WO2010074126A1 - 組換え鶏伝染性コリーザワクチン及びその製造方法 - Google Patents
組換え鶏伝染性コリーザワクチン及びその製造方法 Download PDFInfo
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- WO2010074126A1 WO2010074126A1 PCT/JP2009/071398 JP2009071398W WO2010074126A1 WO 2010074126 A1 WO2010074126 A1 WO 2010074126A1 JP 2009071398 W JP2009071398 W JP 2009071398W WO 2010074126 A1 WO2010074126 A1 WO 2010074126A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Definitions
- the present invention relates to a recombinant chicken infectious coryza vaccine and a method for producing the same. Specifically, a part of the outer membrane protein of A type bacteria and A.pgC type outer membrane protein of A. bacterium paragallinarum (hereinafter also referred to as “A.pg”) The present invention relates to a recombinant chicken infectious coryza vaccine containing a fusion peptide comprising a part of the above as an active ingredient and a method for producing the same.
- chicken infectious coryza An important respiratory organ disease in chickens is chicken infectious coryza which develops due to A.pg infection. Chickens that develop chicken infectious coryza have nasal discharge, facial swelling or lacrimation as the main symptoms. Chicken infectious coryza has a large economic loss because it causes a decrease in the breeding rate of chickens, a delay in the start of spawning, a decrease in the spawning rate or a halt of spawning.
- Page et al. Classify A.pg into three types, A, B, and C, based on differences in serotypes (see, for example, Non-Patent Document 1), and Sawata et al. They were classified into molds (for example, see Non-Patent Document 2). Subsequently, Kume et al. Reported that Page A type A corresponds to Sawata et al type 1 and Page et al C type corresponds to Sawata et al type 2 (see, for example, Non-Patent Documents 3 and 4).
- type A bacteria (hereinafter sometimes referred to as "A.pgA type bacteria") and type C (type 2) bacteria (hereinafter referred to as "A.pgC type bacteria”) Is a major cause of chicken infectious coryza.
- inactivated vaccines in which cells of A.pgA type or A.pgC type cells have been inactivated with formalin, thimerosal, etc. have been widely used in the past, and their preventive effect is good. Although it has achieved results, on the other hand, when these inactivated vaccines are administered, problems of side effects such as formation of necrotic lesions in the inoculated chicken have been pointed out (see, for example, Non-Patent Document 5). . Under such circumstances, research on a recombinant vaccine using a protective antigen obtained by genetic recombination as a vaccine is being conducted as a safe vaccine development effort.
- Tokunaga et al. Isolated and identified a gene encoding a cell outer membrane protein of A.pgA type bacteria (cell outer membrane protein gene), and part of the gene (HPG3.5kbp, HPG4.1kbp) It was found that a peptide obtained by expressing Escherichia coli in Escherichia coli is useful as a protective antigen for chicken infectious coryza. Furthermore, they obtained the outer membrane protein gene of type C bacteria using the DNA fragment as a probe, and compared the base sequence of the open reading frame of the outer membrane protein gene of type A and type C. Analyzed.
- region 1 a region of about 3.4 kbp on the 5 ′ side
- region 3 a region of about 1.2 kbp on the 3 ′ side
- region 2 a region of about 1.5 kbp sandwiched between regions 1 and 3
- Noro et al. Reported that the outer cell membrane protein discovered by Tokunaga et al. Is important as a protective antigen for chicken infectious coryza.
- Noro et al. Immunized chickens with peptides encoded by DNA fragments of 4,801 bp and 5,157 bp, which are part of the outer membrane protein gene of A. pgA type bacteria, so that the peptides were converted to A.pgA type.
- Patent Document 2 HI antibody-inducing ability against bacteria and vaccine effect
- Patent Document 3 HI antibody-inducing ability against bacteria and vaccine effect
- Yamamoto et al. also demonstrated the usefulness of chicken infectious coryza as a vaccine using a polypeptide encoded by a 2,016 bp DNA fragment containing most of the outer membrane protein gene of A.pgA type bacteria.
- about 300 bp at the 3 ′ end of the DNA fragment they reported was significantly different from the base sequence shown by Tokunaga and Noro et al.
- A.pgA type bacteria and A.pgC type bacteria which are the main causative bacteria of chicken infectious coryza, or a peptide composed of a part of the outer membrane protein or part thereof, is a protective antigen of chicken infectious coryza. It has been revealed that it is useful. Therefore, by mixing these infection-protecting antigens, it is possible to efficiently immunize the chicken infectious coryza. However, in the method of simply using a combined vaccine, it is necessary to produce two infection protective antigens separately, which increases the production cost. In general, animal vaccines, unlike human vaccines, are not acceptable to livestock farmers unless they are low in price in addition to quality. Therefore, animal vaccines are required to have production methods with reduced manufacturing costs.
- region 2 of the outer cell membrane protein gene of A.pgA type bacteria and A.pgC type bacteria (3,639 in A.pgA type bacteria).
- a ⁇ 5-1 and C ⁇ 5-1
- AC ⁇ 5-1 a DNA fragment encoding C ⁇ 5-1
- C ⁇ 5-1 is expressed in the soluble fraction when expressed alone, but AC ⁇ 5-1 forms inclusion bodies and is expressed in the insoluble fraction, completing the present invention. It came to.
- the object of the present invention is to provide a fusion in which a peptide fragment containing a specific region of an outer membrane protein of an A.pgA type bacterium and a peptide fragment containing a specific region of an outer membrane protein of an A.pgC type bacterium are combined. It is to provide a chicken infectious coryza vaccine comprising a peptide as an active ingredient and a method for producing the same.
- HMTp210 protein the outer membrane proteins of A.pgA type bacteria and A.pgC type bacteria isolated and identified by Tokunaga et al.
- Patent Document 1 the outer membrane proteins of A.pgA type bacteria and A.pgC type bacteria isolated and identified by Tokunaga et al.
- Patent Document 1 the outer membrane proteins of A.pgA type bacteria and A.pgC type bacteria isolated and identified by Tokunaga et al.
- the present invention is as follows.
- a recombinant chicken infectious coryza vaccine comprising the steps of culturing the host, recovering and purifying the inclusion body fraction from the culture, and preparing a composition containing the purified inclusion body fraction Manufacturing method.
- Peptide A is a sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1, 27, 28, 29, 30, 31, 32, 33, 34 and 35
- peptide C is SEQ ID NO: 2, 3 4.
- the production method according to [1] which is a sequence selected from the group consisting of amino acid sequences represented by 4, 50, 51, 52, 53, 54, 55 and 56.
- [5] The production method according to [3] or [4], wherein peptide A or peptide C has an amino acid sequence in which one or several amino acids are deleted, added or substituted .
- [6] The production according to any one of [1] to [5], wherein the ratio of peptide A to peptide C in the fusion peptide is 1 for peptide A and 1 to 3 for peptide C Method.
- the fusion peptide includes a structure in which peptide C is bound to the C-terminal side of at least one pair of peptides A.
- the fusion peptide has a linker between peptide A and peptide A, between peptide C and peptide C, or between peptide A and peptide C, [1] to [7]
- the production method according to any one of [7].
- the fusion peptide is SEQ ID NO: 8, 9, 10, 11, 12, 13, 41, 42, 43, 44, 45, 46, 47, 48, 49, 61, 62, 63, 64, 65, 66. And 67, the production method according to [3].
- a fusion peptide comprising a peptide fragment derived from the HMTp210 protein of A.pgA type bacteria (peptide A) and a peptide fragment derived from the HMTp210 protein of type A.pgC bacteria (peptide C) Sex coryza vaccine.
- peptide A a peptide fragment derived from the HMTp210 protein of A.pgA type bacteria
- peptide C a peptide fragment derived from the HMTp210 protein of type A.pgC bacteria
- Peptide A is a sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1, 27, 28, 29, 30, 31, 32, 33, 34 and 35
- peptide C is SEQ ID NO: 2, 3
- the vaccine according to [10] which is a sequence selected from the group consisting of the amino acid sequences represented by 4, 50, 51, 52, 53, 54, 55 and 56.
- the fusion peptide is SEQ ID NO: 8, 9, 10, 11, 12, 13, 41, 42, 43, 44, 45, 46, 47, 48, 49, 61, 62, 63, 64, 65, 66.
- the vaccine according to [13] which consists of the amino acid sequence represented by & 67.
- [20] Within the amino acid sequence in which 1 to 200 amino acids are added to the N-terminal side and / or C-terminal side of the amino acid sequence represented by SEQ ID NO: 1, and comprising the amino acid sequence represented by SEQ ID NO: 35 Recombinant chicken infectious coryza vaccine containing the peptide as an active ingredient.
