A fusion protein and vaccine for preventing canine distemper virus infection and its application

Abstract

The present invention relates to a truncated peptide segment, a fusion protein, an immunogenic composition, a vaccine, a recombinant nucleic acid encoding the same, a gene expression cassette, a vector, a host cell, a preparation method, and pharmaceutical uses, etc., which can prevent canine distemper virus infection. From the perspective of developing a CDV nucleic acid vaccine, the present invention makes appropriate truncation of the CDV H protein, and screens out a suitable tetramer functional element to be connected to the N-terminus of the H protein extracellular domain. The fusion protein composed of the preferred tetramer element and the truncated CDV H protein sequence of the present invention has good immunogenicity, can induce a high level of humoral immune response, can also induce cellular immunity, and provides cross-immune protection against different CDV strains, and has a wide range of application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, in particular to the field of immune medicine technology, and specifically relates to a fusion protein and vaccine for preventing canine distemper virus infection and applications thereof. Background Art

[0002] Canine distemper virus (CDV) is a single-stranded negative-sense RNA virus belonging to the Paramyxoviridae family and Morbillivirus genus. It is one of the major pathogens that threaten the health of canines worldwide. CDV not only infects dogs, but also other carnivores, such as foxes, wolves, raccoons, and is even infectious to some primates. Canine distemper is an acute, highly contagious disease caused by CDV, characterized by fever, respiratory symptoms, digestive symptoms, and neurological symptoms.

[0003] Canine distemper virus infection not only directly endangers animal health, but also often forms secondary infections with other pathogens such as bacteria and fungi, aggravating the severity of the disease. In recent years, with the increase in the number of pets and the destruction of wildlife habitats, CDV infection has been on the rise worldwide and has become one of the major issues threatening animal welfare and ecological balance.

[0004] Safe and effective vaccines are considered the best measures to prevent and control CDV infection. The main antigenic proteins of CDV include hemagglutinin protein (H protein), fusion protein (F protein), nucleoprotein (N protein), etc. Among them, H protein is the main glycoprotein on the surface of the virus, responsible for the adsorption of the virus to the host cells, and is the main antigen that induces the production of neutralizing antibodies. The structure of CDV H protein is relatively special, with the N terminus located on the inside of the virus and the C terminus located on the outside of the virus, similar to the class II transmembrane protein of eukaryotic cells.

[0005] The main type of CDV vaccine on the market is live attenuated vaccine. Although live attenuated vaccines weaken the virulence of the virus through multiple passages, there is still a risk of virulence reversal, and the current attenuated live vaccines on the market have poor protection effects. In order to overcome the limitations of existing vaccines, researchers are actively developing new CDV vaccines. For example, the prior art patent CN109867713B (authorization announcement date 2022-07-26) discloses a subunit vaccine of canine distemper virus H protein, and patent CN108704128B (authorization announcement date 2022-06-21) introduces a canine distemper parvovirus bivalent subunit vaccine. These subunit vaccines all use full-length H protein and are expressed and purified in vitro using the insect cell-baculovirus system. This method has the problems of poor cross-immunity protection, high cost, and low yield, and cannot be smoothly promoted.

[0006] Therefore, there is still an urgent need for a new canine distemper vaccine to solve the problems existing in current vaccine development. Compared with traditional vaccine development, nucleic acid vaccine is a better choice. A large number of studies have shown that nucleic acid vaccines have many advantages: (1) They do not carry virulent virus particles and do not have the phenomenon of virulence reversion, which is safer; (2) They can complete the expression of antigen proteins in cells, thereby inducing the body's humoral immunity and cellular immunity to the protein, which can enhance the immune effect and prolong the duration of effective protection without repeated booster immunization; (3) They can restore the spatial conformation of antigens, provide more and more accurate spatial epitopes, and increase immunogenicity; (4) After vaccination, antigen proteins are expressed in cells and can directly bind to the MHCI or II class molecules of the tissue compatibility complex without interference from maternal antigens.

[0007] Therefore, there is an urgent need in the art for a CDV vaccine that provides better conformational epitopes, has cross-immune protection effects, and reduces production costs, so that the vaccine can be more easily promoted and used worldwide. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a new canine distemper virus vaccine molecular architecture design and application, specifically to provide a truncated peptide segment, fusion protein, immunogenic composition, vaccine, recombinant nucleic acid encoding it, gene expression cassette, vector, host cell, preparation method and pharmaceutical use that can prevent canine distemper virus infection. From the perspective of developing CDV nucleic acid vaccines, the present invention makes appropriate truncation of CDV H protein, and screens out suitable tetramer functional elements to be connected to the N-terminus of the extracellular domain of the H protein to promote its self-assembly into a tetramer form during the expression process and restore the natural tetramer conformation. The truncated CDV H protein peptide segment of the present invention can be combined with different elements, and the designed nucleic acid vaccine can be smoothly expressed in eukaryotic cells and has immunogenicity. In addition, the fusion protein composed of the preferred tetramer element and the truncated CDV H protein sequence of the present invention has good immunogenicity, can induce a high level of humoral immune response, can also induce cellular immunity, and provide cross-immune protection against different CDV strains.

