CN101815784A - Production of foreign nucleic acids and polypeptides in plant systems - Google Patents

Production of foreign nucleic acids and polypeptides in plant systems Download PDF

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Publication number
CN101815784A
CN101815784A CN200780007805A CN200780007805A CN101815784A CN 101815784 A CN101815784 A CN 101815784A CN 200780007805 A CN200780007805 A CN 200780007805A CN 200780007805 A CN200780007805 A CN 200780007805A CN 101815784 A CN101815784 A CN 101815784A
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protein
plant
polypeptide
virus
agrobacterium
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维达季·尤西波夫
瓦季姆·梅特
莎伊拉贾·拉宾德拉
穆奈姆·沙劳尔
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Fraunhofer USA Inc
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Fraunhofer USA Inc
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Priority claimed from US11/353,905 external-priority patent/US7692063B2/en
Application filed by Fraunhofer USA Inc filed Critical Fraunhofer USA Inc
Publication of CN101815784A publication Critical patent/CN101815784A/en
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Abstract

The present invention provides systems and methods for producing a nucleic acid or protein in young plants. Typically, the young plants are grown in a contained, regulatable environment. In some embodiments, expression of a pharmaceutically active protein in the young plants is controlled by an exogenously inducible promoter or a viral promoter. In some embodiments, the young plants are edible and may be eaten live or preferably harvested live to preserve the maximal biological activity of the nucleic acid or protein. In some embodiments, the young plant is a young pea plant or a young Nicotiana plant.

Description

The generation of external nucleic acid and polypeptide in the botanical system
Related application
The application's case is a continue case and advocate its rights and interests of the part of No. the 11/353rd, 905, the co-pending U.S. patent application case that is entitled as " external nucleic acid and proteinic generation in the young shoot system (Production of Foreign Nucleic Acids and Polypeptides in Sprout Systems) " of on February 13rd, 2006 application.The complete content of described application case is to be incorporated herein by reference.
The application's case is also advocated co-pending U.S. Provisional Patent Application case the 60/773rd, No. 255 (being entitled as " Bacillus anthracis antigen; vaccine composition and methods involving (Bacillus Anthracis Antigens; Vaccine Compositions; andRelated Methods) "), the 60/773rd, No. 374 (being entitled as " HPV antigen; vaccine composition and methods involving (HPVAntigens; Vaccine Compositions; and Related Methods) ") and the 60/773rd, the rights and interests of each in No. 378 (being entitled as " influenza antigen; vaccine composition and methods involving (Influenza Antigen; Vaccine Compositions; and RelatedMethods) "), each described patent application case is all applied on February 13rd, 2006.The complete content of each all is incorporated herein by reference in these application cases.
The application's case is also advocated the rights and interests of No. the 60/813rd, 955, the co-pending U.S. Provisional Patent Application case that is entitled as " influenza antigen, vaccine composition and methods involving (Influenza Antigen; Vaccine Compositions; and Related Methods) " of on June 15th, 2006 application.The complete content of described application case is to be incorporated herein by reference.
The application's case is also advocated the rights and interests of No. the 60/944th, 770, the co-pending U.S. Provisional Patent Application case that is entitled as " influenza antigen, vaccine composition and methods involving (Influenza Antigen; Vaccine Compositions; and Related Methods) " of on September 15th, 2006 application.The complete content of described application case is to be incorporated herein by reference.
The application's case is also advocated the co-pending U.S. Provisional Patent Application case the 60th that is entitled as " launching carrier (Launch Vector for the Production of Vaccine Antigens in Plants) that is used for producing plant vaccine antigen " of application on January 9th, 2007,879, No. 450 rights and interests.The complete content of each is to be incorporated herein by reference in these application cases.
Technical field
Do not have
Background technology
The quite high and constantly rising of the cost of medicine.Except that the cost height, the supply of some medicines also is restricted, and this makes these medicines to have the patient who needs used for each.Because cost and operability have hindered the distribution of medicine in poor colony, so this is a problem that merits attention especially for developing country.
Some factors cause the expensive of pharmaceutical production, and cause that the human consumer buys the price rising of pharmaceuticals.Cause the shortage of the main factor of this problem for the economized form of the described product of production.For especially true based on the medicine of protein and peptide.For some medicines, another factor is can't oral administration and the medicament for the treatment of significant quantity.Many pharmaceuticals can only by be expelled to the health privileged site throw with.For instance, some immune drugs of treatment allergy or communicable disease need by injection throw with.Such as protein such as human growth hormone and Regular Insulin and peptide pharmaceutical products usually can only by injection throw with.Another factor that causes many cost of drugs is its transmission in hospital or distribution place.Especially at hot weather, transmitting and store medicine needs expensive refrigeration equipment.Because described equipment is difficult to obtain usually, so this is a subject matter in developing country.
Medical protein and peptide in multiple host, have been produced.Many therapeutic proteins in the heterologous expression system that comprises prokaryotic organism (such as intestinal bacteria (Escherichia coli) and Bacillus subtillis (Bacillus subtilis)) and eukaryote (such as yeast, fungi, insect cell, zooblast and transgenic animal), have been produced.The productive rate of relative easy handling of bacterial expression system and product is higher.Yet mammalian proteins needs large-scale posttranslational modification to reach functionally active usually, and this just becomes the limiting factor of bacterial expression system.Comparatively convenient such as cell culture systems such as Mammals, the mankind and insect cell culture systems for producing complex proteins.Yet the delivery date of length, lower product recovery rate, the cause of disease transfer and the higher capital and the production cost that may occur have all proposed serious problem.Transgenic animal can provide the endless supply for complex proteins.Unfortunately, this system is produced novelty and through improving the restriction that spent long period of product and cause of disease are transferred to the risk among the human individual.
In protokaryon and eukaryotic cell, produce the economy of medical protein and peptide and biological chemistry restriction (comprise improper modification in high production cost, low-yield, secretion problem, the protein processing, in proportion expand to the difficulty of comparatively large vol and pollute) ordered about the researchist and studied plant as novel host so that the cost that reduces with expection produces protein and peptide on a large scale.Proteinic generation for example is described in United States Patent (USP) the 5th, 750 in the transgenic plant, and No. 871, the 5th, 565, No. 347, the 5th, 464, No. 763, the 5th, 750, No. 871 and the 5th, 565, in No. 347.Although raised growth of plant and collection are more cheap than protokaryon and eukaryotic cell, external expression of gene is lower usually in the vegetable cell.In addition, herborization for example usually needs by removing blade, stem is separated with root or removing root and destroy plant.Described destruction can cause that usually initial plant part is withered and the process of plant apoptosis.The plant of experience apoptosis can produce the proteolytic enzyme of the protein degradation that causes transgene expression before the beginning protein purification.Even directly consume plant, the activity of expressed medical protein also may reduce because of gathering inductive iuntercellular degradation mechanism.
With produce in the transgenic plant of growth in the open that another relevant subject matter of foreign protein is and the possibility of open-air plant cross-pollination, this is feasible may to make foreign protein enter food chain.Make that around complicacy being difficult to approval is used for agricultural use with novel transgenic plant at the government regulation of the agricultural practices of transgenic plant.In addition, outdoor environment is uncontrollable, makes the suitable growth be difficult to guarantee alien gene, growth and to the regulation and control of its expression.For instance, carry out inducing of thermal induction type, photoinduction type, hormone induction type or chemical inducible promoter in the environment in fact out of doors.Certainly, also uncontrollable outdoor temperature and illumination level.In addition, the hormone or the chemical that are sprayed onto on the plant do not disperse on plant probably, but are distributed in the environment through wind and rain.The cost that sprays the farmland is also quite high.
Need to produce the improvement system of protein or polypeptide.Also need in plant, produce the improvement system of protein or polypeptide especially.For instance, need be used for producing medical protein and the controlled adjustable system of the amount that the expressed proteinic iuntercellular of minimizing is degraded after collection plant.
Summary of the invention
The invention provides in plant protein or polypeptide (and/or nucleic acid), the system of medical protein or polypeptide especially of producing.
On the one hand, the invention provides in plant the system of marking protein rapidly or polypeptide.Therefore, in certain embodiments, the invention provides the expression in seedling of protein or polypeptide.In certain embodiments, described seedling is the germination seedling.In certain embodiments, can consume or gather seedling alive (seedling for example germinates).In certain embodiments, plant is growth (for example from seed growth) in closed adjustable environment (for example indoor).
In certain embodiments, protein or the polypeptide that produces according to the present invention is to construct body surface by the nucleic acid in the artificial introduced plant to reach in vegetable cell.In certain embodiments, protein that produces according to the present invention or polypeptide are by the RNA translation with plant virus feature in vegetable cell.In certain embodiments, these features comprise self-replacation, cell to cell movement, systematicness motion and its combination, or are selected from by self-replacation, cell to cell movement, systematicness motion and its group that forms.In certain embodiments, RNA self-replacation and can cell to cell movement, but can't systematicness motion in plant.
In certain embodiments, protein or the polypeptide that produces according to the present invention is to construct body surface by the nucleic acid that duplicates in Agrobacterium (Agrobacterium) to reach in vegetable cell.In certain embodiments, utilization of the present invention in Agrobacterium, duplicate and also contain guiding have the plant virus feature rna expression promotor construct body.In certain embodiments, described RNA comprises the protein that coding is paid close attention to or the sequence of polypeptide.Therefore, in some embodiments of the invention, in plant by comprise that the method that will duplicate and also contain in the carrier introduced plant cell of promotor of rna expression that guiding has the plant virus feature produces protein or the polypeptide of being paid close attention in Agrobacterium, described RNA can't systematicness move in plant, but can self-replacation and the other protein or the polypeptide of encoding and being paid close attention to of described RNA.
In some embodiments of the invention, when utilize in Agrobacterium, duplicate construct body the time, construct in the body introduced plant cell described by the Agrobacterium osmose process.
Although main reference the present invention herein is used to produce the purposes of medical protein the present invention is described, but the present invention is generally used for producing any basically nucleic acid of paying close attention to (DNA and/or RNA) and/or protein, and is not subjected to the restriction of related nucleic acid or proteinic specific end use.For instance, can be created in the enzyme (for example, the enzyme of degradation of contaminant) that uses in any of multiple commercial run or biological restoration process.Therefore, even do not spell out, but the description in the present invention and claims should be considered as being applicable to any nucleic acid of paying close attention to or protein, comprises having nucleic acid or the protein that treatment is used, and do not have nucleic acid or protein that treatment is used.In certain embodiments, described protein nutritionally is not key protein.Under the NM in this article situation, the present invention can get rid of any specified protein.
In aspect the present invention is any, in its some embodiment, nucleic acid of being paid close attention to or protein in expressing its cell through transcribing after and/or translate post-treatment.In certain embodiments of the invention, the protein of being paid close attention to is by the emiocytosis of expressing it.For instance, described protein can comprise secretory signal sequence usually, and the coding region of nucleic acid that maybe can modify code for said proteins is so that it comprises the part of the secretory signal sequence of encoding.Well-known secretory signal sequence in the affiliated field.
In aspect the present invention is any, in its some embodiment, can change proteinic heterologous sequence that coding pays close attention to use with naturally occurring sequence in the different codon of codon that exists, thereby the expression of improvement in the plant of frequently seen plants and/or specific species.For instance, described stream cipher can be optimized so that express in specific species.Under knownly in the field carry out codon optimized method.
The present invention also provide contain expression as described herein construct the plant of body, contain the composition of described plant, from described plant isolating protein/polypeptide preparation, contain described from described plant isolating protein/polypeptide composition, carry out described isolating method, with use described method through isolated protein/polypeptide (or contain its composition), comprise that the medicine and pharmacology active protein that utilization is expressed treats mammiferous method in plant.
Definition
To having the individuality that needs " throw with " the medicine and pharmacology active protein or the polypeptide meaning to be, so that described proteinic therapeutic efficiency keeps being enough to provide the mode of the time span of required beneficial effect to provide the medicine and pharmacology active protein to described individuality to described host.
All vertebratess (life-form) contained in term " animal ", the mankind, ox, sheep, pig, Canidae, cat family, ferret, rodent, primate, fish, birds (for example poultry) etc.In certain embodiments, described animal is a Mammals.In certain embodiments, described animal is human.
According to the present invention, polypeptide or proteinic " characteristic " contain continuous amino acid elongation or are the protein or the polypeptide of the set of the continuous amino acid elongation of the feature of protein or polypeptide together for having.In certain embodiments, each described continuous elongation will contain at least 2,5,10,15,20 or more a plurality of amino acid usually.In general, characteristic is the part that has at least one functional character except that sequence identity mentioned above with relevant whole protein.In certain embodiments, characteristic can be had a biological activity.
As used herein term, protein or polypeptide " territory " typically refer to, and keep the protein or the peptide sequence section of three-dimensional integrated degree and/or structure during when generation and away from the remainder of protein or peptide sequence.Known as affiliated field, numerous protein and polypeptide have domain structure.In certain embodiments, the territory has activity (for example, in conjunction with, catalytic activity etc.).In certain embodiments, the territory has immunogenicity.The immunogen territory is the same with single epi-position at least big, and bigger than single epi-position usually; In certain embodiments, enough sequences are also contained to guarantee correctly presenting of described epi-position in the immunogen territory except that epi-position.
" expression " is meant native gene or transgenosis transcribing and/or translating in plant (for example, young shoot).Can with or can transgenosis be incorporated in the genomic dna of plant.For instance, " expression " is meant gene the transcribing and/or translating in plant that exists in bacterium, plasmid or the viral nucleic acid, and no matter whether described bacterium, plasmid or viral nucleic acid are incorporated in the genomic dna of plant.Described gene can be for being allogenic gene for bacterium, plasmid or virus.
" expression cassette " or " expression vector " is meant the dna sequence dna (or being the RNA sequence under the situation of RNA viruses or RNA plasmid) that can guide specific nucleotide sequence to express in suitable host cell.For instance, expression cassette can comprise the promotor that is operably connected to the nucleotide sequence of being paid close attention to, and it is operably connected to 3 ' sequence according to circumstances, such as 3 ' regulating and controlling sequence or termination signal.Expression cassette can also comprise the required sequence of suitable translation of carrying out described sequence when any nucleotide sequence of needs usually.The coding region is the protein paid close attention to of coding usually, but the also functional r NA that pays close attention to of codified, for example sense-rna or suppress that specific gene expresses do not translate RNA.The expression cassette that comprises the nucleotide sequence of being paid close attention to can be mosaic, the meaning is meant that described nucleotide sequence comprises the dna sequence dna of an above different sources, it is to merge by recombinant DNA technology, produces natural the existence and non-existent nucleotide sequence in the plant that will express described sequence especially.Expression cassette can also be for natural existence but the expression cassette that is applicable to heterogenous expression that obtains with recombination form.Yet usually expression cassette is allogenic about the host, that is the specific dna sequence of expression cassette is natural in host cell not to be existed and must introduce in the progenitor cell of host cell or host cell by transformation event.The expression of the nucleotide sequence in the expression cassette is subjected to the composition promotor or has only when host cell is exposed to some specific outside stimulus thing just initial inducible promoters of transcribing to control.Under the situation of multi-cell organism (such as plant), promotor also can be to particular organization, organ or etap tool specificity.Usually the nuclear expression box is inserted in the plant nucleus gene group and it can guide the nuclear gene group of plant to express specific nucleotide sequence.Usually the plasmid expression box is inserted in the plant plasmon and it can guide the plasmon of plant to express specific nucleotide sequence.Under the situation of plasmid expression box, for expressing nucleotide sequence, will need other element, for example ribosome bind site, or the 3 ' stem-ring structure that stops the plastid rna polyadenylic acidization and degrade subsequently by plasmon.In certain embodiments of the invention, utilization comprises the expression cassette as the promotor of minimal promoter (such as the TATA element), and the existence of trans activation factor (trans-activating factor) may be expressed, especially guide high level expression essential by the guiding nucleus nucleotide sequence.For instance, expression cassette can comprise the DNA district in conjunction with transcription activating.Described expression cassette and promotor wherein are called " activable ".
" food " or " food " is can be by liquid or solid preparation human or other animal picked-up.Preferred described term comprises can the directly fresh mankind of offering and/or living plant or living plant (for example, the germination seedling) preparation of other animal.The material that obtains from plant is intended to comprise can be by whole edible plants human or other animal picked-up.Described term also can comprise any through processing plant (for example, germination seedling) and the nutrition supporting agent of feeding to human and other animal.Procedure of processing can comprise step commonly used in food or the fodder industry.Described step includes, but is not limited to concentrate the solid matter of plant for example to form particle, to produce mashed prod, drying or freeze-drying; Perhaps can by with plant cutting, smash or grind the various degree that reach to pieces, or make to produce soup juice, syrup or juice by the liquid portion that extracts plant.Procedure of processing also can comprise boiling (for example stifling) plant (seedling for example germinates).
The protein that " gene " paid close attention to for encoding or the sequence of polypeptide (or RNA).Gene can comprise relevant regulating and controlling sequence.The encoding sequence of gene can be transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, just RNA or sense-rna.Yet, understand as one of ordinary skill in the art, " gene " can also be RNA (for example gene in the viral rna vector).The example of " regulating and controlling sequence " is promoter sequence, 5 ' and 3 ' not translation sequences and terminator sequences.In addition, can also comprise intron and exon.In certain embodiments, gene is that encoding sequence and relevant regulating and controlling sequence are heterologous sequence.
As used herein, " heterologous sequence " meaning refers to have different natural origins or synthetic source.For instance, if introduce nucleic acid in the host cell and natural some or all sequence that exists in the described nucleic acid that do not contain of host cell, so just claim that these sequences (and/or nucleic acid) are allogenic about described host cell.The nucleic acid of being introduced can comprise allogeneic promoter, allogeneic coding sequence or allos terminator sequence.In addition, transformed nucleic acid can be allogenic fully or can comprise any possible allos and the combination of endogenous nucleic acid sequence.Similarly, allos is meant that the cell type by identical natural origin obtains and inserts in the cell type of identical natural origin, but exists or nucleotide sequence under different controlling elements controls with non-natural state (for example different copy number).Term " allos " is applicable to cell, comprises plant and bacterial cell, and also is applicable to plasmid, plastid and virus.
" host cell " is for introducing nucleic acid for the cell of expressing.Common described introducing needs manual operation.
" non-activity expression cassette " or " non-activity expression vector " is DNA or the RNA sequence that comprises the foreign nucleus acid sequence of non-activity or silence, and it can guide nucleic acid or the polypeptide expression of being paid close attention to after activation.In general, the non-activity expression cassette has expression cassette characteristic as indicated above, but the nucleic acid that coding is paid close attention to or the sequence of polypeptide are not operably connected to promotor, and for example, it can separate with promotor (or another controlling element) by inserting the nucleic acid district.Can be operatively connected is to take place after recombination event, expresses subsequently thus.Described expression cassette is called as " activable ".
Term " inducible promoters " meaning refers to by the existence of the particular stimulation thing of direct or indirect increase promoter activity or does not have the promotor that opens the beginning.Some limiting examples of described stimulator comprise heat, light, the developmental regulation factor, damage, hormone and chemical (for example small molecules).An example of photoinduction type promotor is ribose-5-phosphoric acid carboxylase promotor.The promotor of chemical induction type also comprises receptor-mediated system, for example from system that other organism obtains, such as steroid-dependent gene expression, Lac repressor system, and being used to from the expression system of fruit bat (Drosophila) by the usp receptor of protecting young tethelin and the mediation of its agonist described in the WO97/13864 (being incorporated herein by reference), and (for example) utilizes the system of acceptor combination described in WO96/27673 (also being incorporated herein by reference).The promotor of other chemical induction type comprises elicitor inductive promotor, safener inductive promotor and can be by some pure and mild ketone inductive alcA/alcR gene activation (WO93/21334 of system; Ka Dike people (1998) nature-biotechnology (Nat.Biotechnol.) 16:177-180 such as (Caddick), its content is to be incorporated herein by reference).The damage inducible promoter comprises the promotor of proteinase inhibitor, for example from the promotor of other related in the proteinase inhibitor II promotor of potato and injury response path plant origin, such as the promotor of polyphenylene oxydase, LAP and TD.For example referring to covering (Gatz) " chemical control of genetic expression (ChemicalControl of Gene Expression) " now, Mol.Biol. (Ann.Rev.Plant Physiol.PlantMol.Biol.) (1997) 48:89-108, it is incorporated herein by reference.Other inducible promoter comprises the promotor of plant origin, such as the promotor in the system acquisition resistance path, for example PR promotor.It should be noted that when discussing inducible promoters in this article can activate promotor and expression cassette can similar type be used for some embodiment of the present invention.