- a chicken infectious coryza vaccine comprising a fusion peptide obtained by binding a peptide fragment of the A.pgA type HMTp210 protein and a peptide fragment of the A.pgC type HMTp210 protein, and a method for producing the same.
- the chicken infectious coryza vaccine of the present invention can simultaneously confer immunity to prevent chicken infectious coryza caused by each of A.pgA type bacteria and A.pgC type bacteria.
- inclusion bodies can be expressed in the insoluble fraction.
- a protective antigen corresponding to each of A.pgA type bacteria and A.pgC type bacteria can be produced by one culture, resulting in a reduction in production cost.
- FIG. 1 is a diagram showing the position of the A ⁇ 5-1 fragment in the HMTp210 gene (HMTp210A gene) of A.pgA type bacteria.
- the base sequence numbers described in the figure correspond to the base sequence numbers disclosed in Patent Document 1 (Tokunaga et al.).
- FIG. 2 is a diagram showing the positions of C ⁇ 4c-1, C ⁇ 5-1, and C ⁇ 6b-1b fragments in the HMTp210 gene (HMTp210C gene) of A. pgC type bacteria.
- the base sequence numbers described in the figure correspond to the base sequence numbers disclosed in Patent Document 1 (Tokunaga et al.).
- FIG. 3 is a photograph showing the results of SDS-PAGE of the supernatant and sediment fractions obtained by centrifuging the crushed product of fusion peptide-producing E. coli.
- M marker
- lane 1 A ⁇ 5-1 / C ⁇ 4c-1 (sediment fraction)
- lane 2 A ⁇ 5-1 / C ⁇ 4c-1 (supernatant fraction)
- lane 3 AC ⁇ 5-1 (sediment fraction)
- lane 4 AC ⁇ 5-1 (supernatant fraction)
- lane 5 A ⁇ 5-1 / C ⁇ 6b-1b (sediment fraction)
- lane 6 A ⁇ 5-1 / C ⁇ 6b-1b (supernatant fraction).
- the arrow indicates the expressed fusion peptide.
- FIG. 4 shows A ⁇ 5-1, A ⁇ 5-2, A ⁇ 5-3, A ⁇ 5-4, A ⁇ 9-2, A ⁇ 9-3, A ⁇ 9-4, C ⁇ 6-2, C ⁇ 6 in the HMTp210 gene (HMTp210A gene) of A.pgA type bacteria.
- FIG. 3 shows the positions of -3 and C ⁇ 6-4 fragments.
- the base sequence numbers described in the figure correspond to the base sequence numbers disclosed in Patent Document 1 (Tokunaga et al.).
- FIG. 5 shows the C ⁇ 5-1, C ⁇ 5-2, C ⁇ 5-4, C ⁇ 9-0, C ⁇ 9-2, C ⁇ 9-4, C ⁇ 6-2 and C ⁇ 6-4 fragments of the A.pgC type HMTp210 gene (HMTp210C gene). It is a figure which shows a position.
- the base sequence numbers described in the figure correspond to the base sequence numbers disclosed in Patent Document 1 (Tokunaga et al.).
- FIG. 6 is a photograph showing a result of SDS-PAGE of a sediment fraction obtained by centrifuging a disrupted product of fusion peptide-producing E. coli.
- M marker
- lane 1 AC ⁇ 5-1 (sediment fraction)
- lane 2 A ⁇ 5-2 / C ⁇ 5-1 (sediment fraction)
- lane 3 A ⁇ 5-3 / C ⁇ 5-1 (sediment fraction)
- lane 4 A ⁇ 5-4 / C ⁇ 5-1 (sediment fraction)
- lane 5 A ⁇ 9-2 / C ⁇ 5-1 (sediment fraction)
- lane 6 A ⁇ 9-3 / C ⁇ 5-1 (sediment fraction)
- lane 7 A ⁇ 9 -4 / C ⁇ 5-1 (sediment fraction)
- lane 8 A ⁇ 6-2 / C ⁇ 5-1 (sediment fraction)
- lane 9 A ⁇ 6-3 / C ⁇ 5-1 (sediment fraction)
- lane 10 A ⁇ 6-4 / C ⁇ 5-1 (sediment fraction).
- FIG. 7 is a photograph showing the results of SDS-PAGE of a sediment fraction obtained by centrifuging a disrupted product of fusion peptide-producing E. coli.
- M marker
- lane 1 AC ⁇ 5-1 (sediment fraction)
- lane 2 A ⁇ 5-1 / C ⁇ 5-2 (sediment fraction)
- lane 3 A ⁇ 5-1 / C ⁇ 5-4 (sediment fraction)
- lane 4 A ⁇ 5-1 / C ⁇ 9-0 (sediment fraction)
- lane 5 A ⁇ 5-1 / C ⁇ 9-2 (sediment fraction)
- lane 6 A ⁇ 5-1 / C ⁇ 9-4 (sediment fraction)
- lane 7 A ⁇ 5 -1 / C ⁇ 6-2 (sediment fraction)
- lane 8 A ⁇ 5-1 / C ⁇ 6-4 (sediment fraction).
- the arrow indicates the expressed fusion peptide.
- FIG. 8 is a photograph showing the results of SDS-PAGE of the supernatant or sediment fraction obtained by centrifuging peptide C-producing Escherichia coli.
- M marker
- lane 1 C ⁇ 5-1-pQE (supernatant fraction)
- lane 2 C ⁇ 5-2-pQE (supernatant fraction)
- lane 3 C ⁇ 5-4-pQE (supernatant fraction)
- lane 4 C ⁇ 9-0-pQE (supernatant fraction)
- lane 5 C ⁇ 9-2-pQE (supernatant fraction)
- lane 6 C ⁇ 9-4-pQE (supernatant fraction)
- lane 7 C ⁇ 6-2 -pQE (sediment fraction)
- lane 8 C ⁇ 6-4-pQE (supernatant fraction).
- the arrow indicates the expressed fusion peptide.
- the feature of the present invention is an inclusion body-forming fusion peptide comprising peptide A derived from HMTp210 protein of Abibacterium paragalinarum (abbreviated as “A.pg”) type A and peptide C derived from HMTp210 protein of type A.pgC
- a method for producing a recombinant chicken infectious coryza vaccine comprising the step of: More specifically, the present invention is characterized by the construction of a host that produces as a inclusion body a fusion peptide composed of a peptide fragment of the HMTp210 protein of A.pgA type bacteria and a peptide fragment of the HMTp210 protein of A.pgC type bacteria.
- HMTp210A gene A. pgA type HMTp210 protein amino acid sequence (SEQ ID NO: 25) encoding the gene (hereinafter also referred to as “HMTp210A gene") and A.pgC type HMTp210 protein amino acid sequence (SEQ ID NO: 26)
- HMTp210C gene A DNA fragment consisting of a part of a gene coding for (hereinafter also referred to as “HMTp210C gene”) can be obtained according to the following method.
- A.pgA type bacteria there are various isolates of A.pgA type bacteria and A.pgC type bacteria, but there is no particular limitation for use in the present invention. So far, as A.pgA type bacteria, for example, 221 strain, O83 strain and W strain, etc., as A.pgC type bacteria, for example, 53-47 strain, modest strain and HK-1 strain, etc. have been isolated, Among strains, mutations in which one to several amino acids are substituted, deleted, or added are observed. Any strain may be used in the present invention.
- A.pgA and A.pgC media containing polypeptone, glucose, casamino acid, sodium glutamate, yeast extract, sodium chloride, chicken water, ⁇ NAD, chicken serum, etc. are used.
- chicken serum-added chicken broth medium 1,000 mL medium, polypeptone S 5 g, casamino acid 1 g, sodium chloride 5 g, sodium L-glutamate 5 g, glucose 1 g, yeast 10 g of extract, 175 mL of chicken water, 25 mL of chicken serum, and 0.025% nicotinamide / adenine / dinucleotide ( ⁇ -NAD) were used.
- the culture conditions are usually set in the range of a temperature of 37 ° C. and a period of 16 to 24 hours, but may be appropriately adjusted according to the purpose of use, the culture form, the amount of planted bacteria, the medium scale, and the like.
- the bacterial cells in the culture solution are collected in the sediment by centrifugation (5,800 g, 20 minutes).
- the HMTp210A gene and the HMTp210C gene (hereinafter sometimes referred to simply as “HMTp210 gene” when there is no need to distinguish between them) are generally described by Sambrook et al.
- genomic DNA extracted from bacterial cells can be prepared according to a conventional genetic recombination technique (Molecular-Cloning, A-Laboratory-Manual-Second-Edition. Cold-Spring-Harbor-Laboratory-Press, NY, A1989).
