WO2005096806A1 - ワクチン遺伝子導入イネ - Google Patents
ワクチン遺伝子導入イネ Download PDFInfo
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- WO2005096806A1 WO2005096806A1 PCT/JP2005/006973 JP2005006973W WO2005096806A1 WO 2005096806 A1 WO2005096806 A1 WO 2005096806A1 JP 2005006973 W JP2005006973 W JP 2005006973W WO 2005096806 A1 WO2005096806 A1 WO 2005096806A1
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- rice
- protein
- gene
- transgenic rice
- ctb
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
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- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention relates to transgenic rice, a vaccine composition using rice harvested from the transgenic rice or a processed product thereof, and a method for inducing an immune response using the vaccine composition. .
- Tissues covered by mucous membranes such as influenza, SARS (severe acute respiratory syndrome), AIDS, and tuberculosis, are responsible for many serious emerging re-emerging infections, such as the nasal cavity, oral cavity, upper bronchi, intestinal tract, and urogenital tract. It is clear from the fact that the mucosal surface protection is most effective in preventing infection. Mucosal immunity centered on secretory immunoglobulin A (S-IgA) forms the defense mechanism on the mucosal surface.
- S-IgA secretory immunoglobulin A
- mucosal vaccines are ideal vaccines because they can establish antigen-specific immunity to both mucosal and systemic immune mechanisms.
- Non-patent Documents 2, 3, and 4 Expression of Escherichia coli heat-labile toxin antigen in tobacco, potato or corn (Non-patent Documents 2, 3, and 4), expression of cholera toxin antigen in potato (Non-patent Document 5), lettuce or Expression of hepatitis B virus antigen in potatoes (non-patented) References 6, 7), expression of Norwalk virus antigen in tobacco or potato (Non-patent Reference 8), expression of rabies virus or RS virus antigen in tomato (Non-patent References 9, 10), cytomegalovirus antigen in tobacco Expression (Non-patent Document 11), expression of the BFO virus antigen in Alfa alpha (Non-patent Document 12), expression of swine infectious gastroenteritis virus antigen in Arabidopsis (Non-patent Document 13), etc. .
- the plant can express the antigenic protein and that the plant expressing the antigenic protein can be used as an "eating vaccine". That is, whether or not a target immune response can be induced by orally administering a plant expressing an antigenic protein depends on the expression amount of the antigenic protein in the plant, the three-dimensional structure of the antigenic protein expressed in the plant. It is determined by various factors, such as the interaction with other components contained in the plant, and the target immune response is immediately obtained because the plant expressing the antigenic protein is orally administered. I can't guide you.
- the target immune response may be different because the three-dimensional structure of the antigenic protein expressed in the plant is different from the original three-dimensional structure. If you can not induce a response (e.g., if the alpha 1-3 fucose, beta 1-2 plant-specific sugar chain added to by different conformation of xylose, etc.), to be administered antigenic proteins and inevitably simultaneously In some cases, other components contained in the target plant may be unable to induce a target immune response due to a disorder.
- a response e.g., if the alpha 1-3 fucose, beta 1-2 plant-specific sugar chain added to by different conformation of xylose, etc.
- the antigen-specific immune response can be induced when a sufficient amount of the purified antigenic protein is orally administered. It is very difficult to predict whether a potential immune response can be induced.
- the expression level of the antigenic protein in the plant and the expression in the plant determine whether or not the target immune response can be induced by oral administration of the plant expressing the antigenic protein.
- Factors such as the three-dimensional structure of the antigenic protein, interaction with other components contained in the plant, the type of plant, the site of expression of the antigenic protein in the plant, the site of accumulation of the antigenic protein in the plant, It is affected by various conditions such as the antigenic protein expression system (eg, promoter), and is capable of inducing a target immune response when orally administering a plant expressing the antigenic protein. Set conditions Is extremely difficult.
- Patent Document 1 International Publication WO90 / 02484
- Non-Patent Document 1 Yuki Y et al., “Reviews in Medical Virology J, 2003, Vol. 13, p.293-310
- Non-Patent Document 2 Haq TA et al., "Science”, 1995, Vol. 268, p.714-716
- Non-Patent Document 3 Mason HS et al., “Vaccine”, 1998, Vol. 16 , P.1336-1343
- Non-patent Document 4 Streatfield SJ et al., "Vaccine", 2001, Vol. 19,
- Non-Patent Document 5 Arakawa T et al., “Nature Biotechnology”, 1998, Vol. 16, p.292-297
- Non-Patent Document 6 Kapusta J et al., "The FASEB JOURNAL", 1999, Vol. 13, p. 1796-1799
- Non-Patent Document 7 Richter LJ et al., “Nature Biotechnology”, 2000, Vol. 18, p.1167-1171
- Non-Patent Document 8 Mason HS et al., "Proceedings of National Academy of Science U.S.A.”, 1996, Vol. 93, p.5335-5340
- Non-Patent Document 9 McGarvey PB et al., "Biotechnology", 1995, Vol. 13, p. 1484-1487
- Non-Patent Document 10 Sandhu JS et al., "Transgenic Research", 2000, Vol. 9, p.127-135
- Non-patent literature l l Tackaberry ES, etc., "Vaccine”, 1999, Vol. 17, p.3020-3029
- Non-Patent Document 12 Dus Santos MJ et al., "Vaccine”, 2002, Volume 20, ⁇ .1141-1147
- Non-Patent Document 13 Gomez ⁇ et al., "Virology”, 1998, Vol. 249, p.352-358
- injectable vaccines need to be purified to an injectable level from the viewpoint of safety, and in consideration of the above-mentioned low-temperature storage and transportation, they must be expensive in terms of cost.
- non-recombinant vaccines using attenuated strains of relatively low-cost pathogenic microorganisms have a problem in terms of safety.
- the method of expressing vaccine antigens in recombinant bacteria and the like which can be developed at relatively low cost and orally administering them has a problem that the vaccine cannot be repeatedly used for the same individual. This is because this method induces a strong immune response against the expected vaccine, and also induces a strong immune response against recombinant microorganisms as a vector that binds, and eliminates the boosted recombinant microorganisms themselves. This is because there is a possibility that
- rice can be mass-produced, can be stored at room temperature for a long time, and can be easily transported to remote locations including developing countries. Therefore, if the development is rice that can be used as a "eat vaccine", it is possible that the force s long-term inter-store a wide variety of large quantities of the vaccine at low cost. Then, it will be possible to achieve global vaccine administration (global vaccine) and global public health (global public health).
- the present invention firstly provides transgenic rice capable of producing rice that can be used as an "eating vaccine", that is, rice that can induce a desired immune response when transmucosally administered by oral administration or the like.
- the purpose is to:
- Another object of the present invention is to provide rice harvested from the above-mentioned transgenic rice or a processed rice product containing an antigenic protein.
- Still another object of the present invention is to provide a vaccine composition containing, as an active ingredient, the rice or the processed rice containing the antigenic protein.
- Still another object of the present invention is to provide a method for inducing an immune response using the above vaccine composition.
- the present inventor has (a) The ability to express various antigenic proteins (vaccine antigens) having a molecular weight of 100,000 daltons or less in rice as rice
- the antigenic protein (vaccine antigen) expressed in rice must be stable at room temperature for at least one year.
