adjuvant lipidoid-substituted lipid nanoparticles to enhance the immunogenicity of SARS-CoV-2 mRNA vaccines. Han X. et al.). Serum samples were inactivated by heating at 56°C for 30 minutes and then diluted with DMEM. Vero E6 cells were cultured in 96-well plates. A VSVΔG-gD-GFP strain was constructed (see Adjuvant lipidoid-substituted lipid nanoparticles to enhance the immunogenicity of SARS-CoV-2 mRNA vaccines. Han X. et al.), and a certain amount of VSVΔG-gD-GFP strain (200 TCID50) was mixed with 50 μL of diluted inactivated serum and incubated at 37°C for 2 hours. 100 μL was added to Vero E6 cells and incubated at 37°C for 2 hours. After two hours, the cells were washed three times with cold PBS and supplemented with complete DMEM medium again. Blank cells were used as negative controls. Incubate at 37°C, 5% CO2 for 48 hours. Calculate the neutralizing antibody content. Figure 5 As shown in B.

[0096] from Figure 5 As can be seen from B, a large amount of neutralizing antibodies were produced after mice were immunized with the positive control vaccine and the sgD mRNA vaccine, indicating that both mRNA vaccines can play a role, and the neutralizing antibody content of the sgD mRNA vaccine was significantly higher than that of the blank control (##: P<0.01), and significantly higher than that of the positive control vaccine (**: P<0.01).

[0097] The survival rate of mice was observed. Figure 6 As shown. Figure 6 It can be seen that two weeks after the virus attack, the survival rate of the two vaccine immunization groups reached 100%, while all the mice in the PBS control group died on the fourth day of the virus attack. The lungs, brains, spleens and kidneys of the mice were observed by pathological sections. Figure 7 As shown. Figure 7 It can be seen that compared with the normal group, the mice showed obvious lesions in the brain, lungs and spleen after infection, and the intervention of both vaccines can produce a certain protective effect.

[0098] The above results show that sgD mRNA vaccine can effectively induce the body to produce high levels of specific anti-gD IgG antibodies and neutralizing antibodies, and its effect is better than commercially available live vaccines.

[0099] Example 6. Pig immune challenge experiment of sgD protein mRNA vaccine

[0100] In order to study the protective effect of the vaccine against pseudorabies virus attack, 12 piglets purchased from Tianjin Bainong Experimental Animal Breeding Technology Co., Ltd. were randomly divided into 4 groups, with 3 pigs in each group. Figure 4 As shown. Group 1 was injected with 1 dose of pseudorabies live vaccine (Keweijing, Keqian Biotechnology) as the positive control group, Group 2 was intramuscularly inoculated with sgD mRNA vaccine (50 μg), and Group 3 was inoculated with 2 ml PBS as the blank control group. Two weeks after the second immunization, serum samples were separated for titer detection, and the PRVHeN1 strain (1×10 6 The 4th group was a negative control group, which was only vaccinated without virus challenge (specific antibody results are not shown). The mortality rate of piglets was counted. After 14 days of virus challenge, the piglets were euthanized, brain, lung and other tissue samples were taken, hematoxylin and eosin (H&E) staining was performed to take serum samples, and the content of piglet-specific antibodies and neutralizing antibodies was determined according to the method of Example 5.

[0101] Specific antibody results such as Figure 8 As shown in A, from Figure 8 It can be seen that the specific anti-sgD antibody titer in the serum of piglets two weeks after the second immunization was significantly higher than that in the blank control group (##: P<0.01), and the titer in the sgD mRNA vaccine group was significantly higher than that in the positive control group (*: P<0.05), indicating that the sgD mRNA vaccine can induce humoral immunity in piglets.

[0102] Neutralizing antibody results Figure 8 As shown in B, a large amount of neutralizing antibodies were produced after piglets were immunized with the positive control vaccine and the sgD mRNA vaccine, indicating that both mRNA vaccines can be effective in pigs, and the neutralizing antibody content of the sgD mRNA vaccine is higher than that of the positive control vaccine.

[0103] The survival rate of piglets was observed. Fig. 9 As shown. Fig. 9 It can be seen that two weeks after the virus attack, the survival rate of the two vaccine-immunized groups reached 100%, while the piglets in the PBS control group all died on the 4th day of the virus attack.

[0104] In addition, the lungs and brains of the piglets were stained with HE for pathological section observation. Fig.10 As shown. Fig.10 It can be seen that compared with the normal group, the pigs showed obvious lesions in the brain and lungs after infection, and the intervention of both vaccines can produce a certain protective effect.

