WO2012011396A1 - Plant de riz transgénique capable d'exprimer des nano-anticorps - Google Patents

Plant de riz transgénique capable d'exprimer des nano-anticorps Download PDF

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WO2012011396A1
WO2012011396A1 PCT/JP2011/065670 JP2011065670W WO2012011396A1 WO 2012011396 A1 WO2012011396 A1 WO 2012011396A1 JP 2011065670 W JP2011065670 W JP 2011065670W WO 2012011396 A1 WO2012011396 A1 WO 2012011396A1
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rice
vhh1
mucorice
mtnfαvhh
nanoantibody
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PCT/JP2011/065670
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Japanese (ja)
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義和 幸
大介 徳原
宏 清野
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国立大学法人東京大学
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Priority to JP2012525370A priority Critical patent/JP5616449B2/ja
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain

Definitions

  • the present invention relates to transgenic rice expressing nanoantibodies, and more particularly to transgenic rice accumulating nanoantibodies in rice in a water-soluble form.
  • Non-patent Documents 1 and 2 when protein is produced in rice, there is a problem that the protein is mainly accumulated in the protein reservoir-I and the accumulated protein becomes hardly soluble.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and an object thereof is to provide transgenic rice in which antibodies are accumulated in a water-soluble form in rice.
  • a further object of the present invention is to provide a method for efficiently producing an antibody using such transgenic rice.
  • Non-patent Document 3 When only the variable region of this double-chain antibody (variable domain of llama heavy-chain; VHH) was fragmented and taken out, it was also found that it has the same binding affinity to the antigen as a normal antibody. This VHH is currently called the “nanoantibody” because it is the smallest antibody with binding affinity for antigen. Nanoantibodies are small, resistant to acid and heat and highly stable (Non-patent Document 4), and are produced as recombinant proteins in various hosts because of their low molecular weight ( Non-patent document 5).
  • the present inventors applied a nanoantibody to a rice expression system that has not succeeded in accumulating proteins in rice in a water-soluble form until now.
  • the present inventors have found that the solubilized nanoantibody retains its original performance of heat resistance and stability.
  • the nanoantibody has a higher neutralizing effect on the antigen than the monomer.
  • the present inventors apply the antibody as a nanoantibody to a rice expression system, thereby allowing the antibody to accumulate in rice in a water-soluble form and without losing the original characteristics of the antibody. It was the first time in the world that the present invention was completed.
  • the present invention provides the following inventions.
  • a composition comprising the rice according to (3), the water-soluble fraction according to (4), or the processed product according to (5).
  • a method for producing a nanoantibody (A) introducing DNA encoding a nanoantibody into rice to produce transgenic rice in which the nanoantibody is expressed in rice; (B) obtaining a water-soluble fraction from the rice of the transgenic rice produced in step (a), Including methods.
  • the present invention makes it possible to accumulate nanoantibodies in rice in a water-soluble form without losing their original characteristics. According to the present invention, a large amount of nanoantibodies can be produced in an inexpensive and stable form.
  • the rice in which nanoantibodies are accumulated, the water-soluble fraction thereof, or the processed product thereof can be applied as a safe composition to medicines, foods and drinks, and the like.
  • MocoRice-VHH1 is a photograph showing the results of detection of VHH1 expression in rice seeds into which VHH1 gene was introduced (hereinafter abbreviated as “MucoRice-VHH1”).
  • A is a photograph of SDS-PAGE and Western blot
  • (b) is a photograph of immunostaining seeds on the 14th day after flowering. It is a figure which shows the result of having analyzed the amino acid sequence of MucoRice-VHH1 using two types of mass spectrometers (QSTAR Elite and LTQ Orbitrap). It is a photograph which shows the result of having examined the VHH1 extraction conditions from MucoRice-VHH1.
  • Lane 1 rVHH1
  • Lane 2 8M Extract from MucoRice-VHH1 using urea-containing PBS
  • Lane 3 Extract from MucoRice-VHH1 using PBS. It is a photograph of a Western blot using SDS-PAGE and an antibody (NKM-16-2-4) having specificity for mouse M cells showing the results of examining scFv extraction conditions from MucoRice-scFv.
  • Lane 1 Extract from MucoRice-scFv using PBS
  • Lane 2 Extract from urea-containing SDS solution from precipitate in PBS extraction of MucoRice-scFv
  • Lane 3 From wild seeds using PBS
  • Lane 4 extract from urea-containing SDS solution from precipitates in PBS extraction of wild-type seeds
  • Lane 5 NKM-16-2-4 antibody
  • Lane 6 MucoRice-scFv using urea-containing SDS solution Extract from lane 7
  • Lane 7 Extract from wild seeds with urea-containing SDS solution.