- Pepsin resistance can be imparted by expressing antigenic proteins (vaccine antigens) in rice
- antigenic proteins vaccine antigens expressed in rice can escape digestion in the stomach, the antigenic proteins required to induce antigen-specific immune responses in the systemic and mucosal systems are identified. Oral dosage should be lower than purified antigenic protein
- the antigenic protein When the antigenic protein (vaccine antigen) is expressed in rice, the antigenic protein becomes a type I storage protein (protein body I) in rice endosperm cells, a type II storage protein (protein body II), etc.
- the antigenic protein S rice In order to confer pepsin resistance to antigenic proteins, it is important that antigenic protein S rice accumulates in type I storage protein (protein body I) in endosperm cells.
- the antigenic protein (vaccine antigen) expressed in rice escapes from digestion in the stomach, reaches the intestinal tract, and has the ability to take up antigens present on the mucous membrane-derived tissue, such as Peyer's patches (eg, M cells)
- the present inventors have found out that an antigen-specific immune response can be induced by incorporation from, and completed the present invention. That is, in order to achieve the above object, the present invention provides the following transgenic rice, rice or processed rice product, a vaccine composition, and a method for inducing an immune response.
- Transgenic rice in which a DNA construct comprising a DNA encoding an antigenic protein and a rice endosperm-specific promoter linked upstream thereof is integrated into genomic DNA so that it can be expressed.
- the molecular weight of the antigenic protein as a monomer is not more than 100,000 daltons ( Transgenic rice according to 1).
- the antigenic protein is a cholera toxin B subunit, a fusion protein of a cholera toxin B subunit and a neutralizing epitope of an AIDS virus, or a botulinum toxin He domain.
- the antigenic protein is a cholera toxin B subunit, a fusion protein of a cholera toxin B subunit and a neutralizing epitope of an AIDS virus, or a botulinum toxin He domain.
- transgene as described in any of (1) to (: 11) above, wherein the DNA construct comprises a rice endosperm-specific terminator linked downstream of the DNA encoding the antigenic protein. Rice.
- the DNA construct comprises a DNA encoding the signal peptide ligated upstream of a DNA encoding the antigenic protein such that a fusion protein comprising a signal peptide of the isolated rice endosperm storage protein is produced.
- the transgenic rice according to any one of the above (1) to (14).
- a vaccine composition comprising, as an active ingredient, the rice or the processed rice product containing the antigenic protein according to (20).
- (22) A method for inducing an immune response in a non-human animal, comprising a step of transmucosally administering the vaccine composition according to (21) to a non-human animal.
- a method for inducing an immune response in a human comprising a step of transmucosally administering the vaccine composition according to the above (21) to a human.
- transgenic rice capable of producing rice that can be used as an “eating vaccine”, that is, rice that can induce a desired immune response when transmucosally administered by oral administration or the like.
- the present invention provides rice harvested from the above-mentioned transgenic rice or processed rice containing an antigenic protein.
- the rice Alternatively, there is provided a vaccine composition comprising a processed rice product containing an antigenic protein as an active ingredient.
- a method for inducing an immune response using the vaccine composition is provided.
- “Rice” means a plant belonging to Orvza ⁇ ik ⁇ , and the variety of rice subject to genetic recombination is not particularly limited as long as it belongs to Qll.
- antigenic includes a property of inducing antibody production and cell-mediated immunity (immunogenicity) and a property of generating an antigen-antibody reaction and a cell-mediated immune reaction.
- the immunogenicity of the antigenic protein is determined by the intravenous, intraperitoneal, subcutaneous, intradermal, oral, nasal, vaginal, anal, pulmonary, etc. administration routes of the antigenic protein alone or together with an immunological adjuvant. When administered to humans or other animals via any of the above administration routes, it is only necessary that the compound be released in vivo.
- immunological adjuvant refers to an antigenic enhancer that enhances the antigenicity of a protein.
- the type of antigenic protein may be intravenous, intraperitoneal, subcutaneous, intradermal, oral, nasal, vaginal, transanal, transpulmonary, etc., with the antigenic protein alone or with an immunological adjuvant.
- an immune response can be induced in vivo when administered to humans or other animals via any of the routes.
- An antigenic protein can induce an immune response when the dosage form, dosage, administration route and the like are set to the most preferable conditions (for example, when a purified and sufficient amount of antigenic protein is administered).
- Immuno response includes various immune responses such as antibody production, antigen-antibody reaction, and cellular immune response, and is used in the present invention to include at least antibody production.
- “Animal” includes humans and other vertebrates (eg, mammals, birds, amphibians, fish, reptiles, etc.).
- Transmucosal administration includes oral administration, buccal administration, nasal administration, transanal administration, vaginal administration, pulmonary administration and the like.
- Antigenic protein also includes glycoproteins.
- the antigenic protein is, for example, a protein derived from a pathogenic microorganism and has no pathogenicity or toxicity per se or an adverse effect on a living body. Proteins that have only weak pathogenicity or toxicity, but retain immunogenicity.
- the type of the pathogenic microorganism is not particularly limited, and examples thereof include cholera, pathogenic Escherichia coli, influenza, pneumococcus, pertussis, diphtheria, plague, tetanus, botulinum, and charcoal.
- typhi Paratyphi, Chlamydia, Ameba dysentery, Legionella, Lyme borrelia, Pathogenic bacteria such as brucellosis (wave-like fever) bacteria; rotavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Norwalk virus, rabies virus, RS virus, cytomegalovirus, roe-foot disease virus, transmission Gastroenteritis virus, rubella virus, ATL virus, adenovirus, mumps (mumps) virus, Russ, enterovirus, herpes virus, smallpox virus, poliovirus, measles virus, Japanese encephalitis virus, Tengu fever virus, yellow fever virus, West Nile virus, SARS (coronavirus), influenza virus, HIV (AIDS virus) ), Pathogenic viruses such as Ebola hemorrhagic fever virus (filovirus), marburda virus (filovirus), Lassa fever virus, hantavirus,
- proteins or peptides constituting the pathogenic microorganism include proteins or peptides constituting the pathogenic microorganism (for example, surface proteins, capsid proteins, ciliary proteins, etc.), and proteins or peptides produced by the pathogenic microorganism (for example, Toxins, enzymes, hormones, immunomodulators, receptors and their ligands, etc.), etc. fragments thereof or domains like et be.
- a protein derived from a pathogenic microorganism which does not itself have pathogenicity or toxicity, or has no adverse effect on living organisms, has only a weak degree, has only pathogenicity or toxicity.
- proteins that retain immunogenicity include, for example, cholera toxin B subunit (C TB), Escherichia coli heat-labile toxin B subunit (LTB), anthrax protective antigen, tetanus toxin Tox C domain Botulinum toxin He domain, influenza virus HA antigen, Y.
- pestis F1 or V antigen malaria SERAn antigen
- rotavirus VP6 or VP7 AIDS virus-derived gp_160, nef, gag, env or tat protein, hepatitis B virus S protein etc Is mentioned.
- the antigenic protein may be a fusion protein (chimeric protein) composed of two or more types of proteins or peptides.
- a fusion protein for example, CTB or LTB having an effect of regulating and enhancing mucosal immune response, and other antigenic proteins or their epitopes (eg, T cell epitopes, B cell epitopes, neutralizing epitopes, etc.)
- a fusion protein consisting of
- the molecular weight of the antigenic protein as a monomer is not particularly limited, but is generally 100,000 daltons or less, preferably 80,000 daltons or less, and more preferably 50,000 daltons or less.
- the antigenic protein can be expressed in rice endosperm cells without losing its antigenicity, and rice or rice collected from transgenic rice can be expressed.