[0009] One aspect of the present invention provides a truncated peptide segment of hemagglutinin glycoprotein (H protein) of canine distemper virus (CDV).

[0010] Furthermore, the amino acid sequence of the truncated peptide segment is shown in SEQ ID NO.5 or SEQ ID NO.9.

[0011] Another aspect of the present invention provides a fusion protein, characterized in that it comprises the truncated peptide segment.

[0012] Furthermore, the fusion protein comprises the following elements from the N-terminus to the C-terminus:

[0013] (a) a tetramer domain; preferably, the tetramer domain is a tetramer domain derived from Staphylothermus; more preferably, the amino acid sequence of the tetramer domain of Staphylothermus is shown in SEQ ID NO.4; and

[0014] (b) The truncated peptide segment.

[0015] Furthermore, it is characterized in that the N-terminus of the fusion protein contains a signal peptide; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.1.

[0016] Furthermore, it is characterized in that the N-terminus of the fusion protein contains an Fc domain; preferably, the amino acid sequence of the Fcdomain is as shown in SEQ ID NO.10.

[0017] Furthermore, it is characterized in that the elements are optionally connected by a linker sequence, preferably, the linker sequence is a GS linker or a GGS linker; preferably, the amino acid sequence of the GS linker is as shown in SEQ ID NO.6, and the amino acid sequence of the GGS linker is as shown in SEQ ID NO.7.

[0018] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the truncated peptide segment and / or fusion protein of the present invention.

[0019] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule.

[0020] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule or the recombinant gene expression cassette.

[0021] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule, or the recombinant gene expression cassette, or the recombinant vector.

[0022] Another aspect of the present invention provides an immunogenic composition or a pharmaceutical composition, characterized in that it comprises one or more of any one of the truncated peptide segments and / or fusion proteins, and / or one or more of the recombinant nucleic acid molecules, and / or one or more of the recombinant gene expression cassettes, and / or one or more of the recombinant vectors, and / or one or more of the recombinant host cells; preferably, the immunogenic composition or the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

[0023] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more of any one of the truncated peptide segments and / or fusion proteins, and / or one or more of the recombinant nucleic acid molecules, and / or one or more of the recombinant gene expression cassettes, and / or one or more of the recombinant vectors, and / or one or more of the recombinant host cells, and / or one or more of the immunogenic compositions or pharmaceutical compositions; preferably, the vaccine is a nucleic acid vaccine.

[0024] Another aspect of the present invention provides a method for preparing one or more of the truncated peptides and / or fusion proteins, characterized in that the truncated peptides and / or fusion proteins are expressed by the recombinant host cells, and the truncated peptides and / or fusion proteins are separated.

[0025] Another aspect of the present invention provides the use of one or more of the truncated peptide segments and / or fusion proteins, and / or one or more of the recombinant nucleic acid molecules, and / or one or more of the recombinant gene expression cassettes, and / or one or more of the recombinant vectors, and / or one or more of the recombinant host cells, and / or one or more of the immunogenic compositions or pharmaceutical compositions, and / or one or more of the recombinant vaccines in the preparation of medicaments for preventing diseases caused by canine distemper virus; preferably, the disease is canine distemper.

[0026] Another aspect of the present invention provides a method for preventing a disease, characterized in that a preventive effective amount of one or more of the truncated peptides and / or fusion proteins, and / or one or more of the recombinant nucleic acid molecules, and / or one or more of the recombinant gene expression cassettes, and / or one or more of the recombinant vectors, and / or one or more of the recombinant host cells, and / or one or more of the immunogenic compositions or pharmaceutical compositions, and / or one or more of the recombinant vaccines is administered to a subject; preferably, the disease is a disease caused by canine distemper virus.

[0027] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0028] 1. The present invention finds that the new fusion molecule has good immunogenicity and can provide immune protection, and can be used as a universal antigenic framework for nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention has good immunogenicity and can provide effective immune protection against canine distemper virus infection. Compared with traditional live vaccines, it has high safety and low preparation cost, which is conducive to market promotion.

[0029] 2. The present invention found that the nucleic acid vaccine designed based on the full-length CDV H protein cannot be expressed in eukaryotic cells and is difficult to activate the humoral immunity of animals; however, the CDV truncated peptide segment based on the present invention can be combined with different elements to form a new fusion molecule, which is smoothly expressed and secreted in eukaryotic cells and can effectively activate the humoral immunity of animals. The truncated CDV H protein truncated peptide segment 1 is the best choice.

[0030] 3. From the perspective of developing a CDV nucleic acid vaccine, the present invention screens a variety of functional elements to be connected to the N-terminus of the CDV H protein truncated peptide segment, and finally selects a tetrameric functional element that restores the natural tetrameric conformation of the H protein. After forming a fusion protein, it can not only be expressed smoothly, but also has strong immunogenicity. The present invention screens from a variety of different tetrameric domains and selects the tetrameric domain of Crenarchaea with the highest secretion efficiency, which is more conducive to antigen presentation and activation of humoral immunity after being fused with the CDV H protein truncated peptide segment.

[0031] 4. Example 4 of the present invention shows that the fusion molecule based on the present invention can effectively promote the smooth expression and secretion of CDV H protein in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes, thereby further improving the immune protection effect and providing new technology for the field of universal vaccine design for measles-like viruses.

