DNA vaccine for mycobacterial disease and method for preventing mycobacterial disease using the same

Abstract

To provide pharmaceutical agents or methods for preventing or treating Cyprinus rubrofuscus "koi" Mycobacterium infection, especially Mycobacterium infection caused by Mycolicibacterium spp..SOLUTION: The problem can be solved by DNA vaccine against Mycobacterium infection containing, as an active ingredient, a DNA containing a nucleic acid sequence encoding an immunogenic polypeptide against Mycolicibacterium spp. or an expression vector containing the DNA, the immunogenic polypeptide being a polypeptide selected from the group consisting of four types of polypeptides containing specific amino acid sequences, or antigenic fragments thereof, or a combination of two or more thereof, or the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a DNA vaccine for mycobacteriosis and a method for preventing mycobacteriosis using the same. TECHNICAL FIELD According to the present invention, mycobacteriosis in fish can be effectively prevented. [Background technology]

[0002] Mycobacteriosis can occur in koi carp farms from autumn to winter. The progression of mycobacteriosis is chronic, and 10% or more of the individuals may die. Bacteria that cause mycobacteriosis include Mycobacterium paragordonae and Mycoolicibacterium spp. (Non-Patent Document 1). Aquafen (active ingredient florfenicol) is effective against Mycobacterium paragordonae, but is almost ineffective against Mycoolicibacterium spp. (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] "PCR detection of causative bacteria of mycobacteriosis in koi carp," Yuichiro Machida et al., Program and Abstracts of the Annual Meeting of the Japanese Society of Fish Pathology, Vol. 2021 Spring, p. 25 (March 20, 2021) [Non-patent document 2] "Development of Antibacterial Therapy for Mycobacteriosis in Koi Carp," Yuichiro Machida et al., Program and Abstracts of the Annual Meeting of the Japanese Society of Fish Pathology, Vol. 2021 Spring, p. 25 (March 20, 2021) Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a medicine or method for preventing or treating mycobacteriosis in carp, particularly mycobacteriosis caused by bacteria of the genus Mycolicibacterium. [Means for solving the problem]

[0005] The present inventors have conducted extensive research into pharmaceuticals or methods for preventing or treating mycobacteriosis in carp, particularly mycobacteriosis caused by Mycobacterium spp., and have surprisingly found that mycobacteriosis caused by Mycobacterium spp. can be prevented or treated by a DNA vaccine containing nucleic acid encoding a specific antigen of Mycobacterium spp. The present invention is based on this finding. Therefore, the present invention provides [1] A DNA vaccine against mycobacteriosis caused by Mycobacterium genus bacteria, comprising as an active ingredient a DNA containing a nucleic acid sequence encoding an immunogenic polypeptide against Mycobacterium genus bacteria, or an expression vector containing said DNA, wherein said immunogenic polypeptide is (1) a polypeptide selected from the group consisting of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide containing the amino acid sequence represented by SEQ ID NO: 4, or an antigenic fragment thereof, or a combination of two or more thereof; (2) an amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence represented by SEQ ID NO: 2, a sequence (3) a DNA vaccine which is a modified polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 4, and which has an effect of protecting against infection with Mycolicibacterium, or an antigenic fragment thereof, or a combination of two or more thereof; or (4) a homologous polypeptide which has an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 4, and which has an effect of protecting against infection with Mycolicibacterium, or an antigenic fragment thereof, or a combination of two or more thereof. [2] The DNA vaccine according to [1], wherein the nucleic acid sequence is a nucleic acid sequence represented by SEQ ID NO: 5, 6, 7, or 8. [3] A mixed DNA vaccine comprising two or more DNA vaccines according to [1] or [2]. [4] A method for preventing or treating mycobacteriosis, comprising a step of administering the DNA vaccine according to any one of [1] to [3] to a fish. [5] (1) a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, or a combination of two or more thereof; (2) a modified polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and which has an effect of protecting against infection with Mycolicibacterium, or a combination of two or more thereof; or (3) a homologous polypeptide which has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and which has an effect of protecting against infection with Mycolicibacterium, or a combination of two or more thereof; [6] A polynucleotide encoding the polypeptide according to [5]. Regarding. [Effects of the Invention]