" marker gene " can select the gene of the characteristic that maybe can screen for coding.
" dietetic food " comprises the composition that eats or drink and described individuality is had therapeutic action for individual.Dietetic food for example comprises plant (for example, germination seedling) or the plant material therefrom that has produced medical protein or polypeptide.Dietetic food can absorb separately or can be in field of medicaments well-known medical composition throw with.Dietetic food also comprises the feed of the equivalence of using for the non-human animal.
" be operably connected " and be meant the association of nucleic acid elements.For instance, the promotor that is operably connected to the allogeneic dna sequence DNA of coded protein promotes the generation corresponding to the functional mRNA of allogeneic dna sequence DNA.If location regulating DNA sequence and coding RNA or protein DNA sequence, so that described regulating DNA sequence influences the expression of described DNA sequences encoding, think so described regulating DNA sequence " be operably connected to " described dna sequence dna or with described dna sequence dna " association ".
" oral administration with " medicine and pharmacology bioactive peptide or protein mainly be meant through port, preferably by edible mode throw with, and it is intended to comprise stomach or the gastral any dispensing that described peptide or protein is provided to the host.In a preferred embodiment, oral administration contacts with intestinal mucosa with causing the medicine and pharmacology active protein.
" medicine and pharmacology active nucleic acid " are the nucleic acid of coding medicine and pharmacology active protein, or himself have the medicine and pharmacology activity, and for example it has one or more medicine and pharmacology activity, such as at the described activity of medicine and pharmacology active protein.For instance, nucleic acid can be one or more chains that RNA disturbs (RNAi) factor.The described factor comprises the short interfering rna (siRNA) of target target transcript (for example transcript of contagium or individual endogenous disease-related transcript), or short hairpin RNA (shRNA), or the siRNA precursor, or microRNA sample RNA.
When throwing with the host with " medicine and pharmacology active protein or polypeptide " with the treatment significant quantity, it is with positive manner help or help host's symptom.Medicine and pharmacology active protein or polypeptide have rehabilitation or alleviate characteristic disease, and can offer medicine with one or more symptoms of improving, alleviate, alleviate, reversing disease or illness, delay its outbreak or alleviate its severity.Medicine and pharmacology active protein or polypeptide can have the prevention characteristic and can be used for delaying seizure of disease, perhaps alleviate the severity of described disease or pathology symptom when disease occurs.Term " medicine and pharmacology active protein or polypeptide " comprises whole protein or polypeptide and can also refer to its medicine and pharmacology active fragments.It can also comprise the medicine and pharmacology active analogue thereof of protein or peptide or the segmental analogue of described protein or peptide.Term " medicine and pharmacology active protein or polypeptide " also can refer to cooperate or act synergistically so that the multiple proteins or the peptide of treatment benefit to be provided.It should be noted that term " medicine and pharmacology active protein or polypeptide " especially comprises the protein that comprises vaccine antigen, that is throwing causes host's part or all of preventative immune response with protein to individuality.In certain embodiments of the invention, immune response prevents that individuality is subjected to contagium (for example virus, bacterium, fungi or protozoon pathogenic agent) influence.The example of vaccine antigen comprises hepatitis B surface antigen(HBsAg) (HbsAg), coli heat-sensitive enterotoxin, rabies virus glycoprotein and norwalk virus capsid protein (Norwalkvirus capsid protein).In other embodiment of the present invention, immune response prevents that individuality is subjected to non-infectious symptom or sickness influence or alleviates at least a symptom of described symptom or disease or the severity of symptom.See that with regard to this point the disease of being paid close attention to includes, but is not limited to cancer and autoimmune disorders.Term " medicine and pharmacology active protein or polypeptide " comprises that individuality is partially or completely tolerated to be exposed to and will to cause the protein of the anaphylactogen of allergy (allergic or anaphylactic) reaction in addition.
Dna sequence dna as used herein, that " promotor " transcribes for initial associated dna sequence.Promoter region also can comprise the element of the regulator (such as activator, enhanser and/or repressor) that serves as genetic expression.
" controlling element " is meant the sequence that relates to the expression of giving nucleotide sequence.Controlling element comprises 5 ' regulating and controlling sequence, such as the promotor that can be connected with the nucleotide sequence of being paid close attention to; 3 ' sequence is such as 3 ' regulating and controlling sequence or termination signal.Controlling element is also contained the suitably required sequence of translation of nucleotide sequence usually.
" small molecules " is usually less than about 1 kilodalton (kilodalton) and be biological, organic or even mineral compound (for example cis-platinum (cisplatin)).Described micromolecular example comprises nutrient, such as sugar and sugar derivatives (comprising phosphate derivative), hormone (such as plant hormone gibberic acid or dormin) and synthesized micromolecule.
" specificity regulation and control " are meant that small molecules preferentially influences a promotor but not the ability of transcribing (such as strengthening or weakening the intracellular overall situation and transcribe) of promotor group as comparing with nonspecific action.
" germination seedling " or " young shoot " are the sprout from seed or root, preferably seeds germinated recently.In certain embodiments, germination seedling of the present invention is edible germination seedling or young shoot (for example clover bud, Semen Phaseoli radiati Germinatus, radish bud, Fructus Hordei Germinatus, leaf mustard bud, spinach bud, Radix Dauci Sativae bud, beet bud, rareripe, garlic, celery bud, rheum officinale bud; Leaf is such as Caulis et Folium Brassicae capitatae bud or lettuce bud, watercress or Chinese celery bud; The medicinal herbs bud is such as parsley or trifolium bud, Cauliflower bud, broccoli sprouting, big bean sprouts, root of Szemao crotalaria bud; The edible bud is such as Sunflower Receptacle bud etc.).According to the present invention, the germination seedling can grow to two leaf phases.Usually, young shoot of the present invention be 2 to 14 days big.
" separate in fact " and repeatedly use in this article, and normally instigate protein or polypeptide and the uncorrelated or isolating partial purification at least of pollution components (for example plant structure albumen and metabolism protein).The method of the affiliated well-known separation in field and protein purification or polypeptide.
" conversion " be meant nucleic acid introduced in the cell, especially with the dna molecular stable integration to pay close attention in the organic genome.
Description of drawings
Fig. 1 is the synoptic diagram of use based on the Different Strategies of the vector expression alien gene of plant virus.
Fig. 2 is AIMV and the genomic synoptic diagram of TMV.
Fig. 3 is the figure of the Western trace that reorganization GFP expresses in Ben Saimushi tobacco (Nicotiana benthamiana) plant of Av/A4 and Av/GFP inoculation.
Fig. 4 is at the figure that produces human growth hormone's (hGH) Western trace in the Ben Saimushi tobacco plant that in vitro the GH transcript infects.
Fig. 5 is the synoptic diagram that body is constructed in the conversion of express recombinant protein in the leafy mustard (Brassica juncea).
Fig. 6 is the western blot figure of the transgenosis leafy mustard of expressing human class tethelin under the control of HSP18.2 promotor.
Fig. 7 describes the synoptic diagram that the various Agrobacteriums that contain target gene construct body.Fig. 7 A: carrier pBIV, wherein target gene is integrated in plant promoter (any plant promoter of IV=, manual activation, or other promotor that in vegetable cell, works, for example, such as the promotor of the plant virus of cauliflower mosaic virus (cauliflower mosaic virus)) and the no terminator between; Fig. 7 B: carrier pBIV-GUS, wherein gus gene inserts among the pBIV.Fig. 7 C:pBIV-virus vector+target, the virus vector or the replicon (for example, viral genome or genome part) that wherein are loaded with target gene insert among the pBIV; Fig. 7 D: carrier pBIV-virus vector+GFP, wherein GFP serves as reasons and inserts the target gene that virus vector among the pBIV or replicon carry.Carrier described in Fig. 7 C and the 7D is considered as " emission " carrier, and this is because behind the introduced plant cell, it " starts " to produce virus sequence (arriving under the situation of specified plant cell wherein its self-replacation at least in the described sequence of emission).
Fig. 8 is presented at the GUS dyeing of carrying out with behind the pBIV-GUS Agrobacterium infiltration young shoot.Fig. 8 A shows the dyeing of Semen Phaseoli radiati Germinatus; Fig. 8 B shows the dyeing of trigonella bud.
Fig. 9 is presented at the GUS dyeing of carrying out with behind the pBIV-GUS Agrobacterium infiltration rape young shoot.Fig. 9 A shows coloration result; Fig. 9 B is a table of listing dissimilar young shoots, test it and express by Agrobacterium and construct the protein that body transmits or the ability of polypeptide, described Agrobacterium constructs body and comprises that guiding is loaded with the virus sequence expression promoter of the gene of protein that coding pays close attention to or polypeptide.
Figure 10 is presented at the expression of GFP in the Ben Saimushi tobacco of passing in time after the pBI121/D4-GFPC3 infiltration.
Figure 11 shows the western engram analysis that is contained hGH expression in the caused plant of Agrobacterium of virus vector (sequence that contains the hGH that encode) by infiltration.Road number: the 1st road: molecular weight standard product (ladder); The 2nd road: 50ng hGH; The 3rd road: through the plant sample of pBI121/D4-HGH 6dpi 1:2 diluent infiltration; The 4th road: through the plant sample of pBI121/D4-HGH6dpi 1:10 diluent infiltration; The 5th road: through the plant sample of pBI121/D4-HGH 6dpi 1:50 diluent infiltration; The 6th road: through the plant sample of pBI121/D4-HGH 6dpi 1:100 diluent infiltration; The 7th road: from the plant sample of health plant; The 8th road: from the plant sample of the blade that permeates pBI121; The 9th road: the plant sample that comes the plant 13dpi of self-infection D4-HGH; The 10th road: the sample that comes clone's root system of self-infection D4-HGH.For plant sample: with a slice blade (about 5mg tissue) grinding in 100 μ L Bu Shi damping fluids (Bradley buffer) and Laplace loading buffer liquid (Laemmli loading buffer).Per pass loads 10 μ L.Therefore, for the 3-6 road, this main sample is through dilution.
Figure 12 shows IA-2ic protein expression in the Ben Saimushi tobacco plant that utilizes the hydroponics growth: the 1st road: IA-2ic standard substance; The 2nd road: magical mark (Magic Marker); The 3rd road: injection has the soil growth plant of Agrobacterium; 4th, 5 roads: permeate back 4 days hydroponics growth plant; 6th, 7 roads: permeate back 6 days hydroponics growth plant.
Figure 13 describes an embodiment who is used for the agrobacterium vector that emission is loaded with the virus sequence of the target protein of being paid close attention to is delivered to the Agrobacterium osmosis system of seedling of according to the present invention utilization.
Figure 14 explanation is detected lichenin enzymic activity in the various pea kinds of the Agrobacterium osmose process that the system that experiences use Figure 13 carries out.
Figure 15 illustrates experience and uses the GFP of the spot pea of the Agrobacterium osmose process that the system of Figure 13 carries out to express and plant strain growth.
Figure 16 illustrates the best fate of tissue specificity and infiltration back that experience is used some spot pea of the Agrobacterium osmose process that the system of Figure 13 carries out.
Figure 17 describes and can be used for the lichenase carrier molecule that merges with protein of being paid close attention to or polypeptide according to certain embodiments of the invention.Figure A represents the b-1 from Clostridium thermocellum (Clostribium thermocellum), 3-1, the schematic description of 4-dextranase.Figure B represents the schematic description of the LicKM carrier of ring-type exchange.In each figure, vertical hachure is corresponding to the N-terminal district of catalytic domain, and frame of broken lines is corresponding to the C-terminal district of catalytic domain.
Figure 18 represents the synoptic diagram of launching carrier pBID4.
Figure 19 is for being optimized to the schematic description of each related the process of purifying end product step from target gene, described end product is easy to store according to one particular embodiment of the present invention.
Figure 20 describes the automatic apparatus operating design of the step that is used for from the plant seeding to the tissue sampling.As described, estimate that described module will occupy about 40 feet and multiply by about 70 feet floor area and be about 32 feet high.Shown in system comprise the following assembly that connects by travelling belt: central frame unit wherein keeps the plant between each step; Sowing unit, vacuum infiltration and rinse unit and tissue sampling unit.
Embodiment
Protein or polypeptide
The present invention is applicable to and produces any protein or polypeptide (or any functional type DNA or RNA molecule) in botanical system.As indicated above, in certain embodiments, the present invention is directed to the generation of medical protein, but the present invention is not subjected to the restriction of nucleic acid or proteinic specific end use.For instance, can be created in the enzyme (for example, the enzyme of degradation of contaminant) that uses in any of multiple commercial run or biological restoration process.Therefore, even do not spell out, but the description in the present invention and claims should be considered as being applicable to any nucleic acid or the protein of being paid close attention to, and comprises having nucleic acid or the protein that treatment is used, and do not have nucleic acid or protein that treatment is used.In certain embodiments, protein is unessential protein nutritionally.Under the NM in this article situation, the present invention can get rid of any specified protein.
Usually, expressed protein or polypeptide are not protein or polypeptide expressed in the plant of occurring in nature.Even according to the present invention, described protein or polypeptide are protein or polypeptide expressed in the plant of occurring in nature, and it is to be higher than existing concentration expression in the suitable tissue of occurring in nature usually in the seedling tissue.
Lift a few examples, the present invention can be used for producing the medical protein of being paid close attention to, include, but is not limited to hormone (Regular Insulin, Triiodothyronine, catecholamine, gonad-stimulating hormone, trop(h)ic hormone, prolactin, pitocin, Dopamine HCL, Trobest, leptin etc.), tethelin (for example human growth hormone), somatomedin (Urogastron for example, nerve growth factor, insulin-like growth factor etc.), growth factor receptors, cytohormone and immune system protein are (for example, interleukin-, G CFS (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), erythropoietin, tumour necrosis factor (TNF), Interferon, rabbit, integrate plain, addressin, select plain, homing receptor, TXi Baoshouti, immunoglobulin (Ig), water-soluble major histocompatibility complex antigen, immuno-activated-antigen is (such as bacterium, parasite or virus antigen or anaphylactogen), autoantigen, antibody), enzyme (histiotype plasminogen activator, streptokinase, cholesterol biosynthesizing or degraded, the steroid synthetic enzyme, kinases, phosphodiesterase, methylase, demethylase, desaturase, cellulase, proteolytic enzyme, lipase, Phospholipid hydrolase, aromatizing enzyme, cytopigment, adenylic acid (AMP) or guanylate cyclase, neuraminidase etc.), acceptor (steroid hormone receptor, the peptide acceptor), conjugated protein (steroid binding protein, tethelin or growth factor bindin etc.), transcribe and translation factor, cancer protein or proto-protein (for example cyclin), myoprotein (myosin or tropomyosin etc.), mycoprotein, neural activity albumen, tumor growth arrestin (angiostatin or Endostatin, the two is angiogenesis inhibiting all), antibacterial protein (sterilization/power/permeability increasing protein), structural protein are (such as collagen protein, silk-protein, Fibrinogen, elastin, tubulin, Actin muscle and myosin), blood protein (zymoplasm, serum albumin, the VII factor, the VIII factor, Regular Insulin, the IX factor, the X factor, the histiotype plasminogen activator, C albumen, the temperature Wei Baishi factor (von Wilebrand factor), Antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified factor VIII, antithrombotics (such as r-hirudin (huridin)) etc.
In some specific embodiments, utilize the present invention to produce antigen protein or polypeptide.For instance, can utilize the present invention to produce by the organic albumen of infectivity (or its part) that is infected individual immune system recognition.Described protein or polypeptide can prevent aspect the vaccine that relevant organism infects particularly useful in research and development.Lift a few examples, can produce from anthrax (anthrax) (Bacillus anthracis (Bacillus anthracis) according to system of the present invention; LF for example, PA), cholera (vibrio cholerae (Vibrio cholerae)), cytomegalovirus (cytomegalovirus), enterotoxin type coli strain (enterotoxigenic strains of E.coli), foot and mouth disease virus (foot-and-mouth disease virus), hepatitis B (for example hepatitis B surface antigen(HBsAg) HBsAg), hepatitis C (for example HCV core protein), human immunodeficiency virus (human immunodeficiency virus) (Tat for example, Rev, Nef, gp160, gp120 etc.), human papillomavirus (E7 for example, E6), influenza virus (HA for example, NA), malaria (malaria) (plasmodium falciparum (Plasmodium falciparum); For example Pfs25, Pfs28, Pfs48/45, Pfs230), Measles virus, norwalk virus, the plague (plague) (plague bacillus (Yersiniapestis); F1 for example, LcrV), Pseudomonas aeruginosa (Pseudomonas aeruginosa), rabies virus (rabies virus), respiratory syncytial virus (respiratorysyncytial virus) (F albumen for example, G albumen), rhinovirus (rhinovirus), rotavirus (rotavirus), streptococcus aureus (Staphylococcus aureus), Transmissible gastroenteritis virus (transmissible gastroenteritisvirus), trypanosome (trypanosomes) (trypanosoma bocagei (Trypanosoma brucei); For example alpha-tubulin, 'beta '-tubulin), the useful antigen protein of tuberculosis (tuberculosis), SARS etc.
In some specific embodiments, utilize the present invention to produce the multimeric protein mixture, for example comprise immunoglobulin (Ig), such as antibody (being monoclonal antibody).Lift a few examples, can system according to the present invention produce anti-PA, anti-LF, anti-NA, anti-TNFa, anti-IL-8 12 etc.More generally, can be produced according to the present invention at antigenic monoclonal antibody such as following disease-related: cancer (acute myeloid leukemia (AML) for example, breast cancer, colorectal carcinoma, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphomas (non-Hodgkin ' s lymphoma), renal cell carcinoma, noumenal tumour), inflammation and autoimmune disorder (asthma for example, clone disease (Crohn ' s disease), diabetes, graft versus host disease (organ rejection), inflammatory bowel, lupus erythematosus, multiple sclerosis, psoriasis, psoriatic arthritis, rheumatic arthritis, thrombosis, vasculitis etc.), communicable disease (anthrax (Bacillus anthracis) for example, cholera (vibrio cholerae), cytomegalovirus, enterotoxin type coli strain, foot and mouth disease virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papillomavirus, influenza virus, malaria (plasmodium falciparum), Snow watt of gram virus of Measles virus, the plague (plague bacillus), Pseudomonas aeruginosa, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, streptococcus aureus, Transmissible gastroenteritis virus, trypanosome (trypanosoma bocagei), tuberculosis, SARS) etc.
In some specific embodiments, utilize the present invention to produce the self antigen IA-2ic of diabetes (for example at), influenza antigen, anthrax antigen, HPV antigen, Antibody of Influenza etc. (referring to example) of hGH, autoimmune disorders.
In some embodiments of the invention, produce the total length heterologous protein.That is to say the protein that complete generation occurring in nature exists or prepares or design in another case in seedling.In some described embodiment, the protein that is produced does not have any other sequence.In other embodiments, only produce a part of protein, be generally and have a known required active part in (for example, active territory of the epi-position of antigen protein, zymoprotein etc.).In certain embodiments, the part that is produced is the characteristic of protein or polypeptide.In certain embodiments, the part that is produced comprises epi-position.In certain embodiments, the part that is produced comprises the immunogen territory.In certain embodiments, the part that is produced comprises the protein territory.
In some embodiments of the invention, produce the syzygy of protein or polypeptide and other protein or peptide sequence.Known as affiliated field, any in can be for a variety of reasons uses syzygy.For instance, can be with protein or polypeptide and one or more convenient detections and/or the separated portions fusion of planning in seedling (seedling for example germinates), to produce.For instance, may merge with the protein paid close attention to or polypeptide and at available antibodies or other ligands specific and the known antigens epi-position that can be used for convenient isolated or purified.In addition or other, protein or the polypeptide paid close attention to and another polypeptide with regulation three-dimensional structure can be merged the protein for example paid close attention to or polypeptide with required three-dimensional arrangement to give.As another example, the protein paid close attention to or polypeptide and one or more will be able to be guided the meromixis of its Subcellular Localization (or secretion) in vegetable cell.Especially work as the protein paid close attention to or polypeptide and be proteantigen when (for example being used to prepare subunit vaccine), will need to produce the protein that merges with carrier molecule usually, for example to strengthen expression, stability and/or immunogenicity and/or to assist isolated or purified.Under known some the exemplary fusion collocation thing in field for example comprise that antibody epitope (for example His label etc.), immunogenic carrier albumen, Toxins,exo-, cholera, thermo-sensitivity enterotoxin etc. are (for example referring to Wella people such as (Vella), biotechnology (Biotechnology) 20:1,1992; Kai Li people such as (Kelly), immunology (Immuology) 113:163,2004; Bart's profit people such as (Buttery), London RCP magazine (JR Coll Phuysicians Lond) 34:163,2000; Jie Kabuxun people such as (Jacobson), Minerva paediatrics magazine (Minerva Peditr.) 54:295,2002; Moral Wuerzburg people such as (Dertzbaugh) infects and Journal of Immunology (Infect Immunol.) 61:48 1993; Na Saer people such as (Nashar), vaccine (Vaccine) 11:235,1993; In outstanding Vista people such as (Liljeqvist), immunization method magazine (J.Immunol.Methods) 210:125,1997; This tal fibre people such as (Stahl), periodical (Proc.Natl.Acad.Sci.USA) 86:6283 of institute of NAS, 1989; Ulrich people such as (Ulrich), virus research progress (Adv.Virus Res.) 50:141,1998; Smith people such as (Smith), virusology (Virology) 348:475,2006; Te Erben people such as (Turpen), biotechnology (Biotechnology) 13:53,1995).