[0032] 5. Example 5 of the present invention shows that in the guinea pig model, the vaccine D of the present invention can induce 4 / 5 individuals to produce neutralizing antibodies after immunization, and the neutralizing antibodies produced by 3 / 5 individuals have cross-immune protection against CDV1 and CDV3, that is, they can provide cross-immune protection against different CDV strains. The recombinant nucleic acid vaccines B and C of the present invention can induce effective immune protection against CDV1 strains in the guinea pig model.

[0033] 6. Example 6 of the present invention shows that in the black raccoon dog (Canidae) model, the fusion protein of the present invention has good immunogenicity, and the nucleic acid vaccine based on the present invention can provide effective immune protection for the target animal black raccoon dog, and has a good preventive effect against canine distemper virus infection. Although the nucleic acid vaccine D based on the present invention detected high levels of neutralizing antibodies in only 4 / 5 black raccoon dogs after two immunizations in Example 5, the challenge experiment in Example 6 showed that all 5 black raccoon dogs survived successfully. It is speculated that vaccine D not only induces humoral immunity, but also induces cellular immunity, and can obtain complete protection when attacked by CDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the design ideas and fusion protein conformation of the present invention.

[0035] Figure 2 The figure is a schematic diagram of the molecular structure of the fusion protein of the present invention.

[0036] Figure 3 Schematic diagram of the molecular structure of the vaccine used in the examples.

[0037] Figure 4 Schematic diagram of a circular plasmid containing the vaccine template of the present invention.

[0038] Figure 5A-5E The quality control peak diagram and purity test results of the vaccine used in the examples are shown in FIG. Figure 5A This is the quality control peak diagram and purity test results of vaccine A. Figure 5B This is the quality control peak diagram and purity test results of vaccine B. Figure 5C This is the quality control peak diagram and purity test results of vaccine C. Figure 5D This is the quality control peak diagram and purity test results of vaccine D. Figure 5E This is the quality control peak diagram and purity test results of vaccine E.

[0039] Figure 6A-6D The WB (Western blot) test results of the expression effect of the vaccine used in the example after in vitro transfection of HEK293T cells; Fig. 6A This is the WB (Western blot) test result of the expression effect of vaccine A after transfection of HEK293T cells in vitro. Figure 6B This is the WB (Western blot) test result of the expression effect of vaccine B after transfection of HEK293T cells in vitro. Figure 6C This is the WB (Western blot) test result of the expression effect of vaccine C after transfection of HEK293T cells in vitro. Fig.6D These are the WB (Western blot) test results of the expression effects of vaccine D and vaccine E after transfection of HEK293T cells in vitro.

[0040] Figure 7A-7C The present invention provides the immunization and sampling process of the vaccine in the guinea pig model, as well as the neutralizing antibody titer in the guinea pig serum after two immunizations; wherein Fig. 7A The immunization and sampling process of the vaccine of the present invention in the guinea pig model is as follows: Figure 7B The neutralizing antibody titer in the guinea pig serum after two immunizations (test strain: CDV1, NJ (12) 3 strains), Figure 7C It is the neutralizing antibody titer in guinea pig serum after two immunizations (test strain: CDV3, CL strain).

[0041] Figure 8A-8C The present invention is a vaccine of the present invention in the black raccoon dog (Canidae) model of immunization and sampling process, as well as the neutralizing antibody titer and challenge protection effect (survival rate) in the black raccoon dog serum after two immunizations; wherein Fig. 8A The immunization and sampling process of the vaccine of the present invention in the black raccoon dog (Canidae) model is as follows: Figure 8B is the neutralizing antibody titer in the black raccoon dog serum after two immunizations, Figure 8C It is the protection effect (survival rate) against virus after two immunizations. DETAILED DESCRIPTION

[0042] Terms and Definitions

[0043] The term "canine distemper virus" refers to Canine Distemper Virus, abbreviated as CDV in English. It belongs to the Paramyxoviridae family and the Morbillivirus genus. It is a single-stranded RNA virus. CDV can infect Canidae, Mustelidae, Skunks, Hyaenidae, Pandas, Procyonidae, Pinnipeds, some Civets and felines other than domestic cats, causing canine distemper.

[0044] The term "canine distemper" refers to Canine distemper, a disease caused by canine distemper virus, also known as canine distemper or dog distemper.

[0045] The term "H protein" refers to the hemagglutinin glycoprotein, specifically the hemagglutinin glycoprotein of canine distemper virus, which enables the virus to attach to cell receptors, thereby initiating infection. H protein binding to cell receptors induces conformational changes, allowing F protein to trigger virus particle / cell membrane fusion.