- the processed product is transmucosally administered to humans or other animals, systemic immunity and mucosal immunity against the antigenic protein can be effectively induced in the living body.
- the lower limit of the molecular weight of the antigenic protein as a monomer is not particularly limited as long as the antigenicity is exhibited, but is usually at least 1,000 daltons, preferably at least 3,000 daltons, and more preferably at least 3,000 daltons. Is more than 5,000 daltons.
- the antigenic protein is a subunit constituting a homomultimer
- a homomultimer is formed when the antigenic protein is expressed in rice endosperm cells.
- the molecular weight of the homomultimer is usually 1,000,000 dalton or less, preferably 600,000 dalton or less, more preferably 300,000 dalton or less.
- the molecular weight of the homomultimer is in the above range, when the rice or its processed product collected from the transgenic rice is transmucosally administered to humans or other animals, the systemic system for the homomultimer in the living body is in vivo. Immunity and mucosal immunity can be effectively induced.
- the antigenic protein is a subunit constituting a heteromultimer
- a heteromultimer is formed when each subunit constituting the heteromultimer is expressed in rice endosperm cells.
- the molecular weight of the heteromultimer is usually 1,000,000 dalton or less, preferably 600,000 dalton or less, more preferably 300,000 dalton or less.
- rice or processed product thereof obtained from transgenic rice is Can effectively induce systemic and mucosal immunity against heteromultimers in vivo when administered to other animals by mucosal administration.
- antigenic proteins composed of homomultimers include cholera toxin B subunit pentameric protein (CTB5), Escherichia coli heat-labile toxin B subunit pentameric protein (LTB5), anthrax protection Heteromeric proteins of antigen (PA7) and the like, and antigenic proteins composed of heteromultimers include nontoxic mutant cholera toxin (mCTA_CTB5) and chimeric chimeric mutant toxin (mCTA_LTB5).
- CTB5 cholera toxin B subunit pentameric protein
- LTB5 Escherichia coli heat-labile toxin B subunit pentameric protein
- PA7 anthrax protection Heteromeric proteins of antigen
- antigenic proteins composed of heteromultimers include nontoxic mutant cholera toxin (mCTA_CTB5) and chimeric chimeric mutant toxin (mCTA_LTB5).
- the nontoxic mutant cholera toxin is S61F or 112 substituted by the 61st Ser (S) force SPhe (F), which is the active center of ADP-ribosyltransferase of the cholera toxin A subunit having toxic activity.
- S 61st Ser
- SPhe force SPhe
- No. 0111 Consists of El2K substituted by the force SLys (K) and cholera toxin B subunit, and is nontoxic, but retains the mucosal immune adjuvant activity of cholera toxin (Yamamoto S et.al. J Exp Med. 185: 1203-10 (1997);
- the nontoxic chimeric mutant toxin is an Escherichia coli heat-labile toxin (LT) having a similar immunoadjuvant activity in place of the cholera toxin detoxifying A subunit E112K and cholera toxin B subunit. It is a combination with the B subunit (LTB) and is nontoxic, but has mucosal immune adjuvant activity (Kweon MN et. Al J Infect Dis. 2002 186: 1261-9 (2002)).
- LT Escherichia coli heat-labile toxin
- the antigenic protein may be any of a natural protein and a mutant protein as long as it has the desired antigenicity.
- "Variant protein” means a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of a native protein, and having the same antigenicity as the native protein. I do.
- the antigenic protein may be a fusion protein with another protein or peptide as long as it has the desired antigenicity.
- the fusion protein examples include a fusion protein containing an antigenic protein and a signal peptide of a rice endosperm storage protein linked to its N-terminus.
- Antigenic protein power Having a signal peptide of S rice endosperm storage protein allows antigenic proteins expressed in rice endosperm cells to efficiently accumulate in rice endosperm cells And a sufficient amount of antigenic protein to induce an immune response can be accumulated in rice endosperm cells.
- Examples of the signal peptide of rice endosperm storage protein include a signal peptide of glutelin, prolamin or globulin.
- the amino acid sequence of the signal peptide of glutelin GluB_1 is shown in SEQ ID NO: 16, and the nucleotide sequence of the DNA encoding it is shown in SEQ ID NO: 15.
- the signal peptide of glutelin, prolamin or globulin is involved in accumulation in a storage protein (protein body) in rice endosperm cells.
- the antigenic protein has a signal peptide of glutelin, prolamin or globulin
- the antigen protein can be efficiently accumulated in storage proteins (protein bodies I and II) in rice endosperm cells. Then, in the storage proteins (protein bodies I and II) in rice endosperm cells, a sufficient amount of antigenic protein to induce an immune response is accumulated without losing its antigenicity.
- transmucosal administration of rice or its processed products from transgenic rice to humans or other animals effectively induces systemic and mucosal immunity to antigenic proteins in vivo. can do.
- Examples of the fusion protein include, for example, a fusion protein containing an antigenic protein and an endoplasmic reticulum localization signal peptide linked to the C-terminus thereof. Since the antigenic protein has an endoplasmic reticulum localization signal peptide, the antigenic protein expressed in rice endosperm cells can be converted into storage proteins in rice endosperm cells (protein bodies I and II).
- endoplasmic reticulum localization signal peptide examples include a peptide having the amino acid sequence KDEL (SEQ ID NO: 2) and a peptide having the amino acid sequence HDEL (His_Asp-Glu_Leu).
- Examples of the fusion protein include, for example, an antigenic protein, a signal peptide of a rice endosperm storage protein linked to the N-terminus of the antigenic protein, and a localization of an endoplasmic reticulum linked to the C-terminus of the antigenic protein. Fusion proteins containing an activating signal peptide.
- Antigenic protein strength S rice endosperm storage protein signal peptide and endoplasmic reticulum localization signal peptide Proteins can be accumulated more efficiently by storage proteins (protein bodies I and II) in rice endosperm cells and a sufficient amount of antigenic protein to induce an immune response is accumulated in rice endosperm cells Can be done.
- codons contained in the DNA encoding the antigenic protein be modified into plant-type codons.
- the expression efficiency of antigenic proteins in rice endosperm cells can be improved, and sufficient amounts of antigenic proteins to induce an immune response are expressed in rice endosperm cells Can be done.
- Rasperm-specific promoter and "rice endosperm-specific terminator” mean a promoter and a terminator that specifically function in rice endosperm cells, and their types are not particularly limited. Les ,.
- the rice endosperm-specific promoter and terminator include, for example, a promoter and a terminator of a gene encoding a rice endosperm storage protein.
- an antigenic protein can be efficiently expressed in rice endosperm cells, and a sufficient amount to induce an immune response.
- Antigenic proteins can be accumulated in storage proteins (protein bodies I and II) in rice endosperm cells.
- Root endosperm storage protein refers to a protein that is specifically expressed in rice endosperm cells and accumulated in rice endosperm cells, and the type thereof is not particularly limited.
- Rice endosperm storage proteins include, for example, gnoretellin, prolamin, globulin and the like.
- the promoter of the gene encoding the rice endosperm storage protein is preferably a promoter of the glutelin GluB-1 gene. Since the promoter of glutelin GluB_l gene has higher open motor activity than the promoters of the genes encoding other rice endosperm storage proteins, it can efficiently express antigenic proteins in rice endosperm cells, resulting in immune response. A sufficient amount of the antigenic protein to induce the expression can be accumulated in storage proteins (protein bodies I and II) in rice endosperm cells.