- a commercially available kit is used. For example, a pure DNA kit (Gentra Systems), Sepagene kit (Sanko Junyaku), ISOPLANT (Wako Junyaku), etc. are used for extraction of chromosomal DNA.
- chromosomal DNA is extracted from the cells collected by centrifugation using a pure gene kit (Gentra® Systems) or the like, and according to the method of Tokunaga et al. Prepare the rally.
- a pure gene kit Genetra® Systems
- Prime® STAR® HS® DNA® Polymerase (Takara Bio Inc.) is used to amplify a DNA fragment of the desired size by PCR according to the attached protocol.
- Primers used for PCR are designed based on the base sequence of HMTp210 gene derived from A.pgA type bacteria and A.pgC type bacteria disclosed by Tokunaga et al. (Patent Document 1).
- PCR primers can be easily obtained by requesting a DNA synthesis contract organization (for example, Sigma Genosys Japan). At this time, base sequences of appropriate restriction enzyme cleavage sites are added to the 5 ′ end of the upstream Primer and the 5 ′ end of the downstream Primer.
- the DNA fragment encoding the fusion peptide of the present invention can be obtained by directly or once cleaving with the restriction enzyme the DNA fragment of the HMTp210A gene and the DNA fragment of the HMTp210C gene obtained above and then binding them with a DNA synthase. .
- a DNA fragment encoding a linker comprising an amino acid sequence of an appropriate size may be added between the DNA fragment of the HMTp210A gene and the DNA fragment of the HMTp210C gene.
- the amino acid neutral amino acids such as glycine and serine having a high degree of freedom are preferably used, and a linker composed of one kind of amino acid or a linker composed of two or more kinds of amino acids is used.
- the size of the linker is generally 5 to 20 amino acids, preferably 10 to 15 amino acids.
- DNA fragments of HMTp210A gene and HMTp210C gene encoding a fusion peptide that has an ability to protect against A.pgA and A.pgC bacteria and forms inclusion bodies are used.
- a DNA fragment for example, a DNA fragment encoding a peptide in region 2 of the outer cell membrane protein, or a peptide to which the region 2 and its N-terminal side and / or C-terminal side amino acid sequence are added is encoded.
- DNA fragment DNA fragment encoding the peptide in which the same region 2 and its N-terminal or C-terminal amino acid sequence are added and the opposite N-terminal or C-terminal amino acid sequence is deleted And a DNA fragment encoding a peptide in which the amino acid sequence at the N-terminal side and / or C-terminal side of 2 is deleted.
- the number of amino acid sequences to be added or deleted is 1 to 200, preferably 30 to 150.
- A consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 .
- SEQ ID NO: 1 SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 .pgA type HMTp210 protein-derived peptide
- SEQ ID NO: 2 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, a DNA fragment encoding a peptide derived from the HMTp210 protein of A.pgC type bacteria consist
- a DNA fragment encoding a mutant peptide in which one to several amino acids of the above peptide are substituted, deleted or added can also be used.
- “mutant peptide in which one to several amino acids are substituted, deleted or added” means that 1, 2, 3, 4 or 5 amino acids are substituted, deleted or added. It refers to a mutant peptide.
- a DNA fragment encoding such a mutant peptide can be hybridized with a DNA fragment having a base sequence complementary to the DNA fragment encoding the above peptide under stringent conditions or by mutagenesis by site-directed mutagenesis. Can be obtained by law. Since all kits are commercially available, these may be used.
- the DNA fragment derived from the HMTp210A gene and the DNA fragment derived from the HMTp210C gene may be in any combination as long as the fusion peptide forms an inclusion body.
- the DNA fragment encoding the fusion peptide may be one obtained by binding a DNA fragment derived from the HMTp210C gene downstream of the DNA fragment derived from the HMTp210A gene, or vice versa.
- a DNA fragment encoding the fusion peptide may be tandemly bound, or two or more DNA fragments derived from the HMTp210C gene may be ligated downstream of two or more DNA fragments derived from the HMTp210A gene. .
- the DNA fragment encoding the fusion peptide is composed of DNA fragments 1 to 3 derived from the HMTp210C gene with respect to the DNA fragment 1 derived from the HMTp210A gene.
- the ratio is 1: 1.
- the base sequence of the obtained DNA fragment is once cloned into pBluescript II SK + (Stratagene) or pCR2.1-TOPO (Invitrogen), and then determined by a DNA sequencer (ABI Prism 377 Applied Biosystems).
- the DNA fragments derived from the A.pgA type bacteria and A.pgC type bacteria obtained as described above or the DNA fragment encoding the fusion peptide are incorporated into an appropriate expression vector and introduced into a host, whereby expression of each DNA fragment can be achieved.
- Done Bacteria, yeast, animal cells, plant cells, insect cells and the like are commonly used for the expression of foreign proteins and peptides, but any host may be used as long as it forms an inclusion body. A known method may be used when transforming a host cell.
- a calcium phosphate method for example, a calcium phosphate method, a DEAE dextran method, a method using lipofectin-based liposomes, a protoplast polyethylene glycol fusion method, an electroporation method, a heat shock method and the like can be used, and an appropriate method may be selected depending on the host cell to be used.
- E. coli capable of expressing a large amount of foreign protein is used.
- E. coli For expression of E. coli, various expression vectors having trp promoter, T7 promoter, cspA promoter, etc. have been developed and marketed. Examples of such expression vectors include pET-11d (Merck) and pQE30 (Qiagen). An appropriate Escherichia coli such as BL21, HMS174, DH5 ⁇ , HB101, JM109 or the like is selected as a host according to the expression vector. Transformation of E. coli can be performed using a commercially available competent cell according to the attached method. In this way, recombinant E. coli that produces the desired polypeptide is obtained. Medium used for culturing E.
- coli eg, LB, SOC, SOB, etc.
- reagent used for selection of transformants eg, ampicillin, etc.
- reagent used for expression induction eg, indole acetic acid (IAA)
- IAA indole acetic acid
- IPTG isopropylthio-beta-D-galactopyranoside
- pH of the medium is used in a range suitable for the growth of E. coli (pH 6-8).
- a surfactant for example, Triton X100
- a chelating agent for example, EDTA
- lysozyme and the like may be appropriately added to distilled water.
- a certain amount of the collected supernatant and sediment is subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue, and then the expression of the target product is confirmed from the molecular size and stained image.
- a method based on an antigen-antibody reaction such as ELISA, Western blot, or dot blot may be used for confirmation (or detection) of the target product. Both are general methods for detecting foreign proteins and polypeptides expressed in E. coli, and may be appropriately selected according to the purpose.
- clones recovered in the sediment that is, fusion peptide-producing clones that form inclusion bodies are selected.
- the following method may be followed.
- the cells are collected using a centrifuge or an appropriately sized MF membrane (manufactured by Asahi Kasei Co., Ltd.).
- the collected bacterial cells are crushed by an appropriate method, and the inclusion body composed of the fusion peptide is released out of the bacterial cells.
- a method of dissolving with a chemical substance, a surfactant, an enzyme, or the like, or a method of physical treatment such as French press or ultrasonic treatment is used, and any method may be used. By combining some of these methods, the cells can be more effectively disrupted.
- the cells recovered with the MF membrane are diluted and concentrated with deionized water to remove the remaining medium components and the metabolites of the cells, an appropriate buffer and lysozyme are added, and the temperature is lowered (4 to 15 ° C).
- the cell walls of the cells are lysed by standing overnight, and this cell-treated solution is subjected to a French press (manufactured by Manton Gorin) under conditions of 500 to 600 kg / cm 2 to crush the cells.
- the type of the buffer solution is not particularly limited as long as it has a buffer capacity in a pH range (7.5 to 9) where lysozyme acts such as Tris buffer.
- the concentration of the buffer solution may be used within a range generally used as a buffer solution (10 to 50 mM).
- Lysozyme is used at a concentration of 0.3 to 1.0 g / L. For example, 20 mM Tris buffer at pH 8.5 is added, lysozyme (0.6 g / L) is added, and the cells are allowed to stand overnight at 4 ° C. to lyse the cell walls of the cells. After disrupting the cells with a French press, most of the cell components are removed by repeatedly diluting and concentrating the disrupted solution with buffer solution, deionized water and MF membrane. In some cases, a surfactant such as Triton-X100 may be added. Inclusion bodies are recovered as precipitates by centrifuging the concentrated inclusion body-containing solution.
- the collected inclusion body is once dissolved in a solution containing a denaturing agent.
- Urea, guanidine hydrochloride and the like can be used as the denaturing agent, but urea is preferred. Such urea and guanidine hydrochloride are used in concentration ranges of 4-8M and 2-6M, respectively. In the present invention, it is preferable to use 8M urea.