[0006] The DNA vaccine for mycobacteriosis of the present invention can prevent or treat mycobacteriosis caused by bacteria of the genus Mycobacterium. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a graph showing cumulative mortality in a challenge test with Mycolicibacterium sp. NGTWS-1803 strain in a PBS-administered group and in a group administered with the DNA vaccine of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0008] [1] DNA vaccine The DNA vaccine of the present invention is a DNA vaccine against mycobacteriosis caused by Mycobacterium bacteria, which contains, as an active ingredient, DNA comprising a nucleic acid sequence encoding an immunogenic polypeptide against Mycobacterium bacteria, or an expression vector comprising said DNA. (1) It may be a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide comprising a combination of two or more of these polypeptides (hereinafter, this may be referred to as polypeptide (1)); (2) It may be a modified polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and which has the effect of protecting against infection with Mycolicibacterium, or a polypeptide comprising a combination of two or more of these polypeptides (hereinafter, this may be referred to as modified polypeptide (2)); or (3) It may be a homologous polypeptide which has 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and which has the effect of protecting against infection with Mycolicibacterium, or a polypeptide comprising a combination of two or more of these polypeptides (hereinafter, this may be referred to as homologous polypeptide (3)). Furthermore, the "polypeptide comprising a combination of two or more polypeptides" may be a polypeptide comprising a combination of two or more polypeptides, namely, polypeptide (1), modified polypeptide (2), and homologous polypeptide (3).

[0009] 《Mycobacterial disease》 Mycobacteriosis in the present invention is not limited to those that occur in fish of the Cyprinidae family, such as carp (especially koi carp), crucian carp (e.g., silver crucian carp or goldfish), bitterling, Japanese amberjack, Japanese amberjack, Japanese minnow, Japanese dace, Japanese minnow, Japanese minnow, minnow, minnow, lotus, silver carp, grass carp, blue fish, bighead carp, and silver carp. The causative bacteria of mycobacteriosis include, but are not limited to, Mycobacterium paragordonae, two Mycolicibacterium spp., Mycolicibacter nonchromogenicus, Mycobacteroides chelonae, and Mycobacteroides abscessus. These bacteria are Gram-positive, aerobic, acid-fast, short rods. Mycobacteriosis occurs from autumn to winter, especially in yearling fish. The causative bacteria are thought to infect internal organs such as the swim bladder, kidneys, and spleen. Infected fish become thin and some have cloudy swim bladder. Infected fish become weak and may even die. Aquafen (active ingredient florfenicol) is effective against mycobacteriosis caused by Mycobacterium paragordonae.

[0010] Polypeptide The immunogenic polypeptide may be (1) a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, or an antigenic fragment thereof, or a polypeptide comprising a combination of two or more of these polypeptides. The polypeptides represented by SEQ ID NOs: 1, 2, and 3 are novel polypeptides that share a certain identity with Antigen 85 (Ag85), a major antigen of other acid-fast bacteria. The polypeptide represented by SEQ ID NO: 4 is a protein known as the WXG100 family type VII secretion target of Mycolicibacterium.

[0011] The polypeptide (1) may be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter sometimes referred to as Ag85L-1), a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as Ag85L-3), a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (hereinafter sometimes referred to as Ag85L-5), or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 (hereinafter sometimes referred to as WXG100), or it may be a polypeptide containing Ag85L-1, Ag85L-3, Ag85L-5, or WXG100. In other words, it may be a fusion polypeptide to which a peptide other than Ag85L-1, Ag85L-3, Ag85L-5, or WXG100 is bound. The other peptides are not particularly limited as long as they do not completely inhibit the immunogenicity of Ag85L-1, Ag85L-3, Ag85L-5, or WXG100. For example, they are not limited as long as they mask B cell epitopes or T cell epitopes of Ag85L-1, Ag85L-3, Ag85L-5, or WXG100 and inhibit the induction of humoral or cellular immunity. Therefore, examples of the other peptides include linker peptides, signal peptides, polypeptides (proteins) such as glutathione S-transferase (GST), or TrpE, and fused polypeptides (fusion proteins) with these polypeptides do not inhibit the immunogenicity of Ag85L-1, Ag85L-3, Ag85L-5, or WXG100. The polypeptide (1) may be a fusion polypeptide that combines two or more of the polypeptides Ag85L-1, Ag85L-3, Ag85L-5, and WXG100. The fusion polypeptide can be obtained by, for example, directly binding the respective polypeptides or by binding them via a linker peptide or the like. The fusion polypeptide may be a combination of two, three, or four of the polypeptides Ag85L-1, Ag85L-3, Ag85L-5, and WXG100. The polypeptide (1) exhibits immunogenicity and can therefore exert protective effects against mycobacterial infections as a DNA vaccine.