  • the dotted arrow in FIG. 4 indicates the scFv protein band.
  • (I) shows the small intestine tissue of a non-infected young mouse
  • (II) shows the small intestine tissue of a young mouse infected with rotavirus
  • (III) shows the infection with rotavirus and administered MucoRice-VHH1
  • (IV) shows the small intestine tissue of a young mouse infected with rotavirus and administered with WT-Rice (Japan fine). It is a graph which shows the result of having evaluated the amount of rotavirus in the small intestine tissue of a young mouse by real-time PCR.
  • Control shows the VP7 RNA copy number in the small intestine tissue of the uninfected young mouse
  • RRV infection only shows the VP7 RNA copy number in the small intestine tissue of the young mouse infected with rotavirus
  • WTrice administration "Indicates VP7 RNA copy number in the small intestine tissue of young mice infected with rotavirus and administered WT-Rice (Japan sunny)”
  • MocoRice VHH1 administration indicates VP7 in the small intestine tissue of young mice administered MucoRice-VHH1 RNA copy number is indicated.
  • the leftmost photo shows the result of analyzing MucoRice-mTNF ⁇ VHH monomer by SDS-PAGE
  • the second photo from the left shows the result of analyzing MucoRice-mTNF ⁇ VHH monomer by Western blot using anti-mTNF ⁇ VHH polyclonal antibody
  • the third photo from the left shows the result of analyzing MucoRice-mTNF ⁇ VHH dimer by SDS-PAGE
  • the rightmost photo shows the result of analyzing MucoRice-mTNF ⁇ VHH dimer by Western blot using anti-mTNF ⁇ VHH polyclonal antibody.
  • Show. “Lane 1” indicates the analysis result of rmTNF ⁇ VHH (recombinant mTNF ⁇ VHH derived from E.
  • “Lane 2” indicates the analysis result of the PBS extract of MucoRice-mTNF ⁇ VHH monomer
  • “Lane 3” indicates MucoRiceVmHNTNH
  • the analysis result of the precipitate after the extraction of the monomer with PBS is shown
  • “Lane 4” shows the analysis result of the PBS extract of MucoRice-mTNF ⁇ VHH dimer
  • “Lane 5” shows the precipitate after the extraction of MucoRice-mTNF ⁇ VHH dimer with PBS The analysis result of a thing is shown.
  • the band surrounded by a square indicates mTNF ⁇ VHH monomer (13.8 kDa) or mTNF ⁇ VHH dimer (28.4 kDa).
  • the photograph on the left shows the result of analyzing MucoRice-mTNF ⁇ VHH etc. by SDS-PAGE, and the photograph on the right shows the result of analyzing MucoRice-mTNF ⁇ VHH etc. in Western blot.
  • rmTNF ⁇ VHH indicates the result of analysis of rmTNF ⁇ VHH
  • MocoRice-mTNF ⁇ VHH indicates the analysis result of the PBS extract of MucoRice-mTNF ⁇ VHH monomer
  • MocoRice-mTNF ⁇ VHHDmMVVHHDmFmMVDHFD The analysis result of the solution is shown
  • WT-Rice shows the analysis result of the PBS extract of WT-Rice (Nipponbare).
  • the band indicated by an arrow indicates mTNF ⁇ VHH monomer (denoted as “VHH (13.8 kDa) in the figure) or mTNF ⁇ VHH dimer (denoted as“ VHH Dimer (28.4 kDa) in the figure) ”. It is a graph which shows the result of having evaluated the inhibition rate of mTNF (alpha) VHH with respect to mTNF (alpha).
  • the rightmost line shows the relationship between the amount of rmTNF ⁇ VHH added and the inhibition rate against mTNF ⁇
  • the middle line shows the amount of added muTRice-mTNF ⁇ VHH monomer-derived PBS extract and mTNF ⁇ of rmTNF ⁇ VHH.
  • the leftmost line “dimer” shows the relationship between the added amount of MucoRice-mTNF ⁇ VHH dimer-derived PBS extract and the inhibition rate of rmTNF ⁇ VHH to mTNF ⁇ .
  • FIG. 1 It is a photograph which shows the result of having analyzed the localization of mTNF (alpha) VHH monomer in a rice seed by the immunoelectron microscope method.