A kind of special fusion collocation thing that the present invention is contained is a heat-stable protein, such as from the lichenase B of Clostridium thermocellum (Clostridium thermocellum) (for example referring to the U.S. Provisional Patent Application case 60/472 of application on May 22nd, 2003, on May 24th, 495 and 2004 application and on March 24th, 2005 with the disclosed PCT application case of WO2005/026375 US04/016452).Total length lichenase (about 35kDa) is made up of signal peptide, catalytic domain, enrichment Pro-Thr frame and grappling territory (referring to Figure 17).According to the present invention, can lack enrichment Pro-Thr frame and grappling territory in the fusion rotein.In addition, the natural signals peptide can be replaced through operator in plant.
Amino acid 32-84) and C-terminal district (C: amino acid 85-246) catalytic domain of described lichenase has ring structure, is divided into two districts: N-terminal district (N:.These two districts can separate at the ring structure place and the ring-type exchange does not influence enzymic activity so that described molecule is easier to accept to insert.Contact place between these two districts introduces multiple clone site (MCS), and 6 * His label and sequence KDEL are put in 3 ' end through exchange carrier respectively with purifying and the reservation of facility in endoplasmic reticulum.Figure 17) illustrate the modified lichenase (LicKM of molecular weight for about 27kDa; The gene pool number of landing DQ776900) synoptic diagram.Target protein or peptide sequence can be expressed as the inside syzygy (allowing to express simultaneously the feature of a plurality of targets) among N or C-terminal syzygy and/or the MCS.
LicKM make syzygy become scale from little peptide to molecule up to about 100kDa or higher full-length proteins.LicKM can keep its enzymic activity down at high temperature (65 ℃), and this is for also being applicable to the characteristic of syzygy usually.Because the thermal treatment of carrying out under 65 ℃ 10 minutes can be removed nearly 50% pollution vegetable-protein, so this feature makes it possible to easily and cost effectively reclaims target protein.During purifying, can follow the trail of the LicKM fusion rotein by monitoring lichenin enzymic activity.LicKM provides other advantage as the purposes of carrier molecule, comprises can be used for strengthening expression and incorporating a plurality of polypeptide (for example, multi-vaccine antigenic determinant) of being paid close attention to into.
Plant
Teaching of the present invention is applicable to multiple different plant.In general, can express any plant of being introduced that constructs body as described herein all is applicable among the present invention.In many examples, needs are used seedling so that improve the generation speed of protein/polypeptide.As described herein, in many examples, utilize the germination seedling.As known in the affiliated field, most of young shoot is grown fast, thereby produces edible plants by the seed of storage.Yet one of ordinary skill in the art will understand, and term " germination seedling " more is usually used in referring to seedling in this article, and no matter whether kind classifies as " young shoot " usually.Thereby the growth sufficiently long time with have sufficient green bio matter and allow to introduce and/or express as the expression that this paper was provided construct body any plant (it will be appreciated that correlation time visual as described in expression construct the transmission of body and/or expression pattern and change) can be considered herein " germination seedling ".
In the many embodiment of the present invention, utilize the edible plants plant of the individual edible (nontoxic) of protein or polypeptide (that is, will the throwing and) to described individuality.
In some preferred embodiment, plant of the present invention can be Btassica or Arabidopsis.Be suitable for transforming and comprise clover, mung bean, radish, wheat, leaf mustard, spinach, Radix Dauci Sativae, beet, onion, garlic, celery, rheum officinale, leafy plant (such as Caulis et Folium Brassicae capitatae or lettuce, watercress or Chinese celery), medicinal herbs (such as parsley, peppermint or trifolium), Cauliflower, Caulis et Folium Brassicae capitatae, soybean, root of Szemao crotalaria, edible flower (such as Sunflower Receptacle etc.) as some edible suitable plants of germination seedling.
Tested the adaptability of various plants species in the present invention's practice.Test multiple different beans and other species by the suitability that Agrobacterium constructs the virus vector generation heterologous protein of body (being introduced by Agrobacterium osmose process as described herein) emission, for example comprised red bean, clover, barley, Caulis et Folium Brassicae capitatae, Bill's jumping bean, buckwheat, Caulis et Folium Brassicae capitatae, Cauliflower, trifolium, kale, trigonella, flax, garbanzo, green soya bean, the Japan spinach, kale, card nurse wheat, wild cabbage, junge Erbsen, mung bean, leaf mustard, Kidney bean, radish, red clover, soybean, the spot pea, Sunflower Receptacle, turnip, (for example referring to example 5 and 8 Fig. 9 etc.) such as yellow folder beans.The invention provides afterclap, promptly some pea kind is particularly suitable for described operation.For instance, according to the present invention, Bill's jumping bean, green soya bean, junge Erbsen, spot pea and/or yellow folder beans are particularly useful in this one side of the present invention.Therefore, in certain embodiments, the invention provides in one or more described plants and to use the related protein that launching code pays close attention to or the virus of polypeptide to construct body (promptly, RNA with plant virus feature) agrobacterium vector produces protein or polypeptide (for example, antigen, antibody and/or other protein).In certain embodiments, RNA has the feature (and/or comprising the AlMV sequence) of AlMV.In certain embodiments, described RNA has the feature (and/or comprising the TMV sequence) of TMV.
Should be appreciated that, on the one hand, the invention provides the target protein that expression pays close attention to or the seedling (for example, germination seedling) of polypeptide.In certain embodiments, seedling is to grow from transgenic seed; The present invention also provides can be by method generation as herein described and/or the seed that utilizes.Can make at the transgenic seed rudiment of any gene of paying close attention to and the protein or the polypeptide of inducing generation to pay close attention to according to circumstances.For instance, can allow to express the seed germination of any gene of being paid close attention to and induce in the following manner: i) virus infection; Ii) Agrobacterium infiltration; Or iii) contain virus genomic bacterium.Can allow to express at the genetically modified seed germination of the heavy chain of any monoclonal antibody or light chain and induce in the following manner: i) virus infection to produce full-length molecule; Ii) Agrobacterium infiltration; Or iii) with containing virus genomic microbionation.Can allow to express genetically modified seed germination, and be used to produce the global function molecule in the following manner: i) virus infection at one or more components in the complicated molecule that comprises a plurality of components (such as sIgA); Ii) Agrobacterium infiltration; Or iii) with containing virus genomic microbionation.Can make from the seed germination of healthy non-transgenic plant and be used to produce target sequence in the following manner: i) virus infection; Ii) Agrobacterium infiltration; Or iii) with containing virus genomic microbionation.
In certain embodiments, seedling is from not genetically modified seed growth.Usually, described seedling will comprise the protein that guiding pays close attention to or the virus sequence of expression of polypeptides.In certain embodiments, described plant also can comprise the Agrobacterium sequence, comprises the sequence of " emission " virus sequence according to circumstances.
The system of marking protein or polypeptide in plant
According to the present invention, can use any marking protein or polypeptide in seedling (seedling for example germinates) in the multiple different system.In certain embodiments, produce transgenic cell line or seed, its rudiment subsequently and growth for some time, (for example in the germination seedling) produces included protein or polypeptide in the transgenic sequence in the seedling tissue thus.The typical technology that produces transgenic plant cells and/or seed comprises transgenosis and the particle bombardment or the electroporation of agrobacterium tumefaciens (Agrobacterium tumefacien) mediation.
In certain embodiments, utilize transient expression system.The typical technology that produces the transient expression of protein or polypeptide in plant tissue utilizes plant virus.It is a kind of conversion plumule comparatively quick and with low cost and the method for germination seedling that virus transforms, and it can be gathered before obtaining product and not have experiment or a hysteresis from generation to generation.On the other hand, the virus of attenuation can not infect other plant, the potential environmental problem that causes.
The invention provides and have the virus expression systems advantage expression system of (for example, express fast, high yield) and Agrobacterium-mediated Transformation advantage (for example controlled dispensing).Specifically, as hereinafter discussing in detail, the invention provides Agrobacterium and construct body (promptly, therefore in Agrobacterium, duplicate and can transmit by Agrobacterium the body of constructing of vegetable cell) comprise the system of plant promoter, described plant promoter is behind introduced plant, guiding is loaded with the expression of virus sequence (for example, comprising the virus replication sequence) of the gene of the protein paid close attention to or polypeptide.This system allows controlled in plant, high-level transient expression protein or polypeptide.
Hereinafter will be independently further discuss the embodiment of a plurality of different expression systems in detail, the some of them expression system produces transgenic plant and other provide transient expression.For any described technology, the one of ordinary skill in the art that read the application's case will understand the scheme of how to regulate and optimizing in seedling tissue (seedling for example germinates) marking protein or polypeptide.
Agrobacterium-mediated Transformation
Agrobacterium is the representative kind of Gram-negative Rhizobiaceae.These species cause canker, such as crown gall and hairly root disease.In dedifferenting plant tissue (this is the feature of tumour), Agrobacterium produces and to be called the amino acid derivative of opine (opines) and through plant katabolism.The bacterial gene of being responsible for the opine expression is the facility source of the controlling elements of chimeric expression box.According to the present invention, can use Agrobacterium transformation system to produce only than the early seedling of gathering of maturation plant (for example, the germination seedling comprises edible germination seedling).Conversion method for agrobacterium is easy to be used for the seedling (for example, germination seedling) of secondary expression pharmaceutical protein.
In general, comprise by cultivating altogether with the Agrobacterium-mediated Transformation plant and transform the vegetable cell that grows in the tissue culture medium (TCM) with the agrobacterium tumefaciens that is loaded with plant/bacteria carrier.Described carrier contains the gene of the pharmaceutical protein of encoding.Agrobacterium is transferred to carrier in the plant host cell and uses antibiotic therapy that it is removed subsequently.To expressing selecting of pharmaceutical protein, make its differentiation and final regeneration become complete shoot (Helen people such as (Hellens), molecular biology of plants (Plant Molecular Biology) (2000) 42 (819-832) through transformed plant cells; Pi Long-Shi Mizi people such as (Pilon-Smits), plant physiology (Plant Physiolog.) (in January, 1999) 119 (1): 123-132; Barfield (Barfield) and general Ah (Pua) vegetable cell report (Plant Cell Reports) (1991) 10 (6/7): 308-314); Rui Wa people such as (Riva), biotechnology magazine (Journal of BiotechnologyI) (on December 15th, 1998) 1 (3) is incorporated herein by reference separately).
The bacillus Expression carrier that is used for the present invention comprise the coding pharmaceutical protein through design with the gene (or expression cassette) in plant, operated and in the sequence of following of described expression cassette upstream and downstream.Follow sequence to be generally plasmid or viral source and provide required feature transferring to from the DNA of bacterium in the required plant host described carrier.
Basis bacterium/plant vector is constructed body preferably provides the protokaryon replication orgin of broad host range, protokaryon selectable marker.Suitable protokaryon selectable marker comprises the resistance to microbiotic (such as Ampicillin Trihydrate (ampicillin) or tsiklomitsin (tetracycline)).Other dna sequence dna of well-known other function of coding in affiliated field also can be present in the carrier.
Agriculture bacillus mediated DNA shifts to plant chromosome needs the agrobatcerium T-DNA sequence.When constructing bacillus Expression and construct body, remove the T-DNA gene of tumor inducing usually, and replace with the sequence of coding pharmaceutical protein or polypeptide.Because the initial T-DNA of T-DNA border sequence district is incorporated in the Plant Genome, so it is kept.If be not easy to detect the expression of pharmaceutical protein, bacterium/plant vector is constructed body and also will be comprised and be suitable for the selectable marker gene of determining whether vegetable cell has transformed so, for example nptII kantlex (kanamycin) resistant gene.
The Ti sequence can be on identical or different bacterium/plant vector (Ti-plasmids).The Ti sequence comprises virulence gene, and it is encoded one group and causes that the T-DNA gene excises, shifts and be incorporated into the protein (Si Xieer (Schell), science (Science) (1987) 237:1176-1183) in the Plant Genome.Be suitable for other sequence that heterologous sequence is incorporated in the Plant Genome can be comprised that also transposon sequence etc. is for homologous recombination.
Some constructs the protein expression box that body will comprise that coding is paid close attention to.One, two or more expression cassettes can be used to specify in the conversion process.Recombinant expression cassettes also contains following at least element except that the sequence of coding pharmaceutical protein: promoter region, plant 5 ' not translation sequences, initiator codon (initiator codon that whether has himself on expressed genes is decided) and transcribe and the translation termination sequence.In addition, can comprise in expression cassette of the present invention or the mosaic gene and transcribing and translation termination.In the time of suitably, allow the signal secretion sequence of protein processing and transposition also can be included in the expression cassette.
Multiple promotor, signal sequence and transcribe with translation termination and all for example be described in the people such as (Lawton) that pauses sieve, molecular biology of plants (Plant Mol.Biol) (1987) 9:315-324 or United States Patent (USP) the 5th, in 888, No. 789, it is incorporated herein by reference.In addition, usually will be at the structure gene of antibiotics resistance with the factor that elects (Fu Leili people such as (Fraley), institute of NAS periodical (Proc.Natl.Acad.Sci., USA) (1983) 80:4803-4807), it is incorporated herein by reference.Described box 5 ' be easy to insert in the carrier that is pre-existing in the Restriction Enzyme site of 3 ' end place uniqueness is feasible.
Other binary vector system description that is loaded with at least one T-DNA border sequence that is used for agriculture bacillus mediated conversion is in PCT/EP99/07414, and it is incorporated herein by reference.The further argumentation of relevant agriculture bacillus mediated conversion is found in Kiel literary composition S.B. (Gelvin, S.B.), " agriculture bacillus mediated Plant Transformation: the biology of " genetically manipulated " instrument behind (Agrobacterium-Mediated Plant Transformation:the Biology behind the " Gene-Jockeying " Tool) ", microbiology and molecular biology summary (Microbiology and MolecularBiology Reviews), 67 (1): 16-37 (2003), (it is incorporated herein by reference), reference wherein, all described documents all are incorporated herein by reference; Lao Lunsi A (Lorence A), Welbert R. (Verpoorte R.), transgenosis in the plant and expression (Gene transfer and expression in plants.), molecular biology method (Methods Mol Biol.), (2004) 267:329-50, it is incorporated herein by reference.
In certain embodiments of the invention, the bacterium of use except that Agrobacterium is with in the nucleotide sequence introduced plant.For example referring to the special W of Bruce strategic point people such as (Broothaerts W), carry out transgenosis (Genetransfer to plants by diverse species of bacteria) by the different bacterium kind to plant, nature (Nature) (2005), 433 (7026): 629-633, it is incorporated herein by reference.
Plant by Agrobacterium (or other bacterium) infection prepares seed, thus the protein that the introducing coding is paid close attention to or the required heterologous gene of polypeptide.Gather described seed, drying, the existence and the expression of cleaning and test vigor and required gene product.Once determining, just be stored under suitable temperature, humidity, health and the safety condition seed stock thing stand-by.Subsequently by the whole plant of protoplast regeneration through cultivating, for example, as Ai Wensi people such as (Evans), culture plant cell handbook (Handbook of Plant Cell Cultures), the 1st volume: New York William McMillan publishing company (MacMillanPublishing Co.New York), 1983; With Vassili I.R. (Vasil I.R.) (volume), culture plant cell and somatic cell genetics (Cell Culture and Somatic Cell Genetics of Plants), the Orlando, the academic press (Acad.Press, Orlando), the I volume, 1984, with the III volume, 1986, it is incorporated herein by reference.
In certain embodiments, plant not regeneration become strain.For instance, in certain embodiments, plant only regenerated to the germination seedling stage.In other embodiments, thus the whole strain plant of regeneration produces the seed stock thing and produces seedling (for example, germination seedling) used according to the invention by the seed of described seed stock thing.
Can go out protoplastis and cultivate all plants that obtain whole strain aftergrowth by Agrobacterium-mediated Transformation is separable according to the present invention, thus the whole strain plant of reclaiming the metastatic gene that contains protein that coding pays close attention to or polypeptide.Known in fact all plants all can be by regenerating through culturing cell or tissue (including, but is not limited to produce all main species of edible young shoot).Some suitable plants comprise clover, mung bean, radish, wheat, leaf mustard, spinach, Radix Dauci Sativae, beet, onion, garlic, celery, rheum officinale, leafy plant (such as Caulis et Folium Brassicae capitatae or lettuce, watercress or Chinese celery), medicinal herbs (such as parsley, peppermint or trifolium), Cauliflower, Caulis et Folium Brassicae capitatae, soybean, root of Szemao crotalaria, edible flower (such as Sunflower Receptacle etc.).
Can be different by mode with the difference of plant species through the transformant aftergrowth.Yet one of ordinary skill in the art will recognize, the suspension through the conversion protoplastis that contains the heterologous gene copy at first is provided usually.Form callus and can grow sprouting and take root subsequently by callus induction.Perhaps, can induce plumule to form by protoplastis suspension.Make that these plumules are the same with natural plumule to germinate to form plant.Be soaked in seed in the water or water sprays seed so that the moisture content of seed is increased between the 35-45%, thus initial germination.Go on for making to germinate, in the air that moisture reaches capacity, preserving seed under controlled temperature and the flow conditions usually.Substratum will contain each seed amino acid and hormone (such as growth hormone and phytokinin) usually.It also is useful in the described substratum that L-glutamic acid and proline are added to, especially for all the more so such as species such as clovers.Grow sprouting and take root and take place simultaneously usually.Effective regeneration will be decided on substratum, genotype and cultivation course.If control this three variablees, regeneration can reproduce and repeat fully so.
Be selfing and identify the unseparated transgenic plant of isozygotying by maturation plant through the transformed plant cells growth.Described selfing plant produces the seed of the metastatic gene that contains protein that coding pays close attention to or polypeptide.These seeds can germinate and grow into seedling (for example, the germination seedling) stage to produce protein or the polypeptide of being paid close attention to.
In related embodiment, transgenic seed (being loaded with the protein that the coding that is incorporated into usually in the genome pays close attention to or the metastatic gene of polypeptide) can form the seed product and follow relevant how to make growth of seedling arrive the suitable stage (for example, growing into the germination seedling stage) gathered and/or offerd medicine or the specification sheets allocated is sold together with as described herein.In other related embodiment, the hybridization or the novel kind that comprise desired characteristic (that is, the protein paid close attention to of coding or the metastatic gene of polypeptide) are to be grown by the selfing transgenic plant.
Blindly connect integration
According to the present invention, by particle bombardment or electroporation with dna fragmentation be integrated directly into also can be used in the vegetable cell genome protein that coding is paid close attention to or polypeptide expression construct in the body introduced plant tissue (for example referring to, Kai Kete J.R. (Kikkert, J.R.), Xiu Misidun people such as (Humiston), in vivo cell and developmental biology (In VitroCellular; Developmental Biology.) plant: tissue culture association magazine (Plant:Journal of the TissueCulture Association.) (in January, 1999/February) 35 (1): 43-50; Doubly special G.W. (Bates, G.W.), the Tallahassee, Florida State, (the Florida State University of Florida State University, Tallahassee, FL.), molecular biotechnology (Molecular Biotechnology) (in October, 1994) 2 (2): 135-145).More particularly, can will contain the carrier of gene of protein that coding pays close attention to or polypeptide by in the various technology introduced plant cells.As indicated above, carrier can comprise the selectable marker that can be used in the vegetable cell.Described carrier also can comprise its sequence of selecting and breed of permission in secondary host, such as the sequence that contains replication orgin and selectable marker.Common secondary host comprises bacterium and yeast.In a preferred embodiment, secondary host is intestinal bacteria, and replication orgin is a colE1 type replication orgin, and selectable marker is the gene of coding Ampicillin Trihydrate resistance.Described sequence be affiliated field well-known and in the market on sale (for example, the many Cologne Imtech of California, USA Paro Austria (Clontech, Palo Alto, CA) or (Stratagene of Si Tante genome company in California, USA La Jolla city, La Jolla, CA)).