[0046] The term "tetrameric domain" refers to a specific structural region in a protein that can promote the polymerization of four subunits or monomers into a tetramer. This domain is usually composed of secondary structural elements such as α-helices or β-folds, which stabilize the formation of tetramers through intermolecular interactions (such as hydrophobic interactions, hydrogen bonds, electrostatic interactions, etc.). The specific tetrameric domain is derived from the tetrameric domain of human vasodilator-stimulated phosphoprotein (VASP), the amino acid sequence of which is shown in SEQ ID NO.11; or from the pII mutant (GCN4-pIItetrameric domain) of the tetrameric domain of yeast transcription factor General Control Nonderepressible 4 protein, the amino acid sequence of which is shown in SEQ ID NO.3; or from the tetrameric domain of Staphylothermus, the amino acid sequence of which is shown in SEQ ID NO.4; or from the p53 tetrameric domain, the amino acid sequence of which is shown in SEQ ID NO.2. Preferably, the tetramerization domain of the present invention is derived from a tetramerization domain of Staphylothermus.

[0047] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and a cellular response in an organism. The immune response can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays that measure T cell activation or proliferation, and / or assays that measure the modulation of one or more cytokine activity or expression.

[0048] The term "administration" or "vaccination" refers to administration based on the nucleic acid vaccine or vaccine composition of the present invention preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injection administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Administration in the neck muscle of the animal is preferred. The administration regimen can be adjusted to provide optimal immunity using an accelerated regimen (boosting regimens).

[0049] 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.

[0050] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0051] The term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0052] The term "mRNA" refers to messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0053] The term "5'-UTR", which refers to the "5' untranslated region" or "5'UTR", is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes modifying the ends, removing introns, capping and / or shearing off individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is usually defined as the region located between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNA can be tens to hundreds of bases in length.

[0054] The term "3'-UTR", meaning "3'-untranslated region" or "3'UTR", refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region typically extends from the stop codon of the translation product to a poly(A) sequence that is typically attached after the transcription process. The 3'-untranslated region of mammalian mRNA typically has a homology region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeats and may fold to produce a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (e.g., RNA binding proteins) that are known to increase RNA stability.

[0055] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0056] The terms "individual", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats, etc.).

[0057] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0058] The term "drug combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that it can be effectively absorbed in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an effective component that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0059] The term "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated by exposing the subject to (e.g., administering) the recombinant vaccine, composition, etc. according to the present invention, compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0060] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0061] The present invention discloses a novel method for preparing a CDV vaccine and its application. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0062] In the fusion protein and encoding nucleic acid and elements thereof, preparation methods and applications provided by the present invention, the raw materials and reagents used can all be obtained commercially.

[0063] The present invention is further illustrated below in conjunction with the examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for preparing a recombinant vaccine.

[0064] Example 1 Construction of the recombinant nucleic acid vaccine of the present invention

[0065] In order to prepare a recombinant nucleic acid vaccine comprising the antigen of the present invention and compare whether the vaccine based on the present invention has a good in vitro expression effect, the vaccine molecular architecture involved in the embodiment is as follows: Figure 2 , Figure 3 As shown. For example, Figure 2 The schematic diagram of the molecular structure of the fusion protein of the present invention comprises the following elements from N-terminus to C-terminus: signal peptide, tetramer domain, CDV H protein truncated peptide segment. The elements can be directly connected or connected through different linkers, such as GS linker. Figure 3 The molecular structures of vaccines A, B, C, D and E used in the comparative experiments in the examples are shown in Figure 1. Figure 3 The recombinant nucleic acid vaccine of the protein shown in the embodiment first constructs a gene expression cassette for expressing the antigen sequence described in the embodiment. The expression cassette comprises, from the 5' end to the 3' end, 5'UTR, CDS region, 3'UTR, PolyA, wherein the CDS region comprises the fusion molecule architecture of the present invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (commissioned to GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector circular plasmid that can be used for in vitro RNA transcription, such as Figure 4 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine is obtained.

[0066] According to the above method, the carrier used in the subsequent examples was prepared:

[0067] (1) Recombinant nucleic acid vaccine A preparation vector

[0068] Step a: Synthesize the "CDV full-length H protein" gene fragment, the amino acid sequence of which is shown in SEQ ID NO: 8.

[0069] Step b: Construct a nucleic acid vaccine framework vector.

[0070] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0071] Step c: preparing recombinant plasmid.

[0072] The gene synthesized in step a is inserted into the vector structure of step b to obtain a recombinant nucleic acid vaccine A preparation vector.

[0073] (2) Preparation of vectors based on the recombinant nucleic acid vaccine B of the present invention

[0074] Step a: synthesizing a "signal peptide-CDV H protein truncated peptide segment 2" fusion gene fragment, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the CDV H protein truncated peptide segment 2 is shown in SEQ ID NO: 9.

[0075] Step b: Construct a nucleic acid vaccine framework vector.

[0076] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0077] Step c: preparing recombinant plasmid.

[0078] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine B based on the present invention.

[0079] (3) Preparation of vector based on the recombinant nucleic acid vaccine C of the present invention

[0080] Step a: Synthesize the "Fc domain (containing a signal peptide at the N-terminus) - CDVH protein truncated peptide segment 2" fusion gene fragment, and connect the two via a GGS linker sequence whose amino acid sequence is shown in SEQ ID NO: 7. The amino acid sequence of Fcdomain (containing a signal peptide at the N-terminus) is shown in SEQ ID NO: 10, and the amino acid sequence of CDV H protein truncated peptide segment 2 is shown in SEQ ID NO: 9.

[0081] Step b: Construct a nucleic acid vaccine framework vector.