  • A shows mTNF ⁇ VHH rice (MucoRice-mTNF ⁇ VHH monomer) localization of mTNF ⁇ VHH monomer (type I protein reservoir (PB-I) and cytoplasm) detected with anti-mTNF ⁇ VHH antibody
  • B shows mTNF ⁇ VHH rice. Shows the results of detecting and observing the localization of mTNF ⁇ VHH monomer (type II protein reservoir (PB-II), cytoplasm, starch (St: Starch), cell wall (CW: Cell Wall)) using anti-mTNF ⁇ VHH antibody .
  • PB-II type II protein reservoir
  • St Starch
  • CW Cell Wall
  • C shows the result of observing PB-I, PB-II and cytoplasm by reacting mTNF ⁇ VHH rice with normal rabbit IgG (“ ⁇ -Rabbit IgG”, negative control), and D shows Nihonsei rice (WT-Rice).
  • A shows the results of detection and observation of mTNF ⁇ VHH dimer localization (PB-I, PB-II, cytoplasm) in mTNF ⁇ VHH dimer rice (MucoRice-mTNF ⁇ VHH dimer) using anti-mTNF ⁇ VHH antibody
  • B shows the results in mTNF ⁇ VHH dimer rice
  • the localization of mTNF ⁇ VHH dimer (PB-I, PB-II, cytoplasm, cell wall) was detected using an anti-mTNF ⁇ VHH antibody, and the observation results are shown.
  • C shows the result of observing PB-I, PB-II, St and cytoplasm after reacting mTNF ⁇ VHH dimer rice with normal rabbit IgG
  • D shows the result of reacting Nippon Harimai (WT-Rice) with anti-mTNF ⁇ VHH antibody.
  • the results of observation of PB-I, PB-II and cytoplasm (“WT Nipponbare”, negative control) are shown.
  • the present invention provides transgenic rice in which a DNA encoding a nanoantibody is introduced and the nanoantibody is expressed in rice.
  • “rice” means a plant belonging to Oryza sativa , and the variety of rice to be genetically modified is not particularly limited as long as it belongs to Oryza sativa .
  • nanoantibody means an antibody that is composed of a variable region of an antibody heavy chain (also referred to as variable domain of a heavy-chain antibody or VHH) and can recognize an antigen.
  • Nanoantibodies are typically antibodies derived from camelids (eg, dromedaries, bactrian camels, llamas, alpaca, vicuna).
  • the nanoantibody in the present invention can use VHH as a monomer, but can also be used as a multiple antibody of dimer or higher.
  • the dimer or higher multi-antibody may be one in which the same VHH is linked (for example, a homodimer in the case of a dimer), and two or more recognize different molecules or different epitopes within the same molecule. (For example, a heterodimer in the case of a dimer) may be used.
  • Such a VHH connection method is not particularly limited, and a known method can be used. For example, a method comprising constructing a vector containing DNA encoding an antibody in which VHHs are linked via a spacer sequence and expressing the vector in rice of transgenic rice can be mentioned.
  • a single VHH can be expressed in rice, purified from a water-soluble fraction, and then the purified VHHs can be bound to each other to produce multiple antibodies of dimers or higher.
  • a nanoantibody against a desired antigen can be used.
  • the antigen include (1) pathogenic viruses (for example, rotavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, noo Oak virus, rabies virus, RS virus, cytomegalovirus, Foot-and-mouth disease virus, contagious gastroenteritis virus, rubella virus, ATL virus, adenovirus, mumps virus, coxsackie virus, enterovirus, herpes virus, smallpox virus, poliovirus, measles virus, Japanese encephalitis virus, proboscis fever Virus, Yellow fever virus, West Nile virus, SARS (coronavirus), Influenza virus, HIV (AIDS virus), Ebola virus (Firovirus), Marburg virus (Firovirus), Lassa fever Will (2) Pathogenic bacteria (eg, Vibrio cholerae, Pathogenic Escherichia coli, Haemophilus), a virus, and viruses,
  • Colon cancer tumor antigens breast cancer tumor antigens such as Her2, prostates such as PSA (prostate specific antigen) N such as a tumor antigen), (9) such as cell antigen (e.g. CD3, CD25, RANKL), (10) Others (eg von Willebrand factor (von Willebrand factor: vWF platelet aggregating factor), and the like).
  • PSA prostate specific antigen
  • N such as a tumor antigen
  • cell antigen e.g. CD3, CD25, RANKL
  • Others eg von Willebrand factor (von Willebrand factor: vWF platelet aggregating factor), and the like).
  • the codon contained in the DNA encoding the nanoantibody is preferably modified to a plant codon.
  • a plant-type codon By changing to a plant-type codon, the expression efficiency of the nano antibody in rice (in rice endosperm cells) can be improved.