- a buffer solution having a pH of 6 to 9 is used. Preferably, the pH is 7-8.
- the buffer solution any one of a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, a carbonate buffer solution having a buffering ability in the above pH range may be used.
- the temperature at the time of dissolution is not particularly limited as long as it is 40 ° C. or lower. The dissolution time may be set while observing the dissolution state of the inclusion body, and is usually stirred for 30 minutes to 1 hour.
- refolding of the fusion peptide that is, normal three-dimensional structure by adding 10 to 20 times the amount of the buffer to the lysate of the inclusion body or dialysis against the buffer. Is rebuilt.
- the type, concentration, and pH of the buffer when refolding may be the same as those used for dissolving the inclusion bodies.
- the temperature at the time of refolding is not particularly limited as long as it is not more than room temperature. Refolding is performed by allowing to stand for 1 to 7 days, preferably 3 to 4 days.
- the fusion peptide-containing solution is subjected to the next purification step as necessary.
- Such purification steps generally include purification methods used in protein chemistry, such as centrifugation, salting-out, ultrafiltration, isoelectric precipitation, electrophoresis, ion exchange chromatography, gel filtration chromatography.
- a method in which a method such as an affinity chromatography method, a hydrophobic chromatography method, or a hydroxyapatite chromatography method is combined is used.
- the amount of the obtained protein or polypeptide is measured using a protein measuring reagent such as BCA-Protein-Assay-Reagent-Kit (Pierce-Biotechnology, Inc) or Protein-Assay-Kit (BIO-RAD, Inc).
- the usefulness of the fusion peptide of the present invention as a chicken infectious coryza vaccine is to immunize chickens with the fusion peptide-containing solution and to examine the antibody titer against A.pgA type bacteria and A.pgC type bacteria in the resulting serum. Or by performing an attack test on the immunized chicken with a virulent bacterium and observing clinical symptoms such as life and death of the chicken, swelling of the face, nasal discharge and lacrimation.
- an immunostimulant (adjuvant) used for formulation described later may be added to the fusion peptide.
- the administration method is not particularly limited, and is usually administered 1 to 3 times at intervals of 2 to 4 weeks, for example, subcutaneously, intradermally, intraperitoneally, or nasally.
- the fusion peptide-containing solution is aseptically filtered with a membrane filter or the like, and if necessary, such as aluminum hydroxide, aluminum phosphate, mineral oil and non-mineral oil.
- An immunostimulant (adjuvant), polysorbate 80, stabilizers such as amino acids and sugars such as lactose and sucrose, and preservatives such as formalin, thimerosal, 2-phenoxyethanol, benzyl alcohol, benzethonium chloride and benzalkonium chloride are added.
- a lyophilized preparation can be prepared by adding sugars such as lactose and sucrose having an effect as an excipient.
- a vaccine containing the fusion peptide of the present invention as an active ingredient is produced.
- the obtained vaccine may be used alone as a chicken infectious coryza vaccine, and other viruses (for example, chicken infectious bronchitis virus, chicken infectious bursal disease virus, chicken encephalomyelitis virus, hypoovulation syndrome) Virus), bacteria (eg Salmonella typhimurium, Salmonella enteritidis, Salmonella prolam) and protozoa (eg leucochitosome cowlery, Eimeria tenella, Eimeria maxima) at least one kind selected from the group It can also be used as a combination vaccine by combining with a vaccine.
- viruses for example, chicken infectious bronchitis virus, chicken infectious bursal disease virus, chicken encephalomyelitis virus, hypoovulation syndrome
- Virus for example, chicken infectious bronchitis virus, chicken infectious bursal disease virus, chicken encephalomyelitis virus, hypoovulation syndrome
- bacteria eg Salmonella typhimurium, Salmonella enteritidis, Salmonella prolam
- protozoa
- Genomic DNA libraries of A.pgA type 221 strain and A.pgC type strain 53-47 were prepared by the method of Tokunaga et al. (Patent Document 1). Briefly, genomic DNA is extracted from the cells recovered by centrifugation (Tomy Seiko, RD-20PIV, 4,400 g, 20 minutes) using a pure gene kit (Gentra Systems), and the DNA is used as a template (template). As described above, PCR was performed using Prime STAR HS DNA Polymerase (Takara Bio Inc.) to amplify DNA fragments of the HMTp210 protein gene of A.pgA type bacteria and A.pgC type bacteria.
- PCR conditions were as follows: reaction at 98 ° C for 1 minute, thermal denaturation (98 ° C for 10 seconds), annealing (55 ° C for 15 seconds), extension reaction (72 ° C for 120 seconds), 15 cycles. The reaction was carried out at 72 ° C. for 7 minutes.
- Table 1 shows the name and sequence number of each DNA fragment and PCR primers used for amplification.
- the NcoI recognition sequence is added to the 5 ′ primer for amplifying the DNA fragment of A.pgA type bacteria
- the BamHI recognition sequence is added to the 3 ′ primer
- the primer for amplifying the DNA fragment of A.pgC type bacteria is A BamHI recognition sequence was added to both the 5 ′ and 3 ′ primers.
- 1 and 2 show the positional relationship of the obtained DNA fragments.
- the sequence number described in the column of the DNA fragment in the table indicates the amino acid sequence encoded by each DNA fragment.
- the expression plasmid was produced as follows. First, A ⁇ 5-1 was digested with NcoI and Bam HI, separated by 0.8% agarose gel electrophoresis, and eluted and recovered using Wizard SV Gel and PCR Clean-Up System (Promega). The obtained fragment was ligated to an expression vector pET-11d (Merck) digested with NcoI and BamHI, and E. coli BL21 (DE3) strain (Merck) was transformed with this. From this transformant, an expression plasmid (pET-11d-A ⁇ 5-1) was extracted using Wizard Plus SV SV Minipreps DNA Purification System (Promega).
- C ⁇ 4c-1, C ⁇ 5-1, and C ⁇ 6b-1b were digested with BamHI, separated by 0.8% agarose gel electrophoresis, and then eluted and collected using the Wizard® SV® Gel® and PCR® Clean-Up® System (Promega). Further, the obtained fragment was ligated to pET-11d-A ⁇ 5-1 previously digested with BamHI in the forward direction, and E. coli BL21 (DE3) strain (Merck Co., Ltd.) was used to transform each of the expression plasmids.
- a ⁇ 5-1 obtained in Example 1 was digested with NcoI and BamHI, separated by 0.8% agarose electrophoresis, and then eluted and collected using Wizard® SV® Gel® and PCR® Clean-Up® System (Promega).
- a DNA fragment (A ⁇ 5-1-L) is obtained by adding a linker sequence (Gly Linker; SEQ ID NO: 24) consisting of a base sequence encoding 10 glycines to the C-terminal side of the obtained fragment with a DNA synthase. It was. By adding this linker sequence, the Bam HI recognition sequence on the C-terminal side of A ⁇ 5-1 disappears, and a new XbaI recognition sequence is generated.
- C ⁇ 4c-1, C ⁇ 5-1, and C ⁇ 6b-1b were digested with XbaI and BamHI, separated by 0.8% agarose gel electrophoresis, and then eluted and collected using Wizard® SV® Gel® and PCR® Clean-Up® System (Promega).
- Each fragment obtained was ligated with A ⁇ 5-1, digested with Bam HI, separated by 0.8% agarose gel electrophoresis, and eluted and collected using WizardWSV Gel and PCR Clean-Up System (Promega). Further, the obtained fragment was inserted into an expression vector pET-11d (Merck Co., Ltd.) digested with NcoI and BamHI, and E.
- a genomic DNA library of A.pgA type 221 strain was prepared in the same manner as in Example 1, and the DNA fragments shown in Table 5 were amplified by PCR. PCR conditions were 98 minutes at 98 ° C, followed by 15 cycles of heat denaturation (98 ° C for 10 seconds), annealing / extension reaction (70 ° C for 120 seconds), and stop reaction (72 ° C for 7 minutes). It was.
- Expression plasmids containing these fragments (pET-11d-A ⁇ 5-1, pET-11d-A ⁇ 5-2, pET-11d-A ⁇ 5-3, pET-11d-A ⁇ 5-4, pET-11d-A ⁇ 9-2, pET- 11d-A ⁇ 9-3, pET-11d-A ⁇ 9-4, pET-11d-A ⁇ 6-2, pET-11d-A ⁇ 6-3 and pET-11d-A ⁇ 6-4) were extracted.
- Table 5 shows the names and sequence numbers of the PCR primers used for amplification of each DNA fragment. An NcoI recognition sequence was added to the 5 ′ primer, and a BamHI recognition sequence was added to the 3 ′ primer.
- FIG. 4 shows the positional relationship of the obtained DNA fragments.