Also carrier modification of the present invention can be become mesophyte to transform plasmid, it contains with agrobacterium tumefaciens carrier homologous zone, from T-DNA frontier district of agrobacterium tumefaciens and mosaic gene or expression cassette mentioned above.Other carrier can comprise and not cause the agrobacterium tumefaciens of canker to induce plasmid.
According to embodiments of the invention, the direct conversion of carrier of the present invention comprise by use micropipet mechanically shift recombinant DNA with described carrier direct microinjection in vegetable cell (for example referring to, this prestige of clo (Crossway), molecular genetics and General Genetics (Mol.Gen.Genet.), 202:179-185,1985, its by reference herein).Also can use polyoxyethylene glycol genetic stocks to be transferred in the vegetable cell (for example referring to, Ke Lunsi people such as (Krens), nature (1982) 296:72-74).The other method of introducing nucleic acid segment for utilize in beads or particle matrix or have on the surface high speed ballistic penetration that the small-particle of nucleic acid carries out (for example referring to Ke Laien (Klein) people of etc.ing, (1987) 327:70-73 naturally; Ke Nuodesen and Muller (Knudsen and Muller) phytology (Planta) (1991) 185:330-336)).Another kind of introducing method is for merging protoplastis and other minicell, cell, lysosome or other other entity that can merge lipid surface body with (for example referring to people such as Fu Leili, periodical (1982) 79:1859-1863 of institute of NAS).Also can be with carrier of the present invention by (for example referring to periodical (1985) 82:5824 of Fromm institutes of people NAS such as (Fromm)) in the electroporation introduced plant cell.According to this technology, construct electroporation plant protoplast under the situation that the plasmid of body exists containing gene.The electricimpulse of high field strengths reversibly permeates microbial film, thereby allows to introduce plasmid.Plant protoplast through electroporation forms cell walls again, and division also forms plant callus, its renewable formation germination seedling of the present invention.One of ordinary skill in the art will understand the vegetable cell that how to utilize these methods conversions to can be used for producing edible germination seedling.
Virus transforms
According to the present invention, use plant viral vector to infect seed, plumule, germination seedling and in wherein producing foreign protein.With regard to this point, infect any method that comprises in viral genome or its part introducing cell, include, but is not limited to natural viral infection method, wearing and tearing, inoculation etc.Described term comprises to be introduced geneome RNA transcript or its cDNA copy in the cell.Viral genome need not to be the complete genome group, is enough to allow the sequence of duplicating but will contain usually.Described genome codified rdrp virus and can contain and duplicate required any cis acting nucleic acid elements.The high level expression (for example by the Tobamovirus carrier) of the alien gene of coding small peptide and big complex proteins for example is described in the Mike horse and examines people such as (McCormick), periodical (1999) 96:703-708 of institute of NAS; Xiong Gu people such as (Kumagai), gene (Gene) (2000) 245:169-174; And Wei Erqi people such as (Verch), immunization method magazine (J.Immunol.Methods) (1998) 220,69-75, described document is incorporated herein by reference separately).So verified ability of plant viral vector with expression small peptide and big complex proteins.
In certain embodiments, utilize host/viral system to produce the seedling (for example, young shoot) of expressing medical protein (such as Regular Insulin, GAD and the IA-2 relevant) with type 1 diabetes.The seedling that is produced by virus infection provides and turns out to be safe protein of being paid close attention to or polypeptide source.For instance, young shoot is not subjected to the animal pathogenic body pollution.For instance, different with tobacco, can under the situation of purifying not, use by per os at least in theory from the protein of edible young shoot, significantly reduce cost thus.
In addition, because genes involved (coding paid close attention to protein or polypeptide) is introduced and can be grown in a couple of days in plant-scale virus, so virus/seedling (for example young shoot) system also is provided for comparatively simple and easy, the cheap approach that enlarges in proportion and make.By contrast, transgenic plant needed to reach 5-7 before sufficient seed or vegetable material can be used for large-scale experiment or commercialization.
According to the present invention, plant RNA virus has some advantage, and this makes it become the most noticeable carrier that is used to express foreign protein.Determine the molecular biology and the pathology of various plants RNA viruses, and had the knowledge of quite a lot of viral biology, genetics and regulating and controlling sequence.Most of plant RNA virus has less genome and infectious CDNA clones can be used for convenient genetic manipulation.After the infectious virus material entered the susceptible host cell, it promptly duplicated and reaches high level and (inoculation back 1 to 10 day) diffusion in whole plant rapidly.Be easy to reclaim virus particle from infected tissue economically.Virus has the host range of broad, makes it possible to use the single body of constructing to infect several susceptible species.These features are easy to transfer to young shoot.
Fig. 1 describes and uses plant virus to express some kinds of Different Strategies of alien gene.Can be by replacing a kind of virogene with required sequence, external sequence being inserted virus genomic appropriate position or expressed external sequence by external peptide being fused in the viral structural protein.In addition, any trans-complementation by viral critical function in these methods capable of being combined is expressed external sequence.Exist multiple different strategy as the instrument that uses tobacco mosaic virus (TMV) (TMV), alfalfa mosaic virus (AlMV) and its mosaic in the plant that is infected by the virus, to express external sequence.
The AlMV genome is the representative of Bromoviridae (Bromoviridae) virus family and forms (Fig. 2) by three geneome RNAs (RNA1-3) and subgenomic RNA (RNA4 ).Geneome RNA 1 and 2 encode respectively virus replication zymoprotein P1 and 2.Geneome RNA 3 Codocytes are to cell movement albumen P3 and coat protein (CP).CP is by the translation of geneome RNA 3 synthetic subgenomic RNAs 4, and required for beginning to infect.Studies confirm that CP relates to multiple function, comprise that genome activates, duplicates, rna stability, symptom forms and the RNA involucrum (for example referring to, bohr people such as (Bol), virusology (Virology) (1971) 46:73-85; Fan Dewosen people such as (Van Der Vossen), virusology (1994) 202:891-903; Excellent Si Bofu people such as (Yusibov), virusology 208:405-407; People such as excellent Si Bofu, virusology (1998) 242:1-5; People such as bohr, (summary (Review), 100 reference (100refs.)). general virology magazine (J.Gen.Virol.) (1999) 80:1089-1102; De Graff (De Graaff), virusology (1995) 208:583-589; Refined scholar primary people such as (Jaspars), virus research progress (Adv.Virus Res) (1974) .19,37-149; Luo Siqi-Fu Laisi (Loesch-Fries), virusology (1985) 146:177-187; Niermann people such as (Neeleman), virusology (1991) 181:687-693; People such as Niermann, virusology (1993) 196:883-887; Fan Deku clothing people such as (Van Der Kuyl), virusology (1991) 183:731-738; People such as Fan Deku clothing, virusology (1991) 185:496-499).
The involucrum of virus particle partly arrives the long most important apart from motion and systemic infection of not inoculation part for virus from the inoculation of seed, plumule or seedling (for example, germination seedling).According to the present invention, inoculation can take place in any stage of development of plants.In plumule and young shoot, the diffusion of virus inoculation should be exceedingly fast.With the AlMV virus particle with unique CP (24kD) involucrum, thereby form the above particle of a class.Size of described particle (30 to 60nm long and 18nm diameter) and shape (spherical, spheroid or shaft-like) are decided on the size of involucrum RNA.Think the N-terminal of when assembling AlMV CP be positioned on the surface of virus particle and as if can the viral interference assembling people such as (, virusology (1971) 6:73-85) bohrs.In addition, the AlMV CP that has extra 38 amino acid whose peptides at N-terminal forms particle in vitro and keeps biological activity people such as (, general virology magazine (1995) 77:567-573) excellent Si Bofu.
AlMV has the host range of broad, and it comprises valuable crop on the various agricultural, comprises plant seed, plumule and young shoot.Generally speaking, these features make AlMV CP become good carrier molecule material standed for, and make AlMV become the most noticeable candidate's carrier of expressing external sequence in the plant in the young shoot stage that is in growth.In addition, when expressing by allos carrier (such as TMV), AlMV CP under the infective situation of viral interference not with TMV genome involucrum (it is incorporated herein by reference for people such as excellent Si Bofu, periodical (1997) 94:5784-5788 of institute of NAS).This allows the vector virus of the AlMV CP of use TMV conduct and the fusion of external sequence.
TMV (prototype of tobacco mosaic virus (TMV)) has the genome of being made up of the single just NRA through 17.0kD CP involucrum, and described 17.0kD CP produces rod shaped particles (300nm is long) (Fig. 2).CP is the unique structural protein of TMV and is viral involucrum and viral in infected host middle and long distance motion required (Sai Tuo people such as (Saito), virusology (1990) 176:329-336).183 and 126kD albumen be to obtain and be virus replication required (Ishikawa people such as (Ishikawa), nucleic acids research (Nucleic Acids Res.) (1986) 14:8291-8308) by geneome RNA translation.30kD albumen is that the cell of virus arrives cell movement albumen (Mei Xi people such as (Meshi), European molecular biology magazine (EMBOJ.) (1987) 6:2557-2563).Motion albumen and coat protein are to obtain (Hunter people such as (Hunter), nature (1976) 260:759-760 by the subgenomic mRNA translation; Bu Enlin people such as (Bruening), virusology (1976) 71:498-517; Bi Qi people such as (Beachy), virusology (1976) 73:498-50 is incorporated herein by reference separately).
The genomic schematic description of AlMV and TMV is illustrated among Fig. 2.The RNA1 of AlMV and 2 encode respectively replicase protein P1 and P2; Geneome RNA 3 Codocytes are to cell movement albumen P3 and virus capsid protein (CP).CP is obtained by 4 translations of geneome RNA 3 synthetic subgenomic RNAs.The 126kD of TMV and 183kD albumen are required for duplicating; 30kD albumen is that virocyte arrives cell movement albumen, and 17kD albumen is the CP of virus.CP and 30kD albumen are to be obtained by the subgenomic RNA translation.The position of arrow indication subgene group promotor.
Flower transforms
Can be used for other method in the gene introduced plant cell of the protein paid close attention to of coding or polypeptide is comprised the flower that transforms plant.Can immerse by flower and realize in the agrobacterium tumefaciens solution that Arabidopis thaliana transforms (Ke Disi (Curtis) and the nurse (Nam) of receiving, transgenic research (Transgenic Research) (August calendar year 2001) 104:363-371 plant; The people such as (Qing) of the Qin, molecular breeding: the New Policy of plant improvement (Molecular Breeding:New Strategies in PlantImprovement) (in February, 2000). (1): 67-72).In the kind subgroup that produces by " immersion " plant, form through transforming plant.When the specified time of flower development, there is aperture in the ovary wall, agrobacterium tumefaciens is entered ovary inside by described aperture.After in entering ovary, the agrobacterium tumefaciens breeding also transforms indivedual ovules (this people such as (Desfeux) of this excellent gram of enlightening, plant physiology (Plant Physiology) (in July, 2000) 123 (3): 895-904).In ovary, follow the typical path that seed forms through transforming ovule.
Agriculture bacillus mediated transient expression
As described herein, in the many embodiment of the present invention, need be in plant the system of (for example instantaneous) marking protein or polypeptide rapidly.Wherein the invention provides the described effective system of expressing fast in plant (especially seedling, seedling for example germinates) of a kind of realization, it utilizes Agrobacterium to construct body and transmits the protein that coding pays close attention to or the virus expression systems of polypeptide.
Specifically, according to the present invention, preparation " launching carrier ", it contains the Agrobacterium sequence that comprises replication sequence and also contains the plant virus sequence (comprising the self-replacation sequence) of the gene that is loaded with protein that coding pays close attention to or polypeptide.Preferably by allowing the systemic in fact Agrobacterium osmose process of transmitting with in the launching carrier introduced plant tissue.For instantaneous conversion, the not integration T-DNA of launching carrier copy temporarily remains resident in the nuclear and transcribes, and causes carrying expression of gene (Ka Peila people such as (Kapila), 1997).Different with virus vector, agriculture bacillus mediated transient expression can't make the expression of gene systematicness diffusion of being paid close attention to.An advantage of this system be to clone the gene of being longer than 2kb with produce can't utilize that virus vector obtains construct body (Wei is because of nit people such as (Voinnet), 2003).In addition, use described technology, can utilize more than one transgenosiss to transform plant, cause and to express and to assemble multimeric protein (for example, the antibody subunit of complex proteins).In addition, can or use mixed culture medium to obtain fine utilization by independent infiltration by permeating the possibility of carrying out multiple transgenosis coexpression altogether with different Agrobacteriums.
In certain embodiments, launching carrier comprises permission is selected (or detecting at least) and selected/detect in Agrobacterium sequence in the infiltration tissue.In addition, launching carrier is usually included in to transcribe in the plant and obtains the sequence that the viral RNA generation produces viral protein subsequently.In addition, the generation of viral protein and viral RNA causes the quick generation of a plurality of RNA copies of the medicine and pharmacology active protein that coding is paid close attention to.Described generation causes the quick target protein of being paid close attention to that produces in the relatively short time.Therefore can produce and be used to produce proteinic efficient system.
Utilize the Agrobacterium infiltration technology of virus expression carrier to can be used for producing the limited amount protein of paying close attention to, so that determining it checks expression level before whether being worth producing transgenic plant.In addition or other, utilize the Agrobacterium infiltration technology of virus expression carrier to be applicable to that quick generation can produce a large amount of proteinic plants as main production platform.Therefore, this transient expression system can use on technical scale.
Provide the multiple different Agrobacterium plasmids that can use in these and other aspects of the invention, in the binary plasmid or derivatives thereof any in addition, such as pBIV, pBI1221, pGreen etc.A large amount of appropriate carriers have been known in the affiliated field, and can be guided and/or modify according to known method in the affiliated field or method as herein described, so that can be used in the described method that this paper provides.
Figure 18 represents the synoptic diagram of particular exemplary launching carrier pBID4.This carrier contains the 35S promoter of cauliflower mosaic virus (DNA plant virus), and it orders about recombinant virus genomes initial transcribing in back in introduced plant; With the no terminator, i.e. the transcription terminator of Agrobacterium rouge alkali synthetase.Described carrier contains in addition and comprises and be used for virus replication (126/183K) and the cell tmv cdna group sequence to the gene of cell movement (MP).Described carrier contains the gene of transcribing the polypeptide that the coding under the control pays close attention to that inserts unique cloning site place in the tmv cdna group sequence and be in coat protein subgenomic mRNA promotor in addition.Because this " target gene " (promptly, the protein that coding is paid close attention to or the gene of polypeptide) replace the encoding sequence of TMV coat protein, so the gained virus vector is than contain the naked RNA of self-replacation that the CP carrier is difficult for the experience reorganization and can't effectively spreads and survive in described environment.A left side and right border sequence (LB and RB) delimited the boundary of transferring to the launching carrier district in the vegetable cell after utilization is loaded with the reorganization Agrobacterium infiltration plant of carrier.In will being loaded with the Agrobacterium introduced plant tissue of this carrier, (undertaken by the Agrobacterium osmose process usually, but can be undertaken by injection or alternate manner in addition) after, a plurality of single stranded DNAs (ssDNA) sequence copy and release in several minutes promptly between LB and RB, produced.Subsequently by virus replication these sequences of having introduced that increase.The translation of target gene causes a large amount of target protein or the polypeptide of accumulation within a short period of time.
In some embodiments of the invention, agriculture bacillus mediated transient expression produces the per kilogram plant tissue up to about 5g or more target proteins.For instance, in certain embodiments, produce the per kilogram plant tissue up to about 4,3,2,1 or the 0.5g target protein.In certain embodiments, produce the per kilogram plant tissue at least about 20-500mg, or about 50-500mg target protein, or about 50-200 or about 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190, or about 200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1500,1750,2000,2500,3000mg or more target proteins.
In some embodiments of the invention, these expression levels are to reach in about 6,5,4,3 or 2 weeks of infiltration.In certain embodiments, these expression levels be from introduce to express constructed body about 10,7,5,4,3,2 days or even 1 day in reach.Therefore, from introduce (for example infiltration) to the time of gathering usually less than about 2 weeks, 10 days, 1 all or still less.In addition, the extremely noticeable aspect of this embodiment of the present invention is, it allows from selecting in about 8 weeks of aminoacid sequence or the shorter time time of " preparation " expression study (even comprise) generation protein.In addition, each batch albumen all can produce in about 8 weeks, 6 weeks, 5 weeks or shorter time usually.One of ordinary skill in the art will recognize, the visual employed vegetation type of these quantity and slightly changing.Most of young shoot (comprising pea) will be in the scope of specified quantity.Yet the Ben Saimushi tobacco is because of grow slow (from much smaller seed), and may grow the long period (especially before infiltration).One of ordinary skill in the art will understand the adjusting of other expection according to the biology of the specified plant that is utilized.
Inventor of the present invention has used the launching carrier system to produce multiple target protein and polypeptide in multiple different seedling.Inventor of the present invention is surprised to find that, for example comprises that some pea kind of junge Erbsen, Bill's jumping bean, yellow folder beans, spot pea and green soya bean is particularly useful for putting into practice this aspect of the present invention (for example referring to example 7).
Inventor of the present invention is surprised to find that also various Nicotiana plants are particularly useful for putting into practice this one side of the present invention, especially comprise the Ben Saimushi tobacco.One of ordinary skill in the art will understand, and the Nicotiana plant is not regarded as " young shoot " usually.However, the present invention is teaching also, and Nicotiana seedling (especially Ben Saimushi tobacco seedling) is applicable to and puts into practice the present invention.In general, when putting into practice this embodiment of the present invention, the Ben Saimushi tobacco plant will be grown one section before infiltration (for example transmitting the Agrobacterium that contains launching carrier) is enough to time of allowing an amount of biomass to grow.Usually, described plant before infiltration growth greater than about 3 weeks, more generally greater than about 4 weeks or between time in about 5-6 week with accumulates biomass.
Inventor of the present invention is surprised to find that also effective although confirmation TMV and AlMV sequence can be constructed in the body at described launching carrier, in certain embodiments, the AlMV sequence is effective especially through transferrin matter or polypeptide for guaranteeing that high level produces.
Therefore, in some specific embodiment of the present invention, in pea seedling or Nicotiana seedling (for example, Ben Saimushi tobacco), produce protein or the polypeptide of being paid close attention to by the launching carrier that guides generation to be loaded with the AlMV sequence of the gene of being paid close attention to.
Body is constructed in expression
Being applicable to that many features of body are constructed in expression among the present invention will be special in the particular expression system to being discussed as mentioned.Yet, will be in describe in more detail herein applicable to some aspect of different expression systems.
Lift an example, in the many embodiment of the present invention, the protein that needs are paid close attention to or the expression of polypeptide (or nucleic acid) can be induced.In many described embodiment, the generation (and/or generation of sense-rna) of the protein that coding is paid close attention to or the RNA of polypeptide is under the control that can induce (for example external source induction type) promotor.But external source inducible promoter response external but not the internal stimulus thing increases or reduce the expression of transcript.Multiple environmental factors can be served as described outside stimulus thing.In certain embodiments of the invention, transcribe the inducible promoter that is heated (such as, heat-shocked promotor) control.
Outside inducible promoter can be specially adapted to situation controlled, adjustable growing environment.For instance, use the heat-shocked promotor, the temperature of the enclosed environment that can raise simply is to induce the expression of relevant transcript.Certainly, should be appreciated that, because outdoor temperature is uncontrollable, so never use thermal induction type promotor out of doors.When outdoor temperature rises to a certain degree, promotor will open the beginning.Similarly, when outdoor temperature reduces, promotor will be closed.Described temperature transition can take place in one day, for example made by day to express to open the beginning and close at night.Thermal induction type promotor (such as promotor as herein described) even can not use in the greenhouse, its influence degree and outdoor situation that is subject to weather transition is roughly the same.The growth phase of genetic engineering modified plant in the greenhouse is when expensive.By contrast, in system of the present invention, each variable can be controlled, thereby the expression of maximum can be when each the collection, realized.
Other outside inducible promoter of utilization comprises photoinduction type promotor according to the present invention.If lamp is opened all the time in the adjustable environment of sealing, can keep photoinduction type promotor so as the composition promotor.Open the expression of beginning associated retroviral thing during specified time that perhaps, can be between the growth period by turning on light simply.