- the rice endosperm-specific promoter may be any of a natural promoter and a mutant promoter as long as it has rice endosperm-specific promoter activity.
- “Mutant Pro” refers to a promoter having a rice endosperm-specific promoter activity consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted or added in the nucleotide sequence of a natural promoter. The same applies to the rice endosperm-specific terminator.
- the nucleotide sequences of the promoter and terminator of rice glutelin GluB-1 gene are shown in SEQ ID NOS: 17 and 18, respectively.
- Specific examples of the mutant promoter include a promoter having a rice endosperm-specific promoter activity consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 17.
- a terminator having a rice endosperm-specific terminator activity comprising a nucleotide sequence in which one or more bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 18 One.
- the DNA construct that can be expressed so as to be integrated into rice genomic DNA contains DNA encoding an antigenic protein and a rice endosperm-specific promoter linked upstream thereof.
- the DNA construct preferably contains DNA encoding each subunit constituting the heteromultimer.
- a heteromultimer can be formed in rice endosperm cells.
- the DNA construct When expressing a fusion protein containing an antigenic protein and a signal peptide of a rice endosperm storage protein linked to its N-terminus, the DNA construct is linked upstream of the DNA encoding the antigenic protein. DNA encoding the signal peptide. The DNA encoding the signal peptide is linked downstream of the rice endosperm-specific promoter.
- the DNA construct When expressing a fusion protein containing an antigenic protein and an endoplasmic reticulum localization signal peptide linked to its C-terminus, the DNA construct is prepared by connecting the DNA construct downstream of the DNA encoding the antigenic protein. Contains DNA encoding the endoplasmic reticulum localization signal peptide. The DNA encoding the endoplasmic reticulum localization signal peptide is linked upstream of one of the rice endosperm-specific terminators.
- Transgenic rice in which a DNA construct has been incorporated into genomic DNA so that it can be expressed
- the delivery method is not particularly limited.
- Transgenic rice can be produced, for example, by introducing a vector containing a DNA construct into rice cells and then culturing the transformed rice cells to regenerate the rice plant.
- the form of the rice cell into which the vector is introduced is not particularly limited as long as it can be regenerated into a plant.Examples include cultured cells, protoplasts, shoot primordia, multiple shoots, hairy roots, calli, etc. .
- a plasmid When a plasmid is used as the vector, it is preferable to contain a drug resistance gene such as hygromycin, tetracycline, and ampicillin so that the rice cells into which the vector has been introduced can be efficiently selected.
- the method for introducing the vector into rice cells is not particularly limited, and examples thereof include an indirect introduction method using Agrobacterium ⁇ mefaciens, Agrobacterium lysogenes (Hiei, Y. et al.). ., Plant J., 6, 271-282, 1994;
- the method for regenerating rice plants by culturing rice cells is not particularly limited, and any known method may be used.
- transgenic rice endosperm cells have a sufficient amount of antigenic protein accumulated without losing their antigenicity to induce an immune response
- transgenic rice By transmucosal administration of the substance to humans or other animals, systemic immunity and mucosal immunity can be induced in the living body. Therefore, the rice or its processed product collected from transgenic rice can be used as an active ingredient of the vaccine composition, and the vaccine composition is transmucosally administered to humans or other animals. Thus, a desired immune response can be induced in the living body.
- accumulation of the antigenic protein in W endosperm cells imparts pepsin resistance to the antigenic protein.
- the antigenic protein accumulated in rice endosperm cells escapes digestion in the stomach and reaches the intestinal tract, whereupon the ability to take up the antigen present in the mucous membrane-derived tissue such as Peyer's patch is obtained.
- a cell having the antigen eg, an M cell
- an antigen-specific immune response can be induced.
- oral dosages of antigenic proteins required to induce systemic and mucosal antigen-specific immune responses are smaller than purified antigenic proteins.
- “Rice” means a tissue containing endosperm or a part thereof, and includes paddy, brown rice, seed, white rice, these parts and the like.
- “Rice products” include any processed products as long as they contain the antigenic protein. Examples of processing applied to rice include threshing, pulverization, extraction of a protein fraction, purification of the extracted protein fraction, and the like.
- the animal to which the rice or the processed product thereof is administered includes, for example, humans and other vertebrates (eg, mammals, birds, amphibians, fish, reptiles) and the like.
- Transmucosal administration includes, for example, oral administration, buccal administration, nasal administration and the like.
- the form of the vaccine composition can be appropriately selected according to the administration route and the like.
- Examples of the form of the peptide composition include a pharmaceutical composition, a food and drink composition, a feed composition and the like.
- Various forms of compositions such as pharmaceutical compositions, food and drink compositions, feed compositions and the like can be prepared according to conventional methods.
- the vaccine composition may be administered with a suitable adjuvant
- the dose of the vaccine composition can be appropriately set according to conditions such as the age, sex, body weight, symptoms, and administration route of the animal to be administered. Per day in terms of:! To 3000 ⁇ g / kg body weight, preferably 5 to: 1000 ⁇ g / kg body weight, particularly preferably 25 to 400 ig / kg body weight. is there.
- the dose may be administered at intervals of several days, or at intervals of weeks to months, for example: at intervals of 12 weeks or 1 to 12 months.
- the number of administrations is suitably several to several tens, but is not particularly limited.
- CTB genes Two types were prepared: a native CTB (nCTB) gene and a plant codon altered CTB (mutated CTB, mCTB) gene.
- KDEL SEQ ID NO: 2 was added to the 3 'end of the nCTB gene as a PCR product.
- the nCTB_KDEL gene to which the coding DNA (SEQ ID NO: 1) was added was obtained.
- the codon of the nCTB gene was changed to a plant-type codon (particularly rice codon) to make it an optimal codon for expression in rice. That is, among the codons of the nCTB gene, 12 codons that are hardly used in plants were changed to plant-type codons (Table 1), and a mutant CTB gene (mCTB gene: SEQ ID NO: 5) was prepared.
- the amino acid sequence of CTB encoded by the nCTB gene and mCTB gene is shown in SEQ ID NO: 6.
- the mCTB gene is composed of two fragments (Fl, F2) having a length of about 170bps.
- the two DNA fragments consisted of an annealed complementary oligo DNA pair (F1-up (SEQ ID NO: 7) and Fl-low (SEQ ID NO: 8) for F1 and F2-low (SEQ ID NO: 8)).
- F2-up (SEQ ID NO: 9) and F2-low (SEQ ID NO: 10), a pair of powerful oligo DNAs) were prepared by PCR using a type II plasmid.
- forward primer Fl-Fd SEQ ID NO: 11
- reverse primers for F1 amplification primers for F1 amplification.
- Fl-Rv (SEQ ID NO: 12) was used, and forward primer F2-Fd (SEQ ID NO: 13) and reverse primer F2_Rv (SEQ ID NO: 14) were used as primers for F2 amplification.
- PCR was performed using the forward primer Fl_Fd (SEQ ID NO: 11) and the reverse primer CTB-KDEL-R (SEQ ID NO: 4).
- the mCTB_KDEL gene to which DNA (SEQ ID NO: 1) encoding KDEL (SEQ ID NO: 2) was added at the 3 'end was obtained.
- Each of the nCTB-KDEL gene or mCTB-KDEL gene obtained as a PCR product is a rice glutelin ligated downstream of the rice glutelin GluB-1 gene promoter (2.3 kbs) (SEQ ID NO: 17).