  • a promoter and terminator that function specifically in rice endosperm.
  • the promoter is linked upstream of the DNA encoding the nanoantibody
  • the terminator is linked downstream of the DNA encoding the nanoantibody.
  • promoters and terminators that function specifically in rice endosperm include promoters and terminators of genes that encode rice endosperm storage proteins.
  • rice endosperm storage protein means 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 protein include glutelin, prolamin, globulin, and the like.
  • the promoter of the gene encoding rice endosperm storage protein is preferably the promoter of the glutelin GluB-1 gene or the prolamin 13K gene.
  • the promoters of these genes are advantageous in that their promoter activities are higher than those of genes encoding other rice endosperm storage proteins.
  • the rice endosperm-specific promoter may be either a natural promoter or a mutant promoter as long as it has rice endosperm-specific promoter activity.
  • the “mutant promoter” means a promoter having a rice endosperm-specific promoter activity, comprising a base sequence in which one or more bases are substituted, deleted or added in the base sequence of the natural promoter. The same applies to the rice endosperm-specific terminator.
  • an expression suppression cassette that suppresses either 13K prolamin or glutelin expression by RNAi method or suppresses both 13K prolamin and glutelin expression (double suppression) is introduced into rice. To do.
  • the method for introducing a vector into rice cells is not particularly limited.
  • Transgenic rice can be produced, for example, by introducing a vector for expressing a DNA encoding a nanoantibody into rice cells, and then culturing the transformed rice cells to regenerate the rice plant body.
  • 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 body, and examples thereof include cultured cells, protoplasts, shoot primordia, polyblasts, hairy roots, and callus. .
  • a plasmid is used as a vector, it is preferable to contain a drug resistance gene such as hygromycin, tetracycline, ampicillin so that rice cells into which the vector has been introduced can be efficiently selected.
  • hygromycin when used as an oral agent for a pharmaceutical product without being purified from the expressed rice, hygromycin that is not particularly safe must be removed by a known method such as a cotransfection method.
  • the present invention is a method for producing a nanoantibody, wherein (a) DNA encoding the nanoantibody is introduced into rice to produce transgenic rice in which the nanoantibody is expressed in rice; and (b) And (a) obtaining a water-soluble fraction from the rice of the transgenic rice produced in step (a). Furthermore, the present invention also provides a water-soluble fraction containing rice and its nanoantibodies collected from the transgenic rice of the present invention.
  • rice means a tissue containing endosperm or a portion thereof, and includes rice bran, brown rice, seeds, white rice, and these portions.
  • the “water-soluble fraction” means a rice fraction that can be eluted with water or a buffer solution that does not contain a drug for solubilizing proteins such as a surfactant and a reducing agent. Accumulating proteins, including antibodies, in rice in a water-soluble form has never been possible, and is the first in the world to succeed in the present invention. This water-soluble characteristic is considered to be caused by the accumulation of nanoantibodies mainly other than the rice protein reservoir (PB) (ie, cytoplasm).
  • PB rice protein reservoir
  • the present invention also provides a processed product of the rice and water-soluble fraction.
  • the “processed product” includes any processed product as long as it contains nanoantibodies. Examples of the processing to be applied include threshing, powdering, extraction of a desired fraction containing nanoantibodies, purification of the extracted fraction, and the like.
  • the rice thus prepared, its water-soluble fraction, or a processed product thereof can be used as a highly safe composition.
  • a pharmaceutical composition for example, as an oral composition.
  • the water-soluble fraction of rice and its processed product can be used as, for example, an injectable composition.
  • animals to be administered or ingested with the composition of the present invention include humans and other vertebrates (for example, mammals, birds, amphibians, fish, reptiles) and the like.
  • composition in the present invention can also be formulated by a known pharmaceutical method.
  • pharmacologically or carriers that are acceptable as foods and drinks and animal feeds, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, Stabilizer, flavor, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffer, coating agent, lubricant, coloring It can be appropriately combined with agents, sweeteners, thickeners, flavoring agents, solubilizers or other additives.
  • the dose or intake is appropriately selected according to the age, weight, type of composition (pharmaceuticals, food and drink, animal feed, etc.) of the subject.
  • the dose or intake per dose is generally from 0.001 mg / kg body weight to 100 mg / kg body weight.
  • VHH1-expressing rice (MucoRice-VHH1) Nano-antibody against rotavirus (VHH1) gene information used publicly known public information (http://igture-archive.libry.uu.nl/dissertations/2004-0419- 094105 / c4.pdf).