In other embodiments, use chemical inducible promoter to induce the expression of relevant transcript.According to these embodiment, chemical can be sprayed simply or be sprayed on seed, plumule or seedling (for example seedling) and go up to induce the expression of relevant transcript.Can optionally accurately control spraying and spray and it is guided on specific seed, plumule or the seedling (for example seedling).Enclosed environment lacks and will make chemical away from expection recipient's dispersive wind or air-flow, so chemical rests on the recipient of expection.
Growing environment
One aspect of the present invention be controlled, under the regulation and control growth conditions, in plant, produce protein or polypeptide (and/or nucleic acid).According to the present invention, with relevant noticeable being characterised in that of generation protein or polypeptide in seedling (for example, the germination seedling), need be than working as the growth time of identical vegetation type at the time weak point that the Adulthood growth will utilize.In addition, in many cases, can utilize than arriving the size space that the space of needs is little that it is grown up fully when plant-growth.In general, in controlled, adjustable environment, express medical protein or polypeptide growing plant can than the engineered plant of growth hormone gene with still less effort, risk and regulation and control item quickly (because can in herborization more at an early age) pharmaceutical product is provided.Employed closed adjustable environment reduces or gets rid of the risk of nature cross-pollinatd plant among the present invention.
In the many embodiment of the present invention, protein or polypeptide are to express in the growing plants in closed system.Described system avoids the risk of environmental pollution and but the adjustable reproducing environment that can realize repeating comparable result also is provided.When in addition, described system need to allow for example by using the adjustable feature of inducible promoters as described herein or other to realize protein or expression of polypeptides is controlled induces.
In certain embodiments of the invention, plant is to grow in closed regulatable environment.In certain embodiments, closed regulatable environment is for can make house unit or the room of seed in indoor growth.All environmental factorss of the closed adjustable environment of may command.Because the growth of young shoot does not have light requirement, and illumination is very expensive, thus In a particular embodiment, make plant under the situation that does not have light in indoor growth (for example, growing into the germination seedling stage).
Other environmental factors that can regulate and control in closed adjustable environment of the present invention comprises temperature, humidity, water, nutrient, gas (for example, O 2Or CO 2Content or air cycle), chemical (small molecules is such as sugar and sugar derivatives, or hormone, such as plant hormone gibberic acid or dormin etc.) etc.
In the many embodiment of the present invention, plant is to grow under hydroponics.The hydroponics growing system provides multiple advantage.For instance, the hydroponics growing system usually need be than coming the plant of growth phase with quantity based on the littler space of the system of soil.Nutrient and other reagent can flow through the hydroponics system usually, make the control growing ratio based on the easier realization of the system of soil.Can make many aspects automatization of hydroponics growth.In addition, the collection of hydroponics growing plant is very usual.Can by being raise from its growth solution, plant be gathered simply.Need not during collection to purify.Can directly gather seedling (for example, germination seedling) from the hydroponics environment under the situation of not washing or cleaning minimizes the destruction of the material of being gathered.Destruction of plant and withered meeting are apoptosis-induced.During apoptosis, some proteolytic ferment becomes and has activity, and expressed medical protein or polypeptide in its degradable germination seedling causes the active reduction of protein therapeutic.The proteolysis of apoptosis induction can significantly reduce proteinic productive rate in the maturation plant.Use method of the present invention, for example owing to before plant extract protein, do not gathering, so preferably never can apoptosis-induced (that is, avoiding apoptosis).
In certain embodiments of the invention, plant (seedling for example germinates) is to grow in the pallet that can be between the growth period waters, sprays or spray any time the time.For instance, described pallet can be furnished with that one or more water, spray, spraying and water-freeing arrangement, its can be during the germination seedling development specified time and transmit and/or remove water, nutrient, chemical etc. when reaching exact amount.For instance, the seed demand suff water makes it keep moist.Excessive moisture is discharged in the waterways in the room floor by the hole in the pallet.Preferably handle draining in due course to remove deleterious chemical before being discharged in the environment again at it.
Another advantage of pallet is that it can be enclosed in the minimum space.Because seedling (seedling for example germinates) growth does not need light usually, so the pallet that contains seed, plumule or seedling (seedling for example germinates) can vertically closely be stacked in the top of another pallet, thereby the per unit floor area provides large number of biological matter in the house facility that makes up at these purposes especially.In addition, can be in house unit piling up along the horizontal line matrix trays.When seedling (has for example grown into stage of being suitable for gathering, about 2 days or 14 days or look the type of plant, the protein that is produced or polypeptide etc. and be decided to be the long period) after, be about to individual tray and move on to by artificial or automatic member (such as travelling belt) and process in the facility.
One of ordinary skill in the art are easy to understand and are applicable to the many aspects of putting into practice growth conditions of the present invention and technology.For instance, but when utilizing the abduction delivering system, need to select the time of abduction delivering so that protein or the polypeptide expression maximization of being paid close attention in the seedling when gathering.The generation maximization of protein of being paid close attention to when the abduction delivering when particular growth stage when specified number of days (for example after germination) in the plumule can cause gathering or polypeptide.For instance, germinate back 4 days the time can cause by the promotor abduction delivering synthetic than after 3 days or the protein of Duoing by the promotor abduction delivering after 5 days.It will be appreciated by those skilled in the art that and to realize the maximization expressed by normal experiment.In a preferred embodiment, gather seedling about 1,2,3,4,5,6,7,8,9,10,11 or 12 day the time after germination, seedling especially germinates.In other embodiments, make seedling, especially the Nicotiana seedling grows several weeks after germination.
But when utilizing composition but not during the abduction delivering system, can be after introducing encoding sequence gather seedling sometime the time.For instance, if, (for example transform the back in the time of about 1,2,3,4,5,6,7,8,9,10,11,12,13 or 14 day) in the time of can after being expressed in conversion, reaching its maximum so and gather the germination seedling growing early stage (for example in the plumule stage) by virus conversion germination seedling.Depend on the germination of seed, also can after conversion, grow 1,2,3 month or seedling (for example young shoot) for more time for seedling.
In general, after the expression of beginning medical protein, make seed, plumule or seedling (seedling for example germinates) growth up to the medical protein of being paid close attention to or the polypeptide of expressing q.s.In certain embodiments, q.s will be for providing the amount of treatment benefit to the patient when eating the biomass of being gathered with former state.Perhaps, q.s is for being concentrated or the described medical protein of purifying or polypeptide and it is deployed into the amount that individuality is provided the medical composition of treatment benefit after dispensing by biomass.
In certain embodiments, behind the inducing medical protein expression, make growth continue the germination seedling stage of gathering the germination seedling up to reaching.In particularly preferred embodiment, gather the germination seedling that lives.Gather the germination seedling that lives and have some benefits, comprise minimum effort and destruction.
Wherein, the invention provides a kind of unique system, it provides again and contains a large amount of expressed protein of being paid close attention to or the seedling biomass of polypeptide (and/or nucleic acid).No matter directly the form of medical composition still is processed in consumption, when seedling (seedling for example germinates) grows, can provide the medical composition in plant biomass and/or biomass source at lower cost to the human consumer in closed regulatable environment.In addition, the fact that can control the condition of growth of seedling makes product quality and purity constant.Closed adjustable environment of the present invention also can be avoided and stop scientist's many safety rules of the EPA of the engineered agricultural prods of growth hormone gene out of doors.
The protein/polypeptide preparation
The invention provides the range protein or polypeptide (and/or active nucleic acid) preparation and the composite that in seedling, produce.
In the many embodiment of the present invention, protein that is produced or polypeptide are not to separate from plant tissue, but provide under the situation of seedling (seedling for example germinates) of living.In certain embodiments, when the plant edible, directly provide the plant tissue that contains through expressed protein or polypeptide for consumption.Therefore, the invention provides the edible seedling biomass (for example, edible germination seedling) that contain through marking protein or polypeptide.
When edible seedling (seedling for example germinates) is expressed the medical protein of q.s or polypeptide and fresh consumption, in certain embodiments, before consumption germination seedling, can never occur gathering.In this way, guarantee before patient's throwing and medical protein, not have the proteolytic degradation of gathering the inductive medical protein to the needs treatment.For instance, the seedling (for example, germination seedling) of preparing consumption can be directly delivered to the patient.Perhaps, genetic engineering modified seed or plumule are passed to the patient who needs treatment and make it grow into the germination seedling stage by the patient.In a preferred embodiment, the doctor that maybe will treat the patient to the patient provides the supply of genetic engineering modified germination seedling, but the therefore continuous reserve of the germination seedling of some required medical protein of culture expression.This is meaningful especially for developing country's population of unable burden or transmission expensive drugs.The convenience of germination seedling of the present invention of growing makes germination seedling of the present invention conform with described developing country population needs especially.
In certain embodiments, before consumption or allotment plant biomass, for example handle by homogeneity, pulverizing, drying or extraction.In certain embodiments, isolated or purified goes out through marking protein or polypeptide and with it to be deployed into medical composition from described biomass.
For instance, can with live seedling (for example young shoot) grinding, pulverizing or fusion in the damping fluid that contains proteinase inhibitor to produce the biomass slurries.Preferred buffer is about 4 ℃.In certain embodiments, with biomass dry air, spraying drying, freezing or lyophilize.With identical in the maturation plant, some described methods (such as dry air) can cause the forfeiture of medical protein or polypeptide active.Yet, because the plant (seedling for example germinates) that is utilized is minimum and have large surface area and volumetric ratio usually, so this situation unlikely takes place.One of ordinary skill in the art will understand, and can use the technology of the collection biomass that many proteolysis that make medical protein or polypeptide minimize and be applied among the present invention.
Dispensing and medical composition
The invention provides the seedling of expressing medicine and pharmacology active protein or polypeptide, described protein or polypeptide still keep its medicinal activity when throwing and the host who needs is arranged.Preferred host comprises vertebrates, and preferred mammal is more preferably human.According to the present invention, the host comprises the beasts individuality, such as ox, sheep, Canidae, cat family etc.In a preferred embodiment, with the edible young shoot with treatment significant quantity oral administration with individual.In other preferred embodiment, provide the medicine and pharmacology active protein of pharmaceutical preparation form as described herein.
Can be in many ways pharmaceutical preparation of the present invention be thrown and the host, such as per os, through intestines, intranasal, without intestines, intramuscular or intravenously, per rectum, transvaginal, part, through eye, through lung or by contact use throw with.In a preferred embodiment, the medical protein oral administration and the host that will in seedling (for example young shoot), express.In another preferred embodiment, extract and/or medicine and pharmacology active protein that purifying is expressed in seedling (for example young shoot) and using it in the preparation of medical composition.According to known technical point in normal condition and the affiliated field from and protein purification.Described technology comprises such as methods such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis.
Composition of the present invention generally includes the medicine and pharmacology active protein of significant quantity or polypeptide and one or more organic or inorganics, the suitable carrier materials of liquid or solid medicine and pharmacology.
The medicine and pharmacology active protein that produces according to the present invention can use such as following formulation: tablet, capsule, lozenge, dispersion liquid, suspension, solution, capsule, emulsifiable paste, ointment, aerosol, powder packaging, liquor, solvent, thinner, tensio-active agent, isotonic agent, thickening material or emulsifying agent, sanitas, solid binder, as long as described proteinic biological activity is not destroyed by described formulation.
Some examples that can be used as the material of pharmaceutically acceptable supporting agent include, but is not limited to sugar, such as lactose, dextrose plus saccharose; Starch is such as W-Gum and yam starch; Mierocrystalline cellulose and its derivative are such as Xylo-Mucine, ethyl cellulose and rhodia; Powdered tragacanth; Fructus Hordei Germinatus; Gelatin; Talcum; Vehicle is such as theobroma oil and suppository wax; Oil is such as peanut oil, Oleum Gossypii semen, Thistle oil, Viscotrol C, sweet oil, Semen Maydis oil and soybean oil; Glycol is such as propylene glycol; Ester is such as ethyl oleate and Laurate ethyl; Agar; Buffer reagent is such as magnesium hydroxide and aluminium hydroxide, alginic acid, pyrogen-free matter water, isotonic saline solution, Ringer's solution (Ringer ' s solution), ethanol and phosphate buffer soln; And other nontoxic compatible lubricant, such as Sodium Lauryl Sulphate BP/USP and Magnesium Stearate; And judgement according to formulator, also can exist toner, releasing agent, coating agent, sweeting agent, seasonings and perfume compound, sanitas and antioxidant in the described composition (also referring to Lei Shi pharmacy complete works (Remington ' s PharmaceuticalSciences), the 15th edition, E.W. Martin (martin) ((the Mack Publishing Co. of company of Pennsylvania, America Easton city Mike press, Easton PA), 1975).For instance, the protein that can mix by routine, granulation, drageeing manufacturing, dissolving, freeze-drying or similar approach provides the medical composition form.
In certain embodiments, the effect that need prolong pharmaceutical preparation by the absorption that slows down through the medical protein of subcutaneous or intramuscular injection or polypeptide.The liquid suspension of water miscible crystallization or amorphous substance was realized a little less than this can have by use.The uptake rate of protein or polypeptide is then decided on its dissolution rate, but and its dissolution rate apparent size and form and decide.
Perhaps, by with protein dissolving or be suspended in realize in the oily mediator without intestines throw and proteinic delayed absorption.Make injectable storage tank form by forming proteinic microcapsule matrix with biodegradable polymer (such as polylactide-poly-glycollide).Opsin matter is than the character of polymer ratio and employed particular polymers and decide the may command rate of release.The example of other biodegradable polymer comprises poly-(ortho ester) and poly-(acid anhydrides).Storage tank formula injectable composite also can by protein is packaged in compatible liposome of bodily tissue or microemulsion in prepare.
Through intestines throw with protein or polypeptide formulations can solid, semisolid, suspension or emulsion form is introduced and can with mix such as any pharmaceutically acceptable supporting agents such as water, suspension agent and emulsifying agents.Also can throw and protein of the present invention or polypeptide by pump or sustained release form, especially all the more so when offeing medicine as preventive measures, so that prevent individual advancing of disease or improvement or delay to take a disease disease.
Protein that produces according to the present invention or polypeptide be particularly suited for medical composition form oral administration with.The characteristic of the seedling alive that can throw and be gathered (for example seedling) or visual desired protein or polypeptide product and the form of required end product and process for example dry air, lyophilize, extraction etc. in many ways.
In certain embodiments, independent oral uptake composition as indicated above or it is absorbed with food or feed or beverage.Oral administration and composition comprise the germination seedling; The extract of seedling (seedling for example germinates) and from the protein or the polypeptide that provide with dry powder, food, water-based or non-aqueous solvent, suspension or emulsion form of seedling purifying.
The example of non-aqueous solvent is propylene glycol, polyoxyethylene glycol, vegetables oil, fish oil and injectable organic ester.Aqueous carrier comprises water, water-alcohol solution, emulsion or suspension, comprise physiological saline, with common without intestines mediator (bufferedmedial parenteral vehicle), comprise that sodium chloride solution, woods Ge Shi dextrose solution (Ringer ' s dextrosesolution), dextrose add the chlorination sodium solution, contain the Ringer's solution of lactose or fixed oil through cushioning.
The example of dry powder comprises any seedling of drying (for example freeze-drying, dry air or spraying drying) (seedling for example germinates) biomass.For instance, can contain less than 5 weight % water content up to biomass by seedling being put into commercially available instrument air dryer dry air under about Fahrenheit 120 degree (120degrees Fahrenheit).The drying plant is further processed with further processing or by being ground into the powder with required size of mesh with the storage of blocks of solid form.Perhaps, lyophilize can be used for product to the dry air sensitivity.Can by product is put into vacuum drier and under vacuum lyophilize come that it is carried out lyophilize and contain less than about 5 weight % water content up to biomass.Can further dry processing material as described herein.
The medicinal herbs preparation has been that affiliated field is well-known.The medicinal herbs preparation that can be used for throwing with seedling of the present invention comprises liquid and solid medicinal herbs preparation.Some examples of medicinal herbs preparation comprise tincture, extract (for example, water solution extract, alcohol extract), decoction, drying agent (for example dry air, spraying drying, freezing or lyophilize), powder (for example freeze-drying powder) and liquid.The medicinal herbs preparation can provide such as the transmission mediator of any standards such as capsule, tablet, suppository, liquid agent.One of ordinary skill in the art can be applicable to understanding the various composites and the mode of the transmission medicinal herbs preparation among the present invention.
One of ordinary skill in the art will understand, and the particularly preferred method that obtains required medicine and pharmacology active protein or polypeptide is an extraction method.Fresh seedling (for example seedling) be can extract from residual biomass, shifting out the desired protein product, thereby the concentration and the purity of described product increased.Also can in buffered soln, extract seedling.For instance, can transfer to the plant of fresh collection a certain amount of for example in the phosphate buffered saline buffer buffered icy water with 1 to 1 weight ratio.Also can optionally add proteinase inhibitor.Can be when being suspended in plant tissue in the buffered soln, by fusion firmly or grind and destroy described plant tissue and by filtering or the centrifugal biomass of being extracted that remove.The protein that can be further purified in the solution to be loaded with by other step, or be converted into dry powder by lyophilize or precipitation.
Also can extract by squeezing.Can extract living plant by squeezing in squeezing machine, perhaps extract by pulverizing during by intensive cylinder when it.Collection is processed from the fluid through pulverizing the plant extrusion and according to the well-known method in affiliated field.Extract the product that allows to discharge denseer form by squeezing.Yet the overall yield of described product is lower than the productive rate that extracts product in solution.
Can also be for there be or do not exist the packing forms of one or more vehicle as indicated above in seedling (seedling for example germinates) extract, powder, drying agent and purified protein etc.The solid dosage of tablet, drageeing, capsule, pill and granule can be utilized such as dressing and shell preparations such as well-known dressings in casing, controlled release coat and other the medical allotment field.In described solid dosage, medical protein can be mixed with at least a inert diluent (such as sucrose, lactose or starch).Identical with common practice in, described formulation also can comprise other material except that inert diluent, and for example compressing tablet lubricant and other compression aids are such as Magnesium Stearate and Microcrystalline Cellulose.Under the situation of capsule, tablet and pill, described formulation also can comprise buffer reagent.It can contain opalizer according to circumstances and also can be make its in an enteron aisle part according to circumstances with the composition of the mode release of active ingredients that delays or preferential release of active ingredients.The example of spendable embedding composition comprises polymeric material and wax.
In other embodiments, with the biomass of expressing the seedling of medicine and pharmacology active protein of the present invention or described seedling with the form oral administration of dietetic food with.If described edible composition is a solid form, so can be by eating consumption raw; If perhaps be liquid form, so by drinking consumption.In a preferred embodiment, directly absorb vegetable material, and need not the previous procedure of processing or the follow-up MIN cooking.For instance, medicine and pharmacology active protein or polypeptide are to express in direct-edible young shoot.For instance, protein is to express in clover bud, Semen Phaseoli radiati Germinatus or spinach or lettuce leaves young shoot, pea seedling etc.In another embodiment, Recycled materials after procedure of processing are processed and absorbed to germination seedling biomass.
The working method that is preferred among the present invention is the method that is usually used in food or the fodder industry.The end product of described method still comprises in a large number the medicine and pharmacology active protein through expressing or polypeptide and preferably edible expediently or drink.Also end product can be mixed with other food or feed form (such as salt, supporting agent, flavour enhancer, microbiotic etc.) and with solid, semisolid, suspension, emulsion or liquid form consumption.In another preferred embodiment, described method comprises the preservation step, preserves and sanitas such as pasteurization (pasteurization), cooking process or interpolation.
Can use and process any plant in the present invention to produce edible or drinkable plant material.Can use the specific antibody of medicine and pharmacology active protein tool in edible or the drinkable young shoot preparation by the standard method in the affiliated field (for example, gel electrophoresis, Elisa method or Western engram analysis), test described proteinic amount.Described assay method can be used for making the proteinic amount stdn of being absorbed.For instance, can be for example by mixed number criticize that the product with different proteins content is measured and regulation and control young shoot juice in the proteinic amount of therapeutic activity, but so that stdn will drink and absorb the juice quantity of single dose.Yet, use closed adjustable environment according to the present invention should make and minimize for the needs that carry out described standardized program.
The medicine and pharmacology active protein or the polypeptide that produce in seedling (seedling for example germinates) and supply the host to eat are to absorb by Digestive tract.An advantage of the young shoot biomass of picked-up germination seedling or germination seedling preparation, especially complete young shoot or bottom line processing is, proteinic encapsulation or separation are provided in vegetable cell.Therefore, protein can be before arriving intestines or small intestine, is protected at least to a certain extent in order to avoid upper gastrointestinal digestion, and the active substance of higher proportion can be used for absorbing.
