JP5616449B2 - Transgenic rice expressing nanoantibodies - Google Patents

Description

  The present invention relates to transgenic rice expressing nanoantibodies, and more particularly to transgenic rice accumulating nanoantibodies in rice in a water-soluble form.

  In recent years, antibodies have been widely used as gene recombinant drugs. However, in the development of antibody drugs, reduction of production costs and improvement of stability are major issues. Therefore, development of a method capable of producing a large amount of antibody in an inexpensive and stable form is expected. Under such circumstances, rice expression systems have attracted attention, and attempts have been made to express antibodies such as double-chain antibodies and single-chain antibodies (scFv) and proteins such as cytokines in a large amount and a stable form in rice. Has been made.

  However, 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 is hardly soluble (Non-patent Documents 1 and 2).

Yoshihiro F. et al. , Protein Expression and Purification 2010; 72: 125-130. Tomonori N, et al. , PNAS 2007; 104: 10986-10991. Hamers-Casterman C, et al. , Nature. 1993; 363: 446-8. van der Linden RH, et al. Biochim Biophys Acta. 1999; 1431: 37-46. Frenken LG, et al. , J Biotechnol. 2000; 78: 11-21.

  This invention is made | formed in view of the subject which the said prior art has, The objective is to provide the transgenic rice in which the antibody is accumulate | stored in the water-soluble form in the rice. A further object of the present invention is to provide a method for efficiently producing an antibody using such transgenic rice.

  Usually, human antibodies are composed of two heavy chains and two light chains, but for camelids such as llamas and camels, not only four-chain antibodies consisting of heavy and light chains, but also heavy chains. It was reported in 1993 that there is a double-chain antibody consisting only of a chain and the target antigen can be recognized and bound in the same manner as a 4-chain antibody (Non-patent Document 3). Furthermore, 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 a binding affinity to an antigen similar to that of 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. Was found to be accumulated in rice in the form of Analysis of the localization of nanoantibodies in rice revealed that the localization of nanoantibodies other than the protein reservoir of rice (cytoplasm) is one of the main factors in this water-soluble property. . Furthermore, the present inventors have found that the solubilized nanoantibody retains its original performance of heat resistance and stability. In addition, the inventors have also found that the nanoantibody has a higher neutralizing effect on the antigen than the monomer. As described above, 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.

More specifically, the present invention provides the following inventions.
(1) Transgenic rice in which DNA encoding a nanoantibody is introduced, the nanoantibody is specifically expressed in rice endosperm cells, and is accumulated in the cytoplasm of the cell in a water-soluble form .
(2) Rice harvested from the transgenic rice according to (1) .
( 3 ) A water-soluble fraction of the rice according to ( 2 ), containing a nanoantibody.
( 4 ) A processed product containing nanoantibodies of the rice according to ( 2 ) or the water-soluble fraction according to ( 3 ).
( 5 ) A composition containing the rice according to ( 2 ), the water-soluble fraction according to ( 3 ), or the processed product according to ( 4 ).
( 6 ) A method for producing a nanoantibody,
(A) introducing a DNA encoding a nanoantibody into rice, and producing a transgenic rice in which the nanoantibody is specifically expressed in rice endosperm cells and accumulated in the cytoplasm of the cell in a water-soluble form ;
(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 properties. 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.