[0082] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0083] Step c: preparing recombinant plasmid.

[0084] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine C based on the present invention.

[0085] (4) Preparation of vector based on the recombinant nucleic acid vaccine D of the present invention

[0086] Step a: synthesize the "signal peptide-Quanarchaea tetramer domain-CDV H protein truncated peptide segment 1" fusion gene fragment, the tetramer domain 1 and the CDV H protein truncated peptide segment 1 are connected by the GSlinker sequence shown in SEQ ID NO: 6, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the Quanarchaea tetramer domain is shown in SEQ ID NO: 4, and the amino acid sequence of the CDV H protein truncated peptide segment 1 is shown in SEQ ID NO.5.

[0087] Step b: Construct a nucleic acid vaccine framework vector.

[0088] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0089] Step c: preparing recombinant plasmid.

[0090] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine D based on the present invention.

[0091] (5) Preparation of vectors based on the recombinant nucleic acid vaccine E of the present invention

[0092] Step a: synthesize the "signal peptide-p53 tetramer domain-CDV H protein truncated peptide segment 1" fusion gene fragment, the tetramer domain 2 and the CDV H protein truncated peptide segment 1 are connected by a GS linker sequence whose amino acid sequence is shown in SEQ ID NO: 6, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the p53 tetramer domain is shown in SEQ ID NO: 2, and the amino acid sequence of the CDV H protein truncated peptide segment 1 is shown in SEQ ID NO.5.

[0093] Step b: Construct a nucleic acid vaccine framework vector.

[0094] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0095] Step c: preparing recombinant plasmid.

[0096] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine E based on the present invention.

[0097] Table 1 Amino acid sequences of proteins involved in the present invention

[0098] Amino acid sequence and sequence number Signal peptide MYKMQLLSCIALTLVLVANS (SEQ ID NO: 1) p53 tetramerization domain EYFTLQIRGRERFEMFRELNEALELKDAQAG (SEQ ID NO: 2) GCN4-pII tetramerization domain RMKQIEDKLEEILSKLYHIENELARIKKLLGER (SEQ ID NO: 3) Crenarchaea tetramer domain IINETADDIVYRLTVIIDDRYESLKNLITLRADRLEMIINDNVSTILA (SEQ ID NO: 4) CDV H protein truncated peptide 1 (SEQ ID NO:5) GS linker GSGSGS (SEQ ID NO: 6) GGS linker GGSGGSGGSG (SEQ ID NO: 7) CDV full-length H protein MLSYQDKVGAFYKDNARANSSKLSLVTEEQGGRRPPYLLFVLLILLIGILALLAITGVRFHQVSTSNMEFSRLLKEDMEKSEAVHHQVIDVLTPLFKIIGDEIGLRLPQKLNEIKQFILQKTNFFNPNREFDFRDLHWCINPPSKIKVNFTNYCDTVGVKKSIASAANPIILSALSGARGDIFPPYRCSGATTSVGRVFPLSVSLSMSLISRTSEIINMLTAISDGVYGKTYLLVPDYIEGEFDSQKIRVFEIGFIKRWLNDMPLLQTTNYMVLPETSKAKVCTIAVGELTLASLCVDESTVLLYHDSNGSQDGILVVTLGIFGATPMDQVEEVIPIAHPSVERIHITNHRGFIKDSIVTWMVPVLVSEKQEEQKNCLESACHRKSYPMCNQTSWEPFGGGQLPSYGRLTLPLDPSIDLQLNISFTYGPVILNGDGMDYYESPLLDSGWLTIPPKNGTVLGLINKASRGDQFTVTPHVLTFAPRESSGNCYLPIQTSQIMDKDVLTESNLVVLPTQNFRYVIATYDISRGDHAIVYYVYDPIRTISYTYPFRLTTKGRPDFLRIECFVWDDDLWCHQFYRFEANITNSTTSVENLVRIRFSCNRSKP (SEQ ID NO: 8) CDV H protein truncated peptide segment 2 PPSKIKVNFTNYCDTVGVKKSIASAANPIILSALSGARGDIFPPYRCSGATTSVGRVFPLSVSLSMSLISRTSEIINMLTAISDGVYGKTYLLVPDYIEGEEFDSQKIRVFEIGFIKR WLNDMPLLQTTNYMVLPETSKAKVCTIAVGELTLASLCVDESTVLLYHDSNGSQDGILVVTLGIFGATPMDQVEEVIPIAHPSVERIHITNHRGFIKDSIVTWMVPVLVSEKQEEQKN CLESACHRKSYPMCNQTSWEPFGGGQLPSYGRLTLPLDPSIDLQLNISFTYGPVILNGDGMDYYESPLLDSGWLTIPPKNGTVLGLINKASRGDQFTVTPHVLTFAPRESSGNCYLP IQTSQIMDKDVLTESNLVVLPTQNFRYVIATYDISRGDHAIVYYVYDPIRTISYTYPFRLTTKGRPDFLRIECFVWDDDLWCHQFYRFEANITNSTTSVENLVRIRFSCNRSKP (SEQ ID NO: 9) Fc domain MESVFCWVFLVVILKGVQGFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHI DLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK (SEQ ID NO:10) VASP tetramerization domain PSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGSP (SEQ ID NO: 11)

[0099] Example 2 Preparation of the recombinant nucleic acid vaccine of the present invention

[0100] (1) Preparation of capped mRNA vaccines

[0101] Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0102] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0103] (2) Preparation of non-capped mRNA vaccines

[0104] Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0105] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction and the template DNA is degraded.