- the sequence number described in the column of the DNA fragment in the table indicates the amino acid sequence encoded by each DNA fragment.
- C ⁇ 5-1 obtained in Example 1 was digested with Bam HI, separated by 0.8% agarose gel electrophoresis, and then eluted and recovered using Wizard SV Gel and PCR Clean-Up System (Promega). Further, the obtained fragments were previously digested with BamHI pET-11d-A ⁇ 5-1, pET-11d-A ⁇ 5-2, pET-11d-A ⁇ 5-3, pET-11d-A ⁇ 5-4, pET-11d-A ⁇ 9 -2, pET-11d-A ⁇ 9-3, pET-11d-A ⁇ 9-4, pET-11d-A ⁇ 6-2, pET-11d-A ⁇ 6-3 and pET-11d-A ⁇ 6-4 in the positive direction, these E.
- coli BL21 (DE3) strain (Merck Co., Ltd.) was transformed with each expression plasmid (pET-11d-A ⁇ 5-1-C ⁇ 5-1, pET-11d-A ⁇ 5-2-C ⁇ 5-1, pET-11d -A ⁇ 5-3-C ⁇ 5-1, pET-11d-A ⁇ 5-4-C ⁇ 5-1, pET-11d-A ⁇ 9-2-C ⁇ 5-1, pET-11d-A ⁇ 9-3-C ⁇ 5-1, pET-11d-A ⁇ 9 -4-C ⁇ 5-1, pET-11d-A ⁇ 6-2-C ⁇ 5-1, pET-11d-A ⁇ 6-3-C ⁇ 5-1 and pET-11d-A ⁇ 6-4-C ⁇ 5-1) were obtained.
- C ⁇ 5-1 was inserted in the forward direction immediately below the peptide A expression gene, and the fusion peptides shown in Table 6 were obtained.
- a genomic DNA library of A. pgC type strain 53-47 was prepared according to the same method as in Example 1, and the DNA fragments shown in Table 7 were obtained. PCR conditions followed the conditions of Example 3. Table 7 shows the names and sequence numbers of the PCR primers used for amplification of each DNA fragment. A BamHI recognition sequence was added to both the 5 ′ and 3 ′ primers. FIG. 5 shows the positional relationship of the obtained DNA fragments. In addition, the sequence number described in the column of the DNA fragment in the table indicates the amino acid sequence encoded by each DNA fragment.
- C ⁇ 5-1, C ⁇ 5-2, C ⁇ 5-4, C ⁇ 9-0, C ⁇ 9-2, C ⁇ 9-4, C ⁇ 6-2 and C ⁇ 6-4 were digested with BamHI, 0.8 After separation by% agarose gel electrophoresis, elution and recovery using Wizard SV Gel and PCR Clean-Up ⁇ System (Promega), the resulting fragment was directed in the positive direction to pET-11d-A ⁇ 5-1 previously digested with BamHI.
- a genomic DNA library of A. pgC type strain 53-47 was prepared according to the same method as in Example 1, and DNA fragments similar to those in Table 7 were obtained. PCR conditions followed the conditions of Example 3. The names and sequence numbers of the PCR primers used for amplification of each DNA fragment are the same as in Table 7. However, in order to efficiently insert the amplified fragment into the expression vector pQE30 (Qiagen, Inc.), restriction enzyme recognition of the 3 ′ primer The sequence was changed from a BamHI recognition sequence to a HindIII recognition sequence.
- C ⁇ 5-1, C ⁇ 5-2, C ⁇ 5-4, C ⁇ 9-0, C ⁇ 9-2, C ⁇ 9-4, C ⁇ 6-2 and C ⁇ 6-4 were digested with BamHI and HindIII and separated by 0.8% agarose gel electrophoresis Subsequently, elution and collection were performed using Wizard SV-Gel-and PCR-Clean-Up System (Promega). The obtained fragment was ligated to an expression vector pQE30 digested with Bam HI and Hind III, and E.
- ⁇ Fusion peptide expression 1 >> E. coli BL21 (DE3) strain (Merck Co., Ltd.) carrying each expression plasmid obtained in Examples 1 and 2 is inoculated into 1 to 5 mL of LB medium containing 50 ⁇ g / mL of ampicillin and shaking at 30 to 37 ° C. The culture was shaken until the OD 600 of the culture reached 0.5, and IPTG was added to a final concentration of 1 mM, followed by further incubation for 3 hours. After centrifugation (Tomy Seiko, MX-300, 9,100 g, 5 minutes), the supernatant was discarded, and a washing buffer (PBS) equal in volume to the original culture was added and suspended until uniform.
- PBS washing buffer
- This suspension was sonicated 10 times for 10 seconds at a power of 10 using a handy sonicator (Tomy Seiko, UR-20P) under ice cooling, and centrifuged at 17,800 g for 15 minutes. After collecting the centrifugation supernatant, the same amount of washing buffer as that of the sonication solution before centrifugation was added to the sediment and suspended again until it became uniform. An equal amount of sample buffer (2 ⁇ SDS) was added to each of the collected centrifugal supernatant and sediment suspension, heated in boiling water for 5 minutes, subjected to SDS-PAGE by a conventional method, and stained with Coomassie brilliant blue. When each fusion peptide was found in the sediment suspension, it was determined that the fusion peptide formed an inclusion body. Each expression pattern is shown in FIG.
- C ⁇ 4c-1 When expressed alone, C ⁇ 4c-1 was not soluble in the soluble fraction and C ⁇ 5-1 was not stable in the expression fraction, but all fusion peptides stably formed inclusion bodies.
- the expression level of A ⁇ 5-1 / C ⁇ 4c-1 was slightly low, and the other two fusion peptides (AC ⁇ 5-1 and A ⁇ 5-1 / C ⁇ 6b-1b) were almost equivalent and showed good expression.
- the expression level of each fusion peptide to which a linker was added was the same as that to which the linker was not added.
- oil adjuvant vaccine oilbucks NB 2 AC, Institute for Chemical and Serum Therapy
- oilbucks NB 2 AC oil adjuvant vaccine
- inactivated cells and a non-inoculated group were provided.
- oil adjuvant vaccine oilbucks NB 2 AC, Institute for Chemical and Serum Therapy
- 0.2 mL of A.pgA type 221 strain 1.0 ⁇ 10 10 CFU / mL
- A.pgC type 53-47 strain 3.0 ⁇ 10 9 CFU / mL
- the flocks to which the fusion peptide was administered showed good protection against attack by A.pgA type 221 strain and A.pgC type strain 53-47, 0.06 Even in ⁇ g / dose, the vaccine effect of more than 1/1000 amount of commercially available oil adjuvant vaccine was shown. Moreover, the same defense result was shown also about the peptide which added the linker. From the above, it was confirmed that the fusion peptide was useful as a vaccine.
- Amount of antigen used for immunization and virulent strain used for attack (A.pgA type 083 strain (1.0 ⁇ 10 9 CFU / mL), A.pgA type B strain W (4.1 ⁇ 10 9 CFU / mL)) and A The method described in Example 7 was followed except that the .pgC type strain Modesto strain (2.8 ⁇ 10 9 CFU / mL) was different.
- the base sequence in region 2 of each strain used in the attack test was analyzed.
- the 083 strain and the W strain were completely identical, and a mutation of 1 base (A / G) between the 221 strain and the 083 strain and between the 221 strain and the W strain (position 1227 of SEQ ID NO: 25) Of glutamic acid was replaced with glycine).
- the Modesto strain had a deletion of 3-base AAG (the 1144th glutamic acid of SEQ ID NO: 26) compared to the 53-47 strain.
- a vaccine was prepared in the same manner as in Example 7 and emulsified by adding an oil adjuvant so that each dose of AC ⁇ 5-1, peptide A and peptide C shown in Table 11 was contained in 0.5 mL of one dose.
- the vaccine was administered once into the leg muscles of 4-week-old SPF chickens and immunized.
- a non-inoculated group was provided as a control.
- the antibody titer was confirmed 4 weeks after immunization by the method of Ushijima et al. (Japanese Patent Application 2008-29589). Specifically, antibody measurement was performed by ELISA.
- the blocking solution was discarded, and the serum was diluted 100-fold with PBS-T containing 10% skim milk, added 50 ⁇ L per well, and allowed to react at room temperature for 1 hour. After removing the reaction solution, the plate was washed 3 times with PBS-T, and anti-chicken IgG-HRP-labeled antibody was diluted 20,000 times with 5% skim milk-added PBS-T and added in 50 ⁇ L aliquots. The reaction was performed in the dark at room temperature for 30 minutes. After removing the reaction solution, the plate was washed 3 times with PBS-T, and 100 ⁇ L of a chromogenic substrate solution (TMB + substrate-chromogen; DAKO) was added and reacted at room temperature for 15 minutes. Color development was stopped by adding 100 ⁇ L of 3M sulfuric acid. The wavelength of 450 nm was measured with a 96-well plate reader (manufactured by Nippon Molecular Device Co., Ltd.).