It is a photograph which shows the result of having detected the expression of VHH1 in the seed of rice which introduce | transduced VHH1 gene (henceforth "MucoRice-VHH1"). (A) is a photograph of SDS-PAGE and Western blot, and (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: Extract from MucoRice-VHH1 using 8M urea-containing PBS, Lane 3: Extract from MucoRice-VHH1 using PBS. It is the photograph of the Western blot using SDS-PAGE and the antibody (NKM-16-2-4) specific to mouse M cell which show the result of having examined the scFv extraction condition 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 Extract 4: Lane 4: Extracted solution with urea-containing SDS solution from the precipitate in PBS extraction of wild-type seeds, Lane 5: NKM-16-2-4 antibody, Lane 6: MucoRice-scFv with urea-containing SDS solution Extract from lane 7, Lane 7: Extract from wild seeds with urea-containing SDS solution. Also, the dotted arrow in FIG. 4 indicates the scFv protein band. It is a photograph which shows the result of having analyzed the localization of VHH1 in a seed endosperm cell by an immunoelectron microscope. It is the photograph (left) and graph (right) which show the result of having evaluated the neutralization effect of MucoRice-VHH1 with respect to rotavirus. It is a photograph which shows the result of having evaluated the stability and distribution of MucoRice-VHH1 in a digestive tract by Western blotting. It is a photograph which shows the result of having evaluated the neutralization effect of MucoRice-VHH1 in the mouse | mouth infected with rotavirus. It is a graph which shows the result of having evaluated the neutralization effect of MucoRice-VHH1 in the mouse | mouth which started administration of MucoRice-VHH1 before infecting rotavirus. It is a graph which shows the result of having evaluated the neutralization effect of MucoRice-VHH1 in the mouse | mouth which started administration of MucoRice-VHH1 after infecting rotavirus. It is a photograph showing the results of microscopic observation of hematoxylin / eosin staining of the small intestine tissue of young mice. (I) shows the small intestine tissue of an uninfected young mouse, (II) shows the small intestine tissue of a young mouse infected with rotavirus, and (III) shows the infection with rotavirus and administered MucoRice-VHH1 (IV) indicates the small intestine tissue of a young mouse infected with rotavirus and administered with WT-Rice (Nihonbare). 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” indicates the VP7 RNA copy number in the small intestine tissue of an uninfected young mouse, “RRV infection alone” indicates the VP7 RNA copy number in the small intestine tissue of a young mouse infected with rotavirus, and “WTrice administration” "Indicates VP7 RNA copy number in the small intestine tissue of young mice infected with rotavirus and administered WT-Rice (Japan sunny)" "MucoRice VHH1 administration" indicates VP7 in the small intestine tissue of young mice administered MucoRice-VHH1. RNA copy number is indicated. It is a graph which shows the result of having evaluated the improvement effect by MucoRice-VHH1 administration with respect to the diarrhea situation (diarrhea score) of the SCID mouse which infected rotavirus. It is a graph which shows the result of having evaluated the influence by mucoRice-VHH1 administration with respect to the amount of rotavirus VP7 RNA in the SCID mouse which infected rotavirus. It is a photograph which shows the result of having analyzed the expression of mTNF (alpha) VHH in PBS extract of MucoRice-mTNF (alpha) VHH and a precipitate by SDS-PAGE and Western blot. The leftmost photo shows the result of analyzing MucoRice-mTNFαVHH monomer by SDS-PAGE, and the second photo from the left shows the result of analyzing MucoRice-mTNFαVHH monomer by Western blot using anti-mTNFαVHH polyclonal antibody. The third photograph from the left shows the result of analyzing MucoRice-mTNFαVHH dimer by SDS-PAGE, and the rightmost photograph shows the result of analyzing MucoRice-mTNFαVHH dimer by Western blot using anti-mTNFαVHH polyclonal antibody. Show. In addition, “Lane 1” indicates the analysis result of rmTNFαVHH (recombinant mTNFαVHH derived from E. coli, positive control), “Lane 2” indicates the analysis result of the PBS extract of MucoRice-mTNFαVHH monomer, and “Lane 3” indicates MucoRiceVmHNFα. 