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WO2009035205A1 - Plante transformée exprimant l'enzyme bace (enzyme de clivage du site β de l'app) - Google Patents

Plante transformée exprimant l'enzyme bace (enzyme de clivage du site β de l'app) Download PDF

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Publication number
WO2009035205A1
WO2009035205A1 PCT/KR2008/003257 KR2008003257W WO2009035205A1 WO 2009035205 A1 WO2009035205 A1 WO 2009035205A1 KR 2008003257 W KR2008003257 W KR 2008003257W WO 2009035205 A1 WO2009035205 A1 WO 2009035205A1
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Prior art keywords
plant
bace
gene
potato
transformed
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PCT/KR2008/003257
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English (en)
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Hyun Soon Kim
Jae Heung Jeon
Jung Won Youm
Hyouk Joung
Young Ho Kim
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Korea Research Institute Of Bioscience And Biotechnology
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Publication of WO2009035205A1 publication Critical patent/WO2009035205A1/fr

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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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)

Definitions

  • the present invention relates to a transformed plant which expresses BACE
  • Oral plant vaccine which has an advantage of a marriage between stability of a recombinant vaccine and easiness and efficiency for injection of an oral vaccine, is prepared by a plant that is transformed with an antigen gene by using a plant expression vector, and by carrying out an oral administration of the transformed plant an immunological reaction can be induced.
  • Alzheimer type dementia is presumed to be 60% of the whole dementia cases.
  • Senile dementia that is typified by Alzheimer's disease is a degenerative neuronal disease, which starts as a cognitive disorder and progresses into a long and devastating degenerative disorder which eventually destroys basic human spirit.
  • a passive way of coping such as keeping a patient in an isolate facility is not enough to deal with the social and economic burden associated with the disorder. For such reason, more aggressive approach is required such as the development of an agent which can be used for the prevention or the treatment of the disorder.
  • ⁇ -Amyloid is a metabolite produced by the action of a proteolyitc enzyme which is originated from typelinternal membrane protein called APP (amyloid precursor protein), and it is a peptide consisting of from 39 to 43 amino acids with an extracellular domain and a membrane domain.
  • APP amyloid precursor protein
  • potato is one of the four major food crops in the world and is cultivated all over the world except very cold or hot regions.
  • a potato vaccine is developed as desired by the inventors of the present invention, it can be easily spread all over the world wherein potato is routinely cultivated.
  • a tremendous need for the potato vaccine will naturally follow.
  • a technology for mass production of potato microtuber which is one of the world's top level technologies with industrially practical use, has been already acquired by the inventors of the present invention, a highly valuable product can be produced in mass amount with combined benefits of a dietary oral vaccine and a potato microtuber.
  • a potato and a tomato plant which can expresss BACE gene is developed by incorporating BACE gene in Agrobacterium using an expression vector plasmid which is used for transformation and comprises BACE gene, and co-culturing the transformed Agrobacterium with potato leaves and cotyledonous tissues of tomato.
  • a transformed potato and tomato plant, which can express BACE gene in massive amounts were obtained.
  • the expression amount of BACE gene in said transformed plants was increased and a condition for stabilizing the protein was optimized, and as a result the present invention was completed.
  • the present invention provides a transformed plant which expresses ⁇ -secretase (BACE) gene.
  • the present invention provides a method for the production of a transformed plant, comprising a step of transforming the plant with a recombinant plant expression vector which includes ⁇ -secretase (BACE) gene.
  • BACE ⁇ -secretase
  • the present invention provides an oral vaccine composition for preventing dementia which comprises said transformed plant as an effective component.
  • the transformed potato and tomato plant of the present invention which can express ⁇ -secretase (BACE) gene to produce ⁇ -amyloid responsible for Alzheimer type dementia in human, can be developed as a type of food-based vaccine which currently is a hot issue in the world.
  • BACE ⁇ -secretase
  • Figure 1 shows the BACE gene that is incorporated in the cloning vector.
  • Figure 2 shows a vector for transformation that is obtained by introducing BACE gene to the vectors for plant transformation including pGBSSP2, pAT and pE8.
  • Figure 3 A shows the transformed potato plant grown from a callus which has been cultured in a culture medium wherein the callus had been induced by inoculation of Agrobacterium and the microtuber formation from the potato plant
  • Figure 3B shows the callus formation in pieces of tomato cotyledonous leaf and culture process of the transformed tomato plant.