- the flock immunized with the fusion peptide AC ⁇ 5-1 showed a high antibody titer at 0.6 ⁇ g / dose, and the seroconversion rate of the antibody was 100 for both A type and C type. %Met.
- the 0.06 ⁇ g / dose group a positive conversion rate of 80% for type A bacteria and 40% for type C bacteria was confirmed, but in the 0.03 ⁇ g / dose group of peptide A or peptide C, 60% and 0%, respectively.
- the fusion peptide AC ⁇ 5-1 is more effective than each peptide before fusion.
- Example 10 ⁇ Immunogenicity of fusion peptide 2>
- an attack test similar to that in Example 7 was carried out using a homotoxic virulent strain (A.pgA type 221 strain). The method described in Example 7 was followed except that the amount of antigen used for immunization and the number of virulent strains used for the challenge (A.pgA type 221 strain 1.2 ⁇ 10 9 CFU / mL) were different.
- the flocks immunized with each fusion peptide showed good protection results when A.pgA type 221 strain 221 was tested, and A ⁇ 6-4 / C ⁇ 5-1 The 80% protection rate was confirmed, and the longer fusion peptide was confirmed to have a protection rate of 100%.
- Example 12 ⁇ Confirmation of protective effect of peptide C>
- a vaccine emulsified by adding an oil adjuvant to contain peptide C having the antigen amount shown in Table 13 was administered once into the leg muscles of 4-week-old SPF chickens, and immunized.
- 0.2 mL of A.pgC type 53-47 (5.2 ⁇ 10 9 CFU / mL) bacterial solution was administered intranasally, and clinical symptoms such as facial swelling, nasal discharge and lacrimation were observed for 1 week. did.
- the present invention can be used to provide a chicken infectious coryza vaccine caused by Abibacterium paragalinarum type A and type C.
Abstract
Description
[1]A.pgA型菌のHMTp210タンパク質由来のペプチド断片(ペプチドA)とA.pgC型菌のHMTp210タンパク質由来のペプチド断片(ペプチドC)からなる融合ペプチドを封入体として産生する宿主を構築する工程、前記の宿主を培養し、培養物から封入体画分を回収、精製する工程、及び前記の精製封入体画分を含有する組成物を調製する工程を含む、組換え鶏伝染性コリーザワクチンの製造方法。
[2]ペプチドA及びペプチドCが、それぞれ600個以下のアミノ酸からなることを特徴とする、[1]に記載の製造方法。
[3]ペプチドAが配列番号1、27、28、29、30、31、32、33、34及び35で示されるアミノ酸配列からなる群より選ばれた配列、並びにペプチドCが配列番号2、3、4、50、51、52、53、54、55及び56で示されるアミノ酸配列からなる群より選ばれた配列であることを特徴とする、[1]に記載の製造方法。
[4]ペプチドAが配列番号35で示されるアミノ酸配列を含み、ペプチドCが配列番号56で示されるアミノ酸配列を含むことを特徴とする、[3]に記載の製造方法。
[5]ペプチドA又はペプチドCが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものであることを特徴とする、[3]又は[4]に記載の製造方法。
[6]融合ペプチドのペプチドAとペプチドCの比率が、ペプチドAが1に対してペプチドCが1~3であることを特徴とする、[1]ないし[5]の何れかに記載の製造方法。
[7]融合ペプチドが、少なくとも一組のペプチドAのC末側にペプチドCを結合させた構造を含むものであることを特徴とする、[1]ないし[6]の何れかに記載の製造方法。
[8]融合ペプチドが、ペプチドAとペプチドAとの間、ペプチドCとペプチドCとの間又はペプチドAとペプチドCとの間にリンカーを有するものであることを特徴とする、[1]ないし[7]の何れかに記載の製造方法。
[9]融合ペプチドが、配列番号8、9、10、11、12、13、41、42、43、44、45、46、47、48、49、61、62、63、64、65、66及び67で示されるアミノ酸配列からなることを特徴とする、[3]に記載の製造方法。
[10]A.pgA型菌のHMTp210タンパク質由来のペプチド断片(ペプチドA)とA.pgC型菌のHMTp210タンパク質由来のペプチド断片(ペプチドC)からなる融合ペプチドを有効成分として含有する組換え鶏伝染性コリーザワクチン。
[11]融合ペプチドが、宿主に生産させるときに封入体を形成する性質を有することを特徴とする、[10]に記載のワクチン。
[12]ペプチドA及びペプチドCが、600個以下のアミノ酸からなることを特徴とする、[10]に記載のワクチン。
[13]ペプチドAが配列番号1、27、28、29、30、31、32、33、34及び35で示されるアミノ酸配列からなる群より選ばれた配列、並びにペプチドCが配列番号2、3、4、50、51、52、53、54、55及び56で示されるアミノ酸配列からなる群より選ばれた配列であることを特徴とする、[10]に記載のワクチン。
[14]ペプチドAが配列番号35で示されるアミノ酸配列を含み、ペプチドCが配列番号56で示されるアミノ酸配列を含むことを特徴とする、[13]に記載のワクチン。
[15]ペプチドA又はペプチドCが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものであることを特徴とする、[13]又は[14]に記載のワクチン。
[16]融合ペプチドのペプチドAとペプチドCの比率が、ペプチドAが1に対してペプチドCが1~3であることを特徴とする、[10]ないし[15]の何れかに記載のワクチン。
[17]融合ペプチドが、少なくとも一組のペプチドAのC末側にペプチドCを結合させた構造を含むものであることを特徴とする、[10]ないし[16]の何れかに記載のワクチン。
[18]融合ペプチドが、ペプチドAとペプチドAとの間、ペプチドCとペプチドCとの間又はペプチドAとペプチドCとの間にリンカーを有するものであることを特徴とする、[10]ないし[17]の何れかに記載のワクチン。
[19]融合ペプチドが、配列番号8、9、10、11、12、13、41、42、43、44、45、46、47、48、49、61、62、63、64、65、66及び67で示されるアミノ酸配列からなることを特徴とする、[13]に記載のワクチン。
[20]配列番号1で示されるアミノ酸配列のN末側及び/又はC末側に1~200のアミノ酸が付加されたアミノ酸配列内であって、配列番号35で示されるアミノ酸配列を含む配列からなるペプチドを有効成分として含有する、組換え鶏伝染性コリーザワクチン。
[21]配列番号1で示されるアミノ酸配列からなるペプチドを有効成分として含有する、[20]に記載のワクチン。
[22]配列番号3で示されるアミノ酸配列のN末側及び/又はC末側に1~200のアミノ酸が付加されたアミノ酸配列内であって、配列番号56で示されるアミノ酸配列を含む配列からなるペプチドを有効成分として含有する、組換え鶏伝染性コリーザワクチン。
[23]配列番号3又は52で示されるアミノ酸配列からなるペプチドを有効成分として含有する、[22]に記載のワクチン。
[24]ペプチドが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものである、[21]又は[23]に記載のワクチン。
A.pgA型菌221株及びA.pgC型菌53-47株のゲノムDNAライブラリーを徳永らの方法(特許文献1)により調製した。簡単には、遠心分離(トミー精工、RD-20PIV、4,400g、20分間)により回収した菌体からピュアジーンキット(Gentra Systems社)を用いてゲノムDNAを抽出し、そのDNAを鋳型(テンプレート)として、Prime STAR HS DNA Polymerase(タカラバイオ株式会社)を用いてPCRを行い、A.pgA型菌及びA.pgC型菌のHMTp210タンパク質遺伝子のDNA断片を増幅した。PCR条件は、98℃で1分反応の後、熱変性(98℃で10秒間)、アニーリング(55℃で15秒間)、伸長反応(72℃で120秒間の反応)を15サイクル実施し、停止反応(72℃で7分間)で行った。