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-mTNFαVHH dimer, and “Lane 5” is the precipitate after the extraction of MucoRice-mTNFαVHH dimer with PBS. The analysis result of a thing is shown. Further, in the figure, the band surrounded by a square indicates mTNFαVHH monomer (13.8 kDa) or mTNFαVHH dimer (28.4 kDa). It is a photograph which shows the result of having analyzed the expression of mTNF (alpha) VHH in PBS extract of MucoRice-mTNF (alpha) VHH by SDS-PAGE and Western blot. The photograph on the left shows the result of analyzing MucoRice-mTNFαVHH etc. by SDS-PAGE, and the photograph on the right shows the result of analyzing MucoRice-mTNFαVHH etc. in Western blot. In the figure, “rmTNFαVHH” indicates the result of analysis of rmTNFαVHH, “MucoRice-mTNFαVHH” indicates the analysis result of the PBS extract of MucoRice-mTNFαVHH monomer, and “MucoRice-mTNFαVHHDmFiMFVDHFD” The analysis result of a liquid is shown, "WT-Rice" shows the analysis result of the PBS extract of WT-Rice (Nipponbare). Further, in the figure, the band indicated by an arrow indicates mTNFαVHH monomer (denoted as “VHH (13.8 kDa) in the figure) or mTNFαVHH 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). In the figure, the rightmost line (rVHH) shows the relationship between the amount of rmTNFαVHH added and the inhibition rate against mTNFα, and the middle line (monomer) shows the amount of added muTRice-mTNFαVHH monomer-derived PBS extract and mTNFα of rmTNFαVHH. The leftmost broken line “dimer” shows the relationship between the added amount of MucoRice-mTNFαVHH dimer-derived PBS extract and the inhibition rate of rmTNFαVHH to mTNFα. It is a graph which shows the relationship between the addition amount of mTNF (alpha), and the survival rate of WEHI164 cell (mouse fibroblast). 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 mTNFαVHH rice (MucoRice-mTNFαVHH monomer) localization of mTNFαVHH monomer (type I protein reservoir (PB-I) and cytoplasm) detected with anti-mTNFαVHH antibody, and B shows mTNFαVHH rice. Shows the results of detecting and observing the localization of mTNFαVHH monomer (type II protein reservoir (PB-II), cytoplasm, starch (St: Starch), cell wall (CW: Cell Wall)) using anti-mTNFαVHH antibody . C shows the result of reacting mTNFαVHH rice with normal rabbit IgG and observing PB-I, PB-II and cytoplasm (“α-Rabbit IgG”, negative control), and D shows Nihonbare rice (WT-Rice). ) Was reacted with anti-mTNFαVHH antibody, and PB-I, PB-II and cytoplasm were observed (“WT Nipponbare”, negative control). It is a photograph which shows the result of having analyzed the localization of mTNFαVHH dimer in rice seeds by immunoelectron microscopy. A shows the results of detecting and observing the localization of mTNFαVHH dimer (PB-I, PB-II, cytoplasm) in mTNFαVHH dimer rice (MucoRice-mTNFαVHH dimer) using anti-mTNFαVHH antibody, and B shows the results in mTNFαVHH dimer rice The localization of mTNFαVHH dimer (PB-I, PB-II, cytoplasm, cell wall) was detected using an anti-mTNFαVHH antibody and observed. C shows the result of observing PB-I, PB-II, St and cytoplasm by reacting mTNFαVHH dimer rice with normal rabbit IgG, and D shows the reaction of Japanese fine rice (WT-Rice) with anti-mTNFαVHH 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 DNA encoding a nanoantibody is introduced and the nanoantibody is expressed in rice.