  • VHH1 is an antigen-binding site of an antibody having only a heavy chain obtained by immunizing rhesus monkey rotavirus (RRV) serotype G3 with a camelid llama. The gene encoding VHH1 was reconstructed and artificially synthesized based on codons that are readily available to rice.
  • the selection marker cassette As shown in the published patent information (WO 2004/056993 A1), the selection marker cassette, the overexpression cassette of the foreign gene, and the expression suppression cassette for the seed endogenous storage protein gene.
  • the method of ligation onto a single T-DNA binary vector (pZH2B / 35SNos) and simultaneous introduction was referred to. That is, first, the selection marker cassette was obtained by linking a hygromycin resistance gene (mHPT) to a CaMV35S promoter and a Nos terminator. Next, a gene encoding VHH1 was inserted between the endosperm cell-specific prolamin 13K promoter / 10K prolamin signal and the prolamin 13K prolamin terminator in the foreign gene cassette.
  • mHPT hygromycin resistance gene
  • the expression suppression cassette for the storage protein gene was constructed to double suppress the expression of 13K prolamin and glutelin by RNAi method. Specifically, a 45-bp portion of 13K prolamin cDNA and a 129-bp portion of glutelin A cDNA were ligated to both ends of a rice aspartic protease (RAP) intron in the sense and antisense directions, and the ubiquitin promoter and Nos terminator were ligated. It was set as the structure which expresses hairpin type RNA by inserting between.
  • RAP rice aspartic protease
  • a T-DNA binary vector for VHH1 gene expression in which all of these were ligated was introduced into rice (Nipponbare) by a conventional method using Agrobacterium to produce VHH1-expressing rice.
  • Example 2 Analysis of VHH1 expression in MucoRice-VHH1 Regarding whether or not rice seeds into which VHH1 gene has been introduced (hereinafter also referred to as “MucoRice-VHH1”) express VHH1, SDS-PAGE, Western blot, and immunostaining techniques were used. And analyzed. The seeds after threshing (MucoRice-VHH1 and wild type) are pulverized, and the rice powder is suspended in a sample buffer (2% SDS, 5% ⁇ -mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8). As an extract and a positive control, recombinant VHH1 derived from E.
  • a sample buffer 2% SDS, 5% ⁇ -mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8.
  • rVHH1 coli coli
  • CBB Clusterassie Brilliant Blue
  • the separated gel was transferred to a PVDF membrane, and Western blot analysis was performed using an anti-VHH1 polyclonal antibody prepared by immunizing a rabbit with rVHH1.
  • VHH1 expression in seeds was analyzed by immunostaining using an anti-VHH1 polyclonal antibody. Specifically, seeds on the 14th day after flowering were collected and cut, immersed and fixed in a 4% paraformaldehyde solution (PFA), frozen and embedded after sucrose soaking, and 3 ⁇ m frozen sections were prepared, anti-VHH1 A polyclonal antibody was reacted, and then an HRP-labeled donkey anti-rabbit IgG antibody was reacted, and color was developed with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. As a result, no wild-type seed-specific staining image was observed, but in MucoRice-VHH1, an anti-VHH1 antibody-specific staining image was observed throughout the seed (b in FIG. 1).
  • PFA paraformaldehyde solution
  • DAB 3,3′-diaminobenzidine
  • Example 3 Structural analysis of VHH1 derived from MucoRice-VHH1
  • sample buffer 2% SDS, 5% ⁇ -mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8
  • polyacrylamide of rVHH1 as a positive control. Separation by electrophoresis using gel. The separated gel was subjected to CBB staining, a band corresponding to VHH1 was cut out, and after CBB decolorization / dehydration, gel digestion with trypsin was performed to extract peptides.
  • the extracted peptides were separated by nanoLC and analyzed using two types of mass spectrometers (QSTAR Elite and LTQ Orbitrap).
  • QSTAR Elite is shown in SEQ ID NO: 1
  • the analysis result of LTQ Orbitrap is shown in SEQ ID NO: 2, and these are collectively shown in FIG.
  • FIG. 2 the complete amino acid sequence could be decoded by combining the results of the two types of mass spectrometers.
  • SR serine + arginine
  • Example 4 Examination of VHH1 extraction conditions from MucoRice-VHH1 VHH1 extraction conditions from MucoRice-VHH1 were examined. Add 1 mL of phosphate buffer (hereinafter abbreviated as PBS) or 1 mL of PBS containing 8M urea to 100 mg of MucoRice-VHH1 rice powder, and after suspension, immediately centrifuge, and add rVHH1 as sample control (2% It was suspended in SDS, 5% ⁇ -mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8), and separated by electrophoresis using 12% polyacrylamide gel. The separated gel was subjected to CBB staining. As a result, as shown in FIG. 3, it was clarified that VHH1 of the same level as PBS containing 8M urea was extracted from MucoRice-VHH1 with PBS alone. This indicates that VHH1 in the rice powder is water-soluble.