[0106] (3) DNA vaccine preparation

[0107] Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0108] Step b: Use an endotoxin-free plasmid extraction and purification kit to extract and purify the target plasmid.

[0109] Example 3 Quality Control of In Vitro Transcription of Recombinant Nucleic Acid and Vaccine Preparation of the Present Invention

[0110] Vaccine A (recombinant nucleic acid vaccine A), vaccine B (recombinant nucleic acid vaccine B based on the present invention), vaccine C (recombinant nucleic acid vaccine C based on the present invention), vaccine D (recombinant nucleic acid vaccine D based on the present invention), and vaccine E (recombinant nucleic acid vaccine E based on the present invention) were prepared by the method for preparing the capped mRNA vaccine in Example 2. The purity of the produced recombinant nucleic acid was tested, and the purity of the recombinant nucleic acid used in the experiment was greater than 80%. The quality control peak diagram of the recombinant nucleic acid based on the present invention is as follows: Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E The specific description is as follows: (1) Recombinant nucleic acid vaccine A, purity is 90.1%, quality control peak diagram and purity test results are shown in Figure 5A ; (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 85.9%. The quality control peak diagram and purity test results are as follows Figure 5B ; (3) The recombinant nucleic acid vaccine C based on the present invention has a purity of 82.7%. The quality control peak diagram and purity test results are as follows Figure 5C ; (4) The recombinant nucleic acid vaccine D based on the present invention has a purity of 90.2%. The quality control peak diagram and purity test results are as follows Figure 5D ; (5) The recombinant nucleic acid vaccine E based on the present invention has a purity of 82%. The quality control peak diagram and purity test results are as follows Figure 5E The above purity meets the quality requirements for cell transfection experiments and vaccine production.

[0111] Example 4 In vitro expression effect of the recombinant nucleic acid of the present invention

[0112] The vaccine in Example 3 was transfected into HEK293T cells using a cell transfection reagent. After 48 hours of in vitro culture, the protein was collected and tested by Western blot. The protein molecular weights of vaccine A, vaccine B, vaccine C, vaccine D, and vaccine E were calculated, as shown in Table 2.

[0113] Table 2 Protein molecular weight of vaccine A, vaccine B, vaccine C, and vaccine D

[0114] vaccine Protein molecular weight (kDa) A 71.4 B 91.8 C 118.9 D 73.1 E 71.5

[0115] Figures 6A-6D The results of in vitro expression WB (Western blot) of HEK293 cells transfected with vaccines A, B, C, D, and E are shown. The protein expressed by vaccine A is the full-length H protein of CDV, which is a transmembrane protein and can theoretically be detected in the lysate protein; the proteins expressed by vaccines B, C, D, and E are secreted proteins and can theoretically be detected in the supernatant.

[0116] like Fig. 6A As shown, however, contrary to theoretical expectations, no target protein signal was detected in the lysate protein of vaccine A, proving that the nucleic acid vaccine designed based on the full-length H protein of CDV could not be normally expressed in eukaryotic cells.

[0117] like Figure 6B , 6C As shown in Figure 6D, the target protein signal was detected in the supernatant of vaccines B, C, D, and E, and the molecular weight was in line with expectations. This proves that compared with the full-length expressed H protein, the fusion protein based on the present invention can not only be smoothly translated and correctly folded in eukaryotic cells, but also has a stable structure and can be secreted outside the cell.

[0118] For the tetramer domain of the fusion protein, the present invention conducted a preliminary screening in the early stage, using the VASP tetramer domain, GCN4-pII tetramer domain, p53 tetramer domain, and Crenarchaea tetramer domain. These four tetramer domains were initially screened. From the perspective of secretion efficiency, the VASP tetramer domain and GCN4-pII tetramer domain with poor effects were removed. The present invention used the p53 tetramer domain and Crenarchaea tetramer domain for subsequent experiments to select the optimal tetramer domain component. The parallel comparison of the expression levels of vaccine D (including the Crenarchaea tetramer domain) and vaccine E (including the p53 tetramer domain) of the present invention showed that the expression level of vaccine D in the supernatant was the highest, proving that compared with the p53 tetramer domain, the secretion efficiency of the Crenarchaea tetramer domain after fusion with the CDV H protein truncated peptide segment was the highest, which was more conducive to antigen presentation and activation of humoral immunity.

[0119] Among the above results, the supernatant expression levels of vaccines B, C, and D were the highest, so vaccines B, C, and D were used in subsequent animal experiments to verify whether the three vaccines can effectively activate the humoral immune response of animals.

[0120] Example 5: Verification of the neutralizing antibodies and cross-immunity effects induced by the recombinant nucleic acid vaccine of the present invention in a guinea pig model

[0121] In order to verify whether the nucleic acid vaccine based on the present invention can activate animal humoral immunity and produce neutralizing antibodies, this example uses vaccines B, C, and D to immunize guinea pigs. After the second immunization, the neutralizing antibody titer in the serum is detected, and whether cross protection can be produced against other genotypes of CDV viruses is detected.