  • Figure 4 shows the PCR result of the regenerated plant of the transformed tomato and the regenerated plant of the transformed potato of the present invention.
  • Figure 5 shows a Northern blot analysis of mRNA of BACE gene that are isolated from the transformed plants selected by PCR determination.
  • Figure 6 shows a Western blot analysis of BACE protein expression by using ABlO antibody.
  • Figure 7 shows a nucleotide sequence of BACE gene (SEQ ID NQ 6) of which nucleotide sequence has been modified to increase the expression level of BACE gene and to improve the stabilization of BACE protein in the transformed plants of the present invention.
  • the present invention provides a transformed plant which expresses ⁇ -secretase (BACE) gene.
  • the ⁇ - secretase (BACE) gene according to the present invention is originated from a human and preferably consists of a nucleotide sequence of SEQ ID NQ 5. Further, variants of the said sequence are within the scope of the present invention. The variants have a different nucleotide sequence but have similar functional characteristics to those of the nucleotide sequence of SEQ ID NQ 5.
  • BACE gene may comprise a nucleotide sequence with at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NQ 5.
  • Plant transformation means any method by which DNA is delivered to a plant. Such transformation method does not necessarily need a period for regeneration and/or tissue culture. Transformation of plant species is now quite general not only for dicot plants but also for monocot plants. In principle, any transformation method can be used for introducing a hybrid DNA of the present invention to appropriate progenitor cells.
  • the method can be appropriately selected from a calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant MoI. Biol. 8, 363-373), an electroporation method for protoplasts (Shillito R.D. et al., 1985 Bio/Technol.
  • a method preferred in the present invention includes Agrobacterium mediated DNA transfer.
  • so- called binary vector technique as disclosed in EP A 120 516 and USP No. 4,940,838 can be preferably adopted for the present invention.
  • the "plant cell” that can be used for the plant transformation in the present invention can be any type of plant cell. It includes a cultured cell, a cultured tissue, a cultured organ or a whole plant, preferably a cultured cell, a cultured tissue or a cultured organ, and more preferably any type of a cultured cell.
  • plant tissue can be either differentiated or undifferentiated plant tissue, including root, stem, leaf, pollen, seed, cancerous tissue and cells having various shape that are used for culture, i.e., single cell, protoplast, bud and callus tissue, but not limited thereto.
  • Plant tissue can be in planta or in a state of organ culture, tissue culture or cell culture.
  • the plant which can be used in the present invention for the transformation to express the gene of ⁇ -secretase ( BACE), which is the enzyme causing Alzheimer type dementia in human can be food crops including rice, wheat, barley, corn, soy bean, potato, red bean, oat and millet; vegetable crops including Arabidopsis thaUana, Chinese cabbage, radish, hot pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, scallion, onion and carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, wild sesame, peanut and rapseed; fruits including apple, pear, date, peach, kiwi, grape, tangerine, orange, persimmon, plum, apricot and banana; flowers including rose, gladiolus, gerbera, carnation, chrysanthemum, lily,
  • Agrobacterium that can be used for the present invention.
  • it is preferably Agrobacterium tumefaciens LB4404.
  • the expression vector which can be used for introducing ⁇ -secretase gene into said Agrobacterium it is preferable to use an expression plasmid consisting of a kanamycin-resistant gene and CaMV 35 promoter, patatin promoter which is specific to potato tuber, or GBSS promoter, but is not limited thereto.
  • an expression plasmid for plant transformation comprising BACE gene was introduced into Agrobacterium, potato leaf pieces cultured in vitro and cotyledon pieces of tomato seedlings which were then approximately a week old after the germination were co-cultured with said Agrobacterium, yielding a transformed plant.
  • leaf pieces were scored with a knife and an organ differentiation was induced from the callus that is formed in the scored region. By doing so, the plant was regenerated.
  • the transformed and regenerated individual plant was propagated according to a culture method that is appropriate for each type of plant.
  • the expression vector for plant transformation which comprises BACE gene and the Agrobacterium cells wherein said expression vector is incorporated are both within the scope of the present invention.
  • These expression vector and the Agrobacterium cells can be used for various crop plants other than potato and tomato that are tested in the examples of the present invention.
  • a plant cell which can be used for the expression of BACE gene is a food crop and more preferably is a potato, a tomato or a lettuce, etc. that can be easily cultivated by the people of Korea.
  • the present invention provides a method for the production of a transformed plant comprising a step of transforming the plant with a recombinant plant expression vector including ⁇ -secretase gene (BACE).
  • BACE ⁇ -secretase gene
  • the plant is the one belonging to Solanaceae family, and more preferably a potato or a tomato.