In the present invention, “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 .

  In the present invention, the “nanoantibody” refers to 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 nanoantibodies in the present invention can use VHH as a monomer, but can also be used as multiple antibodies of dimers 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. In addition, 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.

  As the nanoantibody in the present invention, a nanoantibody against a desired antigen can be used. Examples of 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 influenzae, Streptococcus pneumoniae, Bordetella pertussis, Diphtheria, Pesto, Tetanus, Botulinum Bacteria, anthrax, wild boar, Escherichia coli O157, Salmonella, MRSA (Staphylococcus aureus), VRE (Enterococcus), Mycobacterium tuberculosis, Shigella, Salmonella typhi, Paratyphi, Chlamydia, Amebic dysentery, Legionella, Lyme disease Antigens such as proteins derived from Borrelia bacteria, Brucella disease (wavy fever), etc.), (3) antigens such as proteins derived from rickettsia (eg, Q fever rickettsia, chlamydia etc.), (4) protozoa (protozoa malaria, Antigens such as proteins derived from trypanosoma, etc.) (5) cytokines (eg, TNF-α, IL-1, IL-6, IL-8, IL-12, IL-18, IL-21, INF-γ, and their receptors), (6) chemokines (eg, CCL17 and its receptor CCR4, CXCL10 and its receptor CXCR3, CXCR4, etc.), (7) Growth factors and hormones (for example, EGF (epidermal growth factor), FGF (Fibroblast growth factor), hCG (human hormonal gonadotropin), and their receptors, etc.) (8) Tumor antigens (for example, CEAec Colon cancer tumor antigens, breast cancer antigens such as Her2, and prostate cancer tumors such as PSA (prostate specific antigen) Hara, etc.), (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).

  The codon contained in the DNA encoding the nanoantibody is preferably modified to 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.

  In vectors containing DNA encoding nanoantibodies for expressing nanoantibodies in rice, it is preferable to use promoters and terminators that function specifically in rice endosperm. In the vector, the promoter is linked upstream of the DNA encoding the nanoantibody, and the terminator is linked downstream of the DNA encoding the nanoantibody. Examples of promoters and terminators that function specifically in rice endosperm include promoters and terminators of genes that encode rice endosperm storage proteins. By using a promoter and terminator of a gene encoding rice endosperm storage protein, nanoantibodies can be efficiently expressed in rice. As used herein, “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. Examples of 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 glutelin GluB-1 gene or 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.

  In addition, in order to efficiently accumulate nanoantibodies in rice, it is preferable to perform a treatment that suppresses the expression of rice storage proteins. As such treatment, for example, 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. For example, an indirect introduction method using Agrobacterium tumefaciens, Agrobacterium rhizogenes (Hiei, Y. et al., Plant J. et al.). Takayama, F. et al., Plant Sci. 111, 39-49, 1995; Japanese Patent No. 3141084), electroporation method (Tada, Y. et al. Theor. Appl. Genet, 80, 475, 1990), polyethylene glycol method (Datta, SK et al., Plant MolBiol., 20, 619-629, 1992), particle gun method (Christou, P. et al., Plant) J.2,275 281,1992, Fromm, M.E., Bio / Technology, 8,833-839,1990) direct introduction method may be mentioned of such. The method for regenerating rice plants by culturing rice cells is not particularly limited, and any known method may be followed.

  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. . When 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. When used as an oral preparation for pharmaceuticals without being purified from the expressed rice, hygromycin for which safety is not ensured must be removed by a known method such as a cotransfection method.

  In the rice of transgenic rice produced in this way, the nanoantibodies are in a water-soluble form and without losing their original properties (for example, antigen binding, heat resistance and stability of the nanoantibodies). Accumulated. Accordingly, 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. Here, “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).

  Moreover, this invention also provides the processed material of the said rice and water-soluble fraction. Here, 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. There is no restriction | limiting in particular in the form of a composition, A pharmaceutical composition, food-drinks, animal feed, etc. are contained. The pharmaceutical composition can be used, 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. Examples of 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.

  The composition in the present invention can also be formulated by a known pharmaceutical method. In these formulations, 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. When administering or ingesting the composition of the present invention, 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. For example, the dose or intake per dose is generally 0.001 mg / kg body weight to 100 mg / kg body weight.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

Example 1
Production of VHH1-expressing rice (MucoRice-VHH1) Nano-antibodies 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.