  • PBS phosphate buffer
  • PBS phosphat
  • Example 5 Localization of VHH1 in MucoRice-VHH1 Localization of VHH1 in seeds was examined by immunoelectron microscopy.
  • the seeds on the 14th day after flowering were collected, sections of about 0.5 to 1.0 ⁇ m were prepared, fixed with 4% PFA, and then immersed in LR-white resin. Thereafter, an ultrathin section of 100 ⁇ m was prepared and placed on a grid, reacted with an anti-VHH1 polyclonal antibody, washed and reacted with an 18 nm gold colloid-labeled anti-rabbit IgG antibody.
  • VHH1 was remarkably observed in PB-II and lightly observed in the vicinity of the outer surface of PB-I, but a large amount of VHH1 was accumulated in the cytoplasm other than PB (FIG. 5). It was considered that the water solubility of VHH1 observed in the extraction study of MucoRice-VHH1 was particularly dependent on the localization to the cytoplasm showing high water solubility.
  • Example 6 Evaluation of neutralization effect of MucoRice-VHH1 against rotavirus (cell infection experiment)
  • MA104 cells rhesus monkey kidney-derived cell line
  • RRV rhesus monkey rotavirus
  • MucoRice-VHH1 stored at room temperature for 1 year has a neutralizing effect comparable to that of MucoRice-VHH1 immediately after harvest, and the long-term room temperature storage of MucoRice-VHH1 was proved.
  • Example 7 Evaluation of stability and distribution of MucoRice-VHH1 in the digestive tract After centrifuging a suspension of MucoRice-VHH1 powder and pure water, the supernatant was collected, and 40 ⁇ l of a 100 ⁇ g / mL concentration of VHH1 solution was applied to the Administration into the stomach of Balb / c mice. The contents of stomach, jejunum, ileum, and large intestine were collected after 1, 3, 4, and 9 hours and suspended in 500 uL of PBS.
  • VHH1 derived from rice was found in the entire digestive tract from the stomach to the large intestine until 4 hours after administration. In addition, 9 hours after administration, about 50% of mice had rice-derived VHH1 from the jejunum to the large intestine.
  • Example 8 Evaluation of neutralization effect of MucoRice-VHH1 against rotavirus (infection experiment 1 using juvenile animals)
  • RRV 2 ⁇ 10 7 ffu obtained in MEM 20 ul
  • trypsin 37 ° C., 30 minutes
  • RRV 2 ⁇ 10 7 ffu obtained in MEM 20 ul
  • trypsin 37 ° C., 30 minutes
  • Fig. 8 watery diarrhea was observed after about 24 hours.
  • Fig. 8 spontaneously improves after 3 to 4 days.
  • mucoRice-VHH1 was tested for its neutralizing effect and diarrhea-suppressing effect on RRV.
  • Example 9 Evaluation of neutralization effect of MucoRice-VHH1 against rotavirus (infection experiment 2 using juvenile animals)
  • a rotavirus infection model was prepared using young mice, and instead of the diarrhea state used in Example 8, the state of the small intestine tissue section and the increase in the amount of rotavirus were used as indicators.
  • the neutralizing effect of MucoRice-VHH1 against rotavirus was evaluated.
  • the amount of rotavirus was obtained by extracting total RNA from small intestine tissue, preparing cDNA by a conventional method, and using the method of Pant et al. (Pant, N. et al., J. Infect. Dis., 2006, 194, 1580-1588).
  • the amount of rotavirus increase / decrease was evaluated by quantifying mRNA encoding VP7 of rotavirus by real-time PCR using the method described in the page. The obtained results are shown in FIG.
  • rotavirus infection (2 ⁇ 10 7 ffu RRV) was performed under the same conditions as in Example 8, and in the small intestine tissue of young mice administered with MucoRice-VHH1, epithelium No abnormality was found in the cells (intestinal epithelial cells) (see III in FIG. 11).
  • the small intestine tissue of young mice infected with rotavirus see II in FIG. 11
  • the small intestine tissue of young mice infected with rotavirus and administered WT-Rice (Nipponbare) (see IV in FIG. 11).
  • numerous vacuoles were observed in the epithelial cells of these mice.
