CN1330718A - Therapeutically active proteins in plants - Google Patents

Therapeutically active proteins in plants Download PDF

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Publication number
CN1330718A
CN1330718A CN99813154A CN99813154A CN1330718A CN 1330718 A CN1330718 A CN 1330718A CN 99813154 A CN99813154 A CN 99813154A CN 99813154 A CN99813154 A CN 99813154A CN 1330718 A CN1330718 A CN 1330718A
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plant
protein
gene
promotor
host
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P·B·海费茨
S·A·戈夫
A·B·塔特尔
M·E·格里奥特－温克
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Syngenta Participations AG
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

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  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Environmental Sciences (AREA)
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  • Ophthalmology & Optometry (AREA)
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Abstract

The present invention discloses transgenic plants expressing therapeutically active proteins, preferably from their plastid genome or targeted to the vacuole. The present invention also describes the administration of such transgenic plants to a host in need thereof for the prevention or treatment of diseases. In a preferred embodiment, such plants or matter derived from such plants is administered orally to a host.

Description

Therapeutic activity protein in the plant
The present invention generally relates to the transgenic plant of expression treatment active protein.Particularly, the present invention relates to, preferably vacuole or the plant of expression treatment active protein in plant plastid more preferably subcellular organelle.The present invention also relates to the therepic use of these transgenic plant.
There is multiple disease can torment animal, comprises people, pet and domestic animal.These diseases not only cause great misery, and are converted into bigger financial loss.Ill workman can not work or have relatively poor performance, and the disease of invasion and attack farming animals can significantly reduce farm output.Every kind of human civilization is all used the pharmacological agent disease of oneself, though numerous disease and is still successfully being treated by modern medicine, multiple other diseases still can't be treated or only can partly be treated.
Cost or its limited supply of a large amount of medicines are the problems that influence disease treatment.Therefore, have only a small amount of patient effectively to be treated sometimes, particularly in developing country, expensive treatment is a white elephant for the sanitation system in the worldwide.In addition, in agriculture field, because cost substantially exceeds the benefit that effective treatment obtains, so cause more low-producing some disease fully not treat.An expensive major reason of some drugs is to lack economic production method, particularly for the medicine based on protein or peptide.In the current medical science another be left in the basket sometimes but very important problem be to the host's organ of hope or the medicine of body portion administering therapeutic significant quantity.In fact, it is satisfied fully that current available application process can not be made us sometimes, for example, and for the treatment of transformation reactions or autoimmune disease.Another problem relevant with medicine is its transportation, and particularly when megathermal climate, this needs expensive refrigeration and expensive preparation, thereby has further improved cost.The operability that does not cause expensive transportation or use the high amount of drug of cost will be very favourable.
For the therapeutic activity protein expression, plant is strong candidate, because they are edible, and high-biomass output is arranged.With the isolated cellular compartment of cytoplasmic protein enzyme in the expression treatment active protein be a kind of attracting especially method that in plant, produces therapeutical active compound.
In a word, needing always but obtain yet can be in a large number and the newtype drug that obtains cheaply, and it can be used patient or host by several different methods, and is easy to obtain in the whole world.
The present invention has set forth a large amount of and cheap medicine particularly based on the needs of the supply of proteinic medicine, is used for prevention or treatment disease.Therefore, the invention provides can expression treatment the transgenic plant of active protein, particularly can be in subcellular organelle, preferably in vacuole, or transgenic plant of expression treatment active protein in plant plastid more preferably.Plant of the present invention is particularly useful for suppressing or reducing undesirable immunne response.Can be by multiple application process, preferably per os is applied in expressed protein in the transgenic plant to the host.For example, also as mentioned below in some cases, can do not extract in advance or the situation of purifying under, or through less extraction or purifying, per os is ingested and is contained proteinic plant of therapeutic activity or vegetable material.This is easily by diet prevention of disease or treatment, and can reduce the cost of medicine greatly.In addition, can extract and be applied in expressed protein in the transgenic plant with the well-known method of field of medicaments.
Especially, the present invention relates to from the transgenic plant of its plastom expression treatment active protein.Usually the genetically modified level that surpasses nuclear expression according to the expression level in the plastid of the present invention.This high-caliber expression causes the protein of the high density in every gram plant tissue, thereby has satisfied the long-standing needs that satisfy yet so far by the vegetable material that uses plant or derive from these plants in multiple therepic use.In addition, because the shortage of gene silencing, transgene expression level is still stable through certain hour and since transgenosis by homologous recombination on plastom accurately, the insertion of target location, it is not problem that position effect changes.In addition, expressed protein is isolated in organoid in plant plastid, therefore avoids degraded easily, particularly avoids degrading in Digestive tract when per os is ingested.Isolation in organoid also prevents transgenosis and the kytoplasm environmental interaction that plastid is expressed.If expressed protein also shows the activity to some composition of plant in the plant, then this feature is necessary.In addition, can obtain induction type plastid expression system (referring to WO 98/11235), the time point that the expression of this render transgenic is confined to wish, thereby the potential for a long time detrimental action that limits high transgene expression level.Similarly, lead the avoiding degrading or avoid unfavorable interaction with the kytoplasm environment of other subcellular compartments such as vacuole by nuclear gene group expressed protein is also protected.
The present invention also provides the therapeutic composition that comprises the plant material that derives from these transgenic plant, the novel method of expression treatment active protein and improve the novel method that multiple host comprises the state of an illness of people, domestic animal, pet and other animals.The method of the therapeutical agent of using plant derivation also is provided.
Therefore the present invention provides:
A kind of plant that in plastom, comprises at least a dna molecular, at least a protein that therapeutic activity is arranged when the host is preferably used with the treatment significant quantity the host of needs of this dna molecule encode, wherein this plant can be expressed one or more protein.In a preferred embodiment, therapeutic activity protein accumulates in plant plastid.In a further preferred embodiment, this protein is expressed in the edible part of plant.In a further preferred embodiment, to this protein of host's dosage forms for oral administration.In a further preferred embodiment, this host is a kind of vertebrates, and preferably Mammals more preferably is people, ox, sheep, pig, dog or cat.In a preferred embodiment, therapeutic activity protein is a kind of antigen, preferably a kind of immuno-activated-antigen.Therefore, the invention provides a kind of plant, it contains the preferably dna molecular of immuno-activated-antigen of a kind of antigen of coding in plastom, and wherein this plant can be expressed this antigen.Preferably, this antigen is expressed in this plant, more preferably, after the host absorbs this plant this antigenic immunne response is reduced.
In a further preferred embodiment, antigen can suppress or reduce animal to antigenic immunne response, preferably by inducing the host to this antigenic tolerance, for example, by intestinal mucosa contact or picked-up.A kind of preferred antigen is allergen, preferably airborne allergen, preferably pollen allergen.Preferred allergenic example is Der f I, Der f II, Der p I and Der p II, Can f II, Lol p V, Sor h I, Amb a I, preferably Amb a I.1, Amb a II, Aln g I, Cor a I, Bet v I, Fel d I or rAed a1 and rAed a2.For example, not glycosylation of the allergen of in plant of the present invention, expressing.Another kind of preferred antigen is autoantigen, as collagen protein, preferably receptor binding protein (interphoto receptorbinding protein), acetylcholine receptor, S-antigen, Regular Insulin, glutamate dehydrogenase, islet cells specific antigens or thyroglobulin between I type or III collagen type, II collagen type, myelin basic protein, myelin proteolipid protein, photosensory cell, or transplantation antigen, as heteroplastic transplantation of the same race or xenotransplantation antigen, MHC albumen for example, preferably MHC II proteinoid, preferably a or b chain.In a further preferred embodiment, immuno-activated-antigen energy induced animal is to antigenic immunity.In a further preferred embodiment, therapeutic activity protein is a kind of hematoglobin protein, hormone, somatomedin, cytokine, enzyme, acceptor, conjugated protein, immune system protein, translation or transcription factor, cancer protein or proto-protein, milk-protein, muscle protein, marrow albumen, neuroactive peptide or tumor growth arrestin or peptide, for example angiostatin or endostatin, both all suppress blood vessel and take place these.In another embodiment preferred of the present invention, therapeutic activity protein is a kind of anti-septicopyemia peptide, as BPI (bactericidal power/permeability enhancing albumen).In another embodiment, immune system protein is a kind of antibody.Dna molecular according to the present invention effectively is connected with the promotor that can express this dna molecular in plant plastid.In a preferred embodiment, promotor is the clpP promotor, is 16S r-RNA gene promoter in another preferred embodiment.In a particularly preferred embodiment of the present invention, promotor is a kind of promotor, particularly T7 gene 10 promotors of trans-activator mediation.In addition, the invention provides a kind of plant, it also comprises the allos nuclear expression box of the dna sequence dna that contains a kind of trans-activator of encoding.In a preferred embodiment, trans-activator is the T7 polysaccharase.The present invention further provides a kind of plant, it contains a kind of coding has therapeutic activity when the host of needs is used with the treatment significant quantity protein DNA molecule in the nuclear gene group, wherein this therapeutic activity protein be selected from mosquito allergen r Aed a1 and r Aed a2, bactericidal power/permeability strengthen albumen (BPI) and pollen allergen Amb a I, Amb a I.1, Amb a II, Amb t V, Aln g I, Cor a I, Lol pV, Sor h and Bet v I.
The present invention also provides a kind of plant, and it contains a kind of coding has therapeutic activity when the host to needs uses with the treatment significant quantity protein DNA molecule in the nuclear gene group, and wherein this therapeutic activity protein is directed to the subcellular organelle of this plant.In a preferred embodiment, therapeutic activity protein is directed in the vacuole.The therapeutic activity protein of guiding vacuole preferably is selected from allergen, as Der f I, Der f II, Der p I and Der p II, Can f II, Lol p V, Sor hI, Amb a I, preferably Amb a I.1, Amb a II, Amb t V, Aln g I, Cor aI, Bet v I, Fel d I, or rAed a1 and rAed a2, autoantigen, as collagen protein, preferably I type or III collagen type, the II collagen type, myelin basic protein, myelin proteolipid protein, receptor binding protein between photosensory cell, acetylcholine receptor, S-antigen, Regular Insulin, glutamate dehydrogenase, islet cells specific antigens or thyroglobulin, or transplantation antigen, as heteroplastic transplantation or xenotransplantation antigen, MHC albumen for example, MHC II proteinoid preferably, preferably a or b chain, or come from blood protein, hormone, somatomedin, cytokine, enzyme, acceptor, conjugated protein, immune system protein, translation or transcription factor, cancer protein or proto-protein, milk-protein, muscle protein, marrow albumen, neuroactive peptide or tumor growth arrestin or peptide, for example angiostatin or endostatin, both all suppress blood vessel and take place these.In another preferred embodiment, the therapeutic activity protein of the vacuole that can lead is a kind of anti-septicopyemia peptide, as BPI (bactericidal power/permeability enhancing albumen).
In a preferred embodiment, plant according to the present invention is a kind of edible plant.In another preferred embodiment, this plant is a kind of dicotyledons, preferably tobacco, tomato, soybean or spinach.In another embodiment, this plant is a kind of monocotyledons, preferably corn or rice.
In a preferred embodiment, the expression of protein in plant is adjustable, but Chemical Regulation preferably.In addition, also composing type, tissue-specific or grow and regulate of protein expression.
This also comprises the seed of this kind of plant, and its seed is randomly handled (as filling or bag quilt) and/or packing, for example is put in sack or other containers with working instructions.
Host according to the present invention is a kind of vertebrates, particularly Mammals, more particularly people, ox, sheep, pig, dog or cat.
The present invention further provides:
A kind of composition that contains with good grounds plant of the present invention or derive from the plant material of these plants, wherein said composition contains and a kind ofly treats the described protein of significant quantity and wherein handled a kind of composition of this plant before the host is used.
The present invention further provides method, wherein:
-use according to composition of the present invention to improve the effective host who measures a kind of needs of host's state of an illness
-to this host's dosage forms for oral administration said composition
-this protein is a kind of antigen, and this host is suppressed this antigenic immunne response or reduces thus
Particularly a kind of allergen of-this antigen, autoantigen or transplantation antigen.
The present invention also provides a kind of treatment or prophylactic method, it comprise host to needs use a kind of treat significant quantity according to plant of the present invention or derive from the plant material of this plant.
In a particular embodiment, described disease is transformation reactions, autoimmune disease or transplant rejection.In another particular embodiment, to the described treatment significant quantity of host's dosage forms for oral administration.
The present invention further provides:
As the plant of the present invention of medicine, be preferably used for treatment or preventing disease, for example transformation reactions, autoimmune disease or transplanting, for example by in the patient of needs, inducing tolerance, oral tolerance for example, or be used for host's immunity.
The present invention further provides:
As the plant of the present invention of medical food, be preferably used for treatment or preventing disease, for example transformation reactions, autoimmune disease or transplanting, for example by in the patient of needs, inducing tolerance, oral tolerance for example, or be used for host's immunity.
The present invention further provides:
A kind of plastid conversion carrier that contains a kind of protein DNA molecule of encoding, when the host to needs uses with the treatment significant quantity, this protein has therapeutic activity, and wherein this dna molecular effectively is connected with guiding this dna molecular expression promoter in plant plastid.In a preferred embodiment, this protein accumulates in plant plastid.In a further preferred embodiment, promotor in the plastid conversion carrier be clpP promotor, 16S r-RNA gene promoter, psbA promotor, rbcL promotor or the trans-activator mediation regulated by the nuclear trans-activator promotor (for example, when trans-activator is T7, be T7 gene 10 promotors).
A kind of plastid that contains aforesaid conversion carrier.
A kind of vegetable cell that contains aforesaid plastid, wherein this vegetable cell can produce described protein.
The present invention also provides:
One kind of plant, it comprises:
A kind of allos nuclear expression box, it contains a kind of promotor, inducible promoter for example, wound-induced type or chemical inducible promoter, tobacco PR-1a promotor for example, or organizing specific type or organ Idiotype promotor, or the promotor of growth adjusting, this promotor and coding trans-activator (naturally occurring trans-activator in plant not preferably, preferably RNA polymerase or DNA are conjugated protein, as the T7 RNA polymerase) dna sequence dna effectively connect, this trans-activator randomly merges with plastid targeting sequencing such as chloroplast(id) targeting sequencing (for example, the expression cassette that can express in plant); With
A kind of allos plastid expression cassette, it contains by the trans-activator adjusting and randomly mediates promotor (for example, being the T7 gene promoter when trans-activator is the T7 RNA polymerase) with the trans-activator that coding therapeutic activity protein DNA molecule of the present invention effectively is connected;
The seed that also comprises this kind of plant, its seed are randomly handled (as filling or bag quilt) and/or packing, for example are put in sack or other containers with working instructions.
The present invention further provides:
A kind of composition that contains aforesaid plant or plant material, wherein said composition contains a certain amount of described protein, and it is that treatment is effective when the host to needs uses.In a preferred embodiment, before being used, the host handles plant material.The preferably conventional a kind of processing that is used for food or fodder industry of this processing.In a further preferred embodiment, composition of the present invention contains a certain amount of immune effective antigens.
The present invention further provides:
Comprise the medicinal compositions of plant of the present invention or plant material and comprise the medical food compositions of plant of the present invention or plant material.
The present invention also provides:
A kind of method comprises the plastom that transforms plant with aforesaid conversion carrier, preferably further is included in and expresses a kind of therapeutic activity protein in this plant.In a preferred embodiment, therapeutic activity protein is a kind of antigen, preferably a kind of immuno-activated-antigen.
The present invention further provides:
A kind of method comprises the host of needs is used a kind of aforesaid composition with the amount that is enough to improve host's situation.Preferably, this method comprises the Orally administered said composition to the host.
The present invention further provides:
The method of a kind of treatment or preventing disease such as transformation reactions, autoimmune disease or transplant rejection, for example by in the host of needs, inducing tolerance, or being used for host's immunity, this method comprises the host used a kind ofly treats the plant of the present invention of significant quantity or by its deutero-plant material.
The present invention further provides:
Plant of the present invention production be used for the treatment of or prophylactic medicine in purposes, for example,, be used for the treatment of transformation reactions, autoimmune disease or transplanting, or be used for host's immunity by in the host of needs, inducing tolerance.
The present invention further provides:
Plant of the present invention production be used for the treatment of or prophylactic medical food in purposes, for example,, be used for the treatment of transformation reactions, autoimmune disease or transplanting, or be used for host's immunity by in the host of needs, inducing tolerance.
The present invention further provides:
Plant of the present invention is used for measuring the purposes of the antigen of immunologic competence in production.
The present invention further provides:
A kind of antibody to the antigen-specific of expressing in the plant of the present invention.
A kind of antibody, it disturbs the antibody of antigen-specific and combining of this antigen expressed to expressing in the plant of the present invention.
The present invention further provides:
A kind of food, it contains the plant edible part according to dna molecular of the present invention, and wherein when with the treatment significant quantity host of needs being used, this food has therapeutic activity.
The present invention further provides:
Derive from the plant that contains with good grounds dna molecular of the present invention or the agricultural-food of plant part, wherein when with the treatment significant quantity host of needs being used, these agricultural-food have therapeutic activity.
The present invention also provides:
All product innovations, methods and applications as described here.
Definition
Broadly, " therapeutic activity protein " helps host's patient's condition in positive mode when with the treatment significant quantity host being used.Therapeutic activity protein has healing, treatment or mitigate effects to disease, and can use to improve, to alleviate, to alleviate the severity of disease." therapeutic activity protein " also has prophylactic effect, and is used for prophylactic outbreak or alleviate the severity of disease or illness in when outbreak.Term " therapeutic activity protein " comprises complete protein or peptide, or its therapeutic activity fragment.It also comprises the therapeutic activity analogue of this protein or peptide, or the segmental analogue of this protein or peptide.Term " therapeutic activity protein " also refer to act synergistically multiple proteins or peptide so that therapeutic action to be provided.
Therapeutic activity proteinic " analogue " comprises the protein relevant with having the bioactive protein structure identical with this protein.
" immunne response " is meant the physiological responses that is caused by the antigen activates immunity system.In the present invention, particularly when dosage forms for oral administration antigen, can suppress immunne response by according to the tolerance of contact antigen induction.
" immunocompetence " be meant at this, for example replys by immune stimulatory or suppress or reduce immunne response or inflammation condition, can regulate immunity system,
" antigen " is a kind of and immunity system, and preferably the product with special body fluid or cellular immunization interacts, and stimulates the material of " immunne response ".Antigen is a peptide species preferably, and is a kind of " therapeutic activity protein ".Antigen is included as all or part of polypeptide of polypeptide.These polypeptide portions for example are antigenic epi-position or antigenic determinant.An antigen can comprise one or more epi-position or antigenic determinant.Antigen also comprises antigenic analogue, comprises and has the biological activity identical with this antigen, be i.e. the relevant molecule of the antigenic structure of identical immunologic competence.In specification sheets of the present invention, antigen comprises for example allergen, autoantigen and transplantation antigen.
" epi-position " is an antigenic part, its decision in antigen-antibody interaction with the specific combination site bonded ability of corresponding antibodies.
" antigenic determinant " is the specific antigen part of immunne response of decision antigenic stimulation.
" adjuvant " is a kind of material, oily substance preferably, and when mixing with antigen when using, particularly when mixing with antigen and injecting, the non-enhancing specifically to this antigenic immunne response.A kind of typical adjuvant is complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (people (1981) J.P.immunol.126:1526 such as Lando).
" autoantigen " is any material of usually finding in animal body, and when abnormal conditions such as autoimmune disease, it no longer is the part of animal self by the immune system recognition of this animal, therefore as allogenic material by immune system attack.
" allergen " is to induce the allergic antigen of host.
The host is the aspect that immune stimulatory is replied to " immunity " of pathogenic agent or toxin, is meant that herein the host passes through the protection of induction of immunity system enantiopathy substance or toxin.Preferably, induce direct immunization to reply and immunological memory, quick and secular host's protection preferably is provided.Toxin host's antigen is used for immunity.Inoculation also is contained in the term immunity.
The host of needs " is used " therapeutic activity protein be meant and provide therapeutic activity protein, so that this proteinic therapeutic action keeps being enough to provide for the host time of the advantageous effect of hope the host.
Therapeutic activity proteinic " dosage forms for oral administration " mainly is meant using of per os, preferably by edible, comprises that also stomach or the digestive tube to the host provides these proteinic any using.In a preferred embodiment, dosage forms for oral administration causes contacting of therapeutic activity protein and intestinal mucosa.
" host " uses the proteinic animal of therapeutic activity of the present invention to it.Term " animal " is contained and is had immune all life forms, comprises people, ox, sheep, pig, dog or cat.
" food " or " food " is meant liquid or solid food or food or feed at this, is the plant that can be taken in by people and other animals, plant part or by its deutero-plant material.This term comprises can directly supply the primordial plant or the vegetable material of people and other animal edibles, or supplies any plant material of having processed that contains nutrition carrier of people and other animal edibles.The material that obtains from plant comprises finally by any part of the plant of people or the absorption of other animals.
" medical food " comprises the composition that can be eaten or drink and the host is had therapeutic action by the host.Medical food comprises plant for example of the present invention or by its deutero-plant material.Medical food can be taken in separately, or can use with the well-known medicinal compositions of medical field.Medical food also comprises the equivalent feed of supplying with the non-human animal.
When this used, " agricultural prods " was meant and can as clover bud, Radix Raphani bud, wheat malt etc., or be referred to that give birth to or ripe form by the edible part of the plant of human consumption by whole edible plant.Edible part can be a root, as rutabaga, beet, Radix Dauci Sativae and Ipomoea batatas; Stem tuber or storage stem are as potato, choke and taro; Stem is as asparagus fern and kohlrabi; Bud is as Brussels sprouts; Bulb is as onion and garlic; Petiole is as celery and rheum officinale; Leaf is as Caulis et Folium Brassicae capitatae, lettuce, parsley and spinach; Immature flower is as Cauliflower, asparagus broccoli and choke; Seed; Green fruit is as eggplant, cucumber and sweet corn (corn); Or mellow fruit, as tomato, pepper, apple, pears, banana, orange, berry etc.
" plant " is meant any plant, particularly spermatophyte.
" vegetable cell " is meant structure and the physiology unit of plant, comprises protoplastis and cell walls.Vegetable cell can be the form of isolating unicellular or culturing cell, perhaps as the part of high-level organization unit as plant tissue or plant organ.
" vegetable material " is meant other any parts or the product of leaf, stem, root, flower or flower part, fruit, pollen, pollen tube, ovule, blastular, ovum, zygote, embryo, seed, cutting, cell or tissue culture or plant.
" plant material " is meant arbitrary part of the plant that is in arbitrary etap, these plants that preferably can dosage forms for oral administration.Plant material comprises edible plant part, as leaf, seed, fruit, stem tuber, maybe can give birth to or ripe other plant part of ingesting.Plant material also comprises the part of isolating plant, as subcellular organelle, and for example plastid or vacuole.Plant material also comprises and has stood multiple procedure of processing, the plant part of procedure of processing commonly used in food or the fodder industry particularly, these steps include but not limited to: the concentrating of vegetable solid material, and form for example precipitation, pasty state, drying or freeze dried product, or grind by cutting or mill plant is broken in various degree, or the liquid portion of extracting plant produces soup, syrup or juice.Procedure of processing also can comprise boil plant or plant material.