- the DNA encoding the signal peptide of GluB-1 (SEQ ID NO: 16) (SEQ ID NO: 15) and the terminator of rice glutelin GluB-1 gene (0.6 kbs) (SEQ ID NO: 18) Subcloning was performed using Sacl.
- Sacl the rice glutelin GluB-1 gene promoter, the DNA encoding the signal peptide of rice glutelin GluB-1 linked downstream, the nCTB gene or mCTB gene linked downstream, and the downstream link
- a DNA construct containing the obtained DNA encoding KDEL and a terminator of the rice glutelin GluB-1 gene linked downstream thereof was prepared. This DNA construct was introduced into pGPTV_35S_HPT containing the hygromycin B resistance gene as a HindlllZEcoRI fragment.
- the T-DNA vector incorporating the mCTB gene As described above, the T-DNA vector incorporating the mCTB gene
- Agrobacterium ummefaciens EHA105 strain was added to 10 mL of YEA liquid medium (beef extract 5 g / L, yeast extract lg / L, peptone lg / L, sucrose 5 g / L, 2 mM MgSO 4)
- PGPTV-35S-HPT-2.3kGluBpro-sig-nCTB was carried out by election port method. Apply 50 kL of the plasmid-introduced bacterial mixture containing 3 xg of each plasmid at 2.5 cm, 25 ⁇ F, 200 ⁇ at 0.2 cm distance between the electrodes, thereby amplifying each plasmid. Introduced to Globataterim. To apply electrical noise, use Norad Genepulser II was used.
- the cells were added to 0.2 mL of YEB liquid medium, cultured with shaking at 25 ° C. for 1 hour, and then seeded on a 50 mg / L kanamycin-added YEB solid medium and cultured at 28 ° C.
- the plasmid-introduced strain was selected by culturing for days.
- the final confirmation of plasmid introduction was carried out by cutting the plasmid extracted from the selected strain by the alkaline SDS method with restriction enzymes and comparing the electrophoresis patterns of the cut fragments.
- PGPTV-35S-HPT- 2.3kGluBpro-sig-nCTB-introduced Agrobaterium 'mmefaciens EHA105 strain is again cultured in YEB solid medium at 28 ° C for 2 days, and then in YEB liquid medium at 25 ° C and 180rpm. The culture was centrifuged at 300 rpm for 20 minutes, and the cells were collected by centrifugation at 300 rpm. Acetosyringone 10 mg / L, 2,4_D (2,4 dichlorophenoxyacetic acid) 2 mg / L and sucrose 30 g / L The suspension was suspended in a liquid medium so as to have an OD of 600 nm and SO.1, and used as an agrobacterium for infection.
- CTB gene-transferred rice is prepared according to Japanese Patent No. 3141084 by adding PGPTV-35S-HPT-2.3kGluBpro-sig-mCTB or
- Rice seeds from Orvza sativa cv. Kitaake were sterilized in a 2.5% sodium hypochlorite (NaCIO) solution in an intact state after removing the rice husks. After sufficient washing with water, the rice seeds were subjected to the following aseptic operation.
- NaCIO sodium hypochlorite
- Rice seeds are grown in N6D medium containing 2,4-D (30 g / l sucrose, 0.3 g / l casamino acid, 2.8 g / l proline, 2 mg / l 2,4_D, 4 g / l genorelite, pH 5.8) (This was sown and kept warm at 27 ° C to 32 ° C. 5) During this time, rice seeds germinated.
- 2N6-AS culture After immersing the pre-cultured rice seeds in the Agrobacterium teratium solution for infection, 2N6-AS culture They were transplanted to the ground (30 g / L sucrose, lOg / L glucose, 0.3 g / L casamino acid, 2 mg / L 2, 4 — D, 10 mg / L acetociringone, 4 g / L gellite, pH 5.2). Co-cultivation was performed at 28 ° C for 3 days in the dark.
- agrobacterium was washed away from the seeds using N6D medium containing 500 mg / L carpenicillin. Next, the transformed seeds were selected under the following conditions.
- 2nd selection Seeds were placed on N6D medium containing 2-4 mg / L 2,4-D supplemented with carpenicillin (500 mg ZL) and hygromycin (25 mg ZL) and kept at 27 ° C for 7 days. Incubated at ⁇ 32 ° C.
- the selected transformed seeds were regenerated under the following conditions.
- MS medium (30 g / L sucrose, 30 g / L sonorebitone, 2 g / L casamino acid, 2 mg / L) supplemented with regeneration medium (carpenicillin (500 mg / L) and hygromycin (25 mg / L))
- the selected seeds were placed on L-potency ricetin, 0.002 mg / L NAA, 4 g / L genorelite, pti 5.8), and kept at 27 ° C to 32 ° C for 2 weeks.
- Second round of redifferentiation Using the same regeneration medium used in the first round of redifferentiation, incubated for an additional 2 weeks at 27 ° C to 32 ° C.
- the redifferentiated transformant was transferred to a rooting medium (MS medium supplemented with hygromycin (25 mg ZL) and containing no holmon), and after confirming root growth, the cells were lifted.
- a rooting medium MS medium supplemented with hygromycin (25 mg ZL) and containing no holmon
- forward primer Fl_Fd (SEQ ID NO: 11) and reverse primer CTB-KDEL-R (SEQ ID NO: 4) were used as primers for amplifying the mCTB gene
- forward primer CTB was used as a primer for amplifying the nCTB gene
- -F-NcoI (SEQ ID NO: 3)
- reverse primer CTB-KDEL-R (SEQ ID NO: 4) were used.
- pGPTV-35S-HPT-2.3kGluBpro-sig-mCTB shows the results when type III was used, and (B) shows the results when pGPTV-35S-HPT-2.3kGluBpro-sig-nCTB was type II.
- RNA extract 0.1 mM Tris-HCl (pH 9 0), 0.1 M NaCl, 5 mM EDTA, 1% (WZV) SDS
- phenol / chloroform / isoamyl alcohol 25: 24: 1 was newly added to the aqueous layer collected by centrifugation, followed by vigorous stirring.
- RNA was washed with 2M lithium chloride and 70% (v / v) ethanol and dried. Finally, it was dissolved in 50 / iL of water to obtain a seed total RNA fraction (Tada, Y. et al. (2003) Plant Biotechnology Journal, vol 1, p 411-422).
- RNAlO zg was electrophoresed on a 1% (WZV) agarose denaturing gel containing formaldehyde, transferred to a nylon membrane, and then the PCR amplified fragment of the entire CTB gene region labeled with 32 P was used as a probe to detect CTB. The transcript of the gene was detected.
- transcripts of the CTB gene were confirmed in the seeds of the mCTB gene-transferred rice (Fig. 3A) and the nCTB gene-introduced rice (Fig. 3B). No transcripts of the CTB gene were found in the offspring.
- the amount of RNA analyzed in this experiment was confirmed by visualizing the rRNA by staining the RNA with ethidium bromide.
- Ripe seeds of wild-type rice and nCTB- or mCTB-transgenic rice are pulverized, and room temperature using 50 mM Tris-HCl buffer ( ⁇ 6.8) containing 4% SDS, 8M urea, 5% mercaptoethanol and 20% glycerol. , And centrifuged, and the supernatant was analyzed by 15% acrylamide SDS-PAGE. The experiment was performed in duplicate, one for protein staining using Coomassie Brilliant Blue, and the other for electrotransfer onto PVDF membrane, followed by Western blotting using anti-CTB rabbit serum and HRP-labeled anti-rabbit IgG antibody. Antibody staining was performed.