  As for the design of the transgene, 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. Furthermore, 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. A VHH1 gene expression T-DNA binary vector ligated with all of these 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% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8). As an extract and a positive control, recombinant VHH1 derived from E. coli (hereinafter abbreviated as rVHH1) was separated by electrophoresis using a 12% polyacrylamide gel. The separated gel was stained with CBB (Coomassie Brilliant Blue). As a result, it was confirmed that a protein of the same size as rVHH1 was expressed in MucoRice-VHH1, whereas a protein of the same size as rVHH1 was not found in the seeds of the wild strain (a in FIG. 1). ). Further, 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. As a result, an antibody-specific band was observed at the same position as the recombinant protein (a in FIG. 1). Furthermore, 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 μm 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 development was performed with 3,3′-diaminobenzidine (DAB) and counterstaining with hematoxylin was performed. As a result, a wild-type seed did not show an anti-VHH1 antibody-specific staining image, but in MucoRice-VHH1, an anti-VHH1 antibody-specific staining image was observed throughout the seed (b in FIG. 1).

Example 3
Structural analysis of VHH1 derived from MucoRice-VHH1 In order to clarify the structure of VHH1 expressed in rice, structural analysis was performed. MucoRice-VHH1 rice powder was suspended in sample buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8), and 12% 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). The analysis result of 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. As a result, as shown in FIG. 2, the complete amino acid sequence could be decoded by combining the results of the two types of mass spectrometers. In addition to the complete sequence of VHH1, the results showed that SR (serine + arginine), which is a rice-specific sequence, is present at the N-terminus, but asparagine, serine, etc. by using the rice expression system Modifications such as glycosylation were not observed.

Example 4
Examination of VHH1 extraction conditions from MucoRice-VHH1 VHH1 extraction conditions from MucoRice-VHH1 were examined. To 100 mg of MucoRice-VHH1 rice powder, 1 mL of phosphate buffer (hereinafter abbreviated as PBS) or 1 mL of PBS containing 8M urea is added, and after suspension, immediately centrifuged, and rVHH1 is added to sample buffer (2% as a positive control). It was suspended in SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8) and separated by electrophoresis using a 12% polyacrylamide gel. The separated gel was subjected to CBB staining. As a result, as shown in FIG. 3, it became clear 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.

(Comparative Example 1)
MucoRice expressing single chain antibody and solubilization Monoclonal antibody NKM-16-2-4 with specificity for mouse M cells (T. Nochi et. Al. J. Exp. Med. 204. 2789-2796 (2007)) ) Was cloned, and the gene encoding the single-chain antibody (scFv) was reconstructed and artificially synthesized based on codons that can be easily used by rice. The scFv gene modified for rice expression is inserted under the endosperm cell-specific promoter of the T-DNA binary vector used in Example 1, and finally rice (Nipponbare) is transformed via Agrobacterium. Then, scFv-expressing rice was produced by a conventional method. This rice powder expressing scFv was not solubilized at all in PBS under the conditions of Example 4 as shown in FIG. 4, but was solubilized only with urea SDS.

(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, a section of about 0.5 to 1.0 μm was prepared, fixed with 4% PFA, and immersed in LR-white resin. Thereafter, an ultrathin section of 100 μm 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. Thereafter, it was washed, fixed with 1% glutaraldehyde, electron double-stained with 2% uranyl acetate and a lead solution, and observed with an electron microscope. As a result, VHH1 was remarkably observed in PB-II and slightly 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 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) were seeded in a 96-well plate, and after about 2 days, the cells were sufficiently grown in the wells and washed with serum-free MEM medium. A rhesus monkey rotavirus (hereinafter abbreviated as RRV) solution (using serum-free MEM medium) that had been trypsinized (37 ° C., 30 minutes) was prepared. In addition, 1 mL of serum-free MEM was suspended in 100 mg of MucoRice-VHH1 powder and wild rice powder, and the supernatant obtained after sterilization through a 0.22 um filter was serially diluted. 200 ffu (focus forming units) trypsinized RRV was added to various concentrations of rice extract, and the mixture was added onto MA104 cells in a 96-well plate. Only serum-free MEM medium was added to each well as a negative control, and only RRV was added to each well as a positive control. After reacting in a CO ₂ incubator at 37 ° C. for about 1 hour, washing was repeated with serum-free MEM medium, MEM medium containing 10% FCS was added, and the cells were cultured in a CO ₂ incubator (37 ° C.) for 12 hours. Thereafter, it was washed with a serum-free MEM medium, further washed with PBS, and fixed with 4% PFA. After washing, a mouse monoclonal antibody against VP6 (a kind of RRV constituent protein) known to grow in infected cells was reacted. After washing, HRP-labeled goat anti-mouse IgG polyclonal antibody was reacted, and color was developed after washing. Since the infected cells were colored reddish brown, the number of infected cells was measured under a microscope, and the number of infected cells in a well reacted only with RRV was defined as 100%. As a result, as shown in FIG. 6, MucoRice-VHH1-derived VHH1 significantly reduced the infection rate compared with RRV alone or a mixture of RRV and wild rice extract, and proved a neutralizing effect on RRV. (VHH1 concentration is 1 ug / mL). In addition, it was revealed that 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 property of MucoRice-VHH1 was proved.