  • a preferred example of plant expression vector is Ti-plasmid vector which can transfer a part of itself, i.e., so called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens.
  • Other types of Ti- plasmid vector are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a genome of a plant.
  • Especially preferred form of Ti-plasmid vector is a so called binary vector which has been disclosed in EP 0 120 516 Bl and USP No. 4,940,838.
  • vector that can be used for introducing the DNA of the present invention to a host plant can be selected from a double- stranded plant virus (e.g., CaMV), a single- stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector.
  • Use of said vector can be advantageous especially when a plant host cannot be appropriately transformed.
  • Expression vector would comprise at least one selective marker.
  • Said selective marker is a nucleotide sequence having a property based on that it can be selected by a common chemical method. Every gene which can be used for the differentiation of transformed cells from non-transformed cell can be a selective marker.
  • Example includes, a gene resistant to herbicide such as glyphosate and phosphintricin, and a gene resistant to antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.
  • herbicide such as glyphosate and phosphintricin
  • antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.
  • a promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but not limited thereto.
  • the term “promoter” means a DNA molecule to which RNA polymerase binds in order to initiate its transcription, it corresponds to a DNA region upstream of a structural gene.
  • plant promoter indicates a promoter which can initiate transcription in a plant cell.
  • constitutive promoter indicates a promoter which is active in most of environmental conditions and development states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing a constitutive promoter is not limited herein.
  • any conventional terminator can be used for the present invention.
  • Example includes, nopaline synthase (NOS), rice ⁇ -amylase RAmyl A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto.
  • NOS nopaline synthase
  • rice ⁇ -amylase RAmyl A terminator rice ⁇ -amylase RAmyl A terminator
  • phaseoline terminator a terminator for optopine gene of Agrobacterium tumefaciens, etc.
  • the present invention provides an oral vaccine composition for preventing dementia which comprises the above-described transformed plant as an effective component.
  • the plant is a potato or a tomato, but not limited thereto.
  • the above-described composition can be used as a transformed plant itself or as a powder after drying. In addition, it can be used with other food or food ingredients, and can be used appropriately by following a common method.
  • the content of the transformed plant as an effective component is suitably determined depending on the purpose (prevention or therapeutic treatment, or health improvement, etc.). Since the transformed plant as an effective component usually has no safety problem, it can be used without any maximum limit.
  • Types of health food product which comprises the above-described composition are not specifically limited.
  • Example of the food product to which the transformed plant of the present invention can be incorporated includes, meat, sausage, bread, chocolate, candy, snack, cookie, pizza, ramen and other types of noodles, gum, dairy products including ice cream, various kinds of soup, drink, tea, beverage, alcoholic beverage and vitamin supplement, etc.
  • dairy products including ice cream
  • various kinds of soup, drink, tea, beverage, alcoholic beverage and vitamin supplement etc.
  • it would be evident to a skilled person in the art that they are not limited to said examples.
  • Solanaceae family plant such as potato and tomato was used as a subject plant and the potato plant tested in the present invention was maintained by tissue culture.
  • Desiree seed potato which is free of any pathogens and viruses, was germinated and then cut into tiny pieces including growth point, followed by surface sterilization with 70% ethanol. After the rinse with distilled water, the potato pieces were sterilized again in a solution of sodium hypochloride for 10 min, rinsed with distilled water three times and placed in a basic medium wherein sucrose (3%) was added with MS salt to induce stem growth. Once the stems have grown well in an incubator, tissue culture of the potato plant was maintained by sub- culturing every two weeks.
  • MS basic medium comprising 90g/L sucrose.
  • Culture condition includes light period for eight hours and culture temperature of 17 0 C.
  • lower part of the stems of the potato plant which had been grown from two to three weeks under said culture condition, was selected for the culture. Approximately two weeks after the culture, the storage stems became to swell and approximately eight weeks later, a mature microtuber having a weight of 1 g or more could be obtained.
  • Agrobacterium for transformation The medium used for forming callus was MS medium to which 3% sucrose, 8% agar and 2.0 mg/L 2,4-D (2,4-dichlorophenoxy acetic acid) were added. The medium was adjusted to have pH 5.8.
  • the transformed leaf pieces were cultured for two days in medium for inducing callus formation which comprises 2,4-D, and then transferred to a medium for regeneration which comprises 0.01 mg/L NAA (naphthaleneacetic acid), 0.1 mg/L gibberellin (GA 3 )and 2.0 mg/L zeatin.