- Fig. 5 shows the results. Under non-reducing conditions, most of the bands were observed around 55 to 65 kDa in both protein staining (Fig. 5A) and antibody staining (Fig. 5B), indicating that CTB expressed in rice seeds had almost a pentameric structure. It seems that you are doing.
- the lower molecule side has TPQNI at the N-terminus.
- the N-terminus seemed to be blocked on the polymer side, and it was presumed that the CTB had a glutelin signal sequence whose amino acid sequence could not be analyzed.
- Example 6 SDS-PAGE was performed under the same conditions as in Example 6 using standard CTB derived from 50 to 500 ng of bacteria, and then transferred to PVDF membrane, using anti-CTB rabbit serum and HRP-labeled anti-rabbit IgG antibody. Antibody staining was performed. Thereafter, the relative signal intensity was read using a scanner (ChemiDoc XRC system: manufactured by Biorad Japan), a calibration curve of CTB protein was drawn from the data of standard CTB, and the CTB expression level of the mCTB transgenic rice seed was quantified.
- Figure 6 shows the results.
- FIG. 6 shows the CTB protein accumulation (g / grain) of 3 to 5 seeds collected from the same transformant line of the first generation hybrid (F1).
- the rice husks are removed from the ripe rice seeds, and 5 seeds per threshed rice seed are placed in a metal bead 2 mL tube of a multi-beads shocker (manufactured by Yasui Equipment Co., Ltd.) and set at 2000 rpm for 7 seconds. Vibrated twice to prepare a fine powder.
- the rice seed thus prepared even when suspended in 100 mg / mL PBS, is so fine that it can be passed through a mouse or a disposable oral probe (diameter of about 18G needle). It had been powdered.
- mice A group of Balb / c mice 6 to 8 weeks old (5 to 6 mice) were suspended in 100 mL of ripe rice seeds pulverized in Example 8 in ImL Maylon solution (Otsuka Pharmaceutical, Maylon-P). And mouse Oral immunization was performed 6 times at 5 day intervals using a discarded oral probe (Fuchigami Kiiki).
- m CTB transgenic rice seeds were quantified by the method of Example 7 and prepared to contain 30 / g CTB per 100 mg of weight.
- Another group used as a positive control was 30 ⁇ g of CTB (List Biological Laboratories, USA) prepared using Escherichia coli, and another group used 20 mg of wild-type ripe rice seeds as a negative control. Immunized orally.
- mice Five days after the final immunization, serum and stool or small intestinal washings were collected from the mice in each group. Stool was extracted with lmL of PBS per lOOmg, and the centrifuged supernatant was used as a stool sample. For washing of the small intestine, the small intestine was cut off, the intestinal tract was opened, cut about 2 cm in length, suspended and washed in 5 mL of PBS, and the centrifugal supernatant was used as the small intestine washing solution.
- the antigen-specific antibody was measured according to the method of Y. Yuki et al. (Int. Immunol. 4: 537-545 (1998)). That is, a Falcon microtest assay plate was coated with 0.5 xg / 0.1 mL of CTB, blocked with PBS containing 1% BSA, and diluted serum or feces extract was added at room temperature. After washing the plate with PBS containing 0.01% Tween 20 (TPBS), 1000-fold diluted anti-mouse ⁇ -, ⁇ -, and ⁇ -chain specific antibody-binding peroxidase (Southern Biotechnology Birmingham, USA) was added to each plate.
- TPBS PBS containing 0.01% Tween 20
- mice orally administered wild-type rice seed powder contained serum obtained from mice orally administered mCTB transgenic rice seed powder or bacterial CTB.
- the CTB-specific IgG subclass antibody with a significantly higher antibody titer was present in the order of IgGl, IgG2b, and IgG2a.
- IgG subclass analysis suggested that CTB expressed in rice seeds, like bacterial CTB, could induce a Th2-type immune response by oral immunization of mice.
- CT cholera toxin
- the induced neutralizing antibody titer was quantified using the damaging effect of CHO cells on CT (S. Yam to et al. J. Exp. Med 185: 1203-1210 (1997)). That is, CT (0.01 ⁇ g / mL) is added to the serially diluted serum or small intestine lavage fluid and mixed, and 50 ⁇ L of the CHO cells (4 ⁇ 10 5 / mL) is added to 50 ⁇ L of the mixture. Add 5% FCS for 24 hours at 37 ° C in 5% CO
- the neutralizing antibody titer was defined as the maximum dilution of serum or small intestinal lavage having a complete cytotoxicity-inhibiting effect.
- the serum other than CTB contained in the rice seeds was contained in the serum orally immunized by the method of Example 9.
- micronized wild-type rice and mature seeds of nCTB or mCTB transgenic rice were treated with 4% SDS, 8M urea, 5% mercaptoethanol and 20% glycerol.
- the mixture was extracted with 50 mM Tris-HCl buffer ( ⁇ 6.8) for 15 minutes at room temperature, centrifuged, the supernatant was subjected to 15% acrylamide SDS-PAGE, and then electrically transferred to a PVDF membrane.
- Antibody staining was performed using the obtained mature seed of wild-type rice, mature seed of mCTB-transgenic rice, or mouse serum orally immunized with bacterial standard CTB .
- V3J1 DNA encoding KDEL (SEQ ID NO: 2) added to the 3 'end of DNA encoding SEQ ID NO: 19 (SEQ ID NO: 19) encoding the neutralizing epitope of AIDS virus V3J1 by chemical synthesis Obtained.
- the V3J1-KDEL gene was linked to the rice gnoterin GluB-1 signal, which was linked downstream of the rice glutelin GluB-1 gene promoter (2.3 kbs) (SEQ ID NO: 17). Restriction between DNA (SEQ ID NO: 15) encoding peptide (SEQ ID NO: 16) and mCTB gene linked downstream thereof, and one terminator of rice glutelin GluB-1 gene (0.6 kbs) (SEQ ID NO: 18) Subcloning was performed using the enzymes BamHI and Sacl.
- the DNA (SEQ ID NO: 20) encoding the hinge peptide sequence Gly-Pro-Gly-Pro and the restriction enzyme BamHI site (ggatcc) is located downstream of the mCTB gene.
- the sequence of the V3J1-KDEL gene was designed to be ligated.
- a DNA construct containing the obtained V3J1-KDEL gene and a terminator of the rice gnoreterin GluB-1 gene linked downstream thereof was prepared. This DNA construct was introduced as a HindlllZEcoRI fragment into pGTV-35S-HPT containing the hygromycin B resistance gene.
- T-DNA vector pGTV-35S-HPT-2.3kGluBpro-sig-mCTB-V3J1 incorporating the mCTB gene and the V3J1-KDEL gene linked downstream thereof was constructed.
- T-DNA vector was introduced into agrobacterium in the same manner as in Example 2, and then the AIDS virus neutralizing epitope was added to the C-terminal of CTB in the same manner as in Example 3.
- Transgenic rice producing a chimeric protein (mCTB_V3J1) to which V3J1 was bound was created.
- Ripe seeds of wild-type rice and mCTB-V3J1 transgenic rice are finely powdered, and 50 mM Tris-HCl buffer (PH6.8) containing 4% SDS, 8M urea, 5% ⁇ -mercaptoethanol and 20% glycerol is used. , And centrifuged. The supernatant was subjected to 15% acrylamide SDS-PAGE, and then electrically transferred to a PVDF membrane.