(Example 7)
Evaluation of stability and distribution of MucoRice-VHH1 in the gastrointestinal tract After centrifuging a suspension of MucoRice-VHH1 powder and pure water, the supernatant was recovered, and 40 μl of a VHH1 solution with a concentration of 100 μg / mL was applied on day 4 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. Thereafter, the same solution is suspended in a sample buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8) and separated by electrophoresis using a 12% polyacrylamide gel. Western blot analysis was performed using an anti-VHH1 polyclonal antibody. As a result, as shown in FIG. 7, 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)
When RRV 2 × 10 ⁷ ffu (contained in MEM 20 ul) treated with trypsin (37 ° C., 30 minutes) was intragastrically administered to Balb / c mice on day 4 of age, watery diarrhea was observed after about 24 hours. Is observed (FIG. 8) and spontaneously improves after 3-4 days. Using this infection model, MucoRice-VHH1 was tested for its neutralizing effect and diarrhea suppressing effect on RRV. The supernatant obtained after suspending MucoRice-VHH1 and PBS (VHH1 concentration 200 ug / mL) was administered 20 μL (4 μg of VHH1) 6 hours before or 6 hours after RRV administration, and intragastric administration every 12 hours thereafter Was repeated until day 4 of administration (the day of RRV administration was defined as day 0). The incidence of diarrhea was measured up to the 4th day of administration. As a result, the incidence of diarrhea was significantly low in both groups administered with rice-derived VHH1 before and after RRV administration (FIGS. 9 and 10). From this, it was found that MucoRice-VHH1 has a neutralizing effect and diarrhea suppressing effect on RRV, and the effect is observed not only after prophylactic administration but also after RRV exposure.

Example 9
Evaluation of neutralization effect of MucoRice-VHH1 against rotavirus (infection experiment 2 using juvenile animals)
In the same manner as in Example 8, 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.

  That is, a small intestine tissue section was stained with Hematoxylin / Eosin by a conventional method and observed. The obtained results are shown in FIG.

  The amount of rotavirus was obtained by extracting total RNA from small intestinal 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.

As is apparent from the results shown in FIG. 11, 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). Meanwhile, the small intestine tissue of young mice infected with rotavirus (see II in FIG. 11) and the small intestine tissue of young mice infected with rotavirus and administered with WT-Rice (Nipponbare) (see IV in FIG. 11). In these mice, numerous vacuoles were observed in the epithelial cells of these mice.

  Further, as is clear from the results shown in FIG. 12, the mouse small intestine and rotavirus infected only with virus infection were infected with rotavirus compared to the mouse small intestine administered with WT-Rice (Nipponbare), and MucoRice. -The copy number of VP7 RNA of rotavirus (RRA) in the small intestine of young mice administered with VHH1 was significantly reduced.

(Example 10)
Evaluation of neutralizing effect of MucoRice-VHH1 against rotavirus (infection experiment using immunodeficient animals)
The effect of anti-RRV-VHH1 of MucoRiceVHH1 was evaluated using SCID mice which are immunodeficient mice. That is, after infecting SCID mice with rotavirus (2 × 10 ⁷ ffu RRV), 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. .