[0012] Ag85L-1, Ag85L-3, and Ag85L-5 are considered to be major antigens of Mycolicibacterium. The amino acid identity between Ag85L-1 and Ag85L-3 is 71.3%. The amino acid identity between Ag85L-1 and Ag85L-5 is 64.3%. The amino acid identity between Ag85L-3 and Ag85L-5 is 61.7%.

[0013] The modified polypeptide (2) is a modified polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, and which has the effect of protecting against infection with Mycolicibacterium, or an antigenic fragment thereof, or a polypeptide comprising a combination of two or more of these polypeptides.The modified polypeptide (2) may be a polypeptide consisting of these modified polypeptides or a polypeptide comprising these modified polypeptides.In other words, it may be a fusion peptide to which another polypeptide is bound. In the present invention, "an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, or added" means that the amino acid has been modified by a well-known method such as site-directed mutagenesis, or by substitution of several amino acids at a number that would occur naturally. The number of amino acid modifications is preferably 1 to 15, more preferably 1 to 10, even more preferably 1 to 5, even more preferably 1 to 3, and most preferably 1. An example of a modified amino acid sequence of the polypeptide of the present invention can be an amino acid sequence in which the amino acids have one or several (preferably 1, 2, 3, or 4) conservative substitutions.

[0014] The homologous polypeptide (3) may be a homologous polypeptide having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, 2, 3, or 4, and having a protective effect against infection with Mycolicibacterium bacteria, or an antigenic fragment thereof, or a polypeptide comprising a combination of two or more of these polypeptides. The identity of the amino acid sequence is preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.

[0015] Furthermore, in this specification, identity in amino acid sequences means the identity calculated by comparing and analyzing two types of amino acid sequences using computer analysis software (SDC software) and assuming that the amino acids are identical in two types of sequences if the same type of amino acid is present in the same position.

[0016] The "amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added" or "amino acid sequence with 90% or more identity" to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 is a mutation of the amino acid sequence of SEQ ID NO: 1 to 4 by substitution or the like, but this amino acid sequence substitution is a conservative substitution that maintains the protective effect against Mycolicibacterium bacteria of the present invention. For example, it is common general knowledge that the protective effect of a protein antigen can be reduced by the substitution of a single amino acid. On the other hand, it is also common general knowledge that the protective effect of a protein antigen is not affected at all by the substitution of an amino acid in a protein antigen. This is because substitution at an important amino acid position in a B cell epitope or T cell epitope related to immunogenicity is likely to change immunogenicity, while substitution at an amino acid position not involved in the antigenicity of the B cell epitope or T cell epitope is unlikely to change immunogenicity. Because B cell epitopes or T cell epitopes are located at specific positions, it is highly likely that the protective effect can be maintained even by the substitution of an amino acid.

[0017] Furthermore, "conservative substitutions," in which an amino acid is replaced with one having similar properties, are likely to maintain immunogenicity. "Conservative substitutions" can be achieved by replacing an amino acid residue with another chemically similar amino acid residue, but are not limited to these. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0018] The fact that the modified polypeptide (2) or homologous polypeptide (3) has a "protective effect against Mycolicibacterium" can be confirmed by following the Examples in this specification. That is, those skilled in the art can easily determine the protective effect against Mycolicibacterium of a DNA vaccine encoding a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, or added, or a polypeptide having an amino acid sequence with 90% or more identity, by following the method described in the Examples.

[0019] The antigenic fragments of the polypeptide (1), modified polypeptide (2), and homologous polypeptide (3) encoded by the polynucleotide of the DNA vaccine of the present invention are not particularly limited, so long as they are antigenic (immunogenic) to carp. That is, it is known that even a peptide of a few amino acids can be antigenic, for example, when the antigenic fragment has a B cell epitope or a T cell epitope. Therefore, the length of the amino acid sequence of the antigenic fragment is preferably 5 amino acids or more, more preferably 10 amino acids or more, more preferably 30 amino acids or more, more preferably 50 amino acids or more, more preferably 100 amino acids or more, more preferably 150 amino acids or more, and more preferably 200 amino acids or more. Furthermore, the length of the amino acid sequence of the antigenic fragment is preferably 1 / 20 or more, more preferably 1 / 10 or more, more preferably 1 / 5 or more, more preferably 1 / 4 or more, more preferably 1 / 3 or more, or more preferably 1 / 2 or more of the amino acid length of the full-length polypeptide.