- Anti-V3J1 rabbit serum, HRP-labeled anti-rabbit IgG Western blotting antibody staining was performed using the antibody.
- nCTB gene-transferred rice and mCTB gene-transferred rice was isolated in the same manner as in Example 4, 10 ⁇ g of the DNA was digested with restriction enzyme Sacl, and 0.7% agarose gel was electrophoresed. The sample was subjected to electrophoresis and plotted on a Hybond N + membrane. nCTB remains Detection of gene or MCTB gene, double-stranded DNA encoding nCTB or MCTB - was performed using those labeled with [a 32 P] dCTP as a probe.
- FIG. 13 As a result, as shown in FIG. 13, two bands were confirmed in the nCTB transgenic rice (lane 2 in FIG. 13A) and the mCTB transgenic rice (lanes 1 to 3 in FIG. 13B), and each band was identified in each genome. It was found that at least two copies of the nCTB gene or mCTB gene had been introduced. In the figure, W indicates the results for wild-type rice.
- Example 7 Six of the mCTB transgenic rice seeds quantified in Example 7 were stored at room temperature for 6 months, at room temperature for 12 months, or at 4 ° C for 12 months, CTB content was quantified, and immediately after individual selection It was compared with the CTB content of rice seeds. The quantification was performed in the same manner as in Example 7, and five rice seeds were used for quantification under each storage condition.
- mCTB transgenic rice seed powder (10 mg) was suspended in 0.5 mg / mL pepsin (Sigma) (0.1 mL) dissolved in acetate buffer ( ⁇ 1.7) and reacted at 37 ° C for 1 hour.
- Western blot analysis was performed in the same manner as in Example 6 to detect CTB, glutelin B1, and prolamin.
- 10 mg of wild-type rice seed powder or 15 xg of purified recombinant CTB was similarly treated.
- the detection of CTB, gnoleterin B1 and prolamin was performed using an anti-CTB antibody, an anti-gnoleterin B1 antibody and an anti-13K prolamin antibody, respectively.
- CTB expressed in rice seeds is resistant to pepsin
- the amount of CTB required for inducing antigen-specific antibodies in vivo was higher in CTB expressed in rice seeds than in CTB expressed in rice seeds. Expected to be lower than recombinant CTB.
- the amount of CTB to orally immunize mice should be S30, 10, 3, 1, 0.3, 0.1 ⁇ g. Was dissolved or suspended in water, and the mice were orally immunized three times every other week, and one week after the final immunization, antibody titers in serum and feces were measured.
- the rice seeds into which the mCTB gene had been At a dose of about 1/3 to 1/10 (in terms of CTB amount), the same antigen-specific immune response as that of purified recombinant CTB (anti-CTB serum IgG (systemic immune response, Fig.15A) and anti-CTB fecal IgA) (Mucosal immune response, Fig. 15B)).
- anti-CTB serum IgG systemic immune response, Fig.15A
- anti-CTB fecal IgA Mucosal immune response
- FIG. 16 shows the results. Note that, in the figure, A is an observation diagram for the mCTB transgenic rice seed, and B is an observation diagram for the wild-type rice seed.
- the large white part is starch granules
- the slightly blackish part is the type II storage protein containing the storage protein gnoleterin
- the slightly grayish part is the type I storage protein containing the storage protein prolamin.
- Body, small black dot indicates CTB.
- CTB was observed in the type I storage protein (protein body 1), the type II storage protein (protein body ⁇ ⁇ ), and the gaps between them (cytoplasm, endoplasmic reticulum, etc.).
- FIG. 16B no significant CTB staining was observed in wild-type rice seeds.
- the rice storage protein glutelin B1 was 90% degraded by pepsin treatment, but the rice storage protein 13K prolamin was not degraded at all by pepsin treatment
- the pepsin resistance of CTB expressed in rice was conferred by the presence of CTB in a location other than type II storage protein (particularly, type I storage protein).
- Orally administered mCTB transgenic rice seed escapes digestion in the digestive tract, reaches the intestinal tract, and has cells capable of taking up antigens present in the mucous membrane-derived tissue such as Peyer's patches ( In particular, in order to prove that it is taken up from M cells), the intestinal tract of untreated mice was ligated, and a suspension of mCTB transgenic rice seeds or wild type rice seed powder was added thereto. After taking out and washing, a paraffin section was prepared, and HRP (horseradish 'peroxidase) enzyme staining was performed using an anti-CTB antibody (IgG fraction) and UEA (Ulex Europeus Agglutinin) -1 lectin.
- HRP horseradish 'peroxidase
- M cells are stained with the lectin UEA-1 that recognizes fucose, and goblet cells that exude mucus in the epithelial cell layer and the epithelial cell villus layer adjacent to the special epithelial layer of the Peyer's patch dome called Taribut (crypt)
- the Paneth cells present between them are also stained with the lectin UEA-1.
- FIG. 17 shows the results.
- three columns in the column indicated by "mCTB transgenic rice seed UEA_1" show the results of staining of UEA-1 when mCTB transgenic rice seed was administered, and "Wild rice seed UEA-1".
- the three figures in the column indicated by are the results of UEA-1 staining when wild-type rice seeds were administered.
- the three columns in the column labeled ⁇ Wild-type rice seed anti-CTB antibody '' show anti-CTB antibody staining results when wild-type rice seeds were administered. Is shown.
- the three figures in the row indicated by A show the results of staining of the special epithelial layer of the Peyer's patch dome (arrows indicate M cells in A), and the three figures in the row shown by B show epithelial cell villi Layered staining results (* indicates goblet cells in B).
- the three figures in the row indicated by C show staining results of talibto (crypt) tissue (arrows indicate Paneth cells in C, * indicate goblet cells). Is shown).
- both the mCTB gene-transferred rice seeds and the wild-type rice seeds were treated with UEA-1 staining to obtain the special epithelial layer (A) of the noisyel plate dome and the epithelial cell chorionic layer.
- (B) and Taribut (crypt) tissue (C) were stained.
- CTB GM1 gandarioside
- the same site can be stained with anti-CTB antibody using Miller staining, and the specific epithelial layer of Peyer's patch dome (A) and epithelial cell villus layer ( The presence of CTB in B) and talipto (crypt) tissue (C), especially in UEA-1 lectin-stained M cells in the special epithelial layer of Peyer's patch dome (A)
- C talipto (crypt) tissue especially in UEA-1 lectin-stained M cells in the special epithelial layer of Peyer's patch dome (A)
- CTB talipto (crypt) tissue
- a DNA fragment (hereinafter, referred to as "PlaHcA_KDEL gene") in which a KDEL sequence and a restriction enzyme Sacl recognition sequence are added to the 3 'end of the plant codon mutant HcA (PlaHcA) gene at the 5' end of the HcoA (PlaHcA) gene. It was synthesized (SEQ ID NO: 21). As in Example 1, the codon of the PlaHcA_KDE L gene was optimized for rice expression. The amino acid sequence of the protein encoded by the PlaHcA-KDEL gene is shown in SEQ ID NO: 22.
- PlaHcA-KDEL gene is replaced with a DNA encoding a signal peptide (SEQ ID NO: 16) of rice glutelin GluBl connected downstream of the promoter (SEQ ID NO: 17) of rice glutelin GluBl gene (SEQ ID NO: 16).
- SEQ ID NO: 15 was performed between SEQ ID NO: 15 and one of the terminators of the rice glutelin GluB1 gene (SEQ ID NO: 18) using restriction enzymes Ncol and Sacl.