  • Example 10 Evaluation of neutralization effect of MucoRice-VHH1 against rotavirus (infection experiment using immunodeficient animals)
  • the effect of MucoRice VHH1 on anti-RRV-VHH1 was evaluated using SCID mice which are immunodeficient mice. That is, SCID mice were infected with rotavirus (2 ⁇ 10 7 ffu RRV), and then 200 mg of MucoRice-VHH1 (containing 1.7 mg VHH1) was orally administered twice daily for 7 days. The amount of virus was evaluated by the amount of VP7 RNA by real-time PCR. The obtained results are shown in FIGS.
  • the “diarrhea score” in FIG. 13 is based on the result of evaluation as “0” when diarrhea is not observed, “1” when weak diarrhea is observed, and “2” when diarrhea is observed. .
  • MucoRice-VHH1 is effective for rotavirus infection not only in children as shown in Examples 8 and 9, but also in immunocompromised patients who cannot be administered a vaccine.
  • TNF- ⁇ VHH-expressing rice (MucoRice-mTNF ⁇ VHH)
  • VHH nanoantibodies
  • a gene encoding mouse TNF ⁇ VHH monomer (mTNF ⁇ VHH monomer) was reconstructed and artificially synthesized based on codons that can be easily used by rice.
  • a gene encoding TNF ⁇ VHH dimer (mTNF ⁇ VHH dimer) was prepared by combining the VHH monomers with each other via an appropriate linker (a spacer sequence consisting of an amino acid sequence [GGGGSGGGGSGGGS]).
  • T-DNA binary vector pZH2B / 35SNos
  • Agrobacterium Rice Nipponbare was transformed through um and TNF- ⁇ VHH-expressing rice (MucoRice-mTNF ⁇ VHH: MucoRice-mTNF ⁇ VHH monomer and MucoRice-mTNF ⁇ VHH dimer) was produced by a conventional method.
  • Example 12 Extraction of mTNF ⁇ VHH from MucoRice-mTNF ⁇ VHH For extraction of each of mTNF ⁇ VHH monomer and mTNF ⁇ VHH dimer from MucoRice-mTNF ⁇ VHH dimer and SDS-PAGE using tSter-PAGE.
  • mTNF ⁇ VHH contained in MucoRice-mTNF ⁇ VHH reached an average of 0.82% per rice powder weight in VHH monomers and an average of 0.47% in VHH dimers, and VHH was highly expressed not only in monomers but also in dimers. It was found that the accumulation was high (FIG. 16).
  • Example 14 Evaluation of inhibitory effect of MucoRice-mTNF ⁇ VHH on mTNF ⁇ (cell death suppression test)
  • WEHI164 cells mouse fibroblasts
  • RPMI164 medium containing 10% FCS in a 96-well plate at 2 ⁇ 10 4 cells / well and cultured in a 5% CO 2 incubator (37 ° C.) for 24 hours.
  • the medium is removed, and the medium alone (negative control), the medium added with only 2 ng / mL rmTNF ⁇ (manufactured by R & D systems) (positive control), or 2 ng / mL rmTNF ⁇ and the MucoRice-mTNF ⁇ VHH-derived PBS extract are mixed.
  • the amount of rmTNF ⁇ added (2 ng / mL) was such that the survival rate of WEHI164 cells was 50%. That is, as described above, WEHI164 cells were cultured in a medium supplemented with a predetermined amount (0.5 to 4 ng / mL) of rmTNF ⁇ and subjected to WST-8 assay. From the number of viable cells obtained, the survival rate was 50. % Of rmTNF ⁇ was calculated (see FIG. 18).
  • Example 15 Localization of mTNF ⁇ VHH in MucoRice-mTNF ⁇ VHH (observation by immunoelectron microscope) The localization of mTNF ⁇ VHH monomer and dimer in seeds was analyzed by immunoelectron microscopy. That is, seeds on the 14th day after flowering of the second generation MucoRice-mTNF ⁇ VHH monomer and dimer were collected, and sections of about 0.5 to 1.0 ⁇ m were prepared. Subsequently, these sections were immersed and fixed in 4% PFA while shaking at 4 ° C. for 4 hours.
  • the LR-White resin was gradually infiltrated over 2 days at 4 ° C, and finally immersed and embedded in 100% LR-White resin at 50 ° C overnight. Polymerized. From the polymer block after polymerization, an ultrathin section having a thickness of 80 to 100 nm was prepared by an ultramicrotome, and collected on a nickel grid (mounting net) with a form bar support film attached thereto to obtain a sample for immunostaining.