各DNA断片及び増幅に用いたPCRプライマーの名称と配列番号を表1に示す。A.pgA型菌のDNA断片を増幅するための5'プライマーにNcoI認識配列を、3'プライマーにBamHI認識配列を付加し、A.pgC型菌のDNA断片を増幅するためのプライマーには、5'及び3'プライマーのいずれにもBamHI認識配列を付加した。図1及び2に、得られた各DNA断片の位置関係を示す。なお、表中のDNA断片の欄に記載の配列番号は各DNA断片によりコードされるアミノ酸配列を示す。
リンカー配列を付加するために、実施例1と同様の方法に従ってA.pgのHMTp210タンパク質遺伝子のDNA断片を増幅した。各DNA断片及び増幅に用いたPCRプライマーの名称と配列番号を表3に示す。5'プライマーにはXbaI認識配列を、3'プライマーにはBamHI認識配列を付加した。なお、表中のDNA断片の欄に記載の配列番号は各DNA断片によりコードされるアミノ酸配列を示す。
A.pgA型菌221株のゲノムDNAライブラリーを実施例1と同様の方法に従って調製し、PCRにて表5のDNA断片を増幅した。PCR条件は、98℃で1分反応後、熱変性(98℃で10秒間)、アニーリング・伸長反応(70℃で120秒間)を15サイクル実施し、停止反応(72℃で7分間)で行った。これらの断片を有する発現プラスミド(pET-11d-AΔ5-1、pET-11d-AΔ5-2、pET-11d-AΔ5-3、pET-11d-AΔ5-4、pET-11d-AΔ9-2、pET-11d-AΔ9-3、pET-11d-AΔ9-4、pET-11d-AΔ6-2、pET-11d-AΔ6-3及びpET-11d-AΔ6-4)を抽出した。各DNA断片の増幅に用いたPCRプライマーの名称と配列番号を表5に示す。5'プライマーにNcoI認識配列を、3'プライマーにBamHI認識配列を付加した。図4に、得られた各DNA断片の位置関係を示す。なお、表中のDNA断片の欄に記載の配列番号は各DNA断片によりコードされるアミノ酸配列を示す。
A.pgC型菌53-47株のゲノムDNAライブラリーを実施例1と同様の方法に従って調製し、表7のDNA断片を得た。PCR条件は実施例3の条件に従った。各DNA断片の増幅に用いたPCRプライマーの名称と配列番号を表7に示す。5'及び3'プライマーのいずれにもBamHI認識配列を付加した。図5に、得られた各DNA断片の位置関係を示す。なお、表中のDNA断片の欄に記載の配列番号は各DNA断片によりコードされるアミノ酸配列を示す。
A.pgC型菌53-47株のゲノムDNAライブラリーを実施例1と同様の方法に従って調製し、表7と同様のDNA断片を得た。PCR条件は実施例3の条件に従った。各DNA断片の増幅に用いたPCRプライマーの名称と配列番号は表7と同様であるが、増幅断片を発現ベクターpQE30(株式会社キアゲン)に効率よく挿入するために、3'プライマーの制限酵素認識配列をBamHI認識配列からHindIII認識配列に変更した。
実施例1及び2で得られた、各発現プラスミドを保持する大腸菌BL21(DE3)株(メルク株式会社)をアンピシリン50μg/mL含有LB培地1~5mLに接種し、30~37℃で振盪しながら、培養菌液のOD600が0.5に達するまで振盪培養し、IPTGを終濃度1mMとなるように加え、更に3時間培養した。遠心分離(トミー精工、MX-300、9,100g、5分間)後、上清を捨て、もとの培養菌液量と等量の洗浄バッファー(PBS)を加え、均一になるまで懸濁した。この懸濁液を氷冷下で、ハンディーソニケーター(トミー精工、UR-20P)を用いて、パワー10で10秒間、10回超音波処理し、17,800gで15分間遠心分離した。遠心上清を回収した後、その沈渣に遠心前の超音波処理液と等量の洗浄バッファーを加えて再び均一になるまで懸濁した。回収した遠心上清及び沈渣懸濁液のそれぞれに等量のサンプルバッファー(2×SDS)を加え、煮沸水中で5分間加熱後、定法によりSDS-PAGEにかけ、クマシーブリリアントブルーで染色した。各融合ペプチドが沈渣懸濁液に認められた場合、当該融合ペプチドは封入体を形成していると判断した。各発現パターンを図3に示す。
融合ペプチドのワクチン効果を確認するために、ホモの強毒株を用いて攻撃試験を実施した。実施例6で得たACΔ5-1及びACΔ5-1-Lの沈渣懸濁液中の封入体を8M尿素で可溶化し、さらに透析膜を用いてバッファーをPBS(pH7.4)に置換した。1ドーズ0.5mL中に、表9に示した各抗原量のACΔ5-1を含むようにオイルアジュバントを加えて乳化したワクチンを8週齢SPF鶏の脚部筋肉内に1回投与し、免疫した。対照として、不活化菌体を用いた市販のオイルアジュバントワクチン(オイルバックスNB2AC、財団法人化学及血清療法研究所)投与群と非接種群を設けた。免疫4週後にA.pgA型菌221株(1.0×1010 CFU/mL)、あるいはA.pgC型菌53-47株(3.0×109 CFU/mL)の菌液0.2mLを鼻腔内投与し、1週間、顔面腫脹、鼻汁漏出、流涙などの臨床症状を観察した。
融合ペプチドの作製に用いたA.pgA型菌221株及びA.pgC型菌53-47株以外の菌株(ヘテロ株)に対する融合ペプチドのワクチン効果を確認するために、実施例7と同様の攻撃試験を行った。免疫に用いた抗原量及び攻撃に用いた強毒株(A.pgA型菌083株(1.0×109 CFU/mL)、A.pgA型菌W株(4.1×109 CFU/mL)及びA.pgC型菌Modesto株(2.8×109 CFU/mL))が異なる以外は実施例7に記載の方法に従った。
融合ペプチドACΔ5-1と融合前のペプチドA及びペプチドCのワクチン効果を比較するために、免疫試験を行った。実施例7と同様の方法でワクチンを作製し、1ドーズ0.5mL中に、表11に示した各抗原量のACΔ5-1、ペプチドA及びペプチドCをそれぞれ含むようにオイルアジュバントを加えて乳化したワクチンを4週齢SPF鶏の脚部筋肉内に1回投与し、免疫した。対照として、非接種群を設けた。免疫4週後に牛島らの方法(特願 2008-29589)により抗体価を確認した。具体的にはELISA法で抗体測定を行った。
実施例3及び4で得られた、各発現プラスミドを保持する大腸菌BL21(DE3)株(メルク株式会社)を実施例6と同様の方法で発現させ、発現量を確認した。各発現パターンを図6及び7に示す。ペプチドAについては単独で発現させた場合、AΔ5-4、AΔ9-2、AΔ9-4、AΔ6-2、AΔ6-4は可溶性画分に発現したが、CΔ5-1と融合した結果、AΔ6-4以外の融合ペプチドは安定して封入体を形成した。AΔ6-4/CΔ5-1は主に封入体を形成するが、可溶性画分に発現することもあり、発現が不安定であった。発現量はすべての融合ペプチドでほぼ同等であり、良好な発現を示した。一方、ペプチドCについては単独で発現させた場合、CΔ6-2を除くすべてのペプチドCが可溶性画分に発現したが、AΔ5-1と融合した結果、融合ペプチドは安定して封入体を形成した。発現量はすべての融合ペプチドでほぼ同等であり、良好な発現を示した。
実施例10で得た融合ペプチドのワクチン効果を確認するために、ホモの強毒株(A.pgA型菌221株)を用いて実施例7と同様の攻撃試験を実施した。免疫に用いた抗原量及び攻撃に用いた強毒株の菌数(A.pgA型菌221株1.2×109 CFU/mL)が異なる以外は実施例7に記載の方法に従った。その結果、表12に示すように、各融合ペプチドで免疫された鶏群は、A.pgA型菌221株で攻撃試験を行った場合に良好な防御成績を示し、AΔ6-4/CΔ5-1で80%の防御率を、それより長い融合ペプチドでは100%の防御率を確認した。
実施例5で得られた、各発現プラスミドを保持する大腸菌JM109株(株式会社キアゲン)を実施例6と同様の方法で発現させ、それぞれの菌体における発現量をSDS-PAGEにより確認した(図8)。得られた各ペプチドをそれぞれ、CΔ5-1-pQE、CΔ5-2-pQE、CΔ5-4-pQE、CΔ9-0-pQE、CΔ9-2-pQE、CΔ9-4-pQE、CΔ6-2-pQE及びCΔ6-4-pQEと命名した。CΔ6-2-pQEを除く全てのペプチドCは可溶性画分に発現し、発現量も良好であった。
実施例12で得られたペプチドCのワクチン効果を確認するために、実施例7と同様の攻撃試験を実施した。表13に示した抗原量のペプチドCを含むようにオイルアジュバントを加えて乳化したワクチンを4週齢SPF鶏の脚部筋肉内に1回投与し、免疫した。免疫4週後にA.pgC型菌53-47株(5.2×109 CFU/mL)の菌液0.2mLを鼻腔内投与し、1週間、顔面腫脹、鼻汁漏出、流涙などの臨床症状を観察した。
Claims (24)
- A.pgA型菌のHMTp210タンパク質由来のペプチド断片(ペプチドA)とA.pgC型菌のHMTp210タンパク質由来のペプチド断片(ペプチドC)からなる融合ペプチドを封入体として産生する宿主を構築する工程、前記の宿主を培養し、培養物から封入体画分を回収、精製する工程、及び前記の精製封入体画分を含有する組成物を調製する工程を含む、組換え鶏伝染性コリーザワクチンの製造方法。
- ペプチドA及びペプチドCが、それぞれ600個以下のアミノ酸からなることを特徴とする、請求項1に記載の製造方法。
- ペプチドAが配列番号1、27、28、29、30、31、32、33、34及び35で示されるアミノ酸配列からなる群より選ばれた配列、並びにペプチドCが配列番号2、3、4、50、51、52、53、54、55及び56で示されるアミノ酸配列からなる群より選ばれた配列であることを特徴とする、請求項1に記載の製造方法。
- ペプチドAが配列番号35で示されるアミノ酸配列を含み、ペプチドCが配列番号56で示されるアミノ酸配列を含むことを特徴とする、請求項3に記載の製造方法。