[0122] A total of 5 SPF guinea pigs were selected for the experiment, and each guinea pig was marked with an ear tag. Fig. 7A , as shown in Table 3, the dosage in this table and below refers to the amount of active ingredient.

[0123] Table 3 Grouping and immunization process of guinea pig immunization experiment in Example 5

[0124] vaccine Number of animals Immunization dose Immunization Procedure Recombinant nucleic acid vaccine B based on the present invention 5 10μg / each Day 0, Day 21 left leg intramuscular injection Based on the recombinant nucleic acid vaccine C of the present invention 5 10μg / each Day 0, Day 21 left leg intramuscular injection Recombinant nucleic acid vaccine D based on the present invention 5 10μg / each Day 0, Day 21 left leg intramuscular injection

[0125] Five guinea pigs were immunized twice according to the immunization process in Table 3, and the blood collection time points were Day 0 (before immunization) and Day 28. Each guinea pig was given more than 2 mL of whole blood each time, and the serum obtained by separation was not less than 1 mL. Each serum sample was marked and stored at -80°C until detection.

[0126] Neutralization titer determination: Guinea pig serum was used to test the neutralization titer of CDV1 strain (NJ(12)3 strain) and CDV3 strain (CL strain, EU726268.1) to evaluate the immune protection and cross-protection effects of the vaccine. The test results are as follows: Figure 7B , 7C shown.

[0127] according to Figure 7B , 7C The results show that the recombinant nucleic acid vaccine B based on the present invention can induce 4 / 5 guinea pigs to produce neutralizing antibodies against the CDV1 strain after two immunizations, with the antibody titer ranging from 1:64 to 1:362; and induce 3 / 5 guinea pigs to produce neutralizing antibodies against the CDV3 strain, with the antibody titer ranging from 1:20 to 1:22.6.

[0128] according to Figure 7B , 7C The results show that the recombinant nucleic acid vaccine C of the present invention can induce 2 / 5 guinea pigs to produce neutralizing antibodies against the CDV1 strain after two immunizations, with the antibody titer ranging from 1:40.3 to 1:90.6; no neutralizing antibodies against the CDV3 strain were induced in guinea pigs.

[0129] according to Figure 7B , 7C The results show that the recombinant nucleic acid vaccine D of the present invention can induce 4 / 5 guinea pigs to produce neutralizing antibodies against the CDV1 strain after two immunizations, with the antibody titer ranging from 1:90.6 to 1:724; and induce 5 / 5 guinea pigs to produce neutralizing antibodies against the CDV3 strain, with the antibody titer ranging from 1:22.6 to 1:181.1.

[0130] For CDV virus, a neutralizing antibody titer greater than 1:45 can produce effective protection. Therefore, this result proves that the recombinant nucleic acid vaccines B and C based on the present invention can induce effective immune protection against the CDV1 strain in the guinea pig model; the recombinant nucleic acid vaccine D based on the present invention can induce cross-immune protection against CDV1 and CDV3 strains in the guinea pig model.

[0131] The above raw data of neutralizing antibody titer are shown in Table 4.

[0132] Table 4 Raw data of neutralizing antibody titer determination in guinea pig immunization experiment in Example 5

[0133]

[0134] Analyzing the above results, the antigen sequence selected by the present invention is derived from the H protein of the CDV1 strain, so vaccines B, C, and D can all induce the production of neutralizing antibodies against the CDV1 strain. Among them, the N-terminus of vaccine D is fused with a tetramer domain, so it can self-assemble into a tetramer during the expression process, simulating the natural conformation of the CDV H protein, providing more spatial epitopes, and thus inducing the production of neutralizing antibodies against CDV3.

[0135] Therefore, the above results prove that: (1) based on the vaccine of the present invention, the truncated H protein can provide sufficient linear epitopes to induce animals to produce effective humoral immune responses, and the truncated CDV H protein truncated peptide segment 1 is the best choice; (2) based on the vaccine of the present invention, the strategy of fusing the tetramer domain at the N-terminus can promote the expression of the H protein in a natural conformation, activate the humoral immune response against the spatial epitopes of the CDV H protein, and induce animals to produce neutralizing antibodies against different genotypes of CDV, and the tetramer domain of Crenarchaea is the best choice.

[0136] Example 6 Preventive effect of the recombinant nucleic acid vaccine of the present invention in the black raccoon dog (Canidae) model

[0137] In order to verify whether the nucleic acid vaccine based on the present invention can activate the humoral immunity of the target animal (Canidae) and produce effective immune protection, this example uses vaccine D that induces the highest level of neutralizing antibodies to immunize black raccoon dogs. After the second immunization, the neutralizing antibody titer in the serum is detected, and a challenge experiment is performed to verify the protection effect.

[0138] The experiment selected 8 black raccoon dogs over 2 months old and divided them into two groups, 5 in the vaccine D immunization group and 3 in the PBS control group. Before immunization, the neutralizing antibody of canine distemper virus in black raccoon dogs was measured to be no higher than 1:4, and each black raccoon dog was marked with an ear tag. Fig. 8A , as shown in Table 5, the dosage in this table and below refers to the amount of active ingredient.