[0105] The above results show that sgD mRNA vaccine can play a protective effect in piglets, and the neutralizing antibody content of sgD mRNA vaccine is significantly higher than that of blank control (##: P<0.01), and significantly higher than that of positive control vaccine (**: P<0.01).

[0106] The mRNA vaccine for preventing and / or treating pseudorabies of the present invention and its application are obtained by codon optimizing the gene sequence of pseudorabies virus gD protein antigen to obtain the optimized gene sequence of pseudorabies virus gD protein antigen; the sequence of pseudorabies virus gD protein antigen is also optimized to obtain pseudorabies virus truncated gD protein antigen, and the gene sequence of the truncated gD protein antigen is codon optimized, and experiments show that the expression amount of the optimized pseudorabies virus truncated gD protein antigen is higher; the sequence-optimized pseudorabies virus truncated gD protein is further prepared into an mRNA vaccine, and after using it for immunization of mice and piglets, it is found that the mRNA vaccine of the sgD protein can play an immune protection role in mice and piglets, and is significantly better than the commercially available pseudorabies inactivated vaccine, and is relatively safer.

[0107] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0108] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A polynucleotide comprising a first nucleotide sequence encoding a pseudorabies virus gD protein or an immunogenic fragment thereof, wherein the amino acid sequence of the pseudorabies virus gD protein or an immunogenic fragment thereof has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:

1.

2. The polynucleotide according to claim 1, characterized in that The sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:2 or SEQ ID NO:

3.

3. The polynucleotide according to claim 1 or 2, characterized in that The immunological fragment is a truncated gD protein of pseudorabies virus, and the amino acid sequence of the truncated gD protein of pseudorabies virus has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:

4.

4. The polynucleotide according to claim 3, characterized in that The sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO:5 or SEQ ID NO:

6.

5. The polynucleotide according to any one of claims 1 to 4, characterized in that The 5' end of the first nucleotide is also connected to a second nucleotide sequence encoding a signal peptide; Preferably, the signal peptide is tPA, IL-2, IL-6 or a neuropeptide, and the neuropeptide is preferably VIP, CGRP or NPY; Further preferably, the 5' end of the second nucleotide sequence is connected to a 5'UTR sequence, and the nucleotide sequence of the 5'UTR sequence is preferably as shown in SEQ ID NO: 9, or The 3' end of the first nucleotide is also connected to a 3'UTR sequence, and the end of the 3'UTR sequence is also connected to a PolyA sequence, and the nucleotide sequence of the 3'UTR sequence is preferably as shown in SEQ ID NO: 10; Also preferably, a T7 promoter is further provided at the 5' end of the 5'UTR sequence, and the nucleotide sequence of the T7 promoter is shown as SEQ ID NO:

7. The polynucleotide is preferably provided with a FLAG tag.

6. The polynucleotide according to any one of claims 1 to 5, characterized in that The polynucleotide is DNA or RNA; Preferably, when the polynucleotide is RNA, a 5' capping structure is further provided at the 5' end of the 5' UTR sequence of the RNA, and the 5' capping structure is preferably Cap1, Cap2, Cap3, Cap4 or Cap101.

7. A vector constructed from the polynucleotide according to any one of claims 1 to 6. A cell transformed by the vector according to claim 7. 9 . A composition for preventing and / or treating pseudorabies, comprising the polynucleotide according to any one of claims 1 to 6 or the mRNA transcribed from the vector according to claim 7 .

10. The composition according to claim 9, characterized in that The composition is an mRNA vaccine, which includes mRNA selected from those expressing pseudorabies virus gD protein and / or immunogenic fragments thereof.

11. The composition according to claim 10, characterized in that The sequence of the mRNA expressing the pseudorabies virus gD protein or an immunogenic fragment thereof has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology with SEQ ID NO: 3 or SEQ ID NO:

6.

12. The composition according to claim 11, characterized in that The composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is preferably a liposome, and the mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles of the liposome, and the particle size of the liposome nanoparticles is preferably 50 to 200 nm; Preferably, the liposome nanoparticles are selected from at least one of cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol and DMG-2000; More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts of cationic lipids, 5-15 parts of distearoylphosphatidylcholine (DSPC), 24-52 parts of cholesterol and 1-2 parts of DMG-2000; Further preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts of cationic lipids, 10 parts of distearoylphosphatidylcholine (DSPC), 38.5 parts of cholesterol and 1.5 parts of DMG-2000.

13. Use of the polynucleotide according to any one of claims 1 to 6, the vector according to claim 7, or the composition according to any one of claims 9 to 12 in the preparation of a medicament for preventing and / or treating pseudorabies.

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