- ペプチドA又はペプチドCが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものであることを特徴とする、請求項3又は4に記載の製造方法。
- 融合ペプチドのペプチドAとペプチドCの比率が、ペプチドAが1に対してペプチドCが1~3であることを特徴とする、請求項1ないし5の何れか一項に記載の製造方法。
- 融合ペプチドが、少なくとも一組のペプチドAのC末側にペプチドCを結合させた構造を含むものであることを特徴とする、請求項1ないし6の何れか一項に記載の製造方法。
- 融合ペプチドが、ペプチドAとペプチドAとの間、ペプチドCとペプチドCとの間又はペプチドAとペプチドCとの間にリンカーを有するものであることを特徴とする、請求項1ないし7の何れか一項に記載の製造方法。
- 融合ペプチドが、配列番号8、9、10、11、12、13、41、42、43、44、45、46、47、48、49、61、62、63、64、65、66及び67で示されるアミノ酸配列からなることを特徴とする、請求項3に記載の製造方法。
- A.pgA型菌のHMTp210タンパク質由来のペプチド断片(ペプチドA)とA.pgC型菌のHMTp210タンパク質由来のペプチド断片(ペプチドC)からなる融合ペプチドを有効成分として含有する組換え鶏伝染性コリーザワクチン。
- 融合ペプチドが、宿主に生産させるときに封入体を形成する性質を有することを特徴とする、請求項10に記載のワクチン。
- ペプチドA及びペプチドCが、600個以下のアミノ酸からなることを特徴とする、請求項10に記載のワクチン。
- ペプチドAが配列番号1、27、28、29、30、31、32、33、34及び35で示されるアミノ酸配列からな群より選ばれた配列、並びにペプチドCが配列番号2、3、4、50、51、52、53、54、55及び56で示されるアミノ酸配列からなる群より選ばれた配列であることを特徴とする、請求項10に記載のワクチン。
- ペプチドAが配列番号35で示されるアミノ酸配列を含み、ペプチドCが配列番号56で示されるアミノ酸配列を含むことを特徴とする、請求項13に記載のワクチン。
- ペプチドA又はペプチドCが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものであることを特徴とする、請求項13又は14に記載のワクチン。
- 融合ペプチドのペプチドAとペプチドCの比率が、ペプチドAが1に対してペプチドCが1~3であることを特徴とする、請求項10ないし15の何れか一項に記載のワクチン。
- 融合ペプチドが、少なくとも一組のペプチドAのC末側にペプチドCを結合させた構造を含むものであることを特徴とする、請求項10ないし16の何れか一項に記載のワクチン。
- 融合ペプチドが、ペプチドAとペプチドAとの間、ペプチドCとペプチドCとの間又はペプチドAとペプチドCとの間にリンカーを有するものであることを特徴とする、請求項10ないし17の何れか一項に記載のワクチン。
- 融合ペプチドが、配列番号8、9、10、11、12、13、41、42、43、44、45、46、47、48、49、61、62、63、64、65、66及び67で示されるアミノ酸配列からなることを特徴とする、請求項13に記載のワクチン。
- 配列番号1で示されるアミノ酸配列のN末側及び/又はC末側に1~200のアミノ酸が付加されたアミノ酸配列内であって、配列番号35で示されるアミノ酸配列を含む配列からなるペプチドを有効成分として含有する、組換え鶏伝染性コリーザワクチン。
- 配列番号1で示されるアミノ酸配列からなるペプチドを有効成分として含有する、請求項20に記載のワクチン。
- 配列番号3で示されるアミノ酸配列のN末側及び/又はC末側に1~200のアミノ酸が付加されたアミノ酸配列内であって、配列番号56で示されるアミノ酸配列を含む配列からなるペプチドを有効成分として含有する、組換え鶏伝染性コリーザワクチン。
- 配列番号3又は52で示されるアミノ酸配列からなるペプチドを有効成分として含有する、請求項22に記載のワクチン。
- ペプチドが、1もしくは数個のアミノ酸が欠失、付加又は置換されたアミノ酸配列を有するものである、請求項21又は23に記載のワクチン。
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JPH10500009A (ja) * | 1994-05-18 | 1998-01-06 | スミスクライン・ビーチャム・バイオロジカルス (ソシエテ・アノニム) | Hcmvおよびhsv由来の融合糖蛋白質 |
WO1998012331A1 (fr) | 1996-09-19 | 1998-03-26 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Nouveau polypeptidetire de hemophilus paragallinarum et son procede de production |
JP2004057078A (ja) | 2002-07-29 | 2004-02-26 | Nippon Inst For Biological Science | ヘモフィルス・パラガリナルム由来新規ポリペプチドとその遺伝子及び製法 |
JP2004531540A (ja) * | 2001-04-27 | 2004-10-14 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | Hiv抗原並びにhsv抗原及び/又はhpv抗原を含む多価ワクチン |
JP2005218414A (ja) * | 2004-02-09 | 2005-08-18 | Biseibutsu Kagaku Kenkyusho:Kk | ヘモフィルス・パラガリナラムa型菌由来の感染防御抗原である組み換え蛋白 |
JP2006516183A (ja) * | 2002-08-30 | 2006-06-29 | グラクソスミスクライン バイオロジカルズ ソシエテ アノニム | 突然変異型タンパク質およびリフォールディング方法 |
JP2008029589A (ja) | 2006-07-28 | 2008-02-14 | Fujishoji Co Ltd | 弾球遊技機 |
JP2008156317A (ja) | 2006-12-26 | 2008-07-10 | Biseibutsu Kagaku Kenkyusho:Kk | アビバクテリウム・パラガリナラムc型菌由来の発症防御抗原である新規組換え蛋白 |
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IL99097A0 (en) * | 1990-09-05 | 1992-07-15 | Akzo Nv | Haemophilus paragallinarum vaccine |
PT2255826E (pt) | 2002-08-02 | 2016-06-08 | Glaxosmithkline Biologicals Sa | Composições de vacina de neisseria compreendendo uma combinação de antigénios |
JP3524546B2 (ja) * | 2003-06-18 | 2004-05-10 | 富士通株式会社 | 一覧表画面表示装置 |
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- 2009-12-24 MX MX2011006903A patent/MX2011006903A/es active IP Right Grant
- 2009-12-24 EP EP14197266.1A patent/EP2853270B1/en active Active
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See also references of EP2382985A4 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106267176A (zh) * | 2015-05-12 | 2017-01-04 | 普莱柯生物工程股份有限公司 | 鸡传染性鼻炎疫苗组合物及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
US8647634B2 (en) | 2014-02-11 |
CN102333539B (zh) | 2014-06-25 |
US20120003257A1 (en) | 2012-01-05 |
ES2616533T3 (es) | 2017-06-13 |
KR20110114600A (ko) | 2011-10-19 |
KR101652060B1 (ko) | 2016-08-29 |
JP5568017B2 (ja) | 2014-08-06 |
US20130183330A1 (en) | 2013-07-18 |
EP2382985A4 (en) | 2013-05-29 |
JPWO2010074126A1 (ja) | 2012-06-21 |
CN102333539A (zh) | 2012-01-25 |
EP2382985B1 (en) | 2015-05-06 |
ES2537426T3 (es) | 2015-06-08 |
MX2011006903A (es) | 2011-09-15 |
EP2382985A1 (en) | 2011-11-02 |
EP2853270B1 (en) | 2017-02-08 |
EP2853270A1 (en) | 2015-04-01 |
BRPI0924068A2 (pt) | 2016-07-26 |
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