Treatability or prophylactically throwing and medical composition of the present invention.In some preferred embodiment, can use described combination treatment or preventing disease (for example, delaying seizure of disease).For instance, can treat the individuality of suffering from disease or the development disease risks being arranged.Should be appreciated that, provide at N under the situation of any symptom of described disease, can think that individuality has the risk of development disease.For instance, if individual have to differentiate to be and the relevant specific genetic marker of risk increase of development specified disease to think that so described individuality has the risk of the described disease of development.Similarly, suffer from specified disease (for example cancer), can think that so described individuality will have the risk of the described disease of development if individual family member has diagnosed.
Oral administration and liquid dosage form include, but is not limited to pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.Except that protein of being paid close attention to or polypeptide, inert diluent commonly used in the field under liquid dosage form can contain is such as water or other solvent; Solubilizing agent and emulsifying agent, such as ethanol, Virahol, ethyl-carbonate, ethyl acetate, phenylcarbinol, phenylamino benzoic acid methyl esters, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oil (especially Oleum Gossypii semen, peanut oil, Semen Maydis oil, germ oil, sweet oil, Viscotrol C and sesame oil), glycerine, tetrahydrofuran base alcohol, polyoxyethylene glycol and poly-Sorbitol Powder fatty acid ester and its mixture.Except that inert diluent, oral compositions also can comprise adjuvant, such as wetting agent, emulsifying agent and suspension agent, sweeting agent, seasonings and perfume compound.
Per rectum or vagina throw and composition be preferably suppository, its can by with composition of the present invention and suitable non-irritating excipient or supporting agent (such as theobroma oil, polyoxyethylene glycol, or suppository wax, its be at ambient temperature solid but under body temperature for liquid, and therefore in rectum or vaginal canal fusion and discharge active protein) be mixed with and obtain.
Part or transdermal are thrown and the formulation of medical composition of the present invention comprises ointment, paste, emulsifiable paste, washing lotion, gel, powder, solution, sprays, inhalation or paster.Under aseptic condition, active protein or its preparation and pharmaceutically acceptable supporting agent and optionally any required sanitas or buffer reagent are mixed.The expection eye with composite, [and eye drop also within the scope of the invention.In addition, the use percutaneous plaster is contained in the present invention, and it has the added benefit that health is provided the controlled delivery of medicine and pharmacology active protein.Described formulation can make by the medicine and pharmacology active protein is suspended or is scattered in the suitable medium.Also can use absorption enhancer to increase the flux that the medicine and pharmacology active protein passes skin.Can be by rate controlling membranes being provided or controlling speed by the medicine and pharmacology active protein is scattered in polymeric matrix or the gel.
Composition is to reach the essential amount throwing of required result and and to last the essential time of required result that reaches.As indicated above, in certain embodiments of the invention, the amount of " the treatment significant quantity " of medical composition for effectively treating, alleviate or prevent host disease.Therefore, as used herein, " effectively treat, alleviate or prophylactic amount " is meant nontoxic but is enough to treat, alleviate or prevents the amount of medical composition of any host's disease.Lift an example, " treatment significant quantity " can be treatment, alleviate or the amount of prevent diabetes.
Required exact amount will be looked individual kind, age and general situation, disease stage, specific medical mixture, dispensing pattern etc. with individual different the variation.Preferably seedling of the present invention (seedling for example germinates) and/or its protein formulation are deployed into unit dosage form with facility dispensing and dosage homogeneity.As used herein, statement " unit dosage form " is meant the entity discreteness unit of the medicine and pharmacology active protein that is suitable for patient to be treated.Yet, should be appreciated that, preferably in rational medical judgment scope, determine total every day of the consumption of the present composition by the attending doctor.Any particular patient or organic particular treatment effective dose will be decided on multiple factor, comprise the illness of being treated and the severity of described illness; The activity of employed specific compound; Employed particular composition; Patient's age, body weight, general health situation, sex; Patient's diet; Patient's pharmacokinetics situation; The dispensing time; Dosing way; Discharge rate with employed specific compound; The treatment time length; With combination of employed specific compound or the medicine that uses simultaneously; With the well-known similar factor of medical technical field.
Should also be clear that and medical composition of the present invention can be used for combination treatment, that is to say, can with one or more other required therapeutical agent or medical procedure simultaneously, before it or at it after throwing and medical composition.The particular combinations that is used for the therapy (therapeutical agent or program) of assembled scheme should be considered the tolerability and the required result of treatment of desiring to reach of required therapeutical agent and/or program.Should also be clear that employed therapy can reach required effect (for example, The compounds of this invention and another carcinostatic agent can be thrown simultaneously with) to same illness, or it can reach different-effect.
Test kit
On the other hand, the present invention also provides a kind of medical packaging or test kit, it comprises the seedling (for example, germination seedling) of work of the present invention or contains by the expressed medicine and pharmacology active protein of described seedling or preparation, extract or the medical composition of polypeptide in one or more are filled with the container of one or more compositions of medical composition of the present invention.In certain embodiments, medical packaging or test kit comprise other the granted therapeutical agent as combination treatment.The regulation pharmaceutical prod that can be government organs' prescribed form that links with the described container specification sheets making, use or sell according to circumstances, described specification sheets reflect that the approval of described mechanism is made, used or sells and are used for human dispensing.
The present invention includes from seedling (seedling for example germinates) purifying medical protein, and use the various plants expression system, most preferably viral plant expression system (that is to say, comprise the system of virus component) realizes that producing the available of medical protein by seedling (seedling for example germinates) enlarges in proportion.Be provided at each the proteinic test kit that comprises in the diagnostic kit at heredity and Immunological diseases.In a preferred embodiment, the invention provides the test kit that has in bioprotein specimen and the test patient serum at the reagent of the existence of the antibody of described bioprotein.For instance, test kit can provide in IA-2 and GAD and the test patient serum reagent at the existing of described proteinic excessive antibodies (type 1 diabetes exists or the clear indication of development).
The present invention can expand to the diagnostic reagent that kit form is provided.Lift a limiting examples, the form that proteins insulin, GAD and the IA-2 relevant with the autoreaction of diabetes can oral composite provides and offers medicine to induce these proteinic oral tolerances.Perhaps can the injectable composite or the vaccine form provide one or more treatment protein for throw with.According to preferred embodiment, with being provided for diagnosis is suffered from the individuality (for example diabetic individual) of disease or the medical dosage or relevant its specification sheets of individuality dispensing of the risk of development disease (for example type 1 diabetes) arranged in the test kit.
Equivalent
Following representative example is intended to help explanation the present invention, and the scope that is not intended to or it should be construed as limiting the invention.In fact, one of ordinary skill in the art will be from the complete content of this case (comprise following example and to science and technology that this paper quoted and the reference of patent documentation) and apparent to the various modifications of the present invention of the content that shows except that this paper and describe and its many other embodiment.The content that should also be clear that the reference of being quoted is to be incorporated herein by reference to help the explanation state of the art.Following example contains important Additional Information, example and guidance, and it is used in each embodiment of the present invention and its equivalent and puts into practice the present invention.
Example
Example 1: in by the transgenosis young shoot of Agrobacterium-mediated Transformation preparation, produce pharmaceutical protein or polypeptide
Collection is through the plant seed of Agrobacterium-mediated Transformation, the existence of dry, cleaning and test vigor and required genetic material.Store the seed stock thing stand-by under proper condition.During use, an amount of seed is soaked in the water that contains a certain amount of surperficial degerming agent (for example clo Cross Co. (Clorox)) reaches 20 minutes to 4 hours.Seed is launched on holding tray surface, and the supply and the water-freeing arrangement of growth substance contained on described plane.The pallet that will contain seed is put on the support in the closed adjustable environment that is under controlled temperature, illumination, inlet, air cycle, water source and the water-freeing arrangement.Reach 1 to 30 minute and be spaced apart 30 minutes to 4 hours to the pallet water spray with the atomizer that is equipped with timing register, be enough to make seed to keep moistening.Excessive moisture is discharged in the waterways in the room floor by the hole in the pallet.
Make seed concurrent the educating of under controlled condition, germinateing.With about 2 to 14 days of seed culture, gather subsequently and process.Certain period during culturing process (gathering preceding 4 hours to 7 days), seed is exposed to induces in the envrionment conditions of having introduced DNA promoter sequence or intrinsic DNA promoter sequence, thereby cause that one or more desired protein synthetic increase in the germination seedling tissue.With culture temperature from about 30 ℃ instantaneous be increased to about 37 ℃ to induce the heat-shocked promotor.
After seedling is cultivated 2 to 14 days, gather seedling on travelling belt by individual tray being moved on in the processing facility.Process the seedling that is collected by in the solution of the phosphate buffered that contains proteinase inhibitor, extracting.When being suspended in seedling in the buffered soln by fusion firmly or grind and destroy seedling and by the centrifugal biomass of being extracted that shift out.
Example 2: in the germination seedling of the instantaneous infection of plant virus, producing pharmaceutical protein or polypeptide
Obtain the seed of the required plant of wild type seeds form from commercial grower's contract.Under suitable temperature, humidity, health and safety condition, store the seed stock thing stand-by.During use, an amount of seed is soaked in the water and in pallet as indicated above under controlled condition, cultivates.
Cultivate after 2 to 14 days, the solution that will contain the genetically modified virus of tool is sprayed on the germination seedling, and in addition or simultaneously with causing that plant leaf organizes the material processing of mechanical abrasion.In this example, with the air atomizer scraper blade that contains abrasive particle.Make viral systemic infection plant reach appropriate time section (about 1 to about 10 days) and monitor the expression of required heterologous protein.
After the infection seedling reaches 1 to 10 day, gather seedling described in the example 1 as mentioned.
Example 3: in the Ben Saimushi tobacco plant, express diabetes associated protein and human growth hormone by virus vector (AvA4)
Early hair style 1 type or juvenile diabetes are a kind of children of influence, teenager and young disease.The metabolic signals of the β islet cells response hyperglycemia of pancreatic endocrine system produces Regular Insulin.The potential etiology of described disease is subjected to the attack and the destruction of health autoimmunization system for the β islet cells.
The individuality of suffering from type 1 diabetes produces and sees all individual intravital protein usually (promptly at least three kinds, Regular Insulin, L-Glutamic decarboxylase (GAD) and tyrosine phosphatase sample protein I A-2) antibody (Lesley people such as (Leslie), diabetology (Diabetologia) (1999) 42:30-14).Before seizure of disease, can in 50 to 75% type 1 diabetes patient body, find at IA-2 and the proteic autoantibody of GAD.In addition, go up common proteinic autoantibody at these surfaces just occurred as far back as the classical symptom outbreak relevant with type 1 diabetes in 7 to 8 years before.
Be applicable to such as self antigen such as Regular Insulin, GAD and IA-2 and on these proteinic levels of oral tolerance, in the susceptible individual body, induce at least and prevent or reduction can cause the damage of the islet cells of diabetes development.The autoimmunity form of the spontaneous development diabetes of non-obese diabetic mouse (NOD) (opening people such as (Zahang), periodical (1991) 88:10252-10256 of institute of NAS).Human insulin's gene and the b subunit of cholera toxin gene fusion of will having encoded and in the transgenic Rhizoma Solani tuber osi plant, express gained and construct body.When raising the NOD mouse with these plants, described animal represents delay (waste river people such as (Arakawa), natural biology technology (Nat.Biotechnol) (1998) 16:934-936) of the obvious reduction of pancreas islet inflammation and diabetes progress.Also in potato plants, expressed GAD albumen.When raising the NOD mouse with these transgenic Rhizoma Solani tuber osis, 40 Zhou Houqi also prevent or obviously delay diabetes outbreaks (horse people such as (Ma), nature-medical science (Nature Medicine) (1997) 3:793-796).
In this example, some non-limiting condition of expressing GAD and IA-2 and reclaim GAD and IA-2 from plant is described.Use the His stamp methods to come purifying GAD and IA-2 albumen.Starting material and proteinic generation are to take place in two stages of branch: the small-scale preliminary study (milligram quantities) of 1) carrying out in animal; With 2) medium scale clinical trial (gram amount).
Stable testing and motionFor carrying out virus stability and exercise test, the synthetic body of respectively constructing on a small quantity.Each construct body contain T7 or the SP6RNA polymerase promoter that merges with strict virus genome RNA 5 ' end and in vitro transcribe before be used to make 3 of plasmid linearization ' terminal unique restriction site.T7 or SP6RNA polysaccharase produce run-off transcript (run-off transcript) subsequently, use it for the inoculation plant.In two leaf phases, by existing abrasive (such as silicon carbide powder (320 granularities; (Fisher, Pittsburgh PA) under) the situation, softly wipe inoculum in blade surface and come the mechanical inoculation plant your company of the generation of Pennsylvania Pittsburgh.Construct body inoculation 5 to 10 strain plants and each each rna transcription thing of inoculation use 1-2 μ g for every kind.The severity of plant symptom when monitoring virus is diffused into whole plants, and reclaim product.Infect 10-15 days (dpi) in back, gather leaf sample to evaluate existing of full-scale reorganization IA-2 and GAD from leaf sample through infecting.The material (10 blades) that a part is gathered is freezing down and keep for the selected production of carrying out subsequently of expansion in proportion of constructing body as the seed inoculum at-80 ℃.The remainder of worked structure immediately.
Infect the back 15-20 days, and reclaimed reorganization IA-2 and GAD.With the high-purity product (90-95% purity) of program optimization with the recovery optimum quantity.After recovery has the product of desired size and measures serology identity (using Western blotting and ELISA to discern by specific antibody), test the stability of constructing body by three generations's health plant.By changing infectious condition or, solving recombinant virus and proteinic assembling, recovery or stability problem in employed size range on nucleotide sequence or the amino acid sequence level by using another host plant.
Determine that seed batch and program are for medium-scale productionWhen the 1st stage finished, make each a small amount of (100 μ l) in vitro the synthetic reorganization construct the transcript of body and use it for inoculation 10 strain plants.After inoculation, gather blade in 10-12 days, the existence by Western blotting test GAD or IA-2 and under-70 ℃, storing as seed material.The part (3-4) of this material is used to inoculate 150-200 strain plant (1-2kg flesh tissue).Inoculation back 15 to 20 days is reclaimed recombinant protein and is used it for the research of function.The average every batch of 60mg product of expection.
Plant inoculation and product reclaimUse in vitro transcript of the synthetic recombinant virus that contains GAD and IA-2 of t7 rna polymerase and purified plasmid DNA.Use RNA cap structure analogue m7G (5) ppp (5) G to attach the names of pre-determined candidates to transcript.For inoculation, the mixture of transcription product in vitro is applied to the blade of target host plant, subsequently with silicon carbide scraper blade surface and on blade surface soft friction so that the inoculum diffusion and the described surface of further swiping.Test produces purity and the activity of the plant of IA-1 and GAD.During protein purification and afterwards, produce antigenic antibody binding capacity by the ELISA test plants.
Protein expression in the Ben Saimushi tobacco:For expressing full length protein in the plant of virus infection, the functions of use complementary method is expressed in the Ben Saimushi tobacco plant from the green fluorescent protein (GFP) of jellyfish (Fig. 4).During plant was gone down to posterity to plant, the amount of alfalfa mosaic virus in the infected tissue (Av)/GFP reduced gradually and after shifting for the third time, can only detect Av/A4.(from the viewpoint of environmental safety, this is favourable.) using present method, every gram flesh tissue can be expressed average 100 μ gGFP.The important component alfalfa mosaic virus CP of this system has geneome RNA with uncorrelated virus and wraps into ability in the infectious particle in the infected host, so it is unique.Utilize the hybrid vector of this unique ability of alfalfa mosaic virus CP with engineered selectively targeted selected crop species.
Analyze the expression (referring to Fig. 3) of reorganization GFP in the Ben Saimushi tobacco plant of Av/A4 and Av/A4GFP inoculation by the Western blotting.On the 12%SDS-polyacrylamide gel, separate protein extraction of leaves thing by electrophoretic method, transfer to it in nitrocellulose filter and make it and the protein-specific antibody reaction from systemic infection as described herein.The identification of alfalfa mosaic virus CP specific antibody has desired size in the plant of Av/A4 or Av/A4 and the inoculation of Av/A4GFP mixture protein (24.0kD).The GFP specific antibody is only discerned the protein in the extract of plant of Av/A4 and the Av/A4GFP mixture inoculation of hanging oneself.The GFP specific antibody not with any proteins react in the plant of Av/A4 inoculation only.Alfalfa mosaic virus CP and GFP specific antibody not with from any proteins react in the extract of the plant of Av/GFP inoculation only, this shows the shortage of system motion.
Also used the engineered and expression human growth hormone of this Av/A4 carrier system.Generation with hGH in inoculation Ben Saimushi tobacco plant of transcript in vitro and the monitoring plant.When 5dpi (days post inoculation), do not detect, but when 11dpi, can detect the signal of relevant hGH in the plant in inoculation to described proteinic specific signals.Fig. 4 is the Western trace of the hGH that produces in the Ben Saimushi tobacco plant of the in vitro transcript that infects 125C/hGH.Inoculate back 24 hours analytic samples.Load the pure hGH of 1 μ g as standard substance.MWM is a molecular weight marker.On the arrow points trace of Fig. 4 by the detected hGH bands of a spectrum of hGH specific antibody.
Example 4: anthrax associated protein and human growth hormone's expression in the transgenosis leafy mustard plant
The virulence of anthrax is to be caused by at least two main virulence factors.These factors are for helping polyglutamic acid pod membrane and the three part circulation toxin (three-part circulating toxin) of the bacterium among the protection host.Anthrax toxin is plasmid-encoded and by the receptor binding protein that is called protective antigen (PA) and be called edema factor and two kinds of zymoproteins of lethal gene form that (Bart Na Geer (Bhatnagar) and Bart draw (Batra), microbiology commentary (Crit.Rev.Mirobiol.) (2001) 27 (3): 167-200) by the 184kb that is called pXO1.
In this example, in the blade of leafy mustard, produce two kinds of the non-activity anthrax toxin component of 0.1mg/gm dry weight concentrations, i.e. protective antigen (PA) and lethal genes (LF) at least.Codon with these plant genes uses optimization and changes aminoacid sequence so that the toxicity of two kinds of lps molecules reduces to minimum.Suggestion inserts synthetic DNA can regulate and control in the carrier of genetic expression in the leafy mustard.The PA gene alteration is become at 314 and 315 disappearance phenylalanine residues.Make the LA gene replaced by L-Ala at 686 or 690 s' Histidine (for example referring to, Sean people such as (Singh), journal of biological chemistry (J.Biol.Chem.) (1994) 269:29039-29046; The happy people such as (Klimpel) of Crane handkerchief, molecular microbiology magazine (Mol.Microbiol.) (1994) 13:1093-1097).
Conversion carrier:With binary vector pGREENII0229 (Helen people such as (Hellens), molecular biology of plants (PlantMolecular Biology) (in April, 2000) 42 (6): 819-832) be used for Plant Transformation (referring to Fig. 5).This plasmid comprises following component.1) pGREENII plasmid main chain is included in and duplicates required sequence in intestinal bacteria and the agrobacterium tumefaciens.2) agrobacterium tumefaciens T-DNA left margin and right margin (LB and RB) sequence is for to be incorporated in the Plant Genome all sequences between LB and the RB required.3) the npt gene of coding kalamycin resistance, it is used to select intestinal bacteria and agrobacterium tumefaciens transformant.4) nos-bar gene, its coding is to the resistance (using two third ammonia phosphorus resistances to select transgenic plant) of the two third ammonia phosphorus of weedicide.The nos-bar gene is to transcribe by having the active cauliflower mosaic virus of composition (CAMV) 35S promoter in the plant, and described gene transcription is to use the CaMV terminator to stop.5) expression of vir gene PA and LF is by as shown in Figure 5 the CAMV35S promotor or HSP18.2 promotor (the gene pool accession number #X17295 of Arabidopis thaliana lower molecular weight heat shock protein(HSP), locus At5g59720) drives (people such as (Matsuhara) of Songyuan City, plant magazine: cell and molecular biology (The Plant Journal:for Cell ﹠amp; Molecular Biolog) (in April, 2000) 22 (1): 79-86).Transcription Termination is the terminator mediation by rouge alkali synthetase gene (no).Select 35S and HSP18.2 promotor according to different activities.35S promoter has the composition activity in most of plant tissue.In many cases, 35S promoter drives the proteinic high level expression of being paid close attention to.By contrast, the HSP18.2 promotor is non-activity almost, and the expense of removing excites plant by heat-shocked.Some proteinic high level expressions can only use the inducible promoters system to realize.