  As is apparent from the results shown in FIGS. 13 and 14, the diarrhea situation was significantly improved in the immunodeficient mice by the administration of MucoRice-VHH1, and at the same time the viral load was significantly reduced.

  Therefore, it was shown that 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 administer the vaccine.

(Example 11)
Production of TNF-αVHH-expressing rice (MucoRice-mTNFαVHH) Known information was used for gene information of nanoantibodies (VHH) against TNF-α (Arthritis Rheum. 54, 1856-1866 (2006), J. Biotechnol. 142: 170-178 (2009)). A gene encoding mouse TNFαVHH monomer (mTNFαVHH monomer) was reconstructed and artificially synthesized based on codons that are readily available to rice. A gene encoding TNFαVHH dimer (mTNFαVHH 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]).

  Then, a gene encoding mTNFαVHH monomer or dimer modified for rice expression is inserted under the endosperm cell-specific promoter of the T-DNA binary vector (pZH2B / 35SNos) used in Example 1, and finally Agrobacterium Rice (Nipponbare) was transformed through um, and TNF-αVHH-expressing rice (MucoRice-mTNFαVHH: MucoRice-mTNFαVHH monomer and MucoRice-mTNFαVHH dimer) was produced by a conventional method.

(Example 12)
Extraction of mTNFαVHH from MucoRice-mTNFαVHH For extraction of each of mTNFαVHH monomer and mTNFαVHH dimer from MucoRice-mTNFαVHH dimer and SDS-PAGE using tter.

  That is, first, 1 mL of PBS was suspended in 100 mg of MucoRice-mTNFαVHH rice powder, and then the mixture was stirred for 1 hour at 4 ° C. and centrifuged. The supernatant and positive control rmTNFαVHH were then suspended in sample buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6.8). The precipitate was suspended in a sample buffer containing urea (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, 8M Urea, pH 6.8). Each sample thus obtained was separated by SDS-PAGE electrophoresis using a 12% polyacrylamide gel, and the gel after separation was subjected to CBB staining. Further, the gel after electrophoresis was transferred to a PVDF membrane, and Western blotting was performed using an anti-mTNFαVHH polyclonal antibody obtained by immunizing mice with rmTNFαVHH. As a result, for both VHH monomer and VHH dimer, antibody-specific bands were observed in the supernatant sample, indicating that mTNFαVHH expressed in rice can be solubilized in PBS not only in the monomer but also in the dimer (FIG. 15). ).

(Example 13)
Quantification of the amount of mTNFαVHH protein in MucoRice-mTNFαVHH In order to calculate the amount of mTNFαVHH protein expressed in MucoRice-mTNFαVHH, the amount of mTNFαVHH protein expressed in MucoRice-mTNFαVHH was examined using SDS-PAGE. That is, MucoRice-mTNFαVHH rice powder was solubilized with PBS, and the resulting supernatant and rmTNFαVHH standard product were sample buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-HCl, 20% glycerol, pH 6. It was suspended in 8) and separated by SDS-PAGE electrophoresis using a 12% polyacrylamide gel. Next, the separated gel was subjected to CBB staining. Further, the gel after CBB staining was quantified with a densitometer (manufactured by BIO-RAD). As a result, mTNFαVHH contained in MucoRice-mTNFαVHH reached an average of 0.82% per rice powder weight in VHH monomer and an average of 0.47% in VHH dimer, and VHH was highly expressed not only in monomer but also in dimer. It was found that the accumulation was high (FIG. 16).

(Example 14)
Evaluation of inhibitory effect of MucoRice-mTNFαVHH on mTNFα (cell death suppression test)
WEHI164 cells (mouse fibroblasts) were seeded in 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. After the culture, the medium is removed, and the medium alone (negative control), the medium added with only 2 ng / mL rmTNFα (manufactured by R & D systems) (positive control), or 2 ng / mL rmTNFα and the MucoRice-mTNFαVHH-derived PBS extract are mixed. 100 μL of the prepared medium was added and cultured for 24 hours. Two hours before the end of the culture, 10 μL of WST-8 (manufactured by Dojindo Laboratories) was added and cultured, and the absorbance was measured at 450 nm to evaluate the number of living cells. Moreover, using the obtained measured value, the inhibition rate of mTNFαVHH with respect to mTNFα was evaluated with the inhibition rate of the positive control being 0% and the inhibition rate of the negative control being 100%. The obtained result is shown in FIG.