[0020] Polynucleotide The polynucleotide used in the present invention is not particularly limited as long as it is a polynucleotide that encodes the polypeptide (1), modified polypeptide (2), or homologous polypeptide (3) or an antigenic fragment thereof, but is preferably (1) a nucleotide sequence represented by SEQ ID NO: 5 to 8, (2) a polynucleotide that has 80% or more homology to the polynucleotide represented by SEQ ID NO: 5 to 8 and that provides a DNA vaccine with a protective effect against infection with Mycolicibacterium bacteria, or a polynucleotide that is a partial sequence of polynucleotide (1) or (2) and encodes the antigenic fragment. As used herein, homology in nucleotide sequences refers to the value calculated by comparing and analyzing two nucleotide sequences using computer analysis software (SDC software), and assuming that the nucleotides in the two sequences are identical if the same type of nucleotide is present in the same position.

[0021] <Combined DNA vaccine> The DNA vaccine of the present invention may be a combination vaccine comprising two or more DNA vaccines, including a vaccine encoding an Ag85L-1-related polypeptide (hereinafter sometimes referred to as an Ag85L-1 vaccine), a vaccine encoding an Ag85L-3-related polypeptide (hereinafter sometimes referred to as an Ag85L-3 vaccine), a vaccine encoding an Ag85L-5-related polypeptide (hereinafter sometimes referred to as an Ag85L-5 vaccine), and a vaccine encoding a WXG100-related polypeptide (hereinafter sometimes referred to as a WXG100 vaccine). For example, combinations of two or more of Ag85L-1 vaccine, Ag85L-3 vaccine, and Ag85L-5 vaccine are acceptable, but those including WXG100 vaccine are preferred. Specific examples include combinations of WXG100 vaccine and Ag85L-1 vaccine, combinations of WXG100 vaccine and Ag85L-3 vaccine, combinations of WXG100 vaccine and Ag85L-5 vaccine, combinations of WXG100 vaccine, Ag85L-1 vaccine, and Ag85L-3 vaccine, combinations of WXG100 vaccine, Ag85L-1 vaccine, and Ag85L-5 vaccine, combinations of WXG100 vaccine, Ag85L-3 vaccine, and Ag85L-5 vaccine, and combinations of WXG100 vaccine, Ag85L-1 vaccine, Ag85L-3 vaccine, and Ag85L-5 vaccine, and combinations of WXG100 vaccine, Ag85L-1 vaccine, Ag85L-3 vaccine, and Ag85L-5 vaccine.

[0022] Expression Vector The expression vector used in the present invention is not particularly limited, as long as it is a vector that can be expressed in fish cells. The expression vector used in the present invention is a self-replicating vector, i.e., it exists as an independent entity outside the chromosome and its replication does not depend on chromosomal replication, and can be constructed based on, for example, a plasmid. Furthermore, the expression vector may be incorporated into the host genome when introduced into the host and replicated together with the chromosome into which it has been integrated. Procedures and methods commonly used in the field of genetic engineering can be used to construct expression vectors that can be used in the present invention.

[0023] Transcriptional regulatory sequences that can be used in the present invention include, for example, constitutive promoters, inducible or regulatable promoters, tissue-specific promoters, and promoters derived from the genes of the antigens being expressed, but are not limited thereto, as long as they are capable of expression in fish cells. Constitutive promoters include, for example, promoter sequences derived from cytomegalovirus (CMV), or strong promoters such as those from Rous sarcoma virus (RSV), simian virus-40 (SV-40), muscle β-actin promoters, and herpes simplex virus (HSV). Tissue-specific promoters include, for example, the thymidine kinase promoter. Inducible or regulatable promoters include, for example, growth hormone-regulated promoters, promoters under the control of lac operon sequences, and zinc-inducible metallothionein promoters. The transcriptional regulatory sequences can be operably linked (i.e., so as to regulate expression of) a nucleotide sequence encoding an immunogenic polypeptide.

[0024] The regulatory sequences can include expression control sequences, including promoter (e.g., the inducible or constitutive promoter) DNA sequences, and optionally further include enhancer elements, intron sequences for splicing of transcription or polyadenylation signals (e.g., from Simian Virus-40 (SV-40) or bovine growth hormone), or one or more copies of immunostimulatory DNA sequences known as CpG motifs.