Carrier is constructed and Plant TransformationUse DH5 (laboratory coli strain) to construct carrier.Map and check order to analyze and construct by restriction endonuclease.After determining to construct, use described carrier to transform to lack natural Ti-plasmids and be suitable for TDNA with binary vector and transfer to agrobacterium tumefaciens bacterial strain GV3101 in the plant.By any available method for transformation, the GV3101 that is loaded with conversion carrier introduced transforming the leafy mustard plant in spending.
Introduce the modification in PA and the LF gene.The nucleotide sequence of PA and LF is modified to express in plant.The encoding sequence of natural PA is that 2295bp and natural LF are 2430bp.These sequence modifications are as follows: 1) introduce making PA and the LF sudden change as the toxin non-activity.Make PA disappearance Phe314 and Phe315 and make His686 and His690 is mutated into Ala.2) optimizing codon is used to express in leafy mustard.Because the codon of leafy mustard uses and the GC/AT ratio is uncertain, so the change of introducing among PA and the LF is also few.Change among 4 codons and the LF among the PA and change 7.3) unique restriction site is added to 5 of gene ' and 3 ' end for being cloned in the plant expression vector.[4] concensus sequence of interpolation translation initiation (tall assorted people such as (Joshi), molecular biology of plants (Plant Molecular Biology.) (in December, 1997) 35 (6): 993-1001).The translation initiation site of restriction site and optimization is described among Fig. 5.5 ' end shows the XbaI restriction site of interpolation and the consistent translation initiation site of plant gene, and it comprises initiator codon, is the Ala codon subsequently.3 ' end shows the SacI restriction site that adds.Modified PA and LF gene are by (Regensburg, Germany (Regensburg, the Germany)) chemosynthesis of Entelechon company.
Second to construct also be to produce at PA and LF.Second purpose of constructing body is to be stranded in the accumulation of optimizing PA and LF in the endoplasmic reticulum by the protein in the target endoplasmic reticulum and with it 40(Ha Luofu people such as (Haseloff), institute of NAS prints (Proceedings of theNational Academy of Sciences USA.) (on March 18th, 1997) 94 (6) to target: 2122-2127) with being stranded in the high level accumulation that can produce recombinant protein among the ER.For reaching this purpose, add N-terminal signal sequence and C-terminal ER and be detained sequence.Signal sequence comprises 22 codons deriving from Arabidopis thaliana chitinase gene (people such as Ha Luofu, with above).It is tetrapeptide HisAspGluLeu (Ge Mode people such as (Gomord), plant magazine: cell and molecular biology (The Plant Journal:for Cell that ER is detained sequence; Molecular Biology.) (in February, 1997) 11 (2): 313-325).Splice by the PCR that uses the synthetic gene produced by Entelechon company to carry out and to add signal sequence (soup rice people such as (Tomme), bacteriology magazine (J Bacteriol.) (1995) 177:4356-4363) as template.The PA by using the 3 ' primer incorporated the HisAspGluLeu encoding sequence into and the pcr amplification of LF gene add ER and are detained sequence.With modified PA and LF sequence clone in 35S and HSP18.2 expression vector.Clone PA and each two kinds of modified pattern of LF (amounting to 4 kinds).Generally speaking, 8 kinds of different bodies of constructing of test.
Selection of the plant of express transgenic and analysis.Be loaded with transform the agrobacterium tumefaciens bacterial strain GV3101 construct body and transform leafy mustard after, make plant via its normal development program development.In 25 days, produce mature seed, dry and collection.Planting seed in potted plant soil, and was made growth of seedling 15 days.(FINALE, (Farnam, Inc.), Phoenix, AZ (Phoenix, AZ)) solution is sprayed on the blade of each plant in Farnam Companies Inc. with two third ammonia phosphorus of 0.0058% (w/v) subsequently.Usually in 5 to 7 days, kill the non-transgenic sensitive plant.May be obvious that two third ammonia phosphorus resistance plants by green and healthy appearance.Confirm the transgenic plant of inferring and use a series of schemes to be analyzed.The existence that the polymerase chain reaction of constructing the specific Oligonucleolide primers of body tool is used for definite transgenic plant TDNA that infers will be used.Subsequently by using specificity DNA probing needle (modified PA or LF gene) imprinted genes group DNA to check the existence of TDNA.At last, by SDS-PAGE, use Coomassie blue (Coomassie Blue) dyeing or immunoblotting to check the protein expression of being paid close attention to subsequently.The analytical plan of protein expression is constructed body and difference with what be used to produce transgenic plant.Because by the composition that is expressed as of 35S promoter, so but direct analysis is loaded with the plant that 35S promoter is constructed body.Before analysis, make the vegetable hot shock that is loaded with the HSP18.2 promotor.The kinetics of expression of recombinant proteins may be with different transgenic plant difference.At last, use standard method that each inductive condition is optimized.Yet,, may carry out and will set up the stdn heat-shocked scheme of other Recombinant Protein Expression in the leafy mustard for initial analysis.
Fig. 6 illustrates the western blot figure of the transgenosis leafy mustard of expressing human class tethelin (hGH) under the control of HSP18.2 promotor.The render transgenic leafy mustard grows in potting mixtures.As shown in Figure 6, from plant, peel off the blade of heavy 1 to 3 gram fresh weight, and put it in the petri diss (petri dish) that contains through the filter paper of water-wet.Petri diss covered and it is put in high humidity, 37 ℃ of constant incubators and reach 1.5 hours.The petri diss that will contain described blade takes out from constant incubator subsequently, and puts it into and be in 100 μ mol photon m -2s -1Reach 5 hours in 24 ℃ of growth rooms under the fluorescence of intensity.The herborization material is also by using the immunoblotting that carries out at the monoclonal antibody (sigma chemistry product company (Sigma Chemical Co.), production code member G-8523) of hGH to be analyzed subsequently.Low bands of a spectrum are 16kDa reorganization hGH.Before heat-shocked, do not observe these bands of a spectrum.The 8th road shows from only through the result of carrier transgenic plant transformed.The higher molecular weight bands of a spectrum are the nonspecific reaction of the secondary antibody that is connected with the horseradish peroxidase that is used to detect immunocomplex.Estimate that PA is that about 88kDa and LF are 93kDa.
After the initial screening that anthrax toxin is expressed, carry out analysis in more detail to expressing.Use the ELISA method to come the quantitative expression level.Follow-up plant filial generation is further analyzed.For avoiding the demand for selection transgenic plant in follow-up filial generation, the ultimate demand separation is constructed the unseparated transgenic line of body about TDNA.This can by to former generation transgenic plant carry out self-pollination, s-generation plant growing is ripe and realize about two third ammonia phosphorus resistance discrete testing third generations.Differentiate unseparated s-generation individuality.After this, form the analysis that does not separate the plant filial generation in a large number and use it for industrial scale condition (for example every month 1, the 600Kg dry biomass).To be grown to the growth of plant of seedling and inductive condition optimization to reach industrial scale.At this moment, also need to characterize insertion site and the TDNA copy number and the sign expression on the mRNA expression level of breeding system.
Example 5: in young shoot, construct body surface and reach GUS report by the Agrobacterium that contains virus sequence
The pBI121 that will contain gus reporter gene is transformed in the agrobacterium tumefaciens lba4404.Bacterial cultures is grown whole night in the YEB substratum that contains 50 μ g/ml kantlex, 20 μ M Syringylethanones and 10mM MES (pH5.6).The incubated overnight thing is centrifugal, with it with OD 6002.0 be suspended in the MMA substratum (MS salt, 10mMMES (pH5.6), 200 μ M Syringylethanones and 2% sucrose), and use it for the vacuum infiltration of young shoot.
Under the room temperature, imbibe reaches 24 hours in the water of the seed that makes each kind of plant on the vibration platform, transfers to them in plastic containers with moist filter paper and cultivated 4 to 6 days (deciding on plant species) down in 21 ℃ under 12 hour daytime.
Behind the vacuum infiltration, young shoot was cultivated 48-60 hour and is used the substrate in situ detection GUS of X-gluc histological chemistry activity again under 12 hour daytime under 21 ℃.Dye whole night and plant sample is decoloured in ethanol at 37 ℃.
Use multiple young shoot to test described system.Referring to Fig. 8 A and 8B and Fig. 9.
Example 6: construct body surface by the Agrobacterium that contains virus sequence and reach diabetes associated protein and human growth hormone
In agrobacterium vector, produced carrier: D4-hGH or D4-GFP based on TMV.For this reason, at first must in pBI121, produce a plurality of cloning sites.pBI121-Xba1-BamH1-Sal1-Pac1-BsiW1-Stu1-Xho1-Spe1-Kpn1-Sac1-pBI121。Use suitable primer and PCR, produce pBI121 with these sites.After determining described sequence, then the TMV genome sequence is introduced in this plasmid.Further argumentation about D4-hGH and D4-GFP, also referring to the U.S.S.N.10/770 of on February 3rd, 2004 application, the U.S. Provisional Application case the 60/444th of application on February 3rd, 600 and 2003, No. 615 (being incorporated herein by reference) and U.S.S.N.10/832,603 and U.S. Provisional Application case 60/546,339 (being incorporated herein by reference).Original vector D4 and carrier therefrom are described in executes general Saden people such as (Shivprasad), virusology, and 255 (2): 312-23, in 1999.The gained plasmid is called pBID4.
35S promoter and the TMV sequence of cauliflower mosaic virus are merged.Thus, 35S promoter guiding TMV sequence transcribes.After successfully incorporating in the cell, produce the virus transcription thing.In certain embodiments of the invention, also produce virus replication and the required component of diffusion in whole plants.These components for example comprise replicative enzyme, motion albumen and coat protein, and it can be from other virus (such as alfalfa mosaic virus) among TMV or each embodiment.These components of coding in the TMV sequence that can provide in independent virus vector or plasmid, the plasmid sequence, perhaps plant can be the genetically modified transgenic plant that comprise the described component of encoding.For example referring to the USSN10/770 of on February 3rd, 2004 application, 600, it is incorporated herein by reference.
Transcribing of subgene group TMV promotor guiding hGH sequence in the TMV sequence.Hammerhead ribozyme is introduced 3 of TMV sequence ' end.Described ribozyme is not required for the present invention.Nos terminator (affiliated field is well-known) is 3 of described ribozyme sequence ' end.
Final carrier contains D4-hGH or D4-GFP.Using among the pBI121 these to construct body subsequently transforms agrobacterium tumefaciens and uses through transforming Agrobacterium infiltration plant.Owing to have replication-competent virus, so with 2 weeks of plant culturing.Analyze the generation of hGH in the blade by the Western blotting.By monitoring the GFP expression with long wave ultraviolet light irradiation plant and taking pictures.Result shown in Figure 10 shows whole and exists in the blade GFP to express through infiltration.Use shown in Figure 11 confirms at the Western engram analysis that the antibody of hGH carries out, and describedly constructs the hGH that body works and can detect high level in plant.
Similarly, with IA-2ic engineered in the pBID4 plasmid to produce pBID4-IA-2ic.In addition, produce the carrier that is loaded with GFP based on AMV virus.Use gained plasmid transforms Agrobacterium and uses in the Ben Saimushi tobacco of conversion Agrobacterium infiltration water cultivating and growing.In simple terms, the Ben Saimushi tobacco seed is seeded in the hydroponics condition on rock wool plantation pad (Rockwoolslab) (18 * 18 * 1) wetting in advance in the Hoagland solution (Hoagland solution) as 1/2 concentration of nutrient.In with a kind of solution, hydroponics plants was kept for 4 weeks.Subsequently, be loaded with pBID4-IA-2ic or be loaded with the agrobacterium suspension (OD of GFP based on the carrier of AMV virus 6000.1) in the plant in 4 ages in week is implemented vacuum infiltration.
Make bacterial cultures growth and induce whole night, subsequently with the cell resuspending in MMA substratum (MS salt, 10mMMES (pH5.6), 20g/l sucrose, 200 μ M Syringylethanones) up to OD 600Be 0.1.Under soft vibration, at room temperature the resuspending culture was cultivated 2-3 hour, and hydroponics Ben Saimushi tobacco plant is put into bacterial suspension, at room temperature apply vacuum subsequently and reach 30-60 second.Discharge vacuum rapidly and effectively inject tissue, and will in the Hoagland solution nutrient of infiltration plant, keep 5-7 days in 1/2 concentration to promote bacterium.Perhaps, use the 10cm of needle-less 2Syringe is suppressed bacterial suspension in the epidermis of blade of complete expansion.The infiltration back is gathered blade and is analyzed-80 ℃ of storages or at expression analysis in the time of 3-6 days.Figure 12 shows the western engram analysis of the plant of expressing IA-2ic.Relevant under light the analysis revealed of the plant through construct the body infiltration based on the GFP of AMV in the whole leaf of infiltration plant, exist GFP to express.
Example 7: in the pea kind, construct body surface and reach protein (green fluorescent protein by the Agrobacterium that contains virus sequence; Lichenase)
The Agrobacterium that the utilization of this case description is loaded with virus expression carrier constructs the Agrobacterium infiltration of some pea kind that body carries out.
Figure 13 describes the whole strategy that the Agrobacterium be used to be loaded with the AlMV sequence constructs body.Known AlMV has sectional genome (discussing as mentioned), so use three kinds of different bodies of constructing: be loaded with coding replicative enzyme 1 and 2 the AlMV sequence construct body (pMOG-R1 and R2); What be loaded with AlMV RNA3 sequence constructs body (pBI-RNA3) (comprise the gene of being paid close attention to be expressed according to circumstances, it replaces the natural A lMV coat protein sequence among the RNA3 usually); With the AlMV sequence that is loaded with coding AlMV coat protein construct body (pBI-CP).One of ordinary skill in the art will understand, and strictly speaking, not need to comprise the body of constructing of coding AlMV coat protein.Yet, because coat protein can promote duplicating of virus sequence (and gene of being paid close attention to that therefore it was loaded with) and/or systematicness diffusion, so comprise that the described body of constructing will be useful.
Even guarantee at needs under the situation of AlMV outer casing protein expression, also also nonessentially utilize three kinds to construct body.For instance, perhaps may utilize, therefore need not to provide separately about the genetically modified plant of AlMV coat protein.In addition or other, can utilize about the genetically modified plant of one or both AlMV replicase proteins.In described plant (being genetically modified about the AlMV coat protein in addition according to circumstances), can utilize the single body of constructing that only is loaded with the RNA3 and the sequence gene of paying close attention to reach similar results.
As shown in figure 13, in these specific embodiments, utilize the body of constructing of expressing three kinds of different proteins (that is green fluorescent protein (GFP), lichenase (Lich) and lichenase-anthrax protective antigen syzygy (Li-PA)).
In the related particular experiment of Figure 13, make at least 1 liter of each agrobacterium strains (promptly, each bacterial strain is loaded with 3 kinds and constructs a kind of among body pMOG-R1 and R2, protein that pBI-RNA3/ pays close attention to, the pBI-CP) growth, be made into spherically, and resuspending reaches 1.0 O.D. in MMA.One of ordinary skill in the art will understand different plants may need different O.D..For the pea plant,, need for example higher O.D. in 1.5 scopes usually if technology can reach.
In the related particular experiment of Figure 13, the agrobacterium strains that will be loaded with replicative enzyme (pMOG-R1 and R2), coat protein (pMOG-CP) and RNA3/ target protein (pBI-RNA3/GFP, Lich or Li-PA) reaches the cumulative volume of 1.5L with the ratio combination of 1:1:2.One of ordinary skill in the art will understand, and these ratios and volume are unimportant; The specific ratios of selecting to be utilized is usually directed to the RNA3 content higher than other genome component with the life cycle of reflecting AlMV.In addition, the RNA of the gene (that is, GFP, lichenase or lichenase-protective antigen gene) that needs high level to be loaded with to be paid close attention to.
In the related particular experiment of Figure 13, utilize as known no soil upholder in the affiliated field (being cheese cloth in the case) and make Kidney bean young shoot hydroponics growth.Seedling is immersed in the combination Agrobacterium mixture, and under-28psi, applies vacuum and reach 90 seconds to realize the Agrobacterium infiltration.In some cases, apply once above vacuum.
In the related particular experiment of Figure 13, after Agrobacterium infiltration, the rinsing seedling is 1 time in tap water, and subsequently it is put back to the growing space and water.Then gather seedling, compile and extract.
Figure 14-16 provides every result of experiment of carrying out according to the strategy described in Figure 13.For instance, as shown in figure 14, the body of constructing that produces lichenase is permeated in spot pea (SP), yellow pea (YP) or Bill's jumping bean (BP) seedling by Agrobacterium.Before Agrobacterium infiltration, seedling is in the dark grown 2 days (germinateing allowing) and 8 days (as by shown in " 2 (8) " symbol) of growth under illumination.Carry out Agrobacterium infiltration as indicated above after these 10 days, and make its after inoculation, grow 5 days (dpi), detect the protein that is produced subsequently.As finding out among Figure 14, all three kinds of pea kinds all produce the lichenin zymoprotein, but the rate ratio spot pea of yellow pea is slightly high, and Bill's jumping bean has lower slightly output.Described variation is in the technician's who is familiar with marking protein in plant experience scope; According to these results, the preferred plant kind of any specified protein will only be needed normal experiment to select to produce.Generally speaking, viewed expression level is about 1/3 (not shown) of viewed expression level when expressing associated protein (for example Lich-PA) in the Ben Saimushi tobacco in these pea seedling systems.The advantage that these seedling are better than the Ben Saimushi tobacco is, its edible is because all components of known described plant is harmless for the mankind at least, so it should simplify any required separation at the medicine and pharmacology active protein that is produced.In addition, its growth is rapid and cheap.On the other hand, because seed is than common seedling and especially the seed of pea is much smaller, so that seed is stored is also simple than Ben Saimushi tobacco.
As finding out from reference Figure 14, in these specific embodiments, it is more more successful than similar TMV system (as described herein) for producing the protein of being paid close attention to that above-mentioned AlMV produces system.One of ordinary skill in the art will understand, for producing some specified protein in some specified plant species, with preferred TMV system; And at the following preferably AlMV of other situation (comprising described situation).
Figure 15 illustrates the successful generation of GFP in the spot pea of the different time sections of growing and the yellow pea before Agrobacterium infiltration.In the particular experiment that in Figure 15, is provided, apply three vacuum and respectively last 90 seconds.In addition, the specific resuspending substratum that is utilized is " MMA fully ".One of ordinary skill in the art will understand, can use in addition multiple other infiltration in substratum any (comprising minimum medium MMA, AB etc.) and may increase or reduce the Agrobacterium infiltration and the therefore final efficient of protein or polypeptide generation.
The expression of lichenase in spot pea blade (L), whole seedling (W), root (R) or the plumular axis (H) when Figure 16 illustrates infiltration back different time sections.As finding out, the most of expression occurs in the blade.And the infiltration back is expressed best in this particular system in the time of about 5-7 days.Expect that some variations will be based upon on age of the plant that is utilized and kind and the expressed proteinic basis.
Other similar experiment shows, for instance, the output that the chances are by increase coat protein (CP) increases the proteinic output of being paid close attention to use the pBI-CP that lacks 5/3 ' UTR structure to construct body.In addition, in native system, the expression of plants the best in 9-12 days ages.Generally speaking, protein expression level is higher in young tissue, but than having more biomass in the old tissue.
Other similar experiment proposes plant is exposed to illumination in 24 hours but not 12/12 daytime/circulation at night obviously reduces the Protein content of being paid close attention to.
Other similar experiment shows that during the Agrobacterium infiltration, pea seedling is exposed to weak sanitising agent probably can increase the proteinic output of paying close attention to by efficient and/or the degree that increases successful Agrobacterium infiltration.
Example 8: the extensive modular system that produces protein or polypeptide in plant
A kind of technology platform of this case description, it will make the manufacturing generation great variety of biological medicine, reduce batch production time (reducing to for 5 weeks in some cases).Specific production system described in this example is utilized agrobacterium vector, and the plant virus RNA of the protein of in several minutes being loaded with of very big copy number being paid close attention to or the encoding sequence of polypeptide is delivered in the seedling.Therefore can be in the week in the agrobacterium vector introduced plant, realize the protein paid close attention to or the high-level transient expression of polypeptide with a large amount of plant biomass.