  • NAA naphthaleneacetic acid
  • GA 3 gibberellin
  • Tomato seeds Dotaerang, a tomato variety for general cultivation
  • Tomato seeds were first treated with 70% ethanol for surface sterilization followed by rinsing with distilled water. Then, the seeds were sterilized again in a 10% solution of sodium hypochloride for 10 min, rinsed with distilled water three times and placed in a basic medium wherein sucrose (3%) was added with MS salt to induce germination. About one week after the germination, newly-formed cotyledonous leaves were cut and used for co- culture with Agrobacterium for transformation.
  • the medium used for forming callus was MS medium to which 3% sucrose, 8% agar and 1.0 mg/L of zeatin were added. The medium was adjusted to have pH 5.8.
  • the transformed cotyledonous leaf pieces were cultured for two days in medium for inducing callus formation, and then transferred to a medium for regeneration comprising 2.0 mg/L zeatin.
  • a medium for regeneration comprising 2.0 mg/L zeatin.
  • calluses started to form at the cut area of the leaf pieces, and four weeks later small plants started to appear (see, Figure 3).
  • BACE gene SEQ ID NQ 5, as confirmed in Figure 5
  • potato leaves that have been cultured less than seven days as described in Example 1 and approximately one hundred tomato cotyledonous leaves that had been obtained about one week after the germination were selected and dipped in a culture solution comprising Agrobacterium tumefaciens LBA 4404 for ten minutes. Moisture was completely removed by drying with a sterilized paper.
  • the potato leaf pieces were then placed in a co-culture medium which comprises 2.0 mg/L 2,4-D while the tomato cotyledonous leaf pieces were placed in a co-culture medium which comprises 1.0 mg/L zeatin.
  • the leaf pieces were culture for two days, respectively.
  • GUS gene was removed with restriction enzymes of Kpnland Sac I followed by insertion of BACE gene (SEQ ID NQ 5) to yield the vectors of pATBACE and pGBSSP2BACE, respectively.
  • BamHI and Kpnl were used as a restriction enzyme.
  • These vectors carry kanamycin-resistant nptll gene that was employed as a selection marker.
  • plasmid DNAs comprising each of said vectors were introduced to Agrobacterium tumefaciens LBA 4404 and used for plant transformation of the present invention.
  • a PCR polymerase chain reaction
  • a selection medium comprising kanamycin
  • small plants that could grow in the medium were primarily selected.
  • PCR was carried out to identify the presence of BACE gene in the genomic DNA of the selected plants. Specifically, genomic DNA was first isolated from the leaf pieces of the small plant based on a medium comprising kanamycin, and PCR was carried out by using a primer of BACE gene and NPTII.
  • RNA expression in the transformed potato and tomato plants of the present invention by first confirming the RNA expression in the transformed potato and tomato plants of the present invention, a more precise selection of transformed plant can be achieved.
  • the vectors used in the present invention are specific to either potato tuber or ripening of tomato fruit, transcripts were identified from the regions (e.g., plant organs) in which said specific features are expressed.
  • regions e.g., plant organs
  • transcripts were identified from the regions (e.g., plant organs) in which said specific features are expressed.
  • For potato total RNA was extracted from 1 g of newly formed microtuber.
  • Ig of fully-ripen red pulp was used for RNA analysis. After quantification, 30 ⁇ g of purified RNA was applied to 1% agarose gel comprising 19.8% formaldehyde.
  • RNAs contained in the gel were transferred to a nylon membrane, without any special pre-treatment.
  • UV light was illuminated twice to the membrane (1200x ⁇ J/cnf).
  • ZJACE-specific RNA band was visualized by using a diagnostic kit which can detect DIG. As a result of testing many plants, one individual plant showing the highest expression level was selected for each individual vectors.
  • plant Number 9 for the transformed potato comprising pATBAC ⁇ vector ( Figure 5A) and plant Number 40 for the transformed potato comprising pGBSSBAC ⁇ vector ( Figure 5B) were selected.
  • plant Number 3 for the transformed tomato comprising p ⁇ 8BAC ⁇ vector was also finally selected ( Figure 5C).
  • BACE protein that had been expressed in the transformed potato and tomato plants of the present invention was analyzed by Western blot.
  • Western analysis was carried out for pGBSSBACE plant which had been previously selected.
  • a specific band (-5OkDa) that had not been observed for non-transformed control potato plant was observed ( Figure 6A). From the tuber of an individual transformed potato plant and well-ripen fruits of an individual transformed tomato plant, proteins were extracted, respectively.
  • Extraction buffer used was a mixture of PBS buffer (pH 7.2), 10 mM EDTA, 1 mM proteinase inhibitor cocktail, 0.1% Triton X-100, and 5 mM ⁇ -mercaptoethanol, which was added in a volume half the weight of the sample. All of the extraction processes were carried out quickly at about 4 0 C. For all of the individual transformed plants tested, a specific band ( ⁇ 50kDa, corresponding to ⁇ -secretase potato tuber protein), that had not been observed for non-transformed control potato plant, was observed.