[0025] Furthermore, the expression vector may contain, if desired, a selectable marker such as a bacterial replication origin sequence or an antibiotic resistance (e.g., kanamycin) gene or a non-antibiotic resistance gene (e.g., β-galactosidase gene) for selection.

[0026] [2] Method for preventing or treating mycobacterial disease The method for preventing or treating mycobacteriosis of the present invention comprises the step of administering the DNA vaccine to fish. The bacteria to which the method for preventing or treating mycobacteriosis of the present invention is applied are not limited to those of the genus Mycobacterium. Mycobacterium bacteria contain Ag85L-1, Ag85L-3, Ag85L-5, and WXG100, or antigens thereof, or antigens homologous to these antigens, and the DNA vaccine of the present invention can induce humoral or cellular immunity that acts to protect against infection.

[0027] Fish to which the preventive or therapeutic methods of the present invention can be applied are not particularly limited, as long as they are fish that can be infected with Mycolicibacterium bacteria, and examples include carp (e.g., koi carp), crucian carp (e.g., ginbuna or goldfish), bitterling, Japanese amberjack, Japanese amberjack, Japanese minnow, Japanese dace, Japanese minnow, Japanese dace, brown minnow, Japanese minnow, common minnow, minnow, Japanese amberjack, lotus fish, silver carp, grass carp, Japanese blue fish, bighead carp, and silver carp.

[0028] DNA vaccines can be administered by oral administration, intramuscular injection, intraperitoneal injection, gene gun administration, and immersion, with intramuscular injection or gene gun administration being preferred. Gene gun administration involves coating a plasmid onto gold particles approximately 1 μm in size and then shooting them into the subject's skin, cells, or tissues using high-pressure helium gas in a specialized device, similar to an air gun. Gene gun administration offers several advantages, including the ability to achieve the same immune effect with 1 / 100-1 / 1000th the amount of DNA required for intramuscular injection, and superior reproducibility compared to intramuscular injection.

[0029] The DNA vaccine of the present invention may be administered as is, or may be administered mixed with an adjuvant. Adjuvants stimulate the immune system to enhance immune responses to antigens and are mainly added to vaccines as auxiliary agents. Typical adjuvants include, for example, aluminum compounds, polynucleotides, and bacterial components.

[0030] [3] Use of DNA vaccines The DNA vaccine of the present invention can be used to induce an immune response against mycobacteria, particularly Mycolicibacterium. Fish in which an immune response can be induced include, but are not limited to, carp (e.g., koi carp), crucian carp (e.g., ginbuna or goldfish), bitterling, Japanese amberjack, Japanese amberjack, Japanese minnow, Japanese dace, Japanese minnow, Japanese minnow, Japanese minnow, lotus, Japanese smelt, grass carp, Japanese bluefin tuna, bighead carp, silver carp, etc. Mycobacteriosis can be prevented or treated by inducing an immune response.

[0031] 《Effect》 The mechanism by which the DNA vaccine of the present invention can suppress mycobacteriosis caused by Mycolicibacterium bacteria in carp and other fish has not been investigated in detail, but is presumed as follows. However, the present invention is not limited to the following presumption. The DNA vaccine of the present invention is believed to be capable of inducing humoral and / or cellular immunity against Ag85L-1, Ag85L-3, Ag85L-5, and WXG100, which are believed to be major antigens of Mycolicibacterium bacteria. The induced humoral and / or cellular immunity is believed to prevent infection of fish internal organs such as the swim bladder, kidney, and spleen with Mycolicibacterium bacteria, and to suppress fish mortality by eliminating infected cells. [Example]