[0139] Table 5 Grouping and immunization process of black raccoon dog immunization experiment in Example 6

[0140] vaccine Number of animals Immunization dose Immunization Procedure Recombinant nucleic acid vaccine D based on the present invention 5 25μg / each Day 0, Day 21 Intramuscular injection into the lateral hind limb PBS 3 1ml / piece / time Day 0, Day 21 Intramuscular injection into the lateral hind limb

[0141] Ten black raccoon dogs were immunized twice according to the immunization process in Table 5, and two weeks after the second immunization, that is, on Day 42, they were challenged with CDV1 strain (CDV-HBF-1).

[0142] Blood was collected at Day 0 (before immunization), Day 21 (before the second immunization), and Day 28. No less than 0.5 mL of serum was obtained from each animal each time, and each serum sample was marked and stored at -80°C until detection. After the challenge, the state of the experimental animals was continuously observed, and the survival rate was calculated to verify the protective effect of the vaccine of the present invention.

[0143] according to Figure 8B The results show that the recombinant nucleic acid vaccine D of the present invention can induce 4 / 5 black raccoon individuals to produce neutralizing antibodies against the challenge strain after two immunizations, and the antibody titer ranges from 1:161.4 to 1:256. The original data are shown in Table 6. Judging from the titer level, it can provide effective protection.

[0144] Table 6 Raw data of neutralizing antibody titer determination in black raccoon dog immunization experiment in Example 6

[0145]

[0146] After the infection, the status of the two groups of black raccoon dogs was continuously observed, the deaths were counted, and the survival rate was calculated. Figure 8C The results show that all five black raccoon dogs in the vaccine D immunization group survived, while three black raccoon dogs in the PBS control group died on days 7, 9, and 11 after the challenge. This result proves that the nucleic acid vaccine D based on the present invention can provide a 100% protection rate.

[0147] Analyzing the above results, although high levels of neutralizing antibodies were detected in only 4 / 5 black raccoon dogs after two immunizations based on the nucleic acid vaccine D of the present invention, the challenge experiment showed that all 5 black raccoon dogs survived successfully. It is speculated that vaccine D not only induces humoral immunity, but also induces cellular immunity, and can obtain complete protection when attacked by CDV virulent virus.

[0148] In summary, the CDV vaccine design concept provided by the present invention can induce the model animal guinea pig to produce high levels of neutralizing antibodies and has a cross-immune protection effect. In addition, the nucleic acid vaccine based on the present invention can provide effective immune protection for the target animal black raccoon dog and has a good preventive effect against canine distemper virus infection. Therefore, the present invention can be applied to the production and development of immune drugs, filling the gap in the current field of canine distemper virus universal vaccine research and development, and has extremely high commercial value and broad application prospects.

[0149] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that The fusion protein consists of a signal peptide, a tetramer domain of Crenarchaea, a GS linker, and a truncated peptide segment 1 of CDV H protein from the N-terminus to the C-terminus, wherein the amino acid sequence of the GS linker is shown in SEQ ID NO: 6, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the tetramer domain of Crenarchaea is shown in SEQ ID NO: 4, and the amino acid sequence of the truncated peptide segment 1 of CDV H protein is shown in SEQ ID NO.

5.

2. A recombinant nucleic acid molecule, characterized in that Encodes the fusion protein of claim 1.

3. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 2.

4. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 2, or the recombinant gene expression cassette according to claim 3.

5. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 2, or the recombinant gene expression cassette according to claim 3, or the recombinant vector according to claim 4.

6. An immunogenic composition or pharmaceutical composition, characterized in that Comprising the fusion protein of claim 1, and / or one or more recombinant nucleic acid molecules of claim 2, and / or one or more recombinant gene expression cassettes of claim 3, and / or one or more recombinant vectors of claim 4, and / or one or more recombinant host cells of claim 5.

7. A recombinant vaccine, characterized in that: Comprising the fusion protein of claim 1, and / or one or more recombinant nucleic acid molecules of claim 2, and / or one or more recombinant gene expression cassettes of claim 3, and / or one or more recombinant vectors of claim 4, and / or one or more recombinant host cells of claim 5, and / or one or more immunogenic compositions or pharmaceutical compositions of claim 6.

8. The recombinant vaccine according to claim 7, characterized in that The vaccine is a nucleic acid vaccine.

9. A method for preparing one or more fusion proteins according to claim 1, characterized in that: The recombinant host cell of claim 5 is used to express the fusion protein of claim 1, and the fusion protein of claim 1 is isolated.

10. The fusion protein of claim 1, and / or one or more recombinant nucleic acid molecules of claim 2, and / or one or more recombinant gene expression cassettes of claim 3, and / or one or more recombinant vectors of claim 4, and / or one or more recombinant host cells of claim 5, and / or one or more immunogenic compositions or pharmaceutical compositions of claim 6, and / or one or more recombinant vaccines of claim 7 or 8 for preparing a medicament for preventing a disease caused by canine distemper virus.

11. The use according to claim 10, characterized in that The disease is canine distemper.

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