  • Immunostaining was performed by blocking with 10% goat serum (Goat Serum) for 30 minutes at room temperature, and then reacting with an anti-mTNF ⁇ VHH antibody (rabbit) purified by affinity using a protein G column at a concentration of 5 ⁇ g / ml for 1 hour at room temperature. . Subsequently, after washing, an 18 nm gold colloid-labeled anti-rabbit IgG antibody was reacted. Then, after reacting the secondary antibody, it was washed again, fixed with 1% glutaraldehyde for 5 minutes at room temperature, and then subjected to double electron staining with 2% uranyl acetate for 5 minutes and then with a lead salt for 5 minutes. The sample immunostained in this way was observed with a transmission electron microscope.
  • the water-soluble nature of mTNF ⁇ VHH shown in FIGS. 15 and 16 is mainly due to the fact that both the nanoantibody monomer and dimer accumulate in other than the rice protein reservoir (PB) (ie, cytoplasm). As one of them.
  • PB rice protein reservoir
  • nano-antibodies VHH1 having binding affinity for rotavirus VP4 and VP7 (Dolk E, et al., Proteins. 2005; 59: 555-64.) And TNF- ⁇ nanoantibodies (Pant N, et al., J Infect Dis. 2006; 194: 1580-8.) are expressed in lactic acid bacteria, so that rotavirus infection occurs orally. ⁇ Successful attempts to prevent enteritis such as diarrhea and Crohn's disease.
  • lactic acid bacteria which are often administered to the human body as an intestinal adjuster, it can be an oral drug with less burden on the human body, but when administered to a person with reduced immunity or a premature infant, sepsis and myocarditis caused by lactic acid bacteria There is a risk (Vandenbrooke K, et al., 2010; 3: 49-56., Land MH, et al., Pediatrics. 2005; 115: 178-81., Schlegel L, et al., Eur J Clin Microbiol Infect Dis. 1998; 17: 887-8.0).
  • oral antibody preparations using nano-antibodies are preferably preparations that do not require refrigerated storage and can be stored at room temperature for a long period of time.
  • Rice that has been developed according to the present invention and in which nanoantibodies are accumulated in a water-soluble form can be prepared as an oral antibody preparation capable of storing the nanoantibodies for a long period of time without adding a purification step.
  • the water-soluble fraction extracted from rice and its processed product can also be used as an injection preparation. Therefore, the present invention can greatly contribute to the development of a pharmaceutical preparation such as an oral preparation or an injection preparation which is inexpensive and can be stored at room temperature, which comprises an antibody as an active ingredient.

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Abstract

La présente invention concerne un plant de riz transgénique dans lequel un anticorps s'accumule dans le riz sous forme hydrosoluble, ainsi qu'un procédé de production d'un anticorps hautement efficace qui utilise le plant de riz transgénique. Un nano-anticorps s'exprime à l'aide du système d'expression du plant de riz. De plus, il a été découvert de manière inattendue que ce nano-anticorps s'accumule dans le riz sous forme hydrosoluble. Il a également été observé que ce nano-anticorps conserve sa thermostabilité et sa stabilité lorsqu'il est solubilisé.
PCT/JP2011/065670 2010-07-20 2011-07-08 Plant de riz transgénique capable d'exprimer des nano-anticorps WO2012011396A1 (fr)

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WO2018181866A1 (fr) * 2017-03-31 2018-10-04 国立大学法人東京大学 Anticorps de norovirus
EP3597758A1 (fr) * 2013-04-29 2020-01-22 AgroSavfe nv Compositions agrochimiques comprenant des polypeptides
US10858666B2 (en) 2014-11-05 2020-12-08 Biotalys Transgenic plants expressing a variable domain of a heavy chain antibody (VHH) that binds to a sphingolipid of a fungus

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3597758A1 (fr) * 2013-04-29 2020-01-22 AgroSavfe nv Compositions agrochimiques comprenant des polypeptides
US11028154B2 (en) 2013-04-29 2021-06-08 Biotalys NV Agrochemical compositions comprising antibodies binding to sphingolipids
US10858666B2 (en) 2014-11-05 2020-12-08 Biotalys Transgenic plants expressing a variable domain of a heavy chain antibody (VHH) that binds to a sphingolipid of a fungus
WO2018181866A1 (fr) * 2017-03-31 2018-10-04 国立大学法人東京大学 Anticorps de norovirus
JPWO2018181866A1 (ja) * 2017-03-31 2020-02-13 国立大学法人 東京大学 ノロウイルス抗体
TWI743346B (zh) * 2017-03-31 2021-10-21 國立大學法人東京大學 諾羅病毒抗體
US11530254B2 (en) 2017-03-31 2022-12-20 The University Of Tokyo Norovirus antibody

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