[0032] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0033] Example 1 In this example, the genes for Ag85L-1, Ag85L-3, Ag85L-5, and WXG100 were isolated from bacteria of the genus Mycolicibacterium, and DNA vaccines were prepared. (1) Preparation of PCR primers The following PCR primers were prepared from the DNA base sequences of Ag85L-1 (SEQ ID NO: 5), Ag85L-3 (SEQ ID NO: 6), Ag85L-5 (SEQ ID NO: 7), and WXG100 (SEQ ID NO: 8) of Mycolicibacterium sp. NGTWS-NA01 strain. Ag85L-1-forward: 5'-tggctagttaagcttcgatgcacgtgggacttgc-3' (SEQ ID NO: 9) Ag85L-1-reverse: 5'-ccctctagactcgagggcggtgttctgagccc-3' (SEQ ID NO: 10) Ag85L-3-forward: 5'-tggctagttaagcttgtatgagacttcttaacaa-3' (SEQ ID NO: 11) Ag85L-3-reverse: 5'-ccctctagactcgaggccgatgaggtgcgcct-3' (SEQ ID NO: 12) Ag85L-5-forward: 5'-tggctagttaagcttccatgcgtgcattgactcg-3' (SEQ ID NO: 13) Ag85L-5-reverse: 5'-ccctctagactcgagcctgatggttgcggcta-3' (SEQ ID NO: 14) WXG100-forward: 5'-tggctagttaagcttccatgtcacagatcatgta-3' (SEQ ID NO: 15) WXG100-reverse: 5'-ccctctagactcgaggccccacttggcacctt-3' (SEQ ID NO: 16)

[0034] Chromosomal DNA was extracted from Mycolicibacterium sp. NGTWS-NA01 strain by the phenol-chloroform method. PCR was performed using the obtained DNA as a template and the PCR primers described above in accordance with the attached instructions using a commercially available PCR reaction reagent (ExTaq; TAKARA Bio) to obtain the desired DNA fragment. The amplified DNA fragments were inserted into the pcDNA3.1 / myc-His (+) A vector and sequenced. The sequences were analyzed and confirmed to be the DNA of Ag85L-1, Ag85L-3, Ag85L-5, and WXG100. These expression plasmids were obtained in large quantities using Escherichia coli JM109. The extracted plasmid DNA was used as a DNA vaccine.

[0035] Example 2 In this example, carp were inoculated with the DNA vaccine and challenged with Mycolicibacterium sp. NGTWS-1803 strain. Each plasmid obtained in Example 1 was administered to carp (average body weight: 8.7 g) at a dose of 10 μg / fish into the left dorsal muscle. A test group containing PBS alone was set up as a negative control group. 30 days after vaccination, Mycolicibacterium sp. NGTWS-1803 strain was administered intraperitoneally at a dose of 5.4 × 10 8The challenge test was conducted using the vaccine at a dose of 1000 CFU / fish. Mortality was then monitored for 235 days. The final cumulative mortality rate in the PBS-administered group was 34.5%, while the cumulative mortality rate in either DNA-vaccinated group was reduced. Specifically, the Ag85L-1 + WXG100 group had a 3.3% cumulative mortality rate, while the Ag85L-1 + -3 + -5 + WXG100 group had a 0% cumulative mortality rate. The vaccine efficacy (Relative Percent Survival, RPS) in these two vaccinated groups was 90.3% and 100%, respectively (Table 1, Figure 1).

[0036] [Table 1] [Industrial Applicability]

[0037] The DNA vaccine of the present invention can be used to prevent or treat mycobacteriosis in carp.

Claims

1. A DNA vaccine for mycobacteriosis caused by Mycobacterium genus bacteria, comprising, as an active ingredient, DNA comprising a nucleic acid sequence encoding an immunogenic polypeptide against Mycobacterium genus bacteria, or an expression vector comprising said DNA, the immunogenic polypeptide is (1) A polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, or an antigenic fragment thereof, or a combination of two or more thereof; (2) A modified polypeptide, or an antigenic fragment thereof, or a combination of two or more thereof, which contains an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and which has an effect of protecting against infection with Mycolicibacterium; or (3) A homologous polypeptide having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and having an effect of protecting against infection with Mycolicibacterium, or an antigenic fragment thereof, or a combination of two or more thereof; That is, a DNA vaccine.

2. The DNA vaccine of claim 1 , wherein the nucleic acid sequence is a nucleic acid sequence represented by SEQ ID NO: 5, 6, 7, or 8.

3. A combined DNA vaccine comprising two or more types of DNA vaccines according to claim 1 or 2.

4. A method for preventing or treating mycobacteriosis, comprising the step of administering the DNA vaccine according to any one of claims 1 to 3 to fish.

5. (1) A polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4; (2) A modified polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and having an effect of protecting against infection with Mycolicibacterium; or (3) A homologous polypeptide having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 3, or the amino acid sequence represented by SEQ ID NO: 4, and having an effect of protecting against infection with Mycolicibacterium bacteria. A polypeptide.

6. A polynucleotide encoding the polypeptide of claim 5.

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