- the promoter of the rice glutelin GluB-1 gene, the DNA encoding the signal peptide of rice glutelin GluB-1 linked downstream thereof, the PlaHcA gene linked downstream thereof, and the KDEL linked downstream thereof A DNA construct containing a DNA encoding the sequence and a terminator of the rice glutelin GluBl gene linked downstream thereof was prepared. This DNA construct was introduced into pTL7 (International Publication No. WO2004Z087910) as a Sse8387l / EcoRI fragment to construct pTLGluBl-PlaHcA.
- HPT hygromycin B resistance gene linked between the 35S promoter (35S promoter) and the nopaline synthase terminator (Nos terminator)
- R cauliflower mosaic virus A yeast-derived RZRS site-specific recombination enzyme linked between the 35S promoter and a nopaline synthase terminator
- ipt the downstream terminator of the cytokinin synthase gene
- the Sse8387I fragment flanked by (RS) was introduced into pTLGluBl-PlaHcA.
- a T-DNA vector pTLGluBl_PlaHcA_130HmintrepR (see FIG. 18) incorporating the PlaHcA gene was constructed.
- This structure is a MAT vector (registered trademark) capable of removing a drug resistance gene (International Publication No. W ⁇ 2004 / 087910).
- T-DNA vector is introduced into agrobacterium in the same manner as in Example 2, and then a botulinum toxin He domain (HcA) is produced according to the method described in Example 1 of WO2004Z087910. Transgenic rice was created.
- HcA botulinum toxin He domain
- Ripe seeds of wild-type rice and PlaHcA gene-transferred rice were pulverized in the same manner as in Example 6, and a 50 mM Tris-HCl buffer (pH 6.8) containing 4% SDS, 8 M urea, 5% mercaptoethanol, and 20% glycerol was used.
- a 50 mM Tris-HCl buffer pH 6.8 containing 4% SDS, 8 M urea, 5% mercaptoethanol, and 20% glycerol was used.
- the supernatant was subjected to 15% acrylamide SDS-PAGE, and electrotransferred to PVDF membrane, anti-HcA rabbit serum, HRP-labeled antiserum.
- Western blotting antibody staining was performed using a rabbit IgG antibody. As a result, as shown in FIG.
- FIG. 3 shows the structure of the A vector pGPTV-35S-HPT-2.3kGluBpro-sig-nCTB.
- FIG. 9 shows the results of PCR using NA as type III.
- FIG. 3 is a diagram showing the results of Northern plot analysis on the seeds of rice with mCTB gene-transferred rice (FIG. 3A) and nCTB gene-transferred rice (FIG. 3B).
- FIG. 4 shows the results of protein (FIG. 4A) and antibody staining (FIG. 4B) relating to the seeds of mCTB transgenic rice and nCTB transgenic rice.
- FIG. 5 shows the results of protein staining (FIG. 5A) and antibody staining (FIG. 5B) of mCTB transgenic rice seeds.
- FIG. 6 shows the results of quantification of CTB expression in seeds of mCTB transgenic rice.
- FIG. 7 shows the results of measuring antibody titers of sera obtained from mice orally administered with wild-type rice seed powder, rice seed powder into which mCTB gene was introduced, or bacterial-derived CTB.
- Environment 8 shows the results of measuring the antibody titers of CTB-specific IgG subclass antibodies contained in sera obtained from mice orally administered with wild-type rice seed powder, mCTB gene-transferred rice seed powder or bacterial CTB.
- Antibodies induced in the serum and small intestinal secretions of mice orally administered wild-type rice seed powder, mCTB transgenic rice seed powder or bacterial-derived CTB include neutralizing antibodies that inhibit CT activity FIG.
- FIG. 4 shows the results of Southern blot analysis of separated genomic DNA.
- Fig. 14 shows the results of measuring the digestive enzyme (pepsin) resistance of rice seeds transfected with the mCTB gene.
- FIG. 15 Diagram showing the dose of CTB required to induce antigen-specific immune responses (anti-CTB serum IgG (systemic immune response, Fig. 15A) and anti-CTB fecal IgA (mucosal immune response, Fig. 15B)) It is.
- FIG. 16 is a view showing the localization of CTB in endosperm cells of mCTB gene-transferred rice seeds (FIG. 16A) and wild-type rice seeds (FIG. 16B).
- FIG. 17 is a diagram showing uptake of mCTB into mucosal-derived tissue.
- FIG. 18 is a view showing the structure of a T_DNA vector pTLGluB 1 -PlaHcA-13 OHmintrepR into which a PlaHcA gene has been incorporated.
- FIG. 19 is a view showing the results of Western blotting antibody staining of rice seeds into which botulinum A toxin neutralizing domain gene (PlaHcA gene) has been introduced.
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US11/547,910 US20070192906A1 (en) | 2004-04-09 | 2005-04-08 | Rice plant having vaccine gene transferred thereinto |
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JP2016002034A (ja) * | 2014-06-17 | 2016-01-12 | 国立大学法人 東京大学 | 外来性dnaのゲノムへの導入位置の決定法 |
CN109627306A (zh) * | 2019-01-25 | 2019-04-16 | 华中农业大学 | 水稻籽粒谷蛋白GluA2亚基的抗原表位、其抗体及应用 |
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WO2009126816A1 (en) * | 2008-04-09 | 2009-10-15 | Ventria Bioscience | Production of ospa for lyme disease control |
RU2680598C1 (ru) * | 2018-01-10 | 2019-02-25 | Федеральное казённое учреждение здравоохранения "Иркутский ордена Трудового Красного Знамени научно-исследовательский противочумный институт Сибири и Дальнего Востока" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека | Способ получения препаратов субклеточных фракций туляремийного микроба |
CN117126881A (zh) * | 2023-09-07 | 2023-11-28 | 昆明理工大学 | 一种狂犬病毒抗原的制备方法 |
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WO1999018225A1 (en) * | 1997-10-07 | 1999-04-15 | Loma Linda University | Expression of cholera toxin b subunit in transgenic plants and efficacy thereof in oral vaccines |
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WO1999018225A1 (en) * | 1997-10-07 | 1999-04-15 | Loma Linda University | Expression of cholera toxin b subunit in transgenic plants and efficacy thereof in oral vaccines |
WO2004094637A1 (ja) * | 2003-04-24 | 2004-11-04 | National Institute Of Agrobiological Sciences | アレルゲン特異的t細胞抗原決定基を植物へ集積させる方法、および該抗原決定基を集積させた植物 |
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OKADA A. ET AL: "Accumulation of Japanese cedar pollen allergen, Cry j 1, in the protein body I of transgenic rice seeds using the promoter and signal sequence of glutelin GluB-1 gene", MOLECULAR BREEDING, vol. 12, no. 1, 2003, pages 61 - 70, XP002986376 * |
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Cited By (3)
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JP2016002034A (ja) * | 2014-06-17 | 2016-01-12 | 国立大学法人 東京大学 | 外来性dnaのゲノムへの導入位置の決定法 |
CN109627306A (zh) * | 2019-01-25 | 2019-04-16 | 华中农业大学 | 水稻籽粒谷蛋白GluA2亚基的抗原表位、其抗体及应用 |
CN109627306B (zh) * | 2019-01-25 | 2021-10-15 | 华中农业大学 | 水稻籽粒谷蛋白GluA2亚基的抗原表位、其抗体及应用 |
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