" expression " is meant the transcribing and/or translating in plant of native gene or transgenosis.For example, for antisense constructs, expression can only refer to transcribing of antisense DNA.
" expression cassette " meaning is a kind of dna sequence dna that can guide specific nucleotide sequence to express in suitable host cell as used herein, it contains the promotor that effectively is connected with the purpose nucleotide sequence, and this sequence randomly regulates sequence with 3 ' sequence as 3 ' or termination signal effectively is connected.Expression cassette generally also contains the suitably required sequence of translation of nucleotide sequence.The coding region target protein matter of encoding usually, but also a kind of purpose function RNA of codified, for example sense-rna or untranslatable rna, they suppress the expression of specific gene such as sense-rna so that justice or antisense orientation to be arranged.The expression cassette that contains the purpose nucleotide sequence can be chimeric, the meaning is that this nucleotide sequence is made up of the dna sequence dna of more than one different sources, they merge by recombinant DNA technology, the generation non-natural exists, particularly non-existent nucleotide sequence in by plant transformed.Expression cassette also can be natural existence but obtain with the recombinant forms that is used for heterogenous expression.Yet generally speaking, expression cassette is allogenic for the host, i.e. the specific dna sequence of expression cassette not natural existence in host cell must import among the ancestors of host cell or host cell by transformation event.The expression of nucleotide sequence can be subjected to the control of constitutive promoter or inducible promoter in the expression cassette, and this promotor is initial transcribing just when host cell contacts certain specific outside stimulus thing only.For multicellular organism, as plant, promotor also may be special to particular organization or organ or etap.The nuclear expression box is inserted in the nuclear gene group of plant usually, and can guide the expression of the specific nucleotide sequence of this plant nucleus gene group.The plastid expression cassette is inserted in the plastom of plant usually, and can guide the expression of the genomic specific nucleotide sequence of this plant plastid.For the plastid expression cassette, in order to express nucleotide sequence, may need other elements, i.e. ribosome bind site, or the 3 ' stem-ring structure that stops plastid RNA polyadenylation and degrade subsequently by plastom.
" gene " is meant a kind of encoding sequence or relevant adjusting sequence, and wherein this encoding sequence can be transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, adopted RNA or sense-rna are arranged.The example of regulating sequence is promoter sequence, 5 ' and 3 ' non-translated sequence and terminator sequence.Other elements that may exist are introns for example.
" allogenic " meaning is different natures or synthetic source as used herein.For example, if use, think that then this nucleotide sequence is allogenic for this host cell by non-existent nucleotide sequence transformed host cell in the transformant.The nucleic acid that transforms can contain a kind of allogeneic promoter, allogeneic coding sequence or allos terminator sequence.In addition, the nucleic acid of conversion also can be allogenic fully, perhaps can comprise the arbitrary possible combination of allos and endogenous nucleic acid sequence.Similarly, allos is meant a kind of deriving from and the nucleotide sequence that inserts identical natural, initiating cell type, but it exists with the non-natural state, for example, has different copy numbers, or is under the control of different adjustment element.
" marker gene " is that a kind of selectivity of encoding maybe can be screened the gene of proterties.
Make the adjusting dna sequence dna influence the expression of a kind of RNA of coding or protein DNA sequence if two kinds of sequences are positioned at the somewhere, think that so this adjusting dna sequence dna and this coding DNA acid sequence " effectively connect " or " relevant ".
" regulatory element " is meant and participates in the sequence that nucleotide sequence is expressed.Regulatory element contains a kind of promotor that effectively is connected with the purpose nucleotide sequence, and randomly 3 ' sequence is regulated sequence or termination signal as 3 '.They generally also comprise the suitably required sequence of translation of nucleotide sequence.
" subcellular organelle " is meant organ in the cell with peculiar 26S Proteasome Structure and Function.Subcellular organelle for example is, vacuole, plastid, plastosome, nucleus, endoplasmic reticulum or plasma membrane.
" homogeneity " refers to a kind of plant, plant tissue or vegetable cell, and wherein all plastids all are identical on the genetics.When plastid is transformed, sudden change or otherwise during hereditary change, this is standard state in plant.At different tissues or in the etap, plastid has different forms, for example chloroplast(id), proplastid, etioplast, amyloplast, chromoplast or the like.
" promotor " is meant the dna sequence dna that initial associated dna sequence is transcribed.The promoter region also can comprise the element as genetic expression instrumentality such as activator, toughener and/or repressor.
" inducible promoter " is initial promotor of transcribing just when some particular outer stimulator of plant contact only, and it is different from constitutive promoter or to particular organization or organ or special promotor of etap.Particularly preferably be chemical inducible promoter and wound-induced type promotor for the present invention.Chemical inducible promoter comprises the promotor of plant derivation, as the promotor in the systemic acquired resistance approach, PR promotor for example, as PR-1, PR-2, PR-3, PR-4 and PR-5 promotor, especially tobacco PR-1a promotor and Arabidopis thaliana PR-1 promotor are when plant contact BTH their initial transcribing during with relevant pharmaceutical chemicals.See United States Patent (USP) 5,614,395, be incorporated herein by reference, and WO 98/03536, be incorporated herein by reference.Chemical inducible promoter also comprises as deriving from the receptor-mediated system of other biological, genetic expression as the steroid dependence, glucocorticosteroid for example, progesterone and estrogen receptor system, the genetic expression that copper relies on, as expression based on ACE1, the genetic expression that tsiklomitsin relies on, the Lac that uses fruit bat (Drosophila) usp receptor of protecting young tethelin and antagonists to mediate thereof prevents system and expression system, this states in WO 97/13864, be incorporated herein by reference, and the system that uses the acceptor combination, as described in WO 96/27673, be incorporated herein by reference.Other chemically inducible promoters comprise inducer (elicitor) inductive promotor, safener inductive promotor and can (WO 93/21334 by certain alcohols and ketone inductive alcA/alcR gene activation system; People such as Caddick (1998) Nature Biotechnol (Nat Biotechnol) 16:177-180, its content is incorporated herein by reference).Wound-induced type promotor comprises the promotor of proteinase inhibitor, as proteinase inhibitor II promotor from potato, and the promotor of the plant derivation of other participation wound response pathway, as the promotor of polyphenoloxidase, LAP and TD.Total see C.Gatz, " chemical regulation of genetic expression ", plant physiology and molecular biology of plants annual report, (Annu.Rev.Plant Physiol.PlantMol.Biol.) (1997) 48:89-108, its content is incorporated herein by reference.
" trans-activator " is a kind of protein, itself or with one or more other protein bound, can make transcribing of coding region be subjected to the control of the promotor of corresponding trans-activator mediation.The example of trans-activator system comprises phage t7 gene 10 promotors, and its transcriptional activation depends on special RNA polymerase such as phage t7 RNA polymerase.Trans-activator generally is that a kind of RNA polymerase or DNA are conjugated protein, and it can for example, suppress the expression or the accumulation of aporepressor, initial the transcribing with specific promotor interaction by directly activating promotor or deactivation repressor gene.DNA is conjugated protein can be a kind of chimeric protein that comprises the land (as the GAL4 land) that is connected with suitable transcription activating protein structural domain.Some trans-activator system may have multiple trans-activator, for example not only needs the promotor of polysaccharase but also the specific subunit of needs (the sigma factor) for Promoter Recognition, DNA combination or transcriptional activation.Trans-activator is allogenic for carrying out inductive plant or subcellular organelle or plant cell constituents preferably.
" minimal promoter " comprises promoter element, TATA element particularly, and it is a non-activity, or have the promoter activity that reduces greatly can activate in no upstream the time.In the presence of suitable upstream activating sequence that merges with minimal promoter and corresponding transcription factor, minimal promoter allows to transcribe.
" recombinant DNA technology " is meant the method that is used for connecting dna sequence dna, for example, as people such as Sambrook, 1989, the cold spring port, NY: press of cold spring harbor laboratory is described.
" marker gene that can screen " is meant a kind of gene, and its expression can not be given the transformant selective advantage, but its expression makes transformant be different from unconverted cell on phenotype.
" selected marker " is meant a kind of gene, and its expression in vegetable cell gives this cell a kind of selective advantage.With the growth phase ratio of unconverted cell, the selective advantage that is had with selected marker's cell transformed may be because the ability of its growth in the presence of negative selective agent such as microbiotic or weedicide.Compare with unconverted cell, the selective advantage that transformant has also may be since the compound that its enhanced or new utilization are added as the ability of nutrient, somatomedin or the energy.The selected marker refers to that also the expression in vegetable cell gives the gene or the assortment of genes of negative selection of this cell and positive selective advantage.
" synthetic " is meant the nucleotide sequence that contains non-existent constitutional features in the native sequences.For example, more be similar to the G+C content of dicotyledons and/or monocotyledons gene and the artificial sequence of normal codon distribution and be considered to synthetic.
" conversion " is meant the importing of nucleic acid in cell.Particularly, the stable integration of dna molecular in purpose biological gene group.
Sequence summary in the sequence table
SEQ ID No:1 oligonucleotide
SEQ ID No:2 oligonucleotide
SEQ ID No:3 oligonucleotide
SEQ ID No:4 oligonucleotide
SEQ ID No:5 oligonucleotide
SEQ ID No:6 oligonucleotide
SEQ ID No:7 oligonucleotide
SEQ ID No:8 oligonucleotide
SEQ ID No:9 oligonucleotide
SEQ ID No:10 oligonucleotide
SEQ ID No:11 oligonucleotide
SEQ ID No:12 oligonucleotide
SEQ ID No:13 oligonucleotide
SEQ ID No:14 oligonucleotide
SEQ ID No:15 oligonucleotide
SEQ ID No:16 oligonucleotide
SEQ ID No:17 oligonucleotide
SEQ ID No:18 oligonucleotide
SEQ ID No:19 oligonucleotide
SEQ ID No:20 oligonucleotide
SEQ ID No:21 oligonucleotide
SEQ ID No:22 oligonucleotide
SEQ ID No:23 oligonucleotide
SEQ ID No:24 oligonucleotide
SEQ ID No:25 oligonucleotide
SEQ ID No:26 oligonucleotide
SEQ ID No:27 oligonucleotide T73a_U
SEQ ID No:28 oligonucleotide T73a_L
SEQ ID No:29 oligonucleotide minpsb_U
SEQ ID No:30 oligonucleotide minpsb_L
SEQ ID No:31 oligonucleotide
SEQ ID No:32 oligonucleotide
SEQ ID No:33 oligonucleotide
SEQ ID No:34 oligonucleotide
SEQ ID No:35 oligonucleotide
SEQ ID No:36 oligonucleotide
SEQ ID No:37 oligonucleotide ErspU
SEQ ID No:38 oligonucleotide ErspL
SEQ ID No:39 oligonucleotide ErspovL
SEQ ID No:40 oligonucleotide AmboeU
The invention discloses the particularly antigenic transgenic plant of expression treatment active protein.These protein DNA molecules of encoding are expressed from the plastom of plant, or express in nucleus, and with in this protein targeting cytosol or subcellular organelle such as the vacuole.Plant of the present invention can be with cost-effective mode marking protein, and is easy to obtain.These plants can be expressed a large amount of these protein in cost-effective mode.In addition, the therapeutic activity protein of expressing in plastid is packaged usually, makes purifying and processing easy especially.This compartmentation also makes the treatment molecule be protected in digestive process, thereby is suitable for dosage forms for oral administration.Can be applied in according to the therapeutic activity protein of expressing in the transgenic plant of the present invention the host who comprises people, pet or domestic animal with several different methods, with prevention or treat multiple disease, thereby improve the host's who is treated the state of an illness.Particularly, transgenic plant of the present invention or derive from the vegetable materials of these plants can be by host's oral uptake, and can be used for for example treating transformation reactions, autoimmune disease or prevent transplant rejection preferably by inducing the host to antigenic tolerance.The present invention also discloses the composition that comprises these plants or derive from the plant material of these plants, as medicinal compositions, and by using the method for the composition for improved host state of an illness of the present invention.The therapeutic activity protein of in transgenic plant, expressing
Protein of the present invention preferably can be regulated host's immunne response, has provided embodiment below.Therefore, they can be used for treating or preventing undesirable immunne response, for example, suppress or reduce host's immunne response by inducing tolerance.In addition, the immunity system that they also can stimulation of host, thus help or cause the immunity of host to disease such as bacterium, parasite or virus disease.
In a preferred embodiment, in transgenic plant, express a kind of protein.In this case, if wish that for special treatment some protein is arranged, then use every kind of blended plant of expressing different proteins.In another preferred embodiment, in same transgenic plant, express several different protein.In this case, comprise the dna molecular of the different proteins of encoding in the different expression cassettes that transform plant, perhaps in addition, these dna moleculars are contained in the same expression cassette.For expression, for example, can with the different therapeutic activity protein DNA molecular configuration of coding in a kind of expression cassette, form a kind of polycistronic messenger RNA(mRNA) from plastom.For simple and clear, " a kind of " therapeutic activity protein of mentioning herein is meant " at least a " therapeutic activity protein, and the meaning is one or more protein.The therapeutic activity protein of the present invention of treatment significant quantity also can obtain from a kind of plant or from the plant material that derives from a kind of plant, maybe can obtain from sisters' strain of a plurality of plants such as plant.
Plant transformed can be monocotyledons or dicotyledons according to the present invention, include but not limited to: corn, wheat, barley, rye, sweet potato, Kidney bean, pea, witloof, lettuce, cabbage, Cauliflower, brocoli, radish, Radix Raphani, spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, hemp, zucchini, apple, pears, Wen Bai, muskmelon, plum, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, Chinese sorghum, sugarcane, beet, Sunflower Receptacle, oilseed rape, trifolium, tobacco, Radix Dauci Sativae, cotton, clover, rice, potato, eggplant, cucumber, Arabidopis thaliana (Arabidopsis), turfgrass and ornamental plant and xylophyta such as coniferals and fallen leaves class tree.After the gene transformation specified plant kind of wishing, can utilize traditional raising technology in these species, to breed or transfer in other kinds of same species, particularly including commercial variety.
The present invention also comprises edible algae, as unicell green alga (for example Chlamydomonas (Chlamydomonas)), many cells green alga (for example sea lettuce (Ulva)), unicellular red algae (for example Porphyridium cruentum belongs to (Porphyridium)) and many cells red algae (for example Porphyra (Porphyra)), it contains the plastom that is substantially similar to higher plant, and available similar mode transforms.The expression of therapeutic activity protein in plant plastid
The present invention be more particularly directed to the expression of therapeutic activity protein from the plant plastid genome.In this case, transform the annular plastom of some or all several thousand copies that exist in every kind of plant cell with coding therapeutic activity protein DNA molecule of the present invention.The typical a large amount of transgenosis copy numbers of plastid make expression level substantially exceed the expression level that obtains usually usually from the nuclear expression gene.This high level expression has further reduced the proteinic cost of generation therapeutic activity in plant, and allow these transgenic plant needing in the high-level proteinic purposes at vegetable material to be used for, for example, at per os feeding plant or vegetable material during with treatment transformation reactions, autoimmune disease or prevention transplant rejection.Plastogene is expressed also a large amount of other advantages.For example, because the shortage of gene silencing and position effect change, transgene expression level is still stable through certain hour, polycistronic operon can be expressed by the single promotor or the adjusting sequence of the some protein that allows the generation equimolar amount with cooperative mode, the heredity of single parent's plastogene has prevented that the pollen of foreign DNA in the important crop of most of economy from transmitting, thereby has reduced to possibility wild or the cultivated plant horizontal transfer.The plastid transgenosis is integrated also and is taken place by the homologous recombination process, and the meaning is that accurately the transformation of guiding and gene are replaced and be easy to carry out.In addition, expressed protein is isolated in organoid in plastid, has therefore prevented and the kytoplasm environmental interaction.If expressed protein also shows the active or interaction unfriendly of some composition of antagonism vegetable cell colloidal sol in the plant, then this feature is necessary.
U.S. Patent number 5,451,513,5,545,817,5,545,818 and 5,576,198; PCT application number WO 95/16783 and WO 97/32977; With people such as McBride, among newspaper (the Proc.Natl.Acad.Sci. U.S.) 91:7301-7305 of institute of NAS (1994) the plastid transformation technology has been described extensively, all quote as a reference at this.Transform and in unicell green alga Reinhard chlamydomonas (Chlamydomonas reinhardtii), to carry out at first (people (1988) science (Science) 240:1534-1537 such as Boynton through the plastid of biolistics, be incorporated herein by reference), to utilize very soon and select cis acting antibiotics resistance gene seat (spectinomycin/streptomycin resistance) or non-this method of photosynthesis mutant phenotype complementary to expand to tobacco (Nicotiana tabacum) (institute of people (1990) NAS such as Svab reports 87:8526-8530, is incorporated herein by reference).
The basic fundamental that the tobacco plastid transforms comprises, to the particle bombardment of leaf or callus, or has the plasmid DNA picked-up of PEG mediation in the protoplastis in clone's plastid DNA district that flank is a selectivity antibiotics resistance mark.0.5-1.5kb flanking region is called targeting sequencing, is beneficial to the homologous recombination with plastom, therefore allows the displacement or the modification of 156kb tobacco plastom given zone.During beginning, with giving the plastid 16S rRNA of spectinomycin and/or streptomycin resistance and the point mutation in the rps12 gene as the selected marker (Svab, the Z. that transform, Hajdukiewicz, P. and Maliga, institute of P. (1990) NAS newspaper 87,8526-8530; Staub, J.M. and Maliga, P. (1992) vegetable cell 4,39-45 is incorporated herein by reference).This produces stable homogeneity transformant, and frequency is that per 100 bombardment target leaves produce about one.The existence of cloning site makes and can produce the plastid oriented carrier (P., EMBO be (1993) J.12:601-606, are incorporated herein by reference for Staub, J.M. and Maliga) that is used to import foreign gene between these marks.By separate the recessive rRNA of bacterium aadA gene substitution or the r-albumen antibiotics resistance gene of toxenzyme aminoglycoside-3 '-adenosyl transferase with dominant selectable marker one coding spectinomycin, can realize (the Svab of raising greatly of transformation frequency, Z. and Maliga, P. institute of (1993) NAS newspaper 90,913-917 is incorporated herein by reference).In the past, this mark once was successfully used to the high frequency conversion (4083-4089 is incorporated herein by reference for Goldschmidt-Clermont, M. (1991) nucleic acids research (Nucl.Acids Res.) 19) of green alga Reinhard chlamydomonas plastom.Recently, with the DNA picked-up of polyoxyethylene glycol (PEG) mediation realized the plastid of the protoplastis of tobacco and mosses Physcomitrella patens transform (O ' people (1993) plant magazine 3:729-738 such as Neill; People such as Koop (1996) Planta 199:193-201, both quote as a reference at this).These two kinds of methods of particle bombardment and protoplast transformation all are fit in the present invention.
With coding therapeutic activity protein DNA molecule of the present invention insert comprise a kind of can be in plant plastid in the plastid expression cassette of the promotor of expressible dna molecule.A kind of preferred promoter that can express in plant plastid is from the isolating promotor of plastogene upstream of coding region 5 ' flanking region, it may derive from identical or different species, and its natural product generally is present in most of plastid types and comprises in the plastid that exists in the non-chlorenchyma.Genetic expression in the plastid is different from nuclear gene to be expressed, and relevant with the genetic expression in the prokaryotic organism (as described in people such as Stern (1997) plant science trend (Trends in plant Sciences) 2:308-315, being incorporated herein by reference).The plastid promotor contains prokaryotic promoter typical-35 and-10 elements usually, some plastid promotors are by the intestinal bacteria sample RNA polymerase identification of mainly encoding in plastom, be called PEP (RNA polymerase of plastid coding) promotor, and other plastid promotors are by the RNA polymerase identification (NEP promotor) of nuclear coding.Two types plastid promotor all is applicable to the present invention.The example of plastid promotor comprises the promotor of clpP gene, (WO 97/06250 as tobacco clpP gene promoter, be incorporated herein by reference) and Arabidopis thaliana clpP gene promoter (be contained in the Arabidopis thaliana plastom between the site 71882 and 72371, its sequence can be available from TakakazuKaneko and Satoshi Tabata of the Kazusa DNA Institute, URL:ftp: //genome-ftp.stanford.edu/pub/arabidopsis/chloroplast/).Can be in plant plastid the another kind of promotor of expressible dna molecule derive from the regulatory region (people such as Harris of plastid 16S ribosome-RNA(rRNA) operon, microbiology summary (Microbiol.Rev.) 58:700-754 (1994), people such as Shinozaki, EMBO is (1986) J.5:2043-2049, all quote as a reference at this for two pieces).Can in plant plastid, other examples of the promotor of expressible dna molecule be psbA promotor or rbcL promotor.The plastid expression cassette preferably also contains the plastogene 3 ' non-translated sequence (3 ' UTR) that effectively is connected with dna molecular of the present invention in addition.The effect of non-translated sequence preferably guides 3 ' processing of transcribe rna, rather than Transcription Termination.Preferably, 3 ' UTR is plastid rps16 gene 3 ' non-translated sequence or Arabidopis thaliana plastid psbA gene 3 ' non-translated sequence.In a further preferred embodiment, the plastid expression cassette contains a poly-G tract and replaces 3 ' non-translated sequence.The plastid expression cassette preferably also contains with dna molecular of the present invention and effectively is connected, and the 5 ' non-translated sequence (5 ' UTR) of function is arranged in plant plastid.
The plastid expression cassette is contained in the plastid conversion carrier, and this carrier preferably also contains be useful on the flanking region of integrating by homologous recombination in plastom.The plastid conversion carrier can randomly contain at least one chloroplast(id) replication orgin.The present invention comprises that also wherein dna molecular can be expressed with this plastid conversion carrier plant transformed plastid in plant plastid.The present invention also comprises a kind of plant or vegetable cell that comprises this plant plastid, comprises its offspring.In a preferred embodiment, this plant or vegetable cell comprise its filial generation, are homogeneities for the transgenosis plastid.
Can be in plant plastid other promotors of expressible dna molecule promotor of having trans-activator to regulate, be allogenic preferably for the plant of wherein realizing expressing or subcellular organelle or vegetable cell composition.In these situations, the dna molecular of coding trans-activator is inserted in a kind of suitable nuclear expression box, and it is transformed among the plant nuclear DNA.With plastid transit peptides trans-activator is directed in the plastid.The dna molecular that trans-activator and trans-activator are ordered about gathers together, method is to make the plastid of selection transform system and the transgenic lines hybridization that contains a kind of dna molecular, this dna molecule encode is added with the trans-activator of plastid targeting sequencing, and with one nuclear promotor effectively be connected, the plastid conversion carrier that perhaps will contain the dna molecular that is hopeful directly is transformed in the transgenic lines that contains a kind of dna molecular, this dna molecule encode is added with the trans-activator of plastid targeting sequencing, and effectively is connected with a nuclear promotor.If this nuclear promotor is a kind of inducible promoter, chemical inducible promoter particularly, then the expression of dna molecular in plant plastid by the foliar spray of chemical inducer with and activate.The induction type plastid expression system of this trans-activator mediation preferably can closely be regulated, and no detectable expression before inducing has unusual high protein expression and accumulation after inducing.A kind of preferred trans-activator is a viral rna polymerase for example.But the preferred promoter of the type is the promotor of coverlet subunit RNA polymerase identification, and as T7 gene 10 promotors, it can be discerned by the RNA polymerase that phage t7 DNA relies on.Preferably will encode the gene transformation of T7 polysaccharase in the nuclear gene group, and the T7 polysaccharase will be directed in the plastid with plastid transit peptides.Described hereinafter and be applicable to the promotor of gene as the gene nuclear expression of coding viral rna polymerase such as T7 polysaccharase.The expression of dna molecular in plastid can be composing type or induction type.The expression of dna molecular in plastid also can be that organ or tissue is special.In WO 98/11235, extensively described these different embodiments, be incorporated herein by reference. The proteinic nuclear expression of trans-activator or therapeutic activity
For in specific transgenic plant, expressing, may modify and optimization by needs according to nucleotide sequence of the present invention.Low expression in transgenic plant may be owing to the heterologous nucleotide sequence that contains codon not preferred in plant causes.Known in the art, all biologies all have the special preferences that codon uses, and can change the codon of nucleotide sequence of the present invention, make it to meet the plant preference, and keep amino acids coding thus.In addition, by have at least 35% preferably the encoding sequence of the GC content more than 45% can be implemented in high expression level in the plant best.In addition, may make information remove stable ATTTA primitive and the AATAAA primitive that can cause inappropriate polyadenylation owing to exist, the nucleotide sequence with low GC content may be expressed relatively poor in plant.Though preferred gene order can give full expression in monocotyledons and dicotyledons kind, but can not modify sub-preference of special password and the GC content preference (people such as Murray, nucleic acids research 17:477-498 (1989)) that these sequences adapt to monocotyledons or dicotyledons simultaneously in these preference demonstrations.Also can be at the unconventional splice site that causes information to be blocked have a screening DNA molecule.With publication application EP 0 385 962, EP 0,359 472 and WO 93/07278 described method, utilize well-known site-directed mutagenesis, PCR and synthetic gene constructing technology, the institute that carries out need carrying out in dna molecular as mentioned above changes.
Effectively initial for what translate, the sequence adjacent with initial methionine may need to modify.For example, can comprise known in plant effective sequence.Joshi had once reported the suitable consensus (NAR 15:6643-6653 (1987)) of plant nuclear, and Clontech proposes another kind of total translation initiation district (1993/1994 catalogue, the 210th page).These consensus sequences are suitable for dna molecular of the present invention to be used.These sequences are mixed in the construct that contains this dna molecular, up to and comprise ATG (and second amino acid is not modified), perhaps up to and be included in ATG after GTC (having genetically modified second amino acid whose possibility of modification).
The expression that coding trans-activator or the proteinic nucleotides sequence of therapeutic activity are listed in the transgenic plant is guided by the promotor that shows function in plant.Time and space requirement that expression is depended in the selection of promotor also depend on host species.The expression of dna molecular of the present invention in plant can be composing type or induction type, for example chemical induction type, perhaps the expression of dna molecular can be that organ or tissue is special.Though it also is effective that the multiple promotor that derives from dicotyledons is presented in the monocotyledons, and vice versa, but it is desirable to select the dicotyledons promotor, for Monocotyledon promoter is selected in the expression in monocotyledons for the expression in dicotyledons.Yet, without limits for the source of selected promotor; As long as they express this one side in the cell of guiding nucleus nucleotide sequence in hope effectively just enough.The preferred promoter of constitutive expression comprises CaMV 35S and 19S promotor, derives from the promotor of the gene of coding Actin muscle or ubiquitin.Dna molecular of the present invention also can be expressed under the adjusting of the promotor of Chemical Regulation.In patent application EP 0 332 104 and United States Patent (USP) 5,614,395 in detail, the optimization technique that is used for chemical induction genetic expression has been described in detail.A kind of preferred promoter that is used for chemical induction is a tobacco PR-1a promotor.A kind of preferred promotor is the promotor of wound-induced.The multiple promotor of also expressing at the phytopathogen infection site in wound site had once been described.Ideally, this promotor only has activity in the infection site part.The preferred promoter of this kind comprises the promotor of the following stated: people such as Stanford, molecule General Genetics (Mol.Gen.Genet.) 215:200-208 (1989), people such as Xu, molecular biology of plants (Plant Molec.Biol.) 22:573-588 (1993), people such as Logemann, vegetable cell (Plant Cell) 1:151-158 (1989), Rohrmeier and Lehle, molecular biology of plants 22:783-792 (1993), people such as Firek, people such as molecular biology of plants 22:129-142 (1993) and Warner, plant magazine (Plant J.) 3:191-201 (1993).Preferred tissue specific expression pattern comprises: chlorenchyma specificity, root-specific, stem specificity and flower specific are expressed.Be suitable in chlorenchyma expression promoter and comprise and regulate the multiple promotor that participates in photosynthetic gene, wherein manyly from monocotyledons and dicotyledons, clone.A kind of preferred promotor is the corn PEPC promotor (Hudspeth and Grula, molecular biology of plants 12:579-589 (1989)) that derives from phosphoric acid enol carboxylase gene.A kind of preferred promoter that is used for the root-specific expression is de Framond (FEBS 290:103-106 (1991); EP 0,452 269) described promotor, another kind of preferred root-specific promoter is the promotor that derives from the T-1 gene (people FEBS 290:103-106 (1991) such as de Framond; EP 0 452269).A kind of preferred stem specificity promoter is a United States Patent (USP) 5,625, and 136 is described, the promotor of guiding corn trpA genetic expression.The preferred embodiment of the invention is the transgenic plant with the different mode expressible dna molecule of Gent.Preferred embodiment is the transgenic plant with wound-induced or pathogenic infection inductive mode expressible dna molecule.Other promotors have synthetic promoter, as Gelvin Super MAS promotor (people (1995) plant magazine 7:661-676 such as Ni).
Except the selection of suitable promotor, be used for preferably including the suitable 3 ' sequence that effectively is connected with the heterologous nucleotide sequence downstream at the structure of plant nuclear expressible dna molecule, regulate sequence or transcription terminator as 3 '.Several such terminators are obtainable and known (for example from the tm1 of CaMV with from the E9 of rbcS) in the art.Can use the known arbitrary obtainable promotor that in plant, works in the present invention.Multiple other sequences can be mixed in the expression cassette of dna molecular of the present invention.This comprises that demonstration can strengthen the sequence expressed as (for example deriving from Adh1 and bronze1) intron sequences and (for example, derive from TMV, MCMV and AMV) virus leader sequence.
In another preferred embodiment, the dna molecular of nuclear expression of the present invention is directed to the subcellular location of plant.For example, therapeutic activity protein is secreted into from cell in the apoplast, perhaps with the specific subcellular organelle of therapeutic activity protein targeting, in vacuole or endoplasmic reticulum.Its implementation for example is, with technology well-known in the art suitable targeting sequencing and dna molecular of the present invention is merged.Therefore, for leading in apoplast or vacuole, protein preferably comprises the appropriate signal peptide, and preferably at its N end, it allows the guiding of protein to organoid.In a preferred embodiment, with in the protein targeting vacuole of the present invention.For the guiding of protein in vacuole, except N end signal peptide, protein preferably also comprises as deriving from the vacuole targeting sequencing of tobacco chitinase gene, preferably its C end (institute of people (1991) NAS newspaper 88 such as Neuhaus, 10362-10366).Utilize the suitable plastid transit peptides that preferably is contained in protein N terminal as detailed below, in the protein expression guiding plastid that also can dna molecular of the present invention is coded.Protein of the present invention also can the leading line plastochondria in, for example, by merging, as N.plumbaginifolia F1-ATP enzyme β-subunit people (1994) molecular biology of plants 24:631-641 such as () Chaumont with the plastosome targeting sequencing.
Narrated the carrier that is applicable to Plant Transformation in this manual in addition.For agriculture bacillus mediated conversion, binary vector or the carrier that carries at least a T-DNA border sequence are suitable, and shift for direct gene, and any carrier all is suitable, and the linear DNA that only contains the purpose construct may be preferred.In the situation that direct gene shifts, can use single DNA kind conversion or cotransformation people such as (, biotechnology (Biotechnology) 4:1093-1096 (1986)) Schocher.Shift and agriculture bacillus mediated transfer for direct gene, (but be not must) usefulness can provide the selected marker of microbiotic (kantlex, Totomycin or methotrexate) or weedicide (for example inhibitor of Basta/ phosphinothricin or proporphyrinogen oxidase) resistance to transform usually.Yet the selection of selected marker is unessential for the present invention.The example of expression cassette and conversion carrier is described in greater detail below.
With a large amount of well-known methods dna molecular of the present invention is imported in the vegetable cell.Vegetation type is depended in the selection that it will be appreciated by those skilled in the art that method, promptly prepares the monocotyledons or the dicotyledons that transform.The suitable method of transformed plant cells comprises microinjection (people such as Crossway, biotechnology 4:320-334 (1986)), electroporation (Riggs and Bates, institute of NAS reports 83:5602-5606 (1986)), agriculture bacillus mediated conversion (people such as Hinchee, biotechnology 6:915-921 (1988)), direct gene shifts (people such as Paszkowski, EMBO is (1984) J.3:2717-2722) and utilize available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, the ballistic particle acceleration of the device of Delaware (referring to, for example, United States Patent (USP) 4,945,050; With people such as McCabe, biotechnology 6:923-926 (1988); Referring to people such as Weissinger, (Annual Rev.Genet.) 22:421-477 (1988) is stated in heredity academic year; People such as Sanford, micropartical science and technology (Particulate Science andTechnology) 5:27-37 (1987) (onion); People such as Christou, plant physiology 87:671-674 (1988) (soybean); People such as McCabe, biotechnology 6:923-926 (1988) (soybean); People such as Datta, biotechnology 8:736-740 (1990) (rice); People such as Klein, institute of NAS newspaper, 85:4305-4309 (1988) (corn); People such as Klein, biotechnology 6:559-563 (1988) (corn); People such as Klein, plant physiology 91:440-444 (1988) (corn); People such as Fromm, biotechnology 8:833-839 (1990); With people such as Gordon-Kamm, vegetable cell 2:603-618 (1990) (corn); People such as Koziel (biotechnology 11:194-200 (1993)) (corn); People such as Shimamoto nature 338:274-277 (1989) (rice); The biological technology 9:957-962 (1991) (rice) of people such as Christou; European patent application EP 0 332 581 (orchard grass and other Pooideae); People such as Vasil (biotechnology 11:1553-1558 (1993) (wheat); People such as Weeks (plant physiology 102:1077-1084 (1993) (wheat); People such as Wan (plant physiology 104:37-48 (1994) (barley)); People such as Umbeck, (biotechnology 5:263-266 (1987) (cotton).Therapeutic activity protein is used the host's
An advantage of the invention is that the expressed therapeutic activity protein of transgenic plant can use the host in several ways, for example per os, outside intestines, intranasal, enteron aisle, particularly intramuscular or intravenously, per rectum, part, use etc. through eye, through lung or by contact.In a preferred embodiment, a kind of allergen of in host's dosage forms for oral administration transgenic plant, expressing.
In an embodiment preferred, the therapeutic activity protein of expressing in extraction and the purifying transgenic plant, and be used for preparing medicinal compositions.The therapeutic activity protein positioning of expressing helps this extraction and purifying greatly in plastid.For example, at first the complete plastid of centrifugation makes proteinic extraction of this therapeutic activity and purifying be more prone to.In another embodiment preferred, according to the in the past known normal condition that is used for isolated protein in this area and technical point from protein purification, for example extracting, precipitation, chromatography, affinity chromatography, electrophoresis etc.Such composition generally comprises the pharmaceutically suitable carriers material of the therapeutic activity protein of effective dose and one or more, organic or inorganic, liquid or solid.Therapeutic activity protein produced according to the invention can use with multiple formulation, for example tablet, lozenge, powder, outstanding agent, solvent, capsule, creme, ointment, aerosol, pulvis or liquor is not as long as this formulation is destroyed this proteinic biologic activity and all can.For example, this protein can mix, grind, make by routine drageeing, dissolving, freeze-drying or similarly method make medicinal compositions.This proteinic formulation depends on patient's body weight, age, physique and pharmacokinetics condition so that application process.
If this protein is to use through intestines, then can adopt solid, semisolid, suspension or emulsion form, also can comprise water, suspension agent, emulsifying agent chemical combination with any pharmaceutically acceptable carrier.Protein of the present invention is available pump or use with the slowly-releasing form also, especially when using with the development of prevention patient disease as a kind of prevention method, or is using to improve or when alleviating the disease of completed stroke.
The therapeutic activity protein of producing according to the present invention is specially adapted to as medicinal compositions Orally administered.Oral compositions comprises the protein of dry powder, food, water-based or non-aqueous solvent, suspension or emulsion form.The example of non-aqueous solvent comprises propylene glycol, polyoxyethylene glycol, vegetables oil, fish oil and injection organic ester.Aqueous carrier comprises water, water-alcohol solution, emulsion or suspension, comprises the outer carrier of the medical intestines of salt solution and buffered, comprises that sodium chloride solution, woods Ge Shi glucose solution, glucose add chlorination sodium solution, lactinated Ringer's solution or expressed oil.In a preferred embodiment, the independent per os of this based composition is taken in or is taken in food or feed or beverage.
In another preferred embodiment, the plant material of expressing the proteinic transgenic plant of therapeutic activity of the present invention or deriving from these transgenic plant is as the medicinal foodstuff dosage forms for oral administration.If these edible composition solid forms can pass through edible consumption, if liquid form then can be by drinking consumption.In a preferred embodiment, transgenic plant material is without the preprocessing step or only can directly absorb through the cooking preparation of a little.For example, at direct-edible plant part, as expression treatment active protein in fruit or the vegetables.Preferably, in the plastid of edible plant part, express this protein.All types of plastids all are applicable to the present invention in any plant.For example, in the chloroplast(id) of spinach or lettuce, marking protein in the chromoplast of tomato or in the amyloplast of potato.In an alternative preferred embodiment, process this transgenic plant, and the vegetable material of picked-up processing back recovery.The preferred in the present invention working method of using is the method that is generally used for food or fodder industry.The finished product of these class methods still comprises the protein of fundamental quantity, and can be convenient to eat or drink.This finished product also can with other food or feed form for example salt, carrier, spices, microbiotic etc. mix mutually, and with solid, semisolid, suspension or the consumption of emulsion form.In another preferred embodiment, these methods comprise a conservative step, for example pasteurization, the cooking or interpolation preserving agent and sanitas.Use in the present invention and process any plant to produce edible or drinkable plant material.For example, when using tobacco plant, then be necessary to handle this plant or vegetable material to remove objectionable impurities such as Nicotine wherein according to the present invention.In a preferred embodiment, a kind of tobacco plant of low alkaloid is used for the present invention's protein expression.In a preferred embodiment, measure according to therapeutic activity Protein content in edible of the present invention or the drinkable vegetable material.For example use this proteinic specific antibody and measure this content by Elisa or Western engram analysis method.This mensuration is used for the proteinic amount that stdn is absorbed.The different batches product that for example has the different proteins level by mixing makes the amount of the drinkable fruit juice of each dosage be measured and to be adjusted in the proteinic amount of therapeutic activity in fruit juice such as the tomato juice by stdn.Obviously, the invention provides novel and effective means and produce and use therapeutic activity protein as the expression of plants of medical science food.
In plant, produce and can be absorbed by Digestive tract by the therapeutic activity protein that the host eats.Picked-up plant or vegetable material, especially complete plant or vegetable material, or only be to make this protein to be surrounded in the vegetable cell or isolated with it through a benefit of the vegetable material of appropriateness processing.Therefore this protein can be subjected to the protection to small part, to exempt from upper gastrointestinal destruction before arriving large intestine or small intestine, therefore can absorb the active protein of higher proportion.
When being used to express the proteinic plant of therapeutic activity of the present invention and being tobacco, rough what is called " F2 soluble protein part " for example can be used for the treatment of to be used.Another part F1 produces by precipitation high molecular weight protein from rough tobacco extract.In non-transgenic plant, as United States Patent (USP) 4,347,324 is disclosed, and this F1 part almost is made up of carboxydismutase uniquely.Some therapeutic transgene active protein of producing by method of the present invention also can be assigned in the F1 part.At this moment, this F1 part also can be used for the treatment of and uses.
For preferred host of therapeutic activity protein of the present invention or acceptor is animal.Animal preferably includes vertebrates, preferably Mammals.Preferably Mammals is people, pet or follows animal, as cat, dog, rodent, ferret, primate, fish or bird or farming animals, as ox, pig, poultry etc.Immunity
Several currently used vaccines need expensive refrigeration, therefore not too are applicable to developing country.And, still can not mass production at the effective vaccine of some pathogenic agent, perhaps there are not enough securities for extensive distribution.For improving this situation, hepatitis B virus surface antigen is expressed in plant, and for the inoculation of hepatitis B hepatitis virus is used (for example seeing United States Patent (USP) 5,484,719 and 5,612,487) by edible plants.Yet the gene of this vaccine of encoding is expressed by the nuclear gene group of this plant, only can obtain relatively low vaccine output, therefore, has hindered the production of edible vaccine.Also attempted carrying out at the immunity of pathogenic microorganism, in transgenic plant, expressed the antigenic determinant of pathogenic agent host's dosage forms for oral administration plant (United States Patent (USP) 5,654,184; 5,679,880; 5,689,079).Yet, in this case, also limited the business success of this method from the low-level relatively expression of transgenic plant nuclear gene group.
Therefore a preferred embodiment of the present invention is in transgenic plant, especially in subcellular organelle, preferably in vacuole, more preferably can induce the antigen of host to the pathogenic agent immunity at the phyteral expression in vivo, pathogenic agent such as bacterium, parasite or viral pathogen perhaps make the immunity of host's contratoxin.Plant of the present invention is used for human immunity to following disease, for example: poliomyelitis, measles,mumps,rubella, smallpox, yellow jack, hepatitis B, influenza, rabies, adenovirus infection, Japanese B encephalitis, varicella, dysentery, acute respiratory infection, malaria, Whooping cough, diphtheria, tetanus or introduction stage tetanus.This proteinoid also is advantageously used in immune animal, with preventing disease, as infection, canine distemper, rabies, dog hepatitis, parvovirus, cat leukemia, newcastle disease, rinderpest, hog cholera, bluetongue and foot and mouth disease, brucellosis, fowl cholera, anthrax and the rauschbrand of horse, and protozoon and helminth infection associated diseases.Plant of the present invention also is used for animal, comprises the mankind, and for example snake or bee venom, mosquito saliva, poisonous ivy etc. produce immunity to multiple toxin or stimulator.In administration of antigens, after preferably per os was taken in plant or derived from the plant material of this plant, the host was by immunity.Well-known as field of immunology, adjuvant can a certain amount ofly join and comprise in the antigenic composition of the present invention, in order to the immunologic competence of raising said composition, thereby improves its immune effect.About the information of immunity system and immunological response sees people such as Hood (1984), immunology (Immunology), Benjiamin/Cummings Publishing Co, Inc, Menlo Park, CA.Tolerance
The relevant medical field that suppresses or reduce undesirable immunne response also needs to improve.The immunity system of animal be one to the specialized cell of multiple special signal effect and the complex network of organ.One of its major function is difference " oneself " and " non-own ".This fundamental characteristics can protect the host to avoid invading the infringement of pathogenic agent, and does not evoke the deleterious immune response at host itself.Generally speaking, immunne response is characterised in that the humoral response that some cell causes in the lymphsystem cell response and antibody cause, is met with the not-self antigen determinant and is caused and cause the destruction of " non-" by immunity system.Yet in some cases, the difference between " oneself " and " non-own " thickens, and causes the immune response at self some part, thereby causes autoimmune disease.In other cases, cause undesirable immune effect at the immunne response of special not-self antigen, for example, to the repulsion of non-autologous tissue or organ graft, perhaps at the allergic development of environmental factors.In these cases, the effective inhibition or the reduction of immunne response are wished.Attempt several strategies and reached this purpose.For example, prevented some autoimmune disease (United States Patent (USP) 5,641,473 and 5,641,474) by use autoantigen with aerosol.Other several treatments are attempted being based on the host to antigenic oral tolerance, its mechanism is that the protein taken in has caused (for example seeing Weiner (1994), institute of NAS newspaper 91,10 at the inhibition or the reduction of the immunne response of special foreign matter or autoantigen, 762-10,765; People such as Friedman, (1994), Grandstein RD (volume): " immunoregulation mechanism " chemistry and immunology (Chem.Immunol.), Basel, Karger, 58 volumes, 259-290).Oral tolerance relates to number of mechanisms, the inhibition that two main mechanism are active cells or clone's property anergy, and it is relevant with the absorption of mucous membrane of small intestine that this mechanism is considered to.Antigen is absorbed and is presented to specific mucosal tissue in intestines, Peyer patches cell for example, and this cell is the inlet that enters intestines related immune system.This inductive low reactivity or anergy are relevant with the common food allergy that takes place in most of individualities, and are to cause enter the main path of gastral multiple potential antigenic tolerance as the diet composition.In the treatment field, oral insulin is causing certain alleviation (United States Patent (USP) 5,763 aspect the treatment of type i diabetes, 396), oral I or III collagen type are also obtaining part success (United States Patent (USP) 5,733,547) aspect the arthritic treatment of autoimmunity.In addition, also carried out the trial (United States Patent (USP) 5,593,698) of oral polymorphism MHC II quasi-molecule allosome peptide inhibition of transplant rejection.In these examples, used antigen is by the natural source purifying in the past.Yet, effective for making oral tolerance, must take in a large amount of antigen.So big quantity for a lot of antigens, be difficult to obtain or cost too high, especially for airborne antigen, and be difficult for being prepared in can be oral composition in.Therefore, but lack the success that protein that the per os of q.s takes in has restricted some similar strategies.Recently, attempted some transplantation antigen and the expression of autoantigen in transgenic plant, and intestines are interior or dosage forms for oral administration (WO/95/08347).Yet low-level relatively consideration convey genetic expression may hinder the successful Application of these methods once more.
Therefore, in a preferred embodiment, can suppress or the antigen that reduces host immune response or inflammation in plant, especially in the plastid of plant, express.Preferably, to these antigens of host's dosage forms for oral administration.It is useful especially aspect treatment, prevention or alleviation disease such as transformation reactions, autoimmune disorder or transplant rejection to express such antigenic transgenic plant, preferably by inducing the host that one or more antigenic tolerances are realized.For oral uptake, the plant of antigen expressed or the plant material that derives from this plant both can have been given birth to edible, again can be through processing as through edible after the foregoing procedure of processing or drink.In a preferred embodiment, the result of oral uptake plant or plant material induces the host to this antigenic tolerance.The present invention is by by plant plastid genomic expression antigen, provides abundant and cheap can be used for to produce the antigen of oral tolerance first.At one preferably in the embodiment,, be based on the ideal carrier of the oral tolerance of medicinal foodstuff through transforming with the overexpression antigenic edible plants of specific peptide as described in the present invention.The use of transgenic plant in oral tolerance has many advantages than current approach (biological chemistry purifying or recombinant expressed in cell culture system, and the protein expression in transgenosis milk).These advantages comprise every dose lower production cost, propagate the minimal risk of Mammals pathogenic agent, and do not need when antigenic plant or vegetable material are directly consumed as food or need simple purifying, processing or packing only when containing.
In another preferred embodiment, antigen of the present invention is used the host jointly with the tolerance adjuvant.Such adjuvant such as toxin resemble choleratoxin B subunit (as people such as Arakawa, (1998) Nature Biotechnol, 16:934-8 is described).This toxin sub-unit molecule can strengthen oral tolerance (people such as Sun, PNAS (1996) 93:7196-201) known in the art comparing with mucosal immunity.The another kind of adjuvant of Shi Yonging is the unsettled enterotoxin Bs of intestinal bacteria in the present invention.In a preferred embodiment, the nucleotide sequence of toxin-encoding and the nucleotide sequence of coding for antigens merge, and produce the dna molecular of a kind of fusion rotein of coding.Produce as described herein and contain the plant of this dna molecular, and as described herein the host is used this fusion rotein.In another preferred embodiment, toxin and antigen are expressed in plant with the isolated polypeptide form, and as described herein the host are used.In addition, toxin or antigen are expressed in different plants, and the host are used after combination again.In addition, from different biologies, obtain preferably purified toxins, combine with antigen then, afterwards the host is used.Allergen
Transformation reactions is causing discomfort among the crowd at high proportion, and suspecting also has detrimentally affect to animal.Allergic strength range comprises from slight symptom to heavy type and rapid performance among the crowd, anaphylactic shock for example, general pathological manifestations comprises the reaction of food parasexuality, skin reaction (urticaria or atopic dermatitis), upper airway allergic responses (for example spring fever or rhinallergosis) or lower respiratory tract transformation reactions, this is the major cause (summary that causes asthma, see people such as O ' Hehir, (1991) immunology annual report (Annu.Rev.Immunol.9:67-95).Transformation reactions is generally reacted owing to the over-drastic IgE at all kinds allergen (for example wool, the animal scales of skin that peel off, insect ight soil or dust and food metamorphosis are former), and usually with inflammatory reaction.The pathological manifestations of IgE-AI particularly since mast cell degranulation cause, caused the release of histamine, heparin, leukotriene.The main right and wrong of present pharmacological agent are special, carry out with a kind of processing mode, for example use the antagonist of antihistaminic, suprarenin-mast cell degranulation.Also developed more special treatment, it causes the host to this allergenic desensitization based on the allergen of the inferior abnormal dosage of regular injection.Yet such injection is boring, and desensitization only is devoted to or do not have the t cell responses relevant with transformation reactions at all.Therefore, the often only limited curative effect of desensitization injection.The invention provides novel, special method and treat transformation reactions, method is to express the allergenic dna molecular of coding in plant, and the host is used such plant or derives from the vegetable material of such plant.Such plant or vegetable material are preferably to host's dosage forms for oral administration, and it produces this allergenic tolerance.Baby or young individuals are if its family tree shows that they have the danger of allergic disorder outbreak in may life afterwards, also can the method for employing prevention prevent allergic generation.In addition, according to methods known in the art extract and purifying transgenic plant of the present invention in the allergen of expressing, and the host is desensitized to it by this allergen of the inferior transformation reactions dosage of regular injection.
Be used for suitable allergen of the present invention and comprise that food allergens, medicine allergen, venom allergen, plant allergen, fungi allergen, bacterial allergen, animal allergen and other derive from the allergen and the extract thereof of spontaneous generation or synthetic material.Food allergens comprises, for example seafood, strawberry, fresh fruit and vegetables, peanut and milk.The medicine allergen comprises, for example penicillin and Regular Insulin.The venom allergen comprises, for example honeybee, wasp and mosquito venom.Preferred allergen is meltittin venom peptide PLA-2.The plant allergen comprises, the allergen in pollen source for example is as trees pollen, showy flowers of herbaceous plants powder, weeds pollen.The fungi allergen comprises, for example mould spores.Animal allergen comprises, for example the scales of skin that peel off or the saliva in sources such as dog, cat, horse.The preferred allergen of the present invention is the horse dust mite allergen, as derive from the allergen of dust mite (Dermatophagoidesfarinae) and dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus), be preferably Der f I, Der f II, Der p I and Der p II allergen (United States Patent (USP) 5,552,142,5,770,202 and 5,798,099).Alternative preferred allergen derives from rye grass pollen, for example derives from Lolium perenne, comprises the isolated peptides (United States Patent (USP) 5,710,126) of Lol p V.Further alternative preferred allergen comprises the allergen that derives from Johnson grass pollen, as Sor h I, is the main allergen that derives from Chinese sorghum (Sorghum halepense) (United States Patent (USP) 5,480,972 and %, 710,126).Further preferred allergen comprises the Ambrosia pollen allergen, resembles Amb a I and Amb a II allergen (United States Patent (USP) 5,698,204).Further preferred allergen also comprises the Aln g I allergen (kind of Alder (Alnus)) of alder, the Col a I allergen (kind of Corylus (Corylus)) of hazel and the Bet v I allergen (kind of Betula (Betula)) of birch, as United States Patent (USP) 5,693,495 is described.Other allergens comprise feline antigens, as Feld I allergen (United States Patent (USP) 5,328,991) and dog scales of skin that peel off allergen, as Can f II (United States Patent (USP) 5,939,283).Other allergens also comprise rAed a 1 and rAed a 2 allergens (WO 98/04274) and meltittin venom antigen (Lomnitzer and the Rabson (1986) that is derived from mosquito Aedes aegypti (Aedes aegypty), transformation reactions and clinical immunology magazine (J.AllergyClin.Immunol.78:25-30) or mouse urine protein (people such as Gurka, (1989), transformation reactions and clinical immunology magazine 83:945-954).
In addition, shown that the reaction of anaphylaxis and IgE mediation is separable, and disulfide linkage has with anaphylaxis very big related.Remove in the dust mite Der f II allergen and form relevant cysteine residues with disulfide linkage, demonstrated and greatly to have reduced anaphylaxis and do not change allergic epi-position (people such as Takai, (1997), natural biology learns a skill (NatureBiotechnolohy), 15:754-758).The present invention also comprises the allergenic expression of modifying in the mode of disappearance disulfide linkage.By technology well-known in the art allergenic dna molecular of clones coding in the plastid conversion carrier, produce transgenic plant.Plant of the present invention or vegetable material are used separately the host or are used with combining at allergic other treatment.The antigen of expressing in the transgenic plant
Transgenic plant, especially antigen expressed is a preferred embodiment of the present invention in plant plastid.Preferably, such antigen can be regulated the host's who needs immunity system, reaches desired result of treatment.Express such antigenic transgenic plant in treatment, prevent or improve aspect disease such as transformation reactions, autoimmune disorder or the transplant rejection or useful especially in immune programme for children, preferably by inducing the host one or more antigenic tolerances.These transgenic plant can be used the host by different methods known in the art, and dosage forms for oral administration preferably is preferably by edible or drink plant of the present invention or derive from the plant material of this plant.Autoimmune disease antigen or autoantigen
Autoimmune disease is an animal, comprises the dysfunction of human immunity system, and wherein immunity system can not be distinguished the intravital allogenic material of animal and be the speciality of an animal normal group compound part.Because of no immunological tolerance, T cell or B cell (or both) acceptor appears carrying jointly, and allow its identification and attack self component.This causes the host of containing this type of T cell or B cell to produce autoimmune disease.Multiple after diagnosing disease is to be caused by autoantigen to small part.For example, hemocyte is affected modal cell type, causes the thrombocythemia purpura of disease as the formation antiplatelet antibody, or the agranulocytosis of the autoantibody formation of anti-polymorphonuclear leukocyte is arranged.Haemolysis and anaemia have also reported it is to be caused by the autoantibody of target in erythrocyte surface in some cases.Anti-cell surface receptor antibody also can cause disease to take place by disturbing function of receptors, for example in myasthenia gravis, wherein forms anti-acetylcholine receptor antibodies, has therefore stoped the neuromuscular transmission.Anti-receptor autophosphorylation antibody costimulatory receptor has the opposite effect, also finds in Graves disease or hyperthyroidism.In addition, other examples of suspecting or being known as the disease that immunological tolerance is lost comprise as multiple sclerosis, diabetes, systemic lupus erythematous, polychondritis, systemic sclerosis, the Wegeners granulomatosis, dermatomyositis, chronic active hepatitis, psoriatic, Steve-Johnson syndrome, the primary sprue, autoimmunity inflammatory bowel (comprising) as ulcerative colitis and regional enteritis, sarcoidosis, primary biliary cirrhosis, uveitis (outside of belly and back), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriasis arthropathica and glomerulonephritis (have or do not have nephrotic syndrome, as comprise that idiopathic nephrotic syndrome or the slightest ephrosis change) and sacroiliitis (rheumatic arthritis for example, chronic progrediente sacroiliitis and arthritis deformans).
For autoimmune disease such as transformation reactions, present pharmacotherapy greatly lacks specificity, and often produces unsatisfied side effect, for example host's general immunosuppression.Therefore embodiment preferred of the present invention is exactly to provide novel therapeutic to autoimmune disease by express the fixed autoantigen of autoantibody target in plant plastid.Such plant or the plant material that derives from such plant be to the host of needs dosage forms for oral administration preferably, and be preferably edible or drink by the host.Thereby set up the tolerance of host to special autoantibody, and do not influence this host's overall immune response ability.Plant of the present invention or vegetable material separately and immunosuppressor or anti-inflammatory agent comprise that S-Neoral, rapamycin, FK506 and steroid use the host together.
The preferred autoantigen of the present invention is, for example, collagen protein, be preferably I type or III Collagen Type VI, in order to treatment or prevention sacroiliitis, be preferably rheumatic arthritis (seeing United States Patent (USP) 5,733,547), myelin basic protein is in order to treatment or prevention multiple sclerosis disease, S antigen is in order to treatment or prevention autoimmunity uveoretinitis, and Regular Insulin (is seen United States Patent (USP) 5,763 in order to treatment or prevention type i diabetes, 396), L-Glutamic decarboxylase or islet cells specific antigens are in order to treatment or prevent diabetes, and thyroglobulin is in order to treatment or prevention autoimmune thyroiditis, and acetylcholine receptor is in order to treatment or prevention myasthenia gravis.
With the dna molecular of technology well-known in the art clones coding autoantigen in the plastid conversion carrier, or, produce transgenic plant with carry out consideration conveyization in its importing conversion carrier.Transplantation antigen
Very need graft or transplant tissue or the organ that is badly damaged with replacement.The graft (autograft) that derives from intrasubject is succeedd usually; but if graft is the individuality (heterograft) that derives from the hereditary dissimilar individuality (allograft) of same species or derive from different plant species, if do not use the immunosuppressive drug then generally can be not successful.Without the immunosuppressive drug treatment that continues, transplant organ can be ostracised.Therefore, induce transplant organ tolerance is had very big potentiality to improve general approach of the same race or the heterograft probalility of success, and be elaborated in the present invention.
Transplant rejection is activated by the immunne response at the cell-surface antigens that can distinguish donor and host.Such cell-surface antigens mainly belongs to histocompatibility antigen, especially main histocompatibility complex (MHC).I class or II class mhc gene product are relevant with antigen-presenting.Therefore, they play an important role in transplant rejection by extremely important in the T cell recognition not-self antigen.I class MHC mixture comprise the transmembrane glycoprotein homodimer that an a kind of and b2 microglobulin part is connected (heavy chain, HC) (people (1987) such as Bjorkman, naturally, 329:506).Differentiated three class Ia locus (the mankind is HLA-A, B and C, is H-2K, H-2D, H-2L in mouse) and the coding HC a part several Ib genoid seats (York and Rock (1996), the immunology annual report, 14:369-396).II class MHC be the heterodimer that constitutes by two kinds of glycoprotein a and b chain (people (1993) such as Brown, nature, 364:33).Because the mhc gene seat is vertebrates, especially in Mammals, guard (Klein (1986), " natural history of main histocompatibility complex ", Oxford: Blackwell Sci.), the antigenic dna molecular of coding MHC also can be from multiple Mammals, comprise in the pig and separating that the latter is the preferred donor of xenotransplantation.
Weaken transplant rejection by monoclonal antibody target II quasi-molecule, further confirmed the importance of MHC antigen in transplant rejection.Therefore, in the treatment of the tolerance that relates to the graft acceptor MHC determinant special to donor, MHC antigen is good material standed for.Recently, attempted some transgenosis antigen and the expression of autoantibody in transgenic plant, and intestines are interior or mouth is used (WO/95/08347).Yet the consideration convey genetic expression of relatively low level may be got rid of the successful result of these methods again.
Among the present invention, from plant plastid genome or nuclear gene group, express mhc gene, the II class mhc gene of the gene of the part of the I class of especially encoding MHC and coding a or b chain.Preferably before or after tissue or organ transplantation (of the same race or xenotransplantation), the host is used the transgenic plant that comprise such plastid, tolerate to induce the host.Preferably, to such plant of host's dosage forms for oral administration or derive from the material of such plant, to induce host's oral tolerance.The II class mhc gene of the coding I class MHC different known allotypic dna moleculars of HC gene and coding a or b chain is imported in the transgenic plant.For the application in the special transplanting, determine donor and acceptor both sides' allotype, and preferably the acceptor dosage forms for oral administration is comprised the antigenic plant material of selected donor MHC.The host is used separately or comprises as S-Neoral with immunosuppressor and to use plant of the present invention or plant material.The other treatment active protein
The other treatment active protein of expressing in the plant among the present invention for example, but be not limited in, hematoglobin protein is (as blood coagulation factor VIII and IX, complement factor and complement, oxyphorase or other hematoglobin proteins, serum albumin etc.), hormone is (as Regular Insulin, tethelin, Triiodothyronine, catecholamine, gonad-stimulating hormone, PMSG, trophic hormone, prolactin, pitocin, Dopamine HCL, Trobest, leptin (leptin) etc.), somatomedin is (as EGF, PDGF, NGF, IGF etc.), cytokine is (as interleukin-, CSF, G-CSF, GMCSF, EPO, TNF, TGFa, TCHb, Interferon, rabbit etc.), enzyme (tissue plasminogen activator, streptokinase, cholesterol biosynthesizing or degrading enzyme, the steroid synthetic enzyme, kinases, phosphodiesterase, methylase, demethylase, desaturase, cellulase, proteolytic enzyme, lipase, the phosphide enzyme, aromatase enzyme, cytopigment, adenylic acid (AMP) or guanylate cyclase, neuraminidase etc.), hormone or other acceptors are (as steroid albumen, peptide, lipid or prostaglandin(PG) etc.), conjugated protein (as steroid binding protein, tethelin or growth factor bindin etc.), immune system protein is (as antibody, antibody fragment, chimeric antibody, variable region etc. or mhc gene), antigen is (as bacterium, parasite, viral antigen, allergen, autoimmune disease antigen, transplantation antigen etc.), translation or transcription factor, cancer protein or proto-protein, milk-protein is (as casein, opalescin, whey etc.), muscle protein is (as myosin, tropomyosin etc.), marrow albumen, neuroactive peptide (as enkephalin), collagen protein, anti-septicopyemia peptide (as BPI (bactericidal power/permeability enhancing albumen)) or tumor growth arrestin or peptide, as angiostatin or endostatin, both all can suppress vasculogenesis.Bactericidal power/permeability strengthens the expression of albumen (BPI) in plant
The present invention also relates to BPI or its fragment in transgenic plant, especially in subcellular organelle, preferably in cavity or the more preferably expression in plant plastid.BPI be a kind of separation from Mammals polymorphonuclear leukocyte (PMN) particulate protein, PMN is the necessary hemocyte of microorganism (United States Patent (USP) 5,641,874 of resisting intrusion in the Mammals; People such as Wilde.(1994), journal of biological chemistry, 269:17411-17416).BPI is a kind of active strong sterilant of the anti-Gram-negative bacteria of wide spectrum (see for example United States Patent (USP) 5,753,620,5,827,816 and 5,763,567, be incorporated herein by reference) that has.BPI shows the specificity of height in cytotoxicity, i.e. 10-40nM (0.5-2.0 microgram) sensitive organism is produced killing rate greater than 90%, and the BPI of 100 times of concentration is nontoxic for other microorganisms and eukaryotic cell.Separation has been purified as homogeneity from the BPI of people and rabbit PMN.The molecular weight of people BPI is approximately 58,000 dalton (58kDa), and the molecular weight of rabbit BPI is approximately 50kDa.Owing to accurate selectivity and to the cell no cytotoxicity beyond the Gram-negative bacteria, BPI fragment of the present invention can be used as the specific therapy agent especially.The infection of current Gram-negative bacteria is for example passed through antibiotic therapy, for example penicillin derivative, aminoglycoside antibiotics and paraxin by the infection that intestinal bacteria, multiple Salmonellas, klebsiella or pseudomonas cause.Because of gram negative bacillus tends to present multiple available microbiotic is produced tangible inherent resistance, and owing to the acquisition of resistance factor plasmid is easy to produce another kind of resistance, the microbiotic curative effect is restricted.Yet BPI in reorganization system production has problem always, and if preferably just can reach result of treatment in gram for therapeutic efficiency, purifying is also uneconomical from mammalian cell.A reason of required high dosage is the quick clearance rate of BPI from blood flow.Therefore the preferred embodiment of the present invention is to express coding BPI or the segmental dna molecular of BPI in the plant plastid genome.In the plastid high yield be expressed in especially useful in this case.
When being used for the treatment of because of caused microbemia of Gram-negative bacteria (being that bacterium appears in the blood flow) or Sepsis (bacterial contamination of body fluid), BPI that is produced according to the present invention or BPI fragment are preferably passed through parenteral administration, and with the about 1-1000 μ of each treatment g, the dosage of preferably about 10-250 μ g, most preferably intravenously is used.Treatment the course of treatment can be different because of individuality according to the severity of disease with number of times.Typical treatment regimen can comprise the about 100 μ gBPI fragments of intravenous administration, every day 3 times.For avoiding quick inactivating, BPI or BPI fragment can with physiologically acceptable carrier, for example serum protein of normal presence (as albumin or N,O-Diacetylmuramidase) coupling.BPI of the present invention or BPI fragment also can local be used for the treatment of the Mammals that suffers from skin infections, and this infection betides trouble bedfast patient of decubital ulcer (bedsore) or burn patient, are caused by the Gram-negative bacteria of sensitivity.In a preferred embodiment, each dosage is that BPI of the present invention or the BPI fragment of 1-1000 μ g can be mixed use with microbiotic.In another preferred embodiment of the invention, the mammiferous medicinal compositions of Gram-negative bacteria is infected in treatment, removing the microbiotic such as the penicillin G that contain standard dose (this area is well-known) (can be available from E.R.Squibb and Sons, Inc., Princeton, N.J.) or cynnematin (can be available from Eli Lily ﹠amp; Co., Indianapolis, Ind.) outside, also can comprise BPI fragment of the present invention.In an especially preferred embodiment, BPI fragment of the present invention also can be mixed with the hydrophobicity microbiotic, as Rifampin, and hydrophobicity penicillin, for example penicillin V benzathine (Lederle Labs, PearlRiver, N.Y.).The Gram-negative bacteria permeability that increases after the BPI treatment is estimated to strengthen such the antibiotic curative effect that is difficult for entering non-permeability bacterium.
BPI of the present invention or BPI fragment are estimated to be ideally suited for using the effective any microbiotic of Gram-negative bacteria, immune system cell or the factor, for example co-therapy of T cell or interleukin II, cytotoxic agent etc.Owing to have the increase of pathogenic slick Gram-negative bacteria to BPI fragment susceptibility of the present invention more, BPI fragment of the present invention is estimated to reduce the resistance of this bacterioid to the above-mentioned factor.Basically it is preferred using BPI fragment of the present invention and selected microbiotic simultaneously.In another preferred embodiment, BPI or BPI fragment are preferably used for treating the Digestive tract infectation of bacteria according to aforesaid embodiment oral uptake.This protein is used as medicinal compositions or is absorbed as medicinal food.
BPI is also shown in has activity in the treatment of conditions, in comprising and the anti-freezing collection activity of heparin, suppress blood vessel generation, tumour and endothelial cell proliferation, and the treatment of chronic inflammatory diseases (see U.S. Pat 5807818, be incorporated herein by reference).
Further describe the present invention with reference to following specific embodiment.These embodiment are not intended to restriction except as otherwise noted just in order to illustrate.
Embodiment
Standard recombinant dna of Shi Yonging and molecule clone technology are well known in the art herein, and be described in J.Sambrook, E.F.Fritsch and T.Maniatis, " molecular cloning: lab guide ", cold spring harbor laboratory, the cold spring port, NY (1989) and T.J.Silhavy, M.L.Berman and L.W.Enquist, " gene fusion experiment ", cold spring harbor laboratory, the cold spring port, NY (1984) and Ausubel, people such as F.M., " modern molecular biology method ", GreenePublishing Assoc. and Wiley-Interseience publish (1987).Embodiment 1: increase to the I.1 separation of cDNA of short artemisiifolia (Ambrosia artemisiifolia) pollen Amb a by RT-PCR
From 100mg degreasing pollen (Greer Laboratories), separate total RNA with phenol/SDS method (" modern molecular biology method ").Use oligo (dT) 18Primer synthesizes the first chain cDNA with Advantage RT-for-PCR test kit (Clontech) by the total RNA of 0.5mg.Single stranded DNA is used to use I.1 cDNA (people (1991) journal of biological chemistry 266:1229-1236 such as Rafnar) of Pfu Turbo archaeal dna polymerase test kit (Stratagene) pcr amplification Amb a as template, wherein use following oligonucleotide: " top chain " primer, added a NcoI site (5 '-GCG GCC ATGGGG ATC AAA CAC TGT TGT TA-3 ' to Amb a translation initiation site I.1, SEQ ID NO:1), " end chain " primer, in 3 ' non-translational region, add an XbaI site (5 '-GCG GTCTAG ATC ATT ATA AGT GCT TAG T-3 ', SEQ ID NO:2) after the terminator codon.Be used for subclone with NcoI and XbaI digestion 1.2kb band, global cDNA checked order, and compare with GenBank preserving number M80558.Observe the difference of 7bp, do not form but these differences all change amino acid.Embodiment 2: be used for the structure to the carrier of tobacco plastom homologous recombination
In order to insert mosaic gene, modify the trnV and the rps12/7 intergenic region of tobacco plastom through homologous recombination.By tobacco plastom pcr amplification 1.78kb district (site 139255-141036, people such as Shinozaki (1986) EMBO J.5:2043-2049), and after site 140169, insert a PstI site, produce the 915bp and the 867bp flank plastid DNA that are positioned at PstI insertion site 5 ' and 3 '.Insert a BsiEI site (5 '-TAACGG CCG CGC CCA ATC ATT CCG GAT A-3 ' before being used in site 139255, SEQ ID NO:3) and after site 140169, insert a PstI site (5 '-TAA CTG CAG AAA GAAGGC CCG GCT CCA A-3 ', SEQ ID NO:4) primer is to carrying out pcr amplification (Pfu Turbo archaeal dna polymerase, Stratagene, La Jolla, CA).Insert a PstI site (5 '-CGC CTG CAG TCG CAC TAT TACGGA TAT G-3 ' before also being used in site 140170, SEQ ID NO:5) and the primer that after site 141036, inserts a BsiWI site (5 '-CGC CGT ACG AAA TCC TTC CCG ATA CCT C-3 ', SEQID NO:6) to carrying out pcr amplification.PstI-SacII site with the PstI-BsiEI fragment is inserted pBlueseriptSK+ (Stratagene) produces pAT216, and with the PstI-Acc65I site that the PstI-BsiWI fragment is inserted pBluescript SK+, produces pAT215.PAT218 contains the plastid DNA of true 1.78kb, and it contains the insertion that the PstI site is used for mosaic gene and selected marker, is connected with the 2.7kb PstI-ScaI band of pAT216 by the 2.0kb PstI-ScaI fragment with pAT215 to make up.Embodiment 3: the amplification of the rbs of tobacco 16S rRNA gene promoter and rbcL gene
By tobacco DNA (tobacco cv.Xanthi) pcr amplification 16S rRNA promotor, and merge with the synthetic ribosome bind site (rbs) of tobacco plastid rbcL gene." top chain " primer inserted an EcoRI site (5 '-GCC AGA ATT CGC CGT CGT TCA ATG AGA ATG-3 ', SEQ IDNO:7) at 16S rRNA gene promoter 5 ' end before site 102568." end chain " primer amplification is to the site 102675 of 16S rRNA gene promoter, remove two upstream ATG by changing site 102661 (A changes C into) and 102670 (A changes C into), the rbs (site 57569-57584) that adds the rbcL gene extends as 5 ' of primer, and in 3 ' the terminal BspHI site (5 '-GCC TTC ATG ATC CCT CCCTAC AAC TAT CCA GGC GCT TCA GAT TCG-3 ', SEQ ID NO:8) of inserting of rbs.Gel-purified 142bp amplified production is cut with EcoRI and BspHI enzyme, produces the 128bp fragment contain the tobacco 16S rRNA gene promoter that the rbs with the rbcL gene merges.Embodiment 4: the separation in Arabidopis thaliana (Arabidopsis) 16S rRNA gene promoter district
Gene order in the Arabidopis thaliana plastom with respect to tobacco (Nicotiana tabacum)-known plant of complete plastom-for be the separation that the possibility guarded helps Arabidopis thaliana 16SrRNA gene promoter district.With the closely-related species White Mustard Seed of Arabidopis thaliana (Sinapisalba) in, 16S rRNA gene and Xie Ansuan tRNA direction are as in the tobacco (GenBank preserving number CHSARRN1).Through pcr amplification (PfuTurbo archaeal dna polymerase, Stratagene, La Jolla, CA) separate Arabidopis thaliana 16S rRNA gene promoter district, wherein use total Arabidopis thaliana (cv " Landsberg erecta ") as template, and use the following primer in tobacco and White Mustard Seed, all guard: " top chain " primer (5 '-CAG TTC GAG CCT GATTAT CC-3 ', SEQ ID NO:9) and " end chain " primer (5 '-GTT CTT ACG CGTTAC TCA CC-3 ', SEQ ID NO:10).Make the 379bp amplified production of prediction become flush end, it contains the Arabidopis thaliana 16SrRNA gene promoter district (people (1986) EMBO J 5:2043-2049 such as Shinozaki) corresponding to the Nucleotide 102508-102872 of tobacco plastom, be connected to pGEM5Zf (-) EcoRV site (Promega), and carry out sequential analysis and with the contrast of tobacco 16S rRNA gene promoter.Embodiment 5: the amplification of tobacco plastid rps16 gene 3 ' untranslatable rna sequence (3 ' UTR)
Use following oligonucleotide to by tobacco DNA (tobacco cv.Xanthi) pcr amplification tobacco plastid rps16 3 ' UTR: with " top chain " primer (5 '-CGC GAC TAG TTC AAC CGAAAT TCA AT-3 ', SEQ ID NO:11) after the terminator codon of the plastid rps16 gene that is right after coding ribosomal protein S16, adds a SpeI site, with " end chain " primer (5 '-CGC TCT GCA GTT CAA TGG AAG CAA TG-3 ', SEQ ID NO:12) PstI site of 3 ' terminal adding at rps16 3 ' UTR.The gel-purified amplified production, with SpeI and PstI digestion, generation contains 163bp fragment (the site 4941-5093 of tobacco plastom of tobacco rps16 3 ' UTR, people such as Shinozaki, 1986, EMBO is J.5:2043-2049), its 5 ' flank is a SpeI site, 3 ' is a PstI site.Embodiment 6: the structure that is used for 16S rRNA gene promoter ∷ aadA gene ∷ rps163 ' the UTR box of plastid transformation and selection
The encoding sequence that separates the aadA gene from pRL277, aadA gene are a kind of coding bacterial gene that the aminoglycoside 3 ' adenosyl transferase of spectinomycin and streptomycin resistance is provided (people (1991) gene 103:17-23 such as people (1993) molecular biology 9:77-84 such as Black and Prentki).5 ' major portion of aadA encoding sequence is separated 724bp BspHI-BssHII fragment (initiator codon is positioned at the BspHI site) from pRL227, with pRL277 as template, after terminator codon 20bp, add the 3 ' remainder that the aadA gene is modified in a SpeI site by pcr amplification, wherein use following oligonucleotide right: " top chain " primer (5 '-ACC GTA AGG CTTGAT GAA-3 ', SEQ ID NO:13) and add " end chain " primer (5 '-CCC ACT AGT TTG AAC GAA TTG TTA GAC-3 ', SEQ IDNO:14) in a SpeI site.Gel-purified 658bp amplified production, digest with BssHII, SpeI, 16S rRNA gene promoter and the rbs of rbcL and the pLITMUS28 carrier (New England Biolabs) of EcoRI-SpeI digestion that has on the aadA gene 5 ' part that has on 89bp fragment and the 724bp BspHI-BssHII fragment, the 128bp EcoRI-BspHI pcr amplified fragment is connected, produces pAT223.The PCR fragment of the EcoRI-SpeI 0.94kb fragment of the aadA gene that contains the guiding of 16S rRNA gene promoter-rbs of pAT223, SpeI, PstI digestion that 163bp contains rps16 3 ' UTR is carried out three the tunnel with pUC19 (New England Biolabs) that EcoRI, PstI enzyme are cut is connected, acquisition contains the pAT229 of UTR of 16S rRNA gene promoter of the aadA gene of pilot tape rps 16 3 ' UTR.Embodiment 7: the amplification of phage t7 gene 10 promotors
From pET-3d (Stratagene) pcr amplification phage t7 gene 10 promotors, wherein use following oligonucleotide right: " top chain " primer is at T7 promotor 5 ' terminal EcoRI site (5 '-CCC GAA TTC ATC CCG CGA AAT TAA TA-3 ' of insertion, SEQ ID NO:15), " end chain " primer is in 3 ' the terminal NcoI site (5 '-CGG CCA TGGGTA TAT CTC CTT CTT AAA GTT AAA-3 ', SEQ ID NO:16) of inserting.The gel-purified amplified production is cut with EcoRI, NcoI enzyme, produces the 96bp fragment that contains the T7 promotor.Embodiment 8: the amplification of phage t7 gene 10 terminators
From pET-3d (Stratagene) pcr amplification phage t7 gene 10 terminators, wherein use following oligonucleotide right: " top chain " primer is at 5 ' terminal HindIII site (5 '-GCG AAG CTT GCT GAG CAA TAA CTA GCA TAA-3 ' of insertion of terminator, SEQID NO:17), " end chain " primer is in 3 ' the terminal PstI site (5 '-GCG CTG CAG TCC GGA TAT AGT TCC TCC T-3 ', SEQ ID NO:18) of inserting of terminator.The gel-purified amplified production is cut with the HindIII-PstI enzyme, produces the 86bp fragment that contains the T7 terminator.Embodiment 9: the amplification of Arabidopis thaliana plastid psbA 3 ' untranslatable rna sequence (UTR)
From Arabidopis thaliana DNA (environmental Landsburg erecta) pcr amplification Arabidopis thaliana plastid psbA 3 ' UTR, wherein use following oligonucleotide right: " top chain " primer has added a SpeI site to the 5 ' end of 3 ' UTR, and removed (the 5 '-GCG ACT AGT TAG TGT TAG TCT AAATCT AGT T-3 ' of an XbaI site in the native sequences by G being sported A (underscore marks), SEQ ID NO:19), " end chain " primer has added a HindIII site (5 '-CCG CAA GCT TCT AAT AAA AAA TAT ATAGTA-3 ', SEQ ID NO:20) to the 3 ' end of UTR.Amplification region extends to 1552 from the site 1350 of GenBank preserving number X79898.Gel-purified 218bp PCR product digests with SpeI and HindIII, and cuts the SpeI-PstI site that the PCR fragment is connected to pBluescriptSK-(Stratagene) with the HindIII-PstI enzyme that has the T7 terminator, produces pPH171.X79898 compares with the GenBank preserving number, and the sequential analysis in psbA 3 ' the UTR district of pPH171 is presented at site 1440 and 1452 places have lacked adenine nucleotide.Embodiment 10: with the structure of poly-guanosine tract as 3 ' UTR replacer's carrier
Poly-guanosine tract shows can functionally substitute plastid atpB gene 3 ' UTR people (1996) RNA 2:652-663 such as () Drager in vivo.Contain 18 continuous guanosine residues, the polyG tract that is respectively SpeI, HindIII cohesive end at 5 ' and 3 ' terminal flank is to be assembled into by the oligonucleotide annealing that following two kinds of kinases are handled: (5 '-CTA GTG GGG GGGGGG GGG GGG GGA-3 ', SEQ ID NO:21) and (5 '-AGC TTC CCCCCC CCC CCC CCC CCA-3 ', SEQ ID NO:22).The polyG18 tract that contains SpeI, HindIII cohesive end is connected to the SpeI of pBluescript SK+ (Stratagene) with the PCR fragment of the HindIII that contains the T7 terminator, PstI digestion, and the PstI site produces pAT222.Embodiment 11: contain the I.1 preparation of the mosaic gene of encoding sequence of ragweed pollen allergen Amb a with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR fusion in tobacco plastid conversion carrier
By containing the 96bp EcoRI of T7 promotor, NcoI PCR fragment, contain Amb a 1.2kb NcoI I.1cDNA, XbaI PCR fragment and contain the 295bp XbaI of the pPH171 of Arabidopis thaliana psbA 3 ' UTR, PstI fragment and T7 terminator, EcoRI to pGEM-3Z (Stratagene), in the PstI site four tunnel connects, and makes up I.1 ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box of phage t7 gene 10 promotor ∷ Amb a, produces plasmid pAT230.The 1.1kb HindIII of pAT229 by will containing 16S rRNA gene promoter-rbs ∷ aadA ∷ rps16 3 ' UTR box, the EcoRI fragment, with the 1.6kb EcoRI that has T7 promotor ∷ Amb aI.1 ∷ psbA 3 ' UTR ∷ T7 terminator box, PstI pAT230 fragment, be cloned into the HindIII of pBlueseript SK+ (Stratagene), the PstI site, with the Amb a of T7 promotor guiding I.1 box gene be connected generation plasmid pAT234 with aadA selected marker box.To contain the 2.7kb PstI band of Amb a pAT234 I.1 and the PstI site that the selected marker box is connected to pAT218, and to Amb a I.1 the direction of insertion of gene when transcribing with the rps12/7ORF equidirectional screen, make up plastid conversion carrier pAT238.Embodiment 12: contain the I.1 preparation of the mosaic gene of encoding sequence of ragweed pollen allergen Amb a with phage t7 gene 10 promotors and terminator and the fusion of poly-guanosine tract in tobacco plastid conversion carrier
By containing the 96bp EcoRI of T7 promotor, NcoI PCR fragment contains Amb a 1.2kb NcoI I.1cDNA, XbaI PCR fragment and have poly G 18The 30bp XbaI of the pAT222 of tract, the HindIII fragment, four the tunnel are connected in the 5.9kb EcoRI that contains selected marker, T7 terminator and the pAT238 of flank homology plastomere, HindIII carrier segments, and to Amb a I.1 the direction of insertion of gene when transcribing with the rps12/7ORF equidirectional screen, make up and contain the I.1 plastid conversion carrier pAT239 of ∷ guanosine tract ∷ T7 terminator and 16S rRNA gene promoter-rbs ∷ aadA ∷ rps16 3 ' UTR box of T7 gene 10 promotor ∷ Amb a.Embodiment 13: contain the preparation of the mosaic gene of the dust mite allergen Der f I encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
United States Patent (USP) 5,770 has been described the encoding sequence of Derf I gene in 202.This sequence is used for being designed for the PCR primer by dust mite cDNA amplification Der f I encoding sequence.Obtain cDNA by reverse transcription (I.1) by the total RNA that from dust mite preparation (Greer Laboratories), extracts for Amb a.Phage t7 gene 10 promotor ∷ Der fI ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box is as structure as described in for pAT230.The Der fI box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 14: contain the preparation of the mosaic gene of the dust mite allergen Der f II encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
United States Patent (USP) 5,770 has been described the encoding sequence of Derf II gene in 202.This sequence is used for being designed for the PCR primer by dust mite cDNA amplification Der f II encoding sequence.Obtain cDNA by reverse transcription (I.1) by the total RNA that from dust mite preparation (Greer Laboratories), extracts for Amb a.Phage t7 gene 10 promotor ∷ Der f II ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box is as structure as described in for pAT230.The Der f II box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 15: contain the preparation of the mosaic gene of the dust mite allergen Der p I encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
United States Patent (USP) 5,770 has been described the encoding sequence of Derp I gene in 202.This sequence is used for being designed for the PCR primer by dermatophagoides pteronyssinus cDNA amplification Der p I encoding sequence.The total RNA that extracts from dermatophagoides pteronyssinus preparation (Greer Laboratories) by reverse transcription (for Amb a I.1) obtains cDNA.Phage t7 gene 10 promotor ∷ Der p I ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box is as structure as described in for pAT230.The Der p I box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 16: contain the preparation of the mosaic gene of the dust mite allergen Der p II encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
United States Patent (USP) 5,770 has been described the encoding sequence of Derp II gene in 202.This sequence is used for being designed for the PCR primer by dermatophagoides pteronyssinus cDNA amplification Der p II encoding sequence.The total RNA that extracts from dermatophagoides pteronyssinus preparation (Greer Laboratories) by reverse transcription (for Amb a I.1) obtains cDNA.Phage t7 gene 10 promotor ∷ Der p II ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box is as structure as described in for pAT230.The Der p II box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 17: contain the preparation of the mosaic gene of the Johnson grass pollen allergen Sor h I encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
United States Patent (USP) 5,480 has been described the encoding sequence of Sor h I gene in 972.Make up phage t7 gene 10 promotor ∷ Sor h I ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box.The Sor h I box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 18: contain the preparation of the mosaic gene of the birch pollen allergen Bet V I encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
J.8:1935-1938, people such as Breiteneder (1989) EMBO has described the encoding sequence of Bet V I gene.Make up phage t7 gene 10 promotor ∷ Bet V I ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box.The Bet V I box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 19: contain the preparation of the mosaic gene of the mosquito saliva allergen rAed a1 encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
The encoding sequence of rAed a1 gene has been described among the WO 98/04274.Make up phage t7 gene 10 promotor ∷ rAed a1 ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box.The rAed a1 box gene of T7 promotor guiding is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 20: contain the preparation of the mosaic gene of L-Glutamic decarboxylase (GAD) encoding sequence that merges with phage t7 gene 10 promotors and terminator and Arabidopis thaliana plastid psbA 3 ' UTR in tobacco plastid conversion carrier
The encoding sequence of people GAD gene obtains from Genbank (preserving number L16888).Make up phage t7 gene 10 promotor ∷ GAD ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box.The guiding GAD box gene of T7 promotor is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 21: contain with phage t7 gene 10 promotors and terminator and tobacco plastid conversion carrier in the preparation of mosaic gene of the thyroglobulin encoding sequence that merges of Arabidopis thaliana plastid psbA 3 ' UTR
The encoding sequence of thyroglobulin gene obtains from Genbank (preserving number U93033).Make up phage t7 gene 10 promotor ∷ thyroglobulin ∷ Arabidopis thaliana psbA 3 ' UTR ∷ T7 terminator box.The guiding thyroglobulin box gene of T7 promotor is connected with aadA selected marker box, and inserts the HindIII of pBluescript SK+ (Stratagene), PstI site.Then as structure plastid conversion carrier as described in the embodiment 11.Embodiment 22: the Biolistic of tobacco plastom transforms
Seven of every plates, with 1 " annular array makes the seed germination of tobacco c.v. ' Xanthi nc ' on the T nutrient agar, and after sowing 12-14 days; basic as Svab; Z. and Maliga, P. (1993) PNAS 90,913-917 is described; with 1 μ m tungsten particle (M10; Biorad, Hercules, CA) bombardment of plasmid DNA bag quilt.Rice shoot incubation two days on the T substratum with bombardment downcuts leaf afterwards, and an axle side far away places (350-500 μ mol photon/m under the light up 2/ s), in contain 500 μ g/ml spectinomycin dihydrochlorides (Sigma, St.Louis, on RMOP plate MO) (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87,8526-8530).The resistance seedling that bombardment 3-8 occurred under the bleaching leaf after week to identical selection substratum, is made it to form callus by subclone, separates and second batch of seedling of subclone.The plastom of complete isolating conversion copy (homogeneity state) in the independent subclone is estimated with Southern trace standard technique people (1989) " molecular cloning: laboratory manual " such as (, cold spring harbor laboratory, cold spring port) Sambrook.(Mettler, I.J. (1987) molecular biology of plants report 5 346-349) separates on 1%Tris-boric acid (TBE) sepharose the total cell dna of BamHI/EcoRI digestion, is transferred to (Amersham) on the nylon membrane, uses 32The random primer dna sequence dna of P-mark is detected, and this sequence is corresponding to the 0.7kb BamHI/HindIII dna fragmentation (seeing patent application WO 98/11235) of the pC8 that contains some rps7/12 plastid targeting sequencing.(McBride, people such as KE. (1994) PNAS 91 7301-7305) go up sterile rootage to the seedling of homogeneity, and it is transferred in the greenhouse containing the MS/IBA substratum of spectinomycin.Embodiment 23: the structure of the phage t7 NRA pol gene of the plastid guiding that merges with tobacco PR-1a promotor
Following fragment is connected, make up pPH110: a kind of synthetic oligonucleotide joint, it contains a NcoI restriction site and ATG initiator codon, follow preceding 7 plastid transit peptides codons and endogenous PstI restriction site (top chain: 5 '-CAT GGC TTC CTC AGT TCT TTC CTC TGC A-3 ', SEQ IDNO:23 by the rbcS gene small subunit of carboxydismutase (coding); End chain: 5 '-GAG GAA AGA ACT GAG GAA GC-3 ', SEQ IDNO:24), pCGN4205 (McBride, K.E. wait people (1994), PNAS 91,2.8kb PstI/SacI dna fragmentation 7301-7305), it contains and the phage t7 rna polymerase gene (T7 Pol) of rbcS gene delivery peptide-coding sequence 3 ' part at the framework endomixis, the 0.9kb NcoI/KpnI dna fragmentation of pCIB296, it contains tobacco PR-1a promotor, NcoI restriction site (people (1993) vegetable cell 5 such as Uknes that an introducing is arranged at the initiator codon place, 159-169), with the binary Agrobacterium-mediated Transformation carrier pSGCGC1 (derivative of pGPTV-Hyg, contain the polylinker that derives from pGEM4 (Promega, Madison WI) of being cloned into the SacI/HindIII site) 4.9kb SfiI/KpnI and 6.6kb SacI/SfiI fragment.Embodiment 24: chimeric PR-1a/T7 Pol gene is by the agriculture bacillus mediated importing of leaf dish conversion in tobacco nuclear gene group
The leaf dish of tobacco ' Xanthi ' and " NahG " people (1995) molecular biology of plants 29:959-68 such as () Friedrich is cultivated altogether with the GV3101 Agrobacterium that has the pPH110 binary vector, as stated from thus obtained seedling and be regenerated as hygromycin resistance NT-pPH110 tobacco plant.For each transgenic lines, about 2-3cm 2Double leaf boring in 3ml BTH (5.6mg/10ml) or sterile distilled water, in about 300 μ mol/m 2Incubation is 2 days under the/s irradiation.Results leaf material suddenly freezes, and grinds in liquid nitrogen.Extract total RNA (people (1989) NAR 17,2362 such as Verwoerd), (people (1991) vegetable cell 3 such as Ward 1085-1094) carries out the Northern engram analysis as stating to use radiolabeled T7 rna polymerase gene probe.To show that 19 NT-110X (Xanthi genetic background) of to a certain degree T7 Pol expression and the T1 of 7 NT-110N (NahG genetic background) are that plant is transferred in the greenhouse and self-pollination.The T2 system of isozygotying is selected in the isolating filial generation selfing in 3: 1 of chain hygromycin resistance mark.Embodiment 25: what the artemisiifolia allergen was expressed in the transgenic plant plastid induces
With isozygoty NT-110X and the NT-110N plant that contain PR-1a-T7 RNA Pol construct the pAT238 that carries maternal inheritance and the homogeneity plastid of pAT239 construct are transformed system's pollination.Nt_pAT238 * NT-110X or NT_110N, and Nt_pAT239 * NT-110X or NT_110NF1 filial generation (it is a heterozygosis for PR-1/T7 polysaccharase nuclear expression box, for T7/Amb a I.1 the plastid expression cassette be homogeneity) in soil, germinate.After reaching the height of 20-40cm, spray inducing compounds BTH is positioned plastid with initiation the I.1 expression of the T7 Pol adjusting of gene of Amb a to plant.Results vegetable material when inducing preceding and induce back 8 hours and 1,2,3,7 and 14 or 28 day, anxious freezing in liquid nitrogen.The transgenic plant that contain Der fI, Sor h I, Bet V I and rAed a 1 allergen gene and GAD and thyroglobulin gene are used similar method.Embodiment 26: antigen presentation of transgenic plant and Determination on content
Extract total RNA people (1989) NAR 17,2362 such as () Verwoerd, and as state that (people (1991) vegetable cell 3 such as Ward, 1085-1094) probe with radiolabeled Amb aI.1 gene specific carries out the Northern engram analysis.In order to measure the Amb a amount I.1 that exists in the transgenic plant tissue, according to working instructions and Harlow and Lane (1988) " antibody: lab guide " (Antibodies:A laboratory manual), Cold SpringHarbor Laboratory, Cold Spring Harbor, with the Amb a of I.1 proteic antiserum(antisera) of anti-Amb a and purifying I.1 the protein standard carry out chemoluminescence (Amersham) Western engram analysis.Use similar method for Der f I, Sor h I and Bet V I, rAed a 1 allergen and GAD and thyroglobulin.Embodiment 27: oral tolerance induces in the rat
Use heavy the 150-220g female Lewis or the Wistar Furth rat in (6-8 age in week).Be used in emulsive 50 μ g cavy antigens immune rat in two metapedes pads in the complete Freund's adjuvant (CFA).In some experiments, in emulsive antigen, add 50 μ g ovalbumins (OVA) (Sigma), and injection similarly.In order to induce oral tolerance, rat is with 5 the feeding transgenic plant of the present invention in 3 days intervals.After the immunity 9 days, put to death rat, and take out Qi lymphonodi poplitei.By pressing stainless steel sift to prepare single cell suspension in lymphoglandula.In quadruplicate will be altogether in round bottom 96 hole flat boards (Costar) 105 lymph-node cell (LNC) cultivate with the irradiation (2000 Rads) or the complete LNC that derive from hello rat of specified quantity.Adding volume in nutrient solution is the antigen (50 μ g/ml) of 20 μ l.Culture incubation 80 hours, and at last 16 hours that cultivate with 1 μ Ci[3H] TdR/ hole pulse labelling.Collect culture with the automated cell collector then, and with standardized liquid scintillation counter reading.Calculate the percentage ratio of (primed) LNC (PLNC) propagation that causes then.In all experiments, all use proliferated culture medium RPMI (Gibco).This substratum after adding 2 * 10-5M 2 mercapto ethanol, 1% Sodium.alpha.-ketopropionate, 1% penicillin and Streptomycin sulphate, 1% non-essential amino acid and 1% autoserum, sterile filtration.
Embodiment 28: the serum level of the mensuration A. antibody of antibody
Solid-phase enzyme-linked immunosorbent test (ELISA) is used for measuring at antigenic antibody titers.With every hole 0.1ml 10 μ g antigens/ml double distilled water incubation microtiter plate.Dull and stereotyped 25 ℃ of following incubations 18 hours.After PBS/ tween 20 pH7.5 (Bio-Rad) washing 3 times, dull and stereotyped 3%BSA/PBS incubation 2 hours of under 37 ℃, using.Wash 2 times, add 100 μ l dilute serums in quadruplicate.Dull and stereotyped 37 ℃ of following incubations 2 hours.After PBS/ tween 20 rinsing 3 times, flat board is used among the 1%BSA/PBS mouse IgG of the goat Chinese People's Anti-Japanese Military and Political College antibody (Tago, the U.S.) of 100 μ l/ hole horseradish peroxidase of dilution in 1: 1000 25 ℃ of incubations 1 hour.By being exposed to O-Phenylene Diamine (0.4mg/ml phosphoric acid salt) citrate buffer solution that contains 30%H2O2, pH5.0) obtain color reaction.By adding 0.4N H2SO 4Termination reaction, and read OD 492nm with the ELISA readout instrument.
B. the external test that produces of antibody
Huo De popliteal and spleen LNC from that raise, wild and the rat of attacking, and shine (2000 Rads) separately or with other specified PLNC, with every ml 10 7The concentration of individual cell is inoculated in culture dish.Culture places the proliferated culture medium that contains or do not contain antigen (20 μ g/ml), and in incubator 3 days, results then.The supernatant liquor of dilution is used to measure the external generation and the secretion of IgG antibody, and produces with ELISA test determination antibody as previously mentioned.Embodiment 29: oral tolerance induces in the mouse
Use the 6-8 female Balb/c mouse in age in week.In order to induce oral tolerance, to mouse feeding according to embodiment 22 by the T7/Amb a extract of the tobacco plant of plastid expression cassette express recombinant artemisiifolia I.1.More specifically, with T7/Amb a I.1 component 1 or the component 2 of the tobacco plant that transforms of plastid expression cassette, or the component 1 of unconverted contrast tobacco plant or component 2 are in contrast to mouse feeding.The animal of wild-type is divided into 3 reorganization artemisiifolia raising groups (0.1,1 and 10mg reorganization artemisiifolia/sky) and 1 unconverted tobacco raising group (in contrast).Form by 8 animals for every group.After initial 10 days of animal breeding plant adaptive phase, begin to animal per os administration of antigens.By continuous 5-8 days tube feed H once a day 2O or phosphoric acid buffer saline solution are adjusted to the corresponding tobacco ingredient of 0.2-0.5ml final volume, feeding animals.
Inducing of 4 days postevaluation oral tolerances of allergen that last per os feeding is tobacco expressed.By with all 4 groups of mouse of 2 weekly interval peritoneal injections reorganization artemisiifolia (10 μ g/ animal) sensitization 3 times.Except the reorganization artemisiifolia, every mouse is also to Protalbinic acid (10 μ g/ animals; SigmaChemical, St.Louis, MO, the U.S.) sensitization, can realize other antigenic immunne responses to prove this mouse.Antigen is dissolved in 0.9% salt solution and with aluminium hydroxide and mixed (2: 1; Alu GelS Serva, Feinbiochemica GmbH, Heidelberg, Germany), obtain the concentration of the hope in the 200 μ l/ mouse final volume.
Collect serum specimen 4 times, in order to measure IgE, IgG1 and the IgG2a level of antigen-specific: once before the beginning oral antigen and the appropriate time each time of three immunity.Five equilibrium serum is stored under-20 ℃ up to mensuration.
All serum specimens are all caught solid-phase enzyme-linked immunosorbent test (ELISA), in order to measure reorganization artemisiifolia and special SERUM IgE, IgG1 and the IgG2a level of Protalbinic acid.96 hole microtiter plate (1mmunoplates Maxisorp TMSurface, Nunc, Roskilde, Denmark) be dissolved in 0.015M yellow soda ash sodium bicarbonate buffer liquid (pH9) with every hole 0.1ml corresponding antigens (1-10 μ g/ml) 37 ℃ of following incubations 2 hours, 4 ℃ are spent the night subsequently.All washings are all with the 0.8% salts solution (pH7.4 that contains 0.05% tween solution; Lavation buffer solution; SigmaChemical) carry out.Unless otherwise noted, all with the lavation buffer solution dilution that contains 1% heat-killed foetal calf serum, all incubations all carry out under moist environment for serum specimen, indicative antibody and reagent, and reaction volume is 100 μ l/ holes.Behind the bag quilt, wash plate 3 times with lavation buffer solution.Serum specimen adds in the antigen coated hole with 3 times of serial dilutions, and 37 ℃ of following incubations 2 hours.After lavation buffer solution washing 3 times, the accordingly biotinylated indicative antibody of adding (So.Biotechnology Assoc., Birmingham, AL, the U.S. in plate; Binding Site, Birmingham, Britain) 37 ℃ 1 hour.After the washing, and the avidin D detection immunoreactivity of adding horseradish peroxidase-labeled (1: 3,000,60 minute, 37 ℃; Vector Lab., Burlingame, CA, the U.S.).After the washing, (substrate buffer solution pH5) is washed plate 3 times to use citrate buffer solution in addition.Be dissolved in the substrate buffer solution O-Phenylene Diamine (SigmaChemical) (0.5mg/ml) and hydrogen peroxide (1 μ l/ml) detect enzymic activity, and after 1-15 minute, use 4N H 2SO 4(50 μ l/ holes; Merck KgaA, Darmstadt, Germany) stop.Read plate device spectrophotometry optical density(OD) under 492nm with ELISA.Net result is expressed as with respect to the relative ELISA unit (REU) of standard serum aggregate (pool) (being arbitrarily designated as 100 REU) or absolute protein concn (μ g/ml).
For comprising Der fI, Sorh I, Bet v I and rAed a1 allergen gene and GAD and thyroglobulin gene or other any antigenic transgenic plant use similar approach mensuration oral tolerances of the present invention.
People such as Sato, immunology 95:193-199,1998 have described inducing the oral tolerance of dermatophagoides pteronyssinus extract.People such as Passalacqua, lancet (Lancet), 351:629-632,1998 have described the dermatophagoides pteronyssinus experiment in the human body.Embodiment 30: the expression of the Chemical Regulation of people BPI gene in plant plastid
I. contain the BPI gene under the reactive promoter element control of phage t7 RNA polymerase The structure of plastid conversion carrier
With the encoding sequence of people BPI gene (as United States Patent (USP) 5,198,541 is described) clone for 182bpNcoI/EcoRI restricted fragment (contain the 5 ' part of cDNA and in the NcoI site, mix initiator codon) and 1288 bp EcoRI/XbaI restricted fragments (contain the cDNA sequence 3 ' partly and terminator codon).These fragments of gel-purified are connected with the 7693 bp NcoI/XbaI fragments that derive from plasmid pT7_GUS with three tunnel reactions.Confirm that through dna sequencing and restriction enzyme analysis the plasmid (pT7_BPI) that obtains contains the BPI gene in the T7 gene 10 promotor downstreams that derive from plasmid pET3a (Novagen), it contains chimeric 5 ' the untranslated leader that derives from T7 gene 10 leaders, sequence is 5 '-GGG AGA CCA CAA CGG TTT CCC TCT AG-3 ' (SEQ IDNO:25), derive from plasmid pC8 the StuI joint joint sequence 5 '-CGAGG-3 ' and modify NcoI restriction site (below line out) is contained in the back at the initiator codon place the sequence 5 ' that derives from tobacco plastid psbA gene 5 ' leader-GGG AGT CCC TGA TGA TTA AATAAA CCA AGA TTT TA C CAT GG-3 ' (SEQ ID NO:26).The construction process of pT7_GUS is, the 386bp EcoRI/NcoI fragment of plasmid pPH142 of chimeric T7 promotor/psbA 5 ' leader is connected (International Patent Application WO 98/11235) with the 9241 bp NcoI/EcoRI fragments of the GUS plastid conversion carrier pC8 of dephosphorylation and gel-purified with containing of gel-purified.The construction process of plasmid pPH142 comprises, with BsaBI digested plasmid pPH136 (60 ℃ 2 hours), gel-purified, and digest linearizing plasmid DNA (37 ℃ 1 hour) once more with BsmFI, with alkaline phosphatase treatment (37 ℃ 1 hour, phenol extracting subsequently), and the 3398bp fragment of the gel-purified that obtains is connected with the synthetic double chain oligonucleotide, this double chain oligonucleotide is by with oligonucleotide T73a_U (5 '-CGA TCC CGC GAA ATT AATACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC C-3 ', SEQ ID NO:27) with T73a_L (5 '-TAG AGG GAA ACC GTT GTG GTCTCC CTA TAG TGA GTC GTA TTA ATT TCG CGG GAT CG-3 ', SEQ ID NO:28) annealing is used T4 polynucleotide kinase phosphorylation (37 ℃ 1 hour) and preparation subsequently.Lack the active e. coli strains of dam methylase (DM-1, Stratagene, La Jolla, California) in amplification plasmid pPH136 DNA, digest with BsaBI allowing.Make up pPH136 subsequently, method is that the StuI/NcoI fragment that will derive from the 3428 bp gel-purified of plasmid pPH120 is connected with double stranded synthetic oligonucleotide, this double chain oligonucleotide is by with oligonucleotide minpsb_U (5 '-GGG AGT CCC TGA TGA TTA AAT AAA CCAAGA TTT TAC-3 ', SEQ ID NO:29) prepares with minpsb_L (5 '-CAT GGT AAAATC TTG GTT TAT TTA ATC ATC AGG GAC TCC C-3 ', SEQ IDNO:30) annealing.This oligonucleotide comprises the 38nt part of tobacco psbA gene 5 ' untranslated homing sequence, and that the translation in external plastid translation system of this part and psbA gene product is raised is relevant (Hirose and Sugiura, EMBO J.15:1687-1695,1996).PPH120 contains the modification part of aadA gene of psbA promotor-guiding and the difference T7-lac promotor (deriving from Novagen plasmid pET21d) of plasmid pC8, with the chimeric ribosome bind site that derives from tobacco plastid rbcL gene, wherein inserted the fragment of cerevisiae dna, with blocking-up derive from the peculiar psbA promotor of carrier of plasmid pPRV111A low-level bidirectional transcription activity (people such as AHison, EMBO is Jun 3 J.1996; 15 (11): 2802-9).The structure of pPH120 is the 256bp EcoRI/HincII fragment with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) LEU2 gene, the 2645bp EcoRI/DraIII fragment of pPH119 is mended flat EcoRI fragment with the 569bpDraIII/Klenow-of pPH119 and is connected.Make up pPH119 subsequently, method comprises with StuI digested plasmid pPH118, and reconnects the multiple StuI joint of deriving and existing in the chimeric T7 promotor with the pC8 that removes pPH118.PPH118 contains the 235bp SpeI/NcoI fragment of pC8, separates and this fragment of gel-purified, is connected to then among the cloning vector pGEM5Zf (+) (Promega, Madison WI) with NcoI and SpeI digestion. II: the Biolistic of tobacco plastom is transformed with plasmid pT7 BPI
As carrying out the conversion of plastom as described in the embodiment 22.With bombardment 3-8 after week at resistance seedling subclone that bleaching occurs under the leaf to identical selection substratum, make it to form callus, separate and second batch of seedling of subclone.The plastom of complete isolating conversion copy (homogeneity state) in the independent subclone is estimated with Southern trace standard technique people (1989) " molecular cloning: laboratory manual " such as (, cold spring harbor laboratory, cold spring port) Sambrook.(Mettler, I.J. (1987) molecular biology of plants report 5 346-349) separates on 1%Tris-boric acid (TBE) sepharose the total cell dna of BamHI/NcoI digestion, is transferred to (Amersham) on the nylon membrane, uses 32The random primer dna sequence dna of P-mark is detected, and this sequence is corresponding to the 0.7kbBamHI/HindIII dna fragmentation that derives from pC8 that comprises a part of rps7/12 plastid targeting sequencing.Contain the 1.25kb fragment of expection and lack the segmental homogeneity seedling of wild-type 3.3kb that (McBride, people such as K.E. (1994) PNAS 91 7301-7305) go up sterile rootage, and it is transferred in the greenhouse containing the MS/IBA substratum of spectinomycin. III: transform with plasmid pT7 BPI, and carry containing by the reactive PR-1a promotor control of BTH- Have in the tobacco plant of the phage t7 rna polymerase gene of nuclear gene group plastid targeting sequencing The chemical induction that BPI expresses
With tobacco is Nt_110X6b-5 to the homogeneity Nt_pT7_BPI plant pollination of C35BPI-5B-4 system, and detects seed by the maternal inheritance that transforms the spectinomycin resistance marker that plasmid pT7_BPI carries.Other seeds of these F1 filial generations are germinateed in the soil of 6 inches flowerpots.After sowing for 8 weeks, spray the 1.0mM BTH of foliage applying up to flowing down from leaf to plant.The results tissue, the compartment analysis BPI with 0,1,2,3,7,14,21,28 and 35 day after using BTH accumulates.Control plant spray water or inert fraction to same system only contain plastid BPI gene and make simultaneously, do not contain the C35BPI-5B-4 plant germination of chemical induction type trans-activator, and measure in the same way with identical arrangement of time.Accumulation with Northern hybridization (mRNA) and Western trace (protein) measured by standard techniques BPI.In addition, carry out determination of activity, with the BPI ability that inhibition index is grown in the liquid nutrient medium of Gram-negative bacteria (for example intestinal bacteria) of estimating expression of plants with leaf extract.
Embodiment 31: be used for the structure at the expression cassette of plant center express transgenic
The gene order that preparation is expressed in transgenic plant at first is assemblied in the expression cassette that is arranged in after the suitable promotor and is positioned at suitable transcription terminator upstream.All requirements that the expression of plants box makes up all are applicable to dna molecular of the present invention, particularly encode trans-activator or therapeutic activity protein DNA molecule, and carry out with technology well-known in the art.
Promotor is selected
The selection of the promotor of using in expression cassette will determine the room and time expression pattern of dna molecular in the transgenic plant.The promotor of selecting will specific cellular type (as leaf epidermal cell, mesophyll cell, root chrotoplast) or in particular organization or organ (as root, leaf or flower) the expressible dna molecule, this selection will reflect the location that this genetically modified organism synthetic is wished.In addition, selected promotor can be regulated the genetically modified expression of guiding under the promotor at photoinduced or other times.Another selection is that selected promotor can be by Chemical Regulation.This will provide the possibility of ability inducing DNA developed by molecule when only handling when wishing and with chemical inducer.
3 ' regulates sequence
Many 3 ' regulates sequence can use in expression cassette.In plant nuclear, these sequences for example are responsible for exceeding genetically modified termination of transcribing and correct polyadenylation thereof.Suitable transcription terminator and the known transcription terminator that works in plant comprise CaMV 35S terminator, tml terminator, nopaline synthase terminator, pea rbcS E9 terminator.They can be used for monocotyledons and dicotyledons.
Strengthen or regulate the sequence of expression
Found that many sequences can strengthen the genetic expression in the transcription unit, these sequences can be used in combination with transgenosis to strengthen its expression in transgenic plant.
Multiple intron sequences shows can strengthen expression, especially in monocot plant cell.For example, find that the intron of corn Adh1 gene significantly strengthens the expression of wild type gene under homologous promoter when being imported into maize cell.Find introne 1 with the fusion constructs of chloramphenicol acetyl transferasegene in especially effectively and strengthen and express people such as (, gene development 1:1183-1200 (1987)) Callis.In identical experimental system, the intron that derives from corn bronzel gene has similar effect aspect the expression strengthening people such as (, the same) Callis.Intron sequences is mixed plant conversion carrier by routine, generally in the untranslated leader.The known multiple untranslated leader that derives from virus also can strengthen expression, and it is effective especially in the dicotyledons cell.Particularly, derive from tobacco mosaic virus (TMV) (TMV, " Ω-sequence "), the leader sequence of corn yellows mottle virus (MCMV) and alfalfa mosaic virus (AMV) strengthen aspect the expression be effectively (as people such as Gallie, nucleic acids research 15:8693-8711 (1987); People such as Skuzeski, molecular biology of plants 15:65-79 (1990)).Embodiment 32: the guiding of gene product in the cell
The mechanism that has multiple guiding gene product in the known plants, and characterized the sequence of controlling these machining functions in more detail.For example, gene product is subjected to control at the signal sequence that the multiple proteins aminoterminal is found to the guiding of plastid such as chloroplast(id), and this signal sequence is cut in the chloroplast(id) output procedure, (for example produces mature protein, people such as Comai, journal of biological chemistry 263:15104-15109 (1988)).These signal sequences can merge with heterologous gene products, and realize the input of allos product in chloroplast(id) people's nature 313:358-363 (1985) such as () van den Broeck.Can separate the DNA of coding appropriate signal sequence in the 5 ' end of the cDNA of other multiple proteins of chloroplast(id) from encode RUBISCO albumen, CAB albumen, EPSP synthase, GS2 albumen and known locations.Selected signal sequence should comprise known restriction enzyme site, and the syzygy of structure should consider that enzyme cuts any amino acid after the required restriction enzyme site.Sometimes this needs can be by adding a small amount of amino acid between restriction enzyme site and transgenosis ATG, some amino acid of perhaps replacing in addition in the transgenic sequence is realized.Utilize (people such as Bartlett, in: people such as Edelmann (volume .) " chloroplast(id) molecular biology method " (Methods in Chloroplast MolecularBiology), Elsevier.1081-1091 (1982); People such as Wasmann, molecule General Genetics 205:446-453 (1986)) described technology, the external translation of the construct by in-vitro transcription, external subsequently chloroplast(id) picked-up can detect the chloroplast(id) ingestion efficiency of the syzygy that makes up into the chloroplast(id) input.Therefore, any nucleotide sequences with the coding trans-activator of these signal sequences is merged, so that trans-activator is directed in the chloroplast(id).Above-mentioned cell guiding mechanism not only can be with homologous promoter, and can use with allogeneic promoter, to realize the specific cell purpose that leads under the transcriptional regulatory of the expression pattern promotor different with the promotor that produces targeting signal.
Other gene products are positioned (for example, people such as Unger, molecular biology of plants (Plant Molec.Biol.) 13:411-418 (1989)) in other organoids such as plastosome and the peroxysome.Also can operate the cDNA of these products of coding, to realize the guiding of heterologous gene products to these organoids.The example of these sequences is the ATP enzyme of nuclear coding and the special aspartic transaminase isozyme of plastosome.People such as Rogers (institute of NAS reports 82:6512-6516 (1985)) have described the guiding to the cell protein body.In addition, characterized and made the lead sequence of other cellular compartments of gene product.Amino terminal sequence is responsible for to the guiding of ER, apoplast with by aleurone cell's exocytosis (Koehler and Ho, vegetable cell 2:769-783 (1990)).In addition, amino terminal sequence is responsible for vacuole guiding people such as (, molecular biology of plants 14:357-368 (1990)) Sbinshi of gene product with the carboxyl terminal sequence.
Embodiment 33: the embodiment that expression cassette makes up
The present invention includes the expression of dna molecular of the present invention under any promotor that can express is regulated in plant, no matter and the source of this promotor how.Therefore, with technology well-known in the art dna molecular is inserted in any expression cassette.These expression cassettes easily can be transferred in the plant conversion carrier of the following stated then.In addition, the present invention also comprise the effable promotor of any plant and transgene expression is required or selected other any sequences unite use.These sequences include but not limited to, transcription terminator, strengthen the external sequence (as intron [as the Adh introne 1], virus sequence [as TMV-Ω]) expressed and preparation with the lead sequence of specific cells device and cellular compartment of gene product.
Constitutive expression: CaMV 35S promoter
Being structured among the disclosed patent application EP 0 392 225 of plasmid pCGN1761 described.PCGN1761 contains " two " 35S promoter and tml transcription terminator, has unique EcoRI site between promotor and terminator, and has pUC type skeleton.Made up a kind of derivative of pCGN1761, it contains a polylinker that also comprises the modified in NotI and XhoI site except that existing EcoRI site.This derivative is named as pCGN1761ENX.PCGN1761ENX can be used for the clone of interior cDNA sequence of its polylinker or gene order (comprising the microorganism open reading frame sequence), its objective is under 35S promoter control and expresses in transgenic plant.Complete 35S promoter-the gene order of this structure-tml terminator box can cut from HindIII, SphI, SalI and the XbaI site of promotor 5 ' side and XbaI, BamHI and the BglI site of terminator 3 ' side, is used for shifting to conversion carrier.In addition, can cut by carry out 5 ' with HindIII, SphI, SalI, XbaI or PstI, and carry out 3 ' cutting, downcut two 35S promoter fragments, and replace with another kind of promotor with any polylinker restriction site (EcoRI, NotI or XhoI).
Expression under the Chemical Regulation type promotor
(1) PR1-a promotor
This part has been described with the two 35S promoters among the arbitrary promotor replacement pCGN1761ENX that selects; Chemical Regulation type PR-1a promotor has been described by way of example.Preferably downcut the promotor of selecting from its source, but also can increase in addition with primer PCR with suitable termination restriction site with restriction enzyme.If should carry out pcr amplification, then promotor should check order once more, to check the amplification mistake after the promotor of clonal expansion in targeting vector.Downcut Chemical Regulation type tobacco PR-1a promotor from plasmid pCIB1004 (referring to EP 0 332 104), and transfer among the plasmid pCGN1761ENX.Cut pCIB1004 with the NcoI enzyme, make 3 ' protuberance of the linear fragment that obtains become flush end by handling with the T4 archaeal dna polymerase.Cut this fragment with the HindIII enzyme then, the gelling purifying obtains contains the PR-1a promoter fragment, and it is cloned among the pCGN1761ENX that removes two 35S promoters.Its implementation comprises, cuts with the XhoI enzyme, is processed into flush end with the T4 polysaccharase, cut with the HindIII enzyme subsequently, and separating clone has the bigger carrier terminator fragment that contains of pCIB1004 promoter fragment.This produces a kind of pCGN1761ENX derivative, the polylinker that interleaves that it contains PR-1a promotor and tml terminator and contains unique EcoRI and NotI site.
The nucleotide sequence of coding dna molecular of the present invention is inserted in this carrier, subsequently fusion product (being promotor-gene-terminator) is transferred in selected any conversion carrier, comprise the carrier described in the application's book, thereby the chemical induction type of preparing trans-activator is expressed.
(2) alcohol induced type promotor
Can also be used to provide genetically modified inducible expression of the present invention by some alcohols or ketone such as alcohol induced promotor.This promotor for example is from the alcA gene promoter of Aspergillus nidulans (Aspergillusnidulans) (people (1998) Nature Biotechnol (Nat BiotechnoI) 16:177-180 such as Caddick).In Aspergillus nidulans, alcA genes encoding ethanol dehydrogenase I, its expression is regulated in the presence of chemical inducer by the AlcR transcription factor.For the present invention, with the CAT gene order among the plasmid palcA:CAT-it contains the alcA gene promoter sequence that merges with minimum 35S promoter people (1998) Nature Biotechnol 16:177-180 such as () Caddick-replace with a kind of genetically modified nucleotide sequence of coding, is formed on the expression cassette that the control of alcA gene promoter contains a kind of genetically modified nucleotide sequence of encoding down.This carries out with method well-known in the art.In a preferred embodiment, minimum 35S promoter is replaced with any minimal promoter easily, for example corn Bronze-1 minimal promoter (people (1991) vegetable cell (The plant Cell) 3:317-325 such as Roth).Termination signal among the palcA:CAT also can replace with other termination signals well-known in the art.The construct that produces transforms the plant species of wishing.Also contain the alcR gene in the plant of the nucleotide sequence that contains the coding dna molecular of the present invention that merges with the alcA gene promoter, the alcR expression of gene is the known or any promotor control that is applicable to expression of plants described herein by this area.Therefore, realize the tissue or the organ specificity of alcR gene product, cause genetically modified derivable tissue or organ specificity.Any termination signal known in the art also is applicable to the alcR expression cassette.
(3) glucocorticoid inducible type promotor
Also relate to the expression of utilization based on the system induction dna molecular of the present invention of steroid hormone.For example, use the inducible system (Aoyama and Chua (1997) plant magazine (The plant Journal) 11:605-612) of glucocorticosteroid mediation, and by using glucocorticosteroid, come abduction delivering as synthetic glucocorticoid (preferred dexamethasone), concentration is preferably 0.1mM-1mM, more preferably is 10mM-100mM.For the present invention, the luciferase genes sequence is replaced with a kind of genetically modified nucleotide sequence of coding, to form a kind of expression cassette, it is a kind of and the fusion of 35S minimal promoter that this expression cassette contains coding, the genetically modified nucleotide sequence under the control of the GAL4 of 6 copies upstream activating sequence.This carries out with method well-known in the art.In a preferred embodiment, minimum 35S promoter is replaced with minimal promoter easily, as corn Bronze-1 minimal promoter (people (1991) vegetable cell 3:317-325 such as Roth), the corn termination signal also can be replaced by other termination signals well-known in the art.The construct that obtains transforms the plant species of wishing.The GAL4 DNA that trans-acting factor comprises with the trans-activation domain of protein herpesvirus VP16 and merge people (1988) gene development (gene sDevel.) 2:718-729 such as () Triezenberg, combines the territory fusion with the hormone of rat glucocorticoid receptor people (1988) cell 54:1073-1080 such as () Picard is in conjunction with territory people (1986) science 231:699-704 such as () Keegan.Expression of Fusion Protein is by any promotor control that is applicable to expression of plants well-known in the art or described herein.In the plant of the genetically modified nucleotide sequence that contains the fusion of coding and 6xGAL4/ minimal promoter, also contain this expression cassette.Therefore, realize the tissue or the organ specificity of fusion rotein, cause genetically modified derivable tissue or organ specificity.Any termination signal known in the art also is applicable to and contains the Expression of Fusion Protein box.
Constitutive expression: actin promoter
Several isotypes of known Actin muscle can be expressed in most cell types, so actin promoter is the preferable selection of constitutive promoter.Especially, the promotor of rice Act1 gene is cloned and is characterized (people such as McElroy, vegetable cell 2:163-171 (1990)).The 1.3kb fragment of finding this promotor contains all required regulatory elements of expression in the rice protoplastis.In addition, made up multiple expression vector, to be used in particular for monocotyledons (people such as McElroy, molecule General Genetics 231:150-160 (1991)) based on the Act1 promotor.Wherein mixed the sequence of Actl-introne 1, Adhl 5 ' flanking sequence and (the maize alcohol dehydrogenase gene) Adhl-introne 1 and CaMV 35S promoter.The carrier that shows high expression level is the syzygy of 35S and Actl intron or Actl 5 ' flanking sequence and Actl intron.The optimization of the sequence around (GUS reporter gene) initial ATG also strengthens expression.The described promoter expression cassettes of people such as McElroy (molecule General Genetics 231:150-160 (1991)) can easily be modified for the expression of dna molecular of the present invention, and is particularly useful for the monocotyledons host.For example, can downcut from the McElroy structure and contain promoter fragment, replace two 35S promoters among the pCGN1761ENX with it, this carrier can be used for the insertion of specific gene sequence then.The fusion gene that makes up like this is transferred to suitable conversion carrier.In indivedual reports, also find to contain bootable high expression level people such as (, vegetable cell report (Plant Cell Rep.) 12:506-509 (1993)) Chibbar in the barley cell of cultivating of the rice Actl promotor of first kind of intron.
Dna molecular of the present invention is inserted this promotor downstream, subsequently fusion product (being promotor-gene-terminator) is transferred in any conversion carrier of selection, comprise the carrier described in the application's book.
Constitutive expression: ubiquitin promotor
Ubiquitin is the known another kind of gene product that can accumulate in the various kinds of cell type, its promotor is cloned from several kinds, be used for transgenic plant (as people such as Sunflower Receptacle-Binet, plant science 79:87-94 (1991), people such as corn-Christensen, molecular biology of plants 12:619-632 (1989)).In transgenosis unifacial leaf system, studied the corn ubiquitin, and its sequence and the carrier that transform to make up for monocotyledons are disclosed among the patent disclosure text EP 0 342926.In addition, people such as Taylor (vegetable cell report 12:491-495 (1993)) have described a kind of carrier (pAHC25), it comprises corn ubiquitin promotor and first kind of intron, has high reactivity when microparticle bombardment is introduced in multiple monocot plant cell suspension.The ubiquitin promotor is suitable for expressing nucleotide sequence in transgenic plant especially monocotyledons.Suitable carriers is by introducing pAHC25 derivative or the described any conversion carrier of the application that suitable ubiquitin promotor and/or intron sequences are modified.
Arabidopis thaliana ubiquitin 3 (UBQ3) gene promoter people (1993) molecular biology of plants (Plant Mol Biol) 21:895-906 such as () Norris also is that a kind of being applicable to expressed genetically modified promotor of the present invention in plant.Therefore a kind of genetically modified nucleotide sequence of will encoding inserts in any this class carrier, and transforms plant with fusion product (being promotor-gene-terminator), causes genetically modified constitutive expression.
The expression that Gent is different
Genetically modified a kind of preferred expression pattern of the present invention is that root is expressed.A kind of suitable root promotor is de Framond (FEBS 290:103-106 (1991)) promotor described and that describe in disclosed patent application EP 0 452 269.This promotor is transferred among suitable carriers such as the pCGN1761ENX, and inserts a kind of genetically modified nucleotide sequence of coding in this carrier.Subsequently complete promotor-gene-terminator box is transferred in the purpose conversion carrier. Wound-induced type promotor
The creation inducible promoter is specially adapted to genetically modified expression.Many these class promotors have been described (for example, people such as Xu, molecular biology of plants 22:573-588 (1993), people such as Logemann, vegetable cell 1:151-158 (1989), Rohrmeier and Lehle, molecular biology of plants 22:783-792 (1993), people such as Firek, molecular biology of plants 22:129-142 (1993), people such as Warner, plant magazine 3:191-201 (1993)), and all be applicable to the present invention.People such as Logemann (the same) have described 5 ' upstream sequence of dicotyledons potato wun1 gene.People such as Xu (the same) prove that the wound-induced type promotor (pin2) of dicotyledons potato has activity in the monocotyledons rice.In addition, Rohrmeier and Lehle (the same) have described the clone of corn Wipl cDNA, and it is the wound-induced type, and can be used to use standard technique to separate homologous promoter.Similarly, people's (the same) such as people such as Firek (the same) and Warner have described the wound-induced gene of the medicinal asparagus of monocotyledons (Asparagusofficinalis), and it invades the position expression at local wound and pathogenic agent.Use clone technology well-known in the art, these promotors can be transferred in the suitable carrier, merge, and be used for expressing these genes at insect infringement position with the genetically modified Nucleotide of coding.
The expression of marrow preference
Patent application WO 93/07278 has described the separation of corn trpA gene, and this gene is preferentially expressed in myelocyte.Utilize standard molecular biological technique, this promotor or its part can be transferred in the carrier as pCGN1761, replace 35S promoter, and be used for guiding genetically modified expression of the present invention in the mode of marrow preference.In fact, comprise the promotor of marrow preference or the fragment of its part and can be transferred in any carrier, and can be and be used for transgenic plant and modify.A kind of expression of marrow preference of genetically modified nucleotide sequence of encoding realizes by insert the genetically modified nucleotide sequence of coding in this carrier.
The expression of pollen-specific
Patent application WO 93/07278 has also described the separation of the calcium dependent protein kinase of the corn of expressing (CDPK) gene in pollen cell.This gene order and promotor are extended from transcriptional start point can reach 1400bp.Utilize standard molecular biological technique, this promotor or its part can be transferred in the carrier as pCGN1761, replace 35S promoter, and be used for guiding genetically modified expression of the present invention in the mode of pollen-specific.In fact, comprise the promotor of pollen-specific or the fragment of its part and can be transferred in any carrier, and can be and be used for transgenic plant and modify.
The expression that leaf is special
Hudspeth and Grula (molecular biology of plants 12:579-589 (1989)) have described the corn gene of a kind of coding phosphoric acid enol carboxylase (PEPC).Application standard Protocols in Molecular Biology, the promotor of this gene can be used to guide the expression of transgenosis of the present invention in transgenic plant in the special mode of leaf.
The expression of chloroplast(id) guiding
Chen and Jagendorf (journal of biological chemistry 268:2363-2367 (1993)) had once described the successful Application of importing heterologous transgene with chloroplast transit peptides.Employed peptide is the transit peptides (people such as Poulsen, molecule General Genetics 205:193-200 (1986)) of the rbcS gene of Nicotiana plumbaginifolia.Use restriction enzyme DraI and SphI, or Tsp509I and SphI, can downcut dna sequence dna people such as (, the same) Poulsen of this transit peptides of coding, use with above-mentioned any structure after the operation from plasmid prbcS-8B.The DraI-SphI fragment is from extending to respect to-58 of initial rbcS ATG, and comprise, be right after first amino acid (also being methionine(Met)) of input cleavage site mature peptide afterwards, and the Tsp509I-SphI fragment is from extending to respect to-8 of initial rbcS ATG, and comprise first amino acid of mature peptide.Therefore, these fragments suitably can be inserted in the polylinker of selected arbitrary expression cassette, produce the fusion gene of transcribing that untranslated leader with selected promotor (as 35S, PR-1a, Actin muscle, ubiquitin etc.) merges, and the genetically modified nucleotide sequence of coding is inserted in the correct fusion gene in transit peptides downstream.This structure is conventional in the art.For example, the DraI end was exactly flush end originally, and 5 ' Tsp509I site can handle produce flush end through the T4 polysaccharase, perhaps can be connected with joint or adapter sequence in addition, so that it and the fusion of selected promotor.Can keep 3 ' SphI site like this, perhaps can be connected with the adapter of joint sequence in addition,, make the suitable restriction site that can get can be used for the insertion of the nucleotide sequence of selected subsequently coding trans-activator like this so that it inserts selected carrier.It is desirable to keep the ATG in SphI site, and comprise first ATG of dna molecular.Chen and Jagendorf (ibid) provide the consensus sequence of the ideal cut that is used for the chloroplast(id) input, and methionine(Met) is preferably located in first site of mature protein in all situations.More variation is arranged, and amino acid may be so not crucial on site subsequently.In any situation, people such as available Bartlett (: people such as Edelmann (volume) " chloroplast(id) molecular biology method ", Elsevier, 1081-1091 page or leaf (1982)) and the described method of people (molecule General Genetics 205:446-453 (1986)) such as Wasmann, the external input efficiency of assessment fusion constructs.Generally speaking, the best way may be that the dna molecular that is used in the aminoterminal unmodified produces fusion gene, and only when fusion gene is not obviously imported chloroplast(id) with high-level efficiency, just do not introduce modification, in this case can be according to existing document (Chen and Jagendorf; People such as Wasmann; Ko and Ko, journal of biological chemistry 267:13910-13916 (1992)) modify.
Be transferred to a kind of preferred carrier of structure among the cloning vector pCGN1761ENX/Sph-by DraI-SphI transit peptides encode fragment with prbcS-8B.This plasmid is cut with the EcoRI enzyme, handles making end become flush end through the T4 archaeal dna polymerase.With SphI digested plasmid prbc-8B, be connected to annealed molecule adapter.Make products therefrom 5 ' end phosphorylation by the processing of T4 kinases.Cut with the DraI enzyme subsequently, discharge the transit peptides encode fragment in the flush end ex-EcoRI site that is connected in above-mentioned modification carrier.By order-checking identify have direction for insert fragment 5 ' end with the adjacent clone of 35S promoter 3 ' end.These clones carry the DNA fusion gene of 35S leader sequence and rbcS-8A promotor-transit peptide sequence, it is from extending to the ATG of mature protein with respect to-58 of rbcS ATG, and comprise unique SphI site in this site, new EcoRI site that produces and NotI and the XhoI site of existing pCGN1761ENX.This novel vector is named as pCGN1761/CT.Dna sequence dna is transferred to pCGN1761/C7 with being met frame, and method is with the round pcr amplification, after with suitable enzyme restriction enzyme digestion it is connected into the pCGN1761/CT of SphI cutting, inserts SphI, NsphI or NIaIII site at the ATG place of amplification.Make up for convenience, need to change second amino acid of clone gene, yet in nearly all situation, use PCR, can be structured in the restriction enzyme site of mature protein and near the sequence of the arbitrary hope first methionine(Met) with the standard site-directed mutagenesis.
The construction process of another kind of preferred vector is, replace two 35S promoters of pCGN1761ENX with the BamHI-SphI fragment of prbcS-8A, the former comprises the light of total length and regulates the rbcS-8A promotor, and it is from-1038 (with respect to transcribing the beginning site) first methionine(Met) up to mature protein.Cut the modified pCGN1761 that has destructive SphI site with PstI and EcoRI enzyme, and handle the generation flush end with the T4 archaeal dna polymerase.Cut prbcS-8A with the SphI enzyme, be connected on the annealed molecule adapter of above-mentioned sequence.Products therefrom is handled through the T4 kinases and is made 5 '-end phosphorylation.Cut release promotor-transit peptides with the BamHI enzyme subsequently, it comprises through the T4 archaeal dna polymerase handles the fragment that produces the BamHI flush end.Promotor-transit peptides fragment cloning of so producing in the pCGN1761ENX carrier of preparation, is produced a kind of rbcS-8A of comprising promotor and transit peptides and the structure in a SphI site is arranged at the cleavage site place that is used for inserting heterologous gene.In addition, there are EcoRI (producing again), NotI and XhoI cloning site in downstream, SphI site.This structure is named as pCGN1761rbcS/CT.
Other GS2 chloroplast transit peptide-coding sequences that can use other sources (unifacial leaf is with dicots) and other genes carry out similar operation.
By optimizing the modification of translation initiation site to pCGN1761ENX
For any construct described in this part, can carry out cloning site modification on every side by introducing the sequence that can strengthen translation.When importing derived from the gene of microorganism in the expression of plants box, this was useful especially, because these genes may not contain and be suitable for the adjacent sequence of the initial methionine of translation initiation in the plant.When the clone of the ATG place of expression of plants box derives from the gene of microorganism, modifying its insertion site may be useful with optimization expression.Described the modification of pCGN1761ENX by way of example, mixed one of several majorizing sequences and be used for expression of plants (for example Joshi is the same)
PCGN1761ENX cuts with the SphI enzyme, handles and connection again with the T4 archaeal dna polymerase, thereby has destroyed the SphI site that is positioned at two 35S promoters 5 '.This produces carrier pCGN1761ENX/Sph-.PCGN1761ENX/Sph-cuts with the EcoRI enzyme, and is connected in annealed molecule joint.This produces carrier pCGNSENX, and it has mixed the plant translation initiation sequence TAAA-C of accurate optimization, and this sequence and itself are that the ATG of a SphI site part is adjacent, is suitable for the heterologous gene the clone of its initial methionine place.Downstream-EcoRI, the NotI and the XhoI site that have kept the SphI site.
Make up another kind of carrier, it uses the NcoI site at initial ATG place.This carrier is named as pCGN1761NENX, prepares by inserting an annealed molecule joint in pCGN1761ENX EeoRI site.Therefore, this carrier comprises accurate optimized sequence TAAACC, and it is adjacent with the initial ATG within the NcoI site.The site, downstream is EcoRI, NotI and XhoI.Yet before this operation, with to above to the described similar technology of SphI, perhaps use " inside and outside " PCR (people such as Innes in addition, " PCR method: method and application guide " (PCRProtocols:A guide to methods and applications), Academic Press, New York (1990)) destroy two the NcoI sites (being positioned at the upstream site of 5 ' 35S promoter unit) in the pCGN1761ENX carrier.By insert any plant cDNA or reporter gene sequence in cloning site, conventional expression analysis in plant can be measured any possible disadvantageous effect of this operation to expressing subsequently.
Therefore, a kind of genetically modified nucleotide sequence of will encoding inserts among the pCGN1761ENX, is used for constitutive expression under the control of the CaMV of the translation initiation site that contains optimization 35S promoter.
Embodiment 34: the structure of plant conversion carrier
Many conversion carriers can be used for Plant Transformation, and dna molecular of the present invention can insert in above-mentioned any expression cassette, make it can express this dna molecular under proper condition in the cell of hope.The expression cassette that will contain nucleotide sequence then mixes in the arbitrary suitable conversion carrier of the following stated.
The selection of used carrier will be depended on preferred transformation technology and the target species that are used to transform.For some target species, preferred different microbiotic or weedicide selective markers.The conventional selective marker of using comprises npfII gene (Messing and Vierra, the gene 19:259-268 (1982) that gives kantlex and associated antibiotic resistance in the conversion; People such as Benvan, nature (Nature) 304:184-187 (1983)), the bar gene of conferring herbicide phosphinothricin resistance (people such as White, nucleic acids research 18:1062 (1990), people such as Spencer, theory and applied genetics 79:625-631 (1990)), give hph gene (Blochinger and the Diggelmann of antibiotic hygromycin resistance, molecular cytobiology 4:2929-2931) and give the dhfr gene (people such as Bourouis of methotrexate resistance, EMBO is (7) J.2: 1099-1104 (1983)), with bacterium aadA gene (Goldschmidt-Clermont, 1991, nucleic acids research (Nucl.Acids Res.) 19:4083-4089), its coding aminoglycoside 3 '-adenosyl transferring enzyme and resistance to Streptomycin sulphate or spectinomycin is provided.Bar gene (the people such as White who provides weedicide phosphinothricin resistance is provided spendable other marks, nucleic acids research 18:1062 (1990), people such as Spencer, theory and applied genetics (Theor Appl gene t) 79:625-631 (1990)), sudden change epsp synthase gene (the people such as Hinchee of coding glyphosate resistance, 1988, biotechnology (Bio/Technology) 6:915-922), mutant acetolactate synthase (ALS) gene (people such as Lee of imidazolone or sulfonylurea resistance is provided, 1988, EMBO J.7:1241-1248), the sudden change psbA gene (people such as Smeda of G-30027 resistance is provided, 1993, plant physiology (Plant Physiol.) 103:911-917) or as EP 0 769 059 described Mutagen protoporphyrinogen oxidase genes.
Also use to cause the selective marker just selected, as at the phosphomannose isomerase described in the patent application WO 93/05163.
But also can be by expressing the examination marker gene, as the E.C. 2.3.1.28 of encoding (CAT), beta-glucuronic acid Glycosylase (GUS), luciferase, green fluorescent protein (GFP) or give other protein of the different proterties of transformant phenotype, realize the evaluation of transformant.(1) is applicable to the structure of the carrier of Agrobacterium-mediated Transformation
Many carriers can be used for using the conversion of agrobacterium tumefaciens.They generally carry at least one T-DNA border sequence, and comprise the carrier as pBIN19 (Bevan, nucleic acids research (1984)) and pXYZ.The structure of two kinds of typical carriers has below been described.
The structure of pCIB200 and pCIB2001
Binary vector pCIB200 and pCIB2001 are used to make up the recombinant vectors that Agrobacterium is used, and make up by the following method.By digest pTJS75 (Schmidhauser and Helinski with NarI, bacteriology magazine 164:446-455 (1985)) downcuts tetracycline resistance gene, insert the AccI fragment that has NPTII from pUC4K (Messing and Vierra, a gene 19:259-268 (1982) subsequently; People such as Bevan, natural 304:184-187 (1983); People such as McBride, molecular biology of plants 14:266-276 (1990)), produce pTJS75kan.The XhoI joint is connected with the EcoRV fragment of pCIB7, this fragment comprises a left side and right T-DNA border, plant selectivity nos/nptII mosaic gene and pUC polylinker (people such as Rothstein, and the XhoI digestion fragment is cloned among the pTJS75kan of SalI digestion and produces pCIB200 (referring to EP 0 332 104) gene 53:153-161 (1987)).PCIB200 contains following single polylinker restriction site: EcoRI, SstI, KpnI, BglII, XbaI and SalI.PCIB2001 is the derivative of pCIB200, and it produces by insert other restriction sites in polylinker.The single restriction site of pCIB2001 polylinker is EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI and StuI.Except that comprising these single restriction sites, pCIB2001 also have plant and bacterium kantlex selectivity, be used for agriculture bacillus mediated conversion a left side and right T-DNA border, the RK2 deutero-trfA function that between intestinal bacteria and other hosts, shifts and OriT that also is derived from RK2 and OriV function.The pCIB2001 polylinker is applicable to the clone of the expression of plants box that contains himself conditioning signal.
Preferably use polylinker, above-mentioned any expression of plants box that will contain dna molecular of the present invention inserts among the pCIB2001.
The structure of pCIB10 and hygromycin selection derivative thereof
Binary vector pCIB10 contains gene, the T-DNA right side and the left margin sequence of the required kalamycin resistance of screening in the coded plant, and the sequence of mixing the plasmid pRK252 of wide host range, makes it all reproducible in intestinal bacteria and Agrobacterium.It makes up by people such as Rothstein and describes (gene 53:153-161 (1987)).Make up the multiple derivative of pCIB10, wherein mixed the described hygromycin B phosphotransferase genes of people (gene 25:179-188 (1983)) such as Gritz.These derivatives can make only to be had Totomycin (pCIB743) or Totomycin is being arranged simultaneously and kantlex (pCIB715, pCIB717) time screening transgenic plant cells.This carrier is used for transforming the expression cassette that contains dna molecular of the present invention.(2) be applicable to the structure of the carrier of non-Agrobacterium-mediated Transformation
Do not use the conversion of agrobacterium tumefaciens to avoid needs, so except that the carrier that comprises the T-DNA sequence as mentioned above, also can use the carrier that lacks these sequences to T-DNA sequence in the selected conversion carrier.The transformation technology that does not rely on Agrobacterium comprises that conversion or pollen by particle bombardment, protoplastis picked-up (as PEG and electroporation), microinjection transform (United States Patent (USP) 5,629,283).The selection of carrier depends primarily on the preferred of kind to be transformed.Below, the structure of some typical carriers has been described.
The structure of pCIB3064
PCIB3064 is a kind of pUC derivative vector, is applicable to and weedicide basta (or phosphinothricin) screening bonded direct gene transfer techniques.Plasmid pCIB246 comprises CaMV 35S promoter and the CaMV 35S transcription terminator that merges effectively with the intestinal bacteria gus gene, openly applies for stating among the WO 93/07278 at PCT.The 35S promoter of this carrier comprises two ATG sequences at initiation site 5 ' end.Suddenly change these sites to remove ATG with the standard round pcr, produce restriction site SspI and PvuII.New restriction site is apart from unique SalI site 96bp and 37bp, apart from actual initiation site 101bp and 42bp.The derivative called after pCIB3025 of the pCIB246 that produces.Downcut gus gene by SalI and SacI digestion from pCIB3025 then, make end become flush end, and connect again, produce plasmid pCIB3060.Plasmid pJIT82 is from John Innes Centre, Norwich obtains, cutting-out contains the SmaI fragment of the 400bp of streptomyces viridochromogenes (Streptomyces viridochromo gene s) bar gene, be inserted into the HpaI site (people such as Thompson, EMBO is (1987) J.6:2519-2523) of pCIB3060.This has produced pCIB3064, and it contains the polylinker that is arranged in the bar gene that is used for herbicide screening, the ampicillin resistance gene (being used for screening intestinal bacteria) under CaMV 35S promoter and the terminator control and has single SphI, PstI, HindIII, BamHI site.This carrier is applicable to the clone of the expression of plants box that contains self conditioning signal, is used for guiding the expression of dna molecular of the present invention.
The structure of pSOG19 and pSOG35
PSOG35 is a kind of application bacillus coli gene Tetrahydrofolate dehydrogenase (DHFR) provides the methotrexate resistance as selected marker a conversion carrier.With the pcr amplification 35S promoter (～800bp), the intron 6 of corn Adh1 gene (～550bp) and the GUS untranslated leader of the 18bp of pSOG10.Also the 250bp fragment of pcr amplification coding intestinal bacteria Tetrahydrofolate dehydrogenase II type gene is assembled the SacI-PstI fragment that comprises pUC19 carrier framework and nopaline synthase terminator of these two PCR fragments and pBI221 (Clontech).These segmental assemblings produce pSOG19, and it contains 35S promoter, GUS leader sequence, DHFR gene and the nopaline synthase terminator that merges with intron 6 sequences.The leader sequence that GUS leader sequence among the pSOG19 replaces with corn yellows mottle virus (MCMV) produces carrier pSOG35.PSOG19 and pSOG35 carry the pUC gene of amicillin resistance, and have HindIII, SphI, PstI and the EcoRI site that can be used for cloning exogenous array dna molecular especially of the present invention.
The plastid conversion carrier
Be listed in the clpP gene promoter sub-element control constitutive expression in plant plastid down for nucleotides sequence of the present invention, use plastid conversion carrier pPH143 (embodiment 36 of WO 97/32011).This nucleotide sequence is inserted among the pPH143, replace the PROTOX encoding sequence thus.This carrier is used for then that plastid transforms and according to the screening of spectinomycin resistance to transformant.In addition, also in pPH143, insert this nucleotide sequence, make it replace the gene of aadA under the control of psbA gene promoter.In this case, according to resistance screening transformant to the PROTOX inhibitor.In another embodiment, use the promoter expression PROTOX encoding sequence of the plastid 16S ribosomal RNA gene that derives from tobacco or Arabidopis thaliana.Embodiment 35: the plastid of corn transforms
I type embryo generation callus culture (people (1983) such as Green, in: A.Fazelahmad, K.Downey, J.Schultz, R.W.Voellmy compiles, gene engineering progress: the molecular genetics of Plants and Animals.Miami's symposial in winter, the 20th volume, AcademicPress is N.Y.) from the long immature embryo of the 1.5-2.5mm of greenhouse growth substance.After pollinating about 14 days from the fringe of surface sterilization aseptic cutting-out embryo.Embryo is placed (people (1985) Planta 165:322-332 such as Duncan) on the initial substratum of D callus that contains 2% sucrose and 5mg/L chloramben, or embryo placed contain 3% sucrose and 0.75mg/L 2, on the initial substratum of KM callus that 4-drips (Kao and Michayluk (1975) Planta 126:105-110).In the dark culturing embryo and embryogenesis culture subsequently.Downcut the embryo thing that reacts from explant after about 14 days.The embryo thing that reacts is placed and contains 2% sucrose and 0.5mg/L 2, and the D callus that 4-drips is kept on the substratum, and will place the KM callus that contains 2% sucrose and 5mg/L dicamba 98 to keep on the substratum from the reactant of the initial substratum of KM callus.Keep in the substratum selectivity cultivation of going down to posterity to fresh weekly, 3-8 sets up the intensive embryogenesis culture of high quality after week.Select the target tissue of the embryo generation callus sheet of active growth as gene delivery.Before gene delivery about 4 hours, these callus sheets are placed on the target flat board of keeping substratum that contains 12% sucrose.These callus sheets are with annular array, and radius is apart from dull and stereotyped center 8mm of target and 10mm..As described in the DuPontBiolistics handbook, plasmid DNA is deposited on the golden microcarrier.In the microcarrier preparation of each 6 bombardments, use every kind of plasmid of 2-3 μ g.Send to the target tissue cell with PDS-1000He Biolistics device and to pass gene.Being provided with of Biolistics device is as follows: 8mm between rupture disc and the huge carrier, 10mm between huge carrier and the termination sieve stops 7cm between sieve and the target.Each target is dull and stereotyped with twice of 650psi rupture disc bombardment.Stop placing between sieve and the target tissue one 200 * 200 stainless steel sift (McMaster-Carr, New Brunswick, NJ).
After 5 days, the callus sheet of bombardment is transferred to and contained 2% sucrose and 0.5mg/L 2,4-drips and does not contain amino acid and contain 750 or the keeping in the substratum of 1000nM general formula X VII.Shift after 4-5 hour or the callus sheet placed light frame last 1 hour in second day, in the dark store 5-6 week in 27 ℃.Behind the 5-6 week first screening stage, with yellow to the tissue of white transfer to contain add 500 or the fresh flat board of the same medium of 750nM general formula X VII on.Shift after 4-5 hour or, tissue is placed light frame last 1 hour, in the dark store 3-4 weeks for 27 ℃ at second day.Behind the 3-4 week secondary screening stage, tissue is transferred on the flat board that contains the same medium of adding 500nM general formula X VII.The tissue of healthy growth is placed light frame last 1 hour and 27 ℃ of storages in the dark.Go down to posterity in per two weeks and cultivate once, till colony is during even as big as regeneration.
In this, colony transferred to contain 3% sucrose and do not contain in the modified MS medium (MS3S) of selective agent (Murashige and Skoog (1962) plant physiology 15:473-497), place under the light.In this substratum, add 0.25mg/L ancymidol and 0.5mg/L cell fission and usually induce the sprouting of embryo, or add the 2mg/L benzyladenine.After 2 weeks, do not contain ancymidol and phytokinin or do not contain in the MS3S substratum of benzyladenine for respectively the regenerated colony being transferred to.To take root or the regrowth of unrooted is transferred in the box that contains the MS3S substratum, reclaim the plantlet of taking root at last, and transfer in the soil in the greenhouse.Embodiment 36: be used at the plant nuclear expression, contain the I.1 preparation of the mosaic gene of encoding sequence of ragweed pollen allergen Amb a of merging with composing type Arabidopis thaliana UBQ3 promotor
By with Amb a 5 ' half section 0.83kb NcoI I.1 as pAT230, SalI fragment and Amb a 3 ' end I.1 is as 0.39kb SalI, XbaI PCR produces the NcoI that fragment is inserted pPH121, in the XbaI site, removing the I.1 dam of gene 3 ' the end XbaI site that methylates of Amb a, and make I.1 encoding sequence and Arabidopis thaliana UBQ3 promotor people (1993) molecular biology of plants 21:895-906 such as () Norris fusion of ragweed pollen allergen Amb a.With pAT230 as template and use following primer to carrying out pcr amplification: " top chain " primer (5 '-GTC GCT TTC AACACG TTC AC-3 ', SEQ ID NO:31) and remove " end chain " primer (5 '-GCG CTC TAG ACA TTA TAA GTG CTT AGT-3 ', SEQ ID NO:32) of the A before the XbaI site.Gel-purified 452bp amplified production with SalI and XbaI digestion, and as above connects, and produces pAT240.Contain guiding Amb a I.1 the HindIH-XbaI of the pAT240 of the UBQ3 promotor of gene be connected in the pPH108 fragment of 11.3kb HindIII, XbaI digestion, produce the I.1 binary vector of gene of a kind of Amb a that carries constitutive expression.Embodiment 37: be used at the plant nuclear expression, contain the I.1 preparation of the mosaic gene of encoding sequence of the ripe artemisiifolia allergen Amb a of deduction of merging with composing type Arabidopis thaliana UBQ3 promotor
I.1, Amb a has the signal peptide cutting site of deduction, and the L-Ala in site 26 is predicted as residue+1 (people (1991) journal of biological chemistry 266:1229-1236 such as Rafnar).With pAT230 as template and use following oligonucleotide to modify I.1 encoding sequence of Amb a through pcr amplification, to remove signal peptide: I.1 " top chain " primer has placed an ATG before first codon of albumen at ripe Amb a, NcoI site (5 '-GCA CCA TGG CCG AAG ATC TCC AGG AAA T-3 ' at the preceding 20bp of this mature protein and the new ATG place that produces, SEQ IDNO:33), " end chain " primer (5 '-CTA CCA GCC CAT CAA CAG ACTTAC-3 ', SEQ ID NO:34).Produce 594 bp amplified productions, it contains I.1 5 ' end of gene of Amb a, and does not contain signal sequence.This fragment of gel-purified, with NcoI and ClaI digestion, and with the 0.35kb fragment and as 0.81kb ClaI, the Amb a of XbaI fragment I.1 3 ' end of gene is connected in the NcoI of pAT240, XbaI 4.4kb fragment, produce the Amb a gene I.1 do not contain the signal peptide that merges with Arabidopis thaliana UBQ3 promotor, it is cloned in a kind of binary vector with terminator sequence.Embodiment 38: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the main allergen Der of the dust mite f I encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
To merge as the dust mite allergen Der f I encoding sequence of clone as described in the embodiment 13 and Arabidopis thaliana UBQ3 promotor, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 39: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the main allergen Der of the dust mite f II encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
To merge as the dust mite allergen Der f II encoding sequence of clone as described in the embodiment 13 and Arabidopis thaliana UBQ3 promotor, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 40: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the main allergen Der of the dermatophagoides pteronyssinus p I encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
To merge as the dust mite allergen Der p I encoding sequence of clone as described in the embodiment 13 and Arabidopis thaliana UBQ3 promotor, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 41: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the main allergen Der of the dermatophagoides pteronyssinus p II encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
To merge as the dust mite allergen Der p II encoding sequence of clone as described in the embodiment 13 and Arabidopis thaliana UBQ3 promotor, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 42: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the Johnson grass allergen Sor h I encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
Johnson grass allergen Sor h I encoding sequence and Arabidopis thaliana UBQ3 promotor are merged, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 43: be used at the plant nuclear expression, contain the preparation of the mosaic gene of the birch pollen allergen Bet V I encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
Birch pollen allergen Bet V I encoding sequence and Arabidopis thaliana UBQ3 promotor are merged, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 44: be used at the plant nuclear expression, contain the preparation of the mosaic gene of mosquito saliva allergen rAed a 1 encoding sequence that merges with composing type Arabidopis thaliana UBQ3 promotor
RAed a 1 encoding sequence and Arabidopis thaliana UBQ3 promotor are merged, and as in a kind of binary vector of insertion as described in the embodiment 34.Embodiment 45: contain I.1 encoding sequence of the ragweed pollen allergen Amba that merges with composing type Arabidopis thaliana UBQ3 promotor, be used for the preparation of the mosaic gene that vacuole expresses
With the plasmid pAT240 that contains ragweed pollen allergen gene A mb a 1.1 as pcr template, use Amb a I.1 775 to extend to " top chain " primer (5 '-GCA ACG GTC GCT TTC AAC ACG TTC A-3 ' of 799 with respect to endogenous ATG from the site in the gene, SEQID NO:35) and " end chain " primer (5 '-CGC TCT AGA TTA CAT AGTATC GAC TAA AAG TCC GCA AGG TGC TCC GGG TTG GCA-3 ', SEQ ID NO:36), its sequence and Amb a last 21bp homology I.1, and comprise and derive from tobacco chitinase gene (people such as Shinshi, (1990) molecular biology of plants 14,357-368, institute of people such as Neuhaus (1991) NAS newspaper 88,21 bp vacuole targeting sequencings 10362-10366), the last codon of identical tobacco chitinase gene and XbaI restriction site.Pcr amplification produces the 447bp product, gel-purified, and digest with SalI and XbaI.To contain I.1 383 bp SalI of 3 ' end of the Amb a that merges with tobacco chitinase vacuole targeting sequencing, XbaI fragment with as the 0.83kb NcoI of pAT240, the segmental Amb a of SalI is I.15 ' end is connected to the 4.4kb NcoI of the pAT240 that contains UBQ3 promotor and carrier, XbaI fragment.The Arabidopis thaliana UBQ3 promotor ∷ Amb a that merges with tobacco chitinase vacuole targeting sequencing box I.1 encoding sequence is cloned in a kind of binary vector with terminator sequence.
Plasmid pSCH10 (people such as Shinshi, (1990) molecular biology of plants 14,357-368, institute of people such as Neuhaus (1991) NAS newspaper 88,10362-10366) as the pcr amplification template, use aminoacid sequence and the preceding 22bp of 23 amino acid N end signals of tobacco chitinase peptide and BspHI restriction site homologous " top chain " primer (ErspU 5 '-CGG TCA TGA GGC TTT GTA AAT TCA CAG-3 ' at ATG place, SEQ IDNO:37), with 17 bp homologies behind sequence and the tobacco chitinase N end signal peptide, with Amb a " end chain " primer (ErspL 5 '-TGGAGA TCT TCG GCT GCC GAG GCA GAA AGC A-3 ', SEQ IDNO:38) of merging of the preceding 14bp of the maturation 5 ' end of the deduction of gene I.1.Gel-purified 88 bp amplified productions, cut with BspHI and BglII enzyme, and will contain 23 amino acid N end signals of tobacco chitinase peptide of merging of gene 5 ' end I.1 with Amb a, the 76bp fragment that does not contain the natural signals peptide and the Amb a that contains tobacco chitinase vacuole targeting sequencing be 3 ' end of gene-as BglII I.1, XbaI fragment-connect into the 4.4kb NcoI of the pAT240 that contains UBQ3 promotor and carrier, in the XbaI fragment.I.1 gene, N end tobacco chitinase signal peptide and C end tobacco chitinase vacuole targeting sequencing box are cloned in a kind of binary vector with terminator sequence with the Arabidopis thaliana UBQ3 promotor ∷ Amb a that removes the natural signals peptide.
Plasmid pSCH10 is as the template of pcr amplification, use sequence and preceding 22 bp of 23 amino acid N end signals of tobacco chitinase peptide and BspHI restriction site homologous " top chain " primer (ErspU) at ATG place, with sequence and the last 17bp homologous of tobacco chitinase N end signal peptide " end chain " primer, this signal peptide and Amb a I.1 5 ' extension of preceding 13bp merge (ErspovL 5 '-AGT GTT TGA TCC CTG CCG AGG CAG AAA GCA-3 ', SEQ IDNO:39).Plasmid pAT240 is as the template of pcr amplification, use I.1 initiator codon preceding 25bp homology afterwards of sequence and Amb a, and can with 13bp annealed " top chain " primer (AmboeU 5 '-GGG ATC AAA CAC TGT TGT TAC ATC T-3 ' of primer ErspovL, SEQ ID NO:40) and at Amb a I.1 135 extend to " end chain " primer of 158 with respect to endogenous ATG from the site in the gene.Two kinds of pcr amplification products of gel-purified, the overlapping extension of the performing PCR of going forward side by side is with two kinds of following merging of product: merge every kind of purifying amplified production of 2ml in a PCR reaction tubes, and under the situation of no primer, carry out 10 circulations, with annealing and extension fusion product, then with the template of 5ml reactant as the pcr amplification that uses " outside " primer ErspU and AmboeL.The amplification end product comprises 23 amino acid signal peptides of tobacco chitinase N end, and a BspHI site is arranged at the initiator codon place, and with Amb a from first amino acid behind the ATG to 155bp downstream gene frame endomixis I.1.This product of gel-purified, with BspHI and BglII digestion, and with as BglII, the Amb a that contains tobacco chitinase vacuole targeting sequencing of XbaI fragment I.1 3 ' end of gene is connected in the 4.4kbNcoI of the pAT240 that contains UBQ3 promotor and carrier, in the XbaI fragment.I.1 gene, N end tobacco chitinase signal peptide and C end tobacco chitinase vacuole targeting sequencing box are cloned in a kind of binary vector with terminator sequence with Arabidopis thaliana UBQ3 promotor ∷ Amb a.Embodiment 46: contain I.1 encoding sequence of the ragweed pollen allergen Amb a that merges with tobacco PR-1a promotor, be used for the preparation of the mosaic gene that vacuole expresses
Scale off and come from PR-1aDXhoNco (people (1993) such as Uknes, vegetable cell 5, PR-1a promotor 159-169) is as 903bp XhoI, the NcoI fragment, with its with contain the I.1 NcoI of gene, N end housing polysaccharidase signal peptide and C end tobacco chitinase vacuole targeting sequencing of Amba, XbaI fragment is connected in the XhoI of pLITMUS28 (New England Biolabs), XbaI site.I.1 gene, N end tobacco chitinase signal peptide and C end tobacco chitinase vacuole targeting sequencing box are cloned in a kind of binary vector with terminator sequence with PR-1a promotor ∷ Amb a.Also I.1 gene, N hold tobacco chitinase signal peptide and C to hold the NcoI of tobacco chitinase vacuole targeting sequencing box with containing the Amb a that removes the natural signals peptide for Xho, NcoI PR-1a promoter fragment, XbaI fragment is connected in the XhoI of pLITMUS28, the XbaI site.I.1 gene, N end tobacco chitinase signal peptide and C hold tobacco chitinase vacuole targeting sequencing box to be cloned in a kind of binary vector with terminator sequence to the PR-1a promotor ∷ Amb a that the natural signals peptide has been removed.After reaching the 20-40cm height, spray inducing compounds BTH, to cause I.1 expression of gene of Amb a to the transgenic plant that contain above-mentioned mosaic gene.Results vegetable material when inducing preceding and induce back 8 hours and 1,2,3,7 and 14 or 28 day, anxious freezing in liquid nitrogen.
Above disclosed embodiment is illustrative.Disclosure of the present invention will make those skilled in the art grasp multiple variation of the present invention.All these obvious and predictable variations all are included in additional claims.

Claims (58)

1. plant that in its plastom, contains a kind of dna molecular, a kind of protein that therapeutic activity is arranged when the host to needs uses with the treatment significant quantity of this dna molecule encode, wherein this plant can be expressed this protein.
2. according to the plant of claim 1, wherein to the described protein of described host's dosage forms for oral administration.
3. according to the plant of claim 1, wherein said protein is a kind of antigen.
4. according to the plant of claim 3, wherein said antigen can suppress or reduce the host to this antigenic immunne response or inflammatory conditions.
5. according to the plant of claim 4, wherein said antigen is a kind of allergen.
6. according to the plant of claim 5, wherein said allergen is a kind of airborne allergen.
7. according to the plant of claim 5, wherein said allergen is a kind of pollen allergen.
8. according to the plant of claim 7, wherein said pollen allergen be selected from artemisiifolia allergen Amb a I, Amb a I.1, Amb t V and Amb a II, alder allergen Aln g I, hazel allergen Cor a I, rye grass allergen Lol p V, Johnson grass allergen Sor h and birch allergen Bet v I.
9. according to the plant of claim 5, wherein said antigen is selected from feline antigens Fel d I, dog allergen Can f II, mosquito allergen rAed a1 and rAed a2, mite allergen Der f I, Der fII, Der p I and Der p II, bee venom allergen peptide PLA-2 and mouse urine protein.
10. according to the plant of claim 3, wherein said antigen is a kind of autoantigen.
11. according to the plant of claim 10, wherein said autoantigen is selected from receptor binding protein between collagen protein, myelin basic protein, myelin proteolipid protein, photosensory cell, acetylcholine receptor, S-antigen, Regular Insulin, L-Glutamic decarboxylase, islet cells specific antigens and thyroglobulin.
12. according to the plant of claim 3, wherein said antigen is a kind of transplantation antigen.
13. according to the plant of claim 12, wherein said transplantation antigen is a MHC albumen.
14. according to the plant of claim 12, wherein said transplantation antigen is a MHC II proteinoid.
15. according to the plant of claim 12, wherein said transplantation antigen is MHC II class a or b chain.
16. according to the plant of claim 3, wherein said antigen derives from a kind of pathogenic agent, wherein this antigen can make the host to this pathogenic agent immunity.
17. according to the plant of claim 1, wherein said protein is a kind of blood protein, hormone, somatomedin, cytokine, enzyme, acceptor, conjugated protein, immune system protein, translation or transcription factor, cancer protein or proto-protein, milk-protein, muscle protein, marrow albumen, neuroactive peptide or tumor growth arrestin.
18. according to the plant of claim 1, wherein said protein is a kind of anti-septicopyemia peptide.
19. according to the plant of claim 18, wherein said anti-septicopyemia albumen is that a kind of bactericidal power/permeability strengthens albumen.
20. according to the plant of claim 1, wherein said immune system protein matter is a kind of antibody.
21. according to the plant of claim 1, wherein said dna molecular effectively is connected with the promotor that can express this dna molecular in the plastid of this plant.
22. according to the plant of claim 21, wherein said promotor is the clpP promotor.
23. according to the plant of claim 21, wherein said promotor is a 16S r-RNA gene promoter.
24. according to the plant of claim 21, wherein said promotor is the promotor of trans-activator mediation.
25. according to the plant of claim 24, the promotor of wherein said trans-activator mediation is T7 gene 10 promotors.
26. according to the plant of claim 24, it further contains a kind of allos nuclear expression box that contains the dna sequence dna of the trans-activator of encoding.
27. according to the plant of claim 25, wherein said trans-activator is the T7 polysaccharase.
28. plant that in its nuclear gene group, contains a kind of dna molecular, this dna molecule encode has the protein of therapeutic activity when the host of needs is used with the treatment significant quantity, wherein this therapeutic activity protein is selected from: mosquito allergen rAed a1 and rAed a2, bactericidal