  The amount of rmTNFα 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 rmTNFα, subjected to WST-8 assay, and the viability was 50 from the number of live cells obtained. % Of rmTNFα was calculated (see FIG. 18).

  As is clear from the results shown in FIG. 17, mTNFαVHH derived from MucoRice-mTNFαVHH markedly inhibited mTNFα. In addition, it was revealed that the inhibitory effect of VHH dimer is 50 to 100 times higher than that of VHH monomer.

(Example 15)
Localization of mTNFαVHH in MucoRice-mTNFαVHH (observation by immunoelectron microscope)
The localization of mTNFαVHH monomer and dimer in seeds was analyzed by immunoelectron microscopy. That is, seeds on the 14th day after flowering of the second-generation MucoRice-mTNFαVHH monomer and dimer were collected, and sections of about 0.5 to 1.0 μm were prepared. Subsequently, these sections were immersed and fixed in 4% PFA while shaking at 4 ° C. for 4 hours. After dehydration in the ethanol (EtOH) series, the LR-White resin was gradually infiltrated over 2 days at 4 ° C., and finally immersed in 100% LR-White resin, and overnight at 50 ° C. Polymerized. From the polymer block after polymerization, an ultrathin section of 80 to 100 nm was prepared by an ultramicrotome, and collected on a nickel grid (mounting net) on which a form bar support film was pasted 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-mTNFαVHH antibody (rabbit) purified by affinity using a protein G column at a concentration of 5 μg / 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.

  As a result, in mTNFα-VHH rice, both mTNFαVHH monomer and dimer were remarkably localized in the cytoplasm, and these localizations were also observed inside PB-II (Proteinbody-II), which is a rice protein reservoir. (A and B in FIG. 19 and A and B in FIG. 20). On the other hand, in mTNFα-VHH rice (FIG. 19C, FIG. 20C) reacted with control rabbit IgG and WT Nippon Haremai (FIG. 19D, FIG. 20D) reacted with mTNFαVHH antibody, the cytoplasm MTNFαVHH was not observed in any of PB-II and PB-II.

  Therefore, the water-soluble nature of mTNFαVHH 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.

  Development of oral antibody preparations using nanoantibodies has been conventionally performed, for example, nanoantibodies (VHH1) having binding affinity for rotavirus VP4 and VP7 (Dolk E, et al., Proteins. 2005; 59: 555-64.) And TNF-α 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. By using 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 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). In consideration of outbreak countermeasures and manufacturing costs, 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. Moreover, 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.

SEQ ID NO: 1
<223> MucoRice-VHH1 (analysis by Q-STAR Elite)
SEQ ID NO: 2
<223> MucoRice-VHH1 (analysis by Orbitrap)

Claims (6)

Transgenic rice in which DNA encoding a nanoantibody is introduced, the nanoantibody is expressed specifically in rice endosperm cells, and is accumulated in the cytoplasm of the cell in a water-soluble form . Rice harvested from the transgenic rice according to claim 1 . A water-soluble fraction of the rice according to claim 2 containing nanoantibodies. A processed product containing nanoantibodies of the rice according to claim 2 or the water-soluble fraction according to claim 3 . A composition comprising the rice according to claim 2 , the water-soluble fraction according to claim 3 , or the processed product according to claim 4 . A method for producing a nanoantibody comprising:
(A) introducing a DNA encoding a nanoantibody into rice, and producing a transgenic rice in which the nanoantibody is specifically expressed in rice endosperm cells and accumulated in the cytoplasm of the cell in a water-soluble form ;
(B) obtaining a water-soluble fraction from the rice of the transgenic rice produced in step (a),
Including methods.