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Abstract

La présente invention concerne une plante transformée qui exprime le gène BACE. Plus précisément, la présente invention concerne une plante transformée qui exprime le gène de la β-sécrétase (BACE), un procédé de production de ladite plante et une composition de vaccin oral permettant de prévenir la démence qui contient ladite plante transformée en tant que composant efficace.
PCT/KR2008/003257 2007-09-13 2008-06-11 Plante transformée exprimant l'enzyme bace (enzyme de clivage du site β de l'app) WO2009035205A1 (fr)

Applications Claiming Priority (2)

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KR10-2007-0093041 2007-09-13
KR1020070093041A KR20090027877A (ko) 2007-09-13 2007-09-13 Ｂｅｔａ－ｓｉｔｅ ＡＰＰ ｃｌｅａｖｉｎ ｇｅｎｚｙｍｅ（ＢＡＣＥ）를 발현하는 형질전환 식물체

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627739B1 (en) * 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2004083417A1 (fr) * 2003-03-21 2004-09-30 Korea Research Institute Of Bioscience And Biotechnology Cellule vegetale transformee exprimant des sequences repetees en tandem du gene beta-amyloide et plante obtenue avec
US20070092517A1 (en) * 2005-08-10 2007-04-26 Oklahoma Medical Research Foundation Truncated memapsin 2 compositions and treatments

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627739B1 (en) * 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2004083417A1 (fr) * 2003-03-21 2004-09-30 Korea Research Institute Of Bioscience And Biotechnology Cellule vegetale transformee exprimant des sequences repetees en tandem du gene beta-amyloide et plante obtenue avec
US20070092517A1 (en) * 2005-08-10 2007-04-26 Oklahoma Medical Research Foundation Truncated memapsin 2 compositions and treatments

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J.W.YOUM,ET AL.: "Transgenic potato expressing A beta reduce A beta burden in Alzheimer`s disease mouse model", FEBS LETTERS, vol. 579, 21 November 2005 (2005-11-21), pages 6737 - 6744 *
W.-P. CHANG,ET AL.: "Amyloid-beta reduction by memapsin 2 (beta-secretase) immunization", FASEB J., vol. 21, 10 May 2007 (2007-05-10), pages 3184 - 3196 *

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