Technology platform described in this example is applicable to multiple monomer or multimeric protein, comprises vaccine antigen, monoclonal antibody and other therapeutical agent (especially) and can carry out in the various plants species.Expression ratio in the plant is expressed in some other systems and is more suitable for providing correct folding and solvability, and has the added benefit of no animal pathogen.In this particular instance, focus on engineered and generation vaccine antigen and monoclonal antibody in the seedling.
Described in this example, the protein of being paid close attention to encoding or the gene clone of polypeptide have in the launching carrier of Agrobacterium and plant RNA virus sequence element to combination.Use Agrobacterium millions of launching carriers to be copied in the introduced plants subsequently by vacuum infiltration.In plant tissue, in addition by a large amount of amplification vector sequences of virus replication.Subsequently after introducing carrier, in less than the time in a week by these virus transcription deposits yields target proteins.Should be appreciated that also nonessential (but also not forbidding) is incorporated into agrobacterium vector in the vegetable cell.On the contrary, the Agrobacterium element of carrier only promotes finally to produce the rapid system transmission (via the Agrobacterium osmose process) of proteinic viral RNA.
Can use described system to produce a large amount of (for example hundreds of milligram quantities) target proteins of per kilogram fresh plant tissue.In addition, owing to be easy to obtain a large amount of seeds (as described, it need not to genetically modified), so can realize high capacity.
As described herein, in some cases, produce the target protein or the polypeptide that merge with carrier molecule, for example to make described protein stabilization and/or to promote downstream processing.Described carrier is particularly useful for producing antigen protein.The carrier that merits attention especially for example comprises thermostability albumen, such as the USSN60/472 of on May 22nd, 2003 application, application in Mays 24 in 495 and 2004 year and on March 24th, 2005 with the protein described in the disclosed PCT/US04/016452 of WO05/026375.It should be noted that from this point described thermostability carrier molecule is the β-1 from Clostridium thermocellum, 2-1, the engineered pattern of 4-dextranase (LicKM).Can with target sequence rapidly engineered be N-terminal, C-terminal or inner fusions with LicKM.The existence of external sequence can not cause the forfeiture of molecular heat stability among the LicKM.This characteristic promotes easily and cost reclaims target protein effectively.Brief heat treating step (for example under 65 ℃ 10 minutes at the most) can cause removing most of host protein.In addition, more than one target proteins and each LicKM molecule can be merged, but use a plurality of insertions site simultaneously.
Specifically, make from the seed of the plant of genetic modification not and under the hydroponics condition, germinate, and in closed indoor plant facility, keep for some time (for example, for about at the most usually 10 days of seedling; For the Ben Saimushi tobacco is 2-6 week sometimes), carry out vacuum infiltration then.Under these conditions, the productive rate of every square feet of plant biomass be generally utilization resulting productive rate of growing plants in soil 4-5 doubly.Use seedling (for example, the germination seedling), can produce every square feet of intensive canopy (canopy) with a large amount of (for example about 100-1000kg) chlorenchymas.Lift a few examples, utilize young shoot can produce every square feet of canopy usually with about 700g chlorenchyma such as pea; Utilize the Ben Saimushi tobacco to produce every square feet of canopy usually with about 200g chlorenchyma.One of ordinary skill in the art will understand other plant may be between this tittle.
Can be by going up support with seedling (for example seedling) and be stacked on another described support and realize high capacity in that the frame of supporting lighting unit (1000 square feet growing spaces will provide in less than two weeks nearly 4 tonnes of biomass) being provided.The Agrobacterium that will comprise launching carrier is by using and all airborne parts of snap-out release vacuum infiltration plant subsequently.This forces Agrobacterium infiltration to have in the whole canopy of the plant tissue that leaf almost completely covers.Subsequently, target protein is accumulated the time (for example, about 2 arrive about 14 days, about in certain embodiments 2 to 10 days, 3 to 7 days, 4 to 6 days etc.) of a couple of days.
Whole process from seed to the tissue of being gathered needs several weeks usually (for example, less than 6,5,4,3 or 2 weeks; For common about 2 weeks of seedling).Because seed material is not genetically modified, thus the generation of seed and store cheap and be easily understood, and for the no any restriction of expansion effectively in proportion.Therefore might in general several weeks, produce a large amount of recombinant proteins.
In case of necessity, the separable target protein that produces.Heating steps can be used for separating the target that merges with the thermostability carrier.Other method of purification can comprise various separation and/or stratographic analysis step.For instance, can use such as ammonium sulfate precipitation, pH value dependency separate, steps such as the size exclusion look general, ion-exchange chromatography and affinity chromatography.
The whole process that is contained in this example comprises 1) gene optimization and synthetic; 2) be cloned in the launching carrier; 3) preparation of vegetable material and Agrobacterium culture; 4) vacuum infiltration of germination seedling; 5) target is expressed; 6) tissue sampling; With 7) the target recovery.For realizing high-throughput production, make most of automatization of this process.Specifically, ferment to homogeneity, clarify and (referring to Figure 19) automatization in steps that the volume of the vegetable material that homogenizes reduces making from non-transgenic planting seed and Agrobacterium culture.To design and build module with 1.5 tonnes of plant biomass abilities of a collection of generation.Specifically, the equipment that concept nature shows among Figure 20 will be made up.
Equipment design described in Figure 20 is used the notion of pylon, thereby whole module can be assemblied in about 5000 square feet space.Central authorities frame unit provides essential space, illumination and water source with the 1.5 tonnes of plant biomass of growing.Automatically monitoring and controlling moisture, temperature and illumination.In design shown in Figure 20, plant will be of a size of 1 ' * 2 ' pallet in the hydroponics growth.Therefore, will use about 6000 pallets.Can make up module by 36 stackable individual memory cell.Each storage unit will be held 6 * 7 * 4 pallets.The modularity of design allows to enlarge production.There are two automations (robot) that pallet is put into frame and taken out from frame, one of each side of frame.Automatically sowing is to carry out on the material that is suitable for the hydroponics growth.After plant had reached suitable ripening degree, travelling belt was transported to permeation unit, and it has vacuum chamber, and described vacuum chamber has the capacity of 1.5 tonnes of plant biomass of infiltration in about 8 hours.Rinse unit will be removed excessive Agrobacterium culture.Behind the rinsing plant, it is sent back the target accumulation of central frame unit for a couple of days.Subsequently, pallet is sent to herborization and with its collecting unit that homogenizes.The plant milk extract that will homogenize subsequently is transported to the downstream machining cell.
Example 9: construct body surface by the Agrobacterium that contains virus sequence and reach HPV antigen
Can utilize the agriculture bacillus mediated transient expression system that obtains by the Agrobacterium osmose process (Te Erben people such as (Turpen), 1993, virological method magazine (J.Virol.Methods), 42:227).Utilization contains the healthy leaves through engineered Agrobacterium rhizogenes infiltration Ben Saimushi tobacco with the virus vector of expressing LicKM-E7 or LicKM-E7GGG.
Agrobacterium rhizogene strain A4 (ATCC43057) or agrobacterium tumefaciens (GV3103) are transformed with constructing body pBI-D4-PRACS-LicKM-E7-KDEL, pBI-D4-PRACS-LicKM-E7VAC, pBI-D4-PRACS-LicKM-E7GGG-KDEL and pBI-D4-PRACS-LicKM-E7GGG-VAC.Make Agrobacterium culture growth as described and induce (people such as Ka Peila, 1997, plant science (Plant Sci.), 122:101).Under 28 ℃, make 2ml cause culture (starter-culture) (from fresh bacterium colony, choosing) at the YEB with 25 μ g/ml kantlex (5g/l beef extract, 1g/l yeast extract, 5g/l peptone, 5g/l sucrose, 2mM MgSO 4) middle growth whole night.To cause culture with 1:500 be diluted in 500ml have 25 μ g/ml kantlex, 10mM2-4 (morpholinyl) ethane sulfonic acid (MES) (pH5.6), the extra MgSO of 2mM 4In the YEB of 20 μ M Syringylethanones.Make subsequently through the dilution culture 28 ℃ down growth whole night to reach about 1.7 O.D.600.With cell with 3, centrifugal 15 minutes of 000 * g and with its resuspending in MMA substratum (MS salt, 10mM MES (pH5.6), 20g/l sucrose, 200 μ M Syringylethanones) to reach 2.4 O.D.600, at room temperature keep 1 hour, and use it for the Agrobacterium infiltration.Use the needle-less disposable syringe that agrobacterium suspension is injected the Ben Saimushi tobacco leaf.Permeate the blade of gathering infiltration in back 6 days.
Example 10: construct body surface by the Agrobacterium that contains virus sequence and reach anthrax antigen
Can utilize the agriculture bacillus mediated transient expression system that obtains by the Agrobacterium osmose process to produce anthrax antigen.Utilization contains the healthy leaves through engineered Agrobacterium rhizogenes infiltration Ben Saimushi tobacco with the virus vector of expressing LicKM or LicKM-PAD4.Employed carrier is pBI-D4, promptly introduces the virus expression carrier D4 pattern among the agrobacterium vector pBI121.(old people such as (Chen), 2003, molecular breeding (Mol.Breed.), 11:287.) 35S promoter is blended in 5 of virus sequence ' end.The carrier sequence is between the BamHI and SacI site of pBI121.With hammerhead ribozyme be put in 3 of virus sequence ' end (Te Erben people such as (Turpen), 1993, virological method magazine (J.Virol.Methods), 42:227).These construct sequence and the fusions of coding from sequence, 6 * His label and the ER delay anchor series KDEL (referring to SEQ ID NO.:10) of the proteic signal peptide of tobacco PR-1a that body comprises coding LicKM-PAD4 or LicKM.
Agrobacterium rhizogene strain A4 (ATCC43057) is transformed with constructing body pBI-D4-PRLicKM-PAD4K and pBI-D4-PRLicKMK.Make Agrobacterium culture growth as described and induce (people such as Ka Peila, 1997, plant science (Plant Sci.), 122:101).Under 28 ℃, make 2ml cause culture (from fresh bacterium colony, choosing) at the YEB with 25 μ g/ml kantlex (5g/l beef extract, 1g/l yeast extract, 5g/l peptone, 5g/l sucrose, 2mM MgSO 4) middle growth whole night.To cause culture with 1:500 be diluted in 500ml have 25 μ g/ml kantlex, 10mM2-4 (morpholinyl) ethane sulfonic acid (MES) (pH5.6), the extra MgSO of 2mM 4In the YEB of 20 μ M Syringylethanones.Make subsequently through the dilution culture 28 ℃ down growth whole night to reach about 1.7 O.D.600.With cell with 3, centrifugal 15 minutes of 000 * g and with its resuspending in MMA substratum (MS salt, 10mM MES (pH5.6), 20g/l sucrose, 200 μ M Syringylethanones) to reach 2.4 O.D.600, at room temperature keep 1 hour, and use it for the Agrobacterium infiltration.Use the needle-less disposable syringe that agrobacterium suspension is injected the Ben Saimushi tobacco leaf.Permeate the blade of gathering infiltration in back 6 days.
Example 11: construct body surface by the Agrobacterium that contains virus sequence and reach influenza antigen
Can utilize the agriculture bacillus mediated transient expression system that obtains by the Agrobacterium osmose process to come the expression of influenza virus antigen.(people such as Te Erben, (1993) contain transfection (Transfection of whole plants from wounds inoculated with Agrobacteriumtumefaciens containing cDNA of tobacco mosaic virus) (virological method, the 42:227 of whole plants of agrobacterium tumefaciens inoculation of the cDNA of tobacco mosaic virus (TMV) by the wound utilization.) utilize the healthy leaves contain through engineered Agrobacterium rhizogenes infiltration Ben Saimushi tobacco with the virus vector of expressing LicKM-HA or LicKM-NA.
Agrobacterium rhizogene strain A4 (ATCC43057) is transformed with constructing body pBI-D4-PRACS-LicKM-HA-KDEL, pBI-D4-PRACS-LicKM-HA-VAC, pBI-D4-PRACS-LicKM-NA-KDEL and PBI-D4-PRACS-LicKM-NA-VAC.As described in people such as Ka Peila, make the growth of Agrobacterium culture and induce (Ka Peila J. (Kapila J.), Drake R. (De Rycke R.), your G. (Angenon G.) (1997) of model Montague M. (Van Montagu M.) and Anji is at agriculture bacillus mediated transient gene expression system (the AnAgrobacterium-mediated transient gene expression system for intact leaves.) plant science (Plant Sci.) 122 of intact leaf, 101-108).Under 28 ℃, make 2ml cause culture (from fresh bacterium colony, choosing) at the YEB with 25 μ g/ml kantlex (5g/l beef extract, 1g/l yeast extract, 5g/l peptone, 5g/l sucrose, 2mM MgSO 4) middle growth whole night.To cause culture with 1:500 be diluted in 500ml have 25 μ g/ml kantlex, 10mM2-4 (morpholinyl) ethane sulfonic acid (MES) (pH5.6), the extra MgSO of 2mM 4In the YEB of 20 μ M Syringylethanones.Make subsequently through the dilution culture 28 ℃ down growth whole night to reach about 1.7 O.D.600.With cell with 3, centrifugal 15 minutes of 000 * g and with its resuspending in MMA substratum (MS salt, 10mM MES (pH5.6), 20g/l sucrose, 200 μ M Syringylethanones) to reach 2.4 O.D.600, at room temperature keep 1-3 hour, and use it for the Agrobacterium infiltration.Use the needle-less disposable syringe that agrobacterium suspension is injected the Ben Saimushi tobacco leaf.Permeate the blade of gathering infiltration in back 6 days.Can be by the existence screening plant that the evaluation of active calibrating of lichenase and immunoblotting assay is expressed at target antigen.
Enzyme spectrum analysis is disclosed in the expression that has HA and NA chimeric protein in the Ben Saimushi tobacco transgenosis root of being tested.Described expression is relevant with the lichenin enzymic activity.The active bands of a spectrum relevant with fusion rotein show its molecular weight than lichenase contrast high and with agroinfection after the molecular weight of the expressed product of plant identical, confirm the existence of complete fusion product.
Example 12: construct body surface by the Agrobacterium that contains virus sequence and reach Antibody of Influenza
Other embodiment
One of ordinary skill in the art will understand, aforementioned representative some preferred embodiment of the present invention and it should be interpreted as limiting the spirit or scope of the present invention as by following claims defined.

Claims (45)

1. method that in seedling, produces medicine and pharmacology active protein or polypeptide, it comprises following steps:
Pea seedling is constructed body with the Agrobacterium that comprises the control virus sequence promotor of transcribing merge, described virus sequence is loaded with the protein that coding pays close attention to or the encoding sequence of polypeptide;
Thereby thereby cultivate the time that described fusion pea seedling is enough to allow to produce from described promoter transcription the virus transcription thing for one being enough to allow to produce under the condition of virus transcription thing from described promoter transcription, described virus transcription thing comprises the protein that coding is paid close attention to or the sequence of polypeptide;
Cultivate one period that is enough to allow to produce described protein or polypeptide of described fusion pea seedling being enough to allow to produce under the condition of described protein or polypeptide.
2. method according to claim 1, wherein said Agrobacterium constructs the virus sequence that body comprises tobacco mosaic virus (TMV), alfalfa mosaic virus, cauliflower mosaic virus or above-mentioned any combination.
3. method according to claim 1, wherein said Agrobacterium constructs the virus sequence that body comprises tobacco mosaic virus (TMV).
4. method according to claim 1, wherein said Agrobacterium constructs the virus sequence that body comprises alfalfa mosaic virus.
5. method according to claim 1, wherein said Agrobacterium constructs the virus sequence that body comprises cauliflower mosaic virus.
6. method according to claim 1, wherein said virus sequence are under the control of 35S promoter of cauliflower mosaic virus.
7. method according to claim 6, the 35S promoter of wherein said cauliflower mosaic virus the initial of rear drive recombinant virus genomes in being fused to described pea seedling transcribes.
8. method according to claim 1, the promotor that wherein said control virus sequence is transcribed is the composition promotor.
9. method according to claim 1, the promotor that wherein said control virus sequence is transcribed is an inducible promoters.
10. method according to claim 9, wherein said inducible promoters are the heat-shocked promotor.
11. method according to claim 9, wherein said inducible promoters are the light inducible promoters.
12. method according to claim 9, wherein said inducible promoters are chemical inducible promoters.
13. method according to claim 1, wherein said Agrobacterium are constructed body and are comprised transcription terminator.
14. method according to claim 13, wherein said transcription terminator are the transcription terminator of Agrobacterium (Agrobacterium) rouge alkali synthetase.
15. method according to claim 1, wherein said Agrobacterium are constructed body and are comprised the gene that one or more are used for virus replication.
Comprise one or more and be used for cell 16. method according to claim 1, wherein said Agrobacterium are constructed body to the gene of cell movement.
17. method according to claim 1, wherein said protein of paying close attention to or polypeptide are in transcribing under the control of virus mRNA promotor.
18. method according to claim 17, wherein said virus mRNA promotor are coat protein subgenomic mRNA promotor.
19. method according to claim 1, wherein said Agrobacterium are constructed body and are comprised left margin and right border sequence.
20. method according to claim 19, wherein said left margin and right border sequence delimited after pea seedling and described Agrobacterium are constructed the body fusion and transferred to the boundary that the described Agrobacterium in the described pea seedling cell constructs the tagma.
21. method according to claim 1, wherein said pea seedling and the multiple body of constructing merge.
22. method according to claim 1, wherein said pea seedling are selected from the group that is made up of junge Erbsen, Bill's jumping bean, yellow folder beans, spot pea and junge Erbsen.
23. method according to claim 1, wherein said protein of paying close attention to or polypeptide are Regular Insulin.
24. method according to claim 1, wherein said protein of paying close attention to or polypeptide are L-Glutamic decarboxylase.
25. method according to claim 1, wherein said protein of paying close attention to or polypeptide are tyrosine phosphatase sample protein I A-2.
26. method according to claim 1, wherein said protein of paying close attention to or polypeptide are green fluorescent protein.
27. method according to claim 1, wherein said protein of paying close attention to or polypeptide are the human growth hormone.
28. method according to claim 1, wherein said protein of paying close attention to or polypeptide are one or more components of anthrax toxin.
29. method according to claim 28, the component of wherein said anthrax toxin are protective antigen.
30. method according to claim 29 has wherein introduced making the sudden change of described protective antigen as the toxin non-activity.
31. method according to claim 28, the component of wherein said anthrax toxin are lethal gene.
32. method according to claim 31 has wherein introduced making the sudden change of described lethal gene as the toxin non-activity.
33. method according to claim 1, wherein said protein of paying close attention to or polypeptide comprise the lichenin enzyme sequence.
34. method according to claim 1, wherein said fusion pea seedling is to cultivate under the hydroponics growth conditions.
35. method according to claim 1, wherein every kilogram of plant tissue produces at least 5 described protein of gram or polypeptide.
36. method according to claim 1, wherein every kilogram of plant tissue produces at least about 1 described protein of gram or polypeptide.
37. method according to claim 1, wherein every kilogram of plant tissue produces at least about 0.5 described protein of gram or polypeptide.
38. method according to claim 1, wherein every kilogram of plant tissue produces at least about described protein of 20-500 milligram or polypeptide.
39. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 6 weeks for construct body from described Agrobacterium.
40. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 3 weeks for construct body from described Agrobacterium.
41. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 2 weeks for construct body from described Agrobacterium.
41. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 1 week for construct body from described Agrobacterium.
42. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 3 days for construct body from described Agrobacterium.
43. method according to claim 1, the wherein said time that is enough to allow to produce described protein or polypeptide merges at least about 1 day for construct body from described Agrobacterium.
44. method according to claim 1, wherein said seedling are pea seedling.
45. a method that produces medicine and pharmacology active protein or polypeptide in Nicotiana (Nicotiana) plant, it comprises following steps:
The Nicotiana seedling is constructed body with the Agrobacterium that comprises the control virus sequence promotor of transcribing merge, described virus sequence is loaded with the protein that coding pays close attention to or the encoding sequence of polypeptide;
Thereby thereby cultivate the time that described fusion Nicotiana seedling is enough to allow to produce from described promoter transcription the virus transcription thing for one being enough to allow to produce under the condition of virus transcription thing from described promoter transcription, described virus transcription thing comprises the protein that coding is paid close attention to or the encoding sequence of polypeptide;
Cultivate described one period that is enough to allow to produce described protein or polypeptide of fusion Nicotiana seedling being enough to allow to produce under the condition of described protein or polypeptide.
CN200780007805A 2006-02-13 2007-02-13 Production of foreign nucleic acids and polypeptides in plant systems Pending CN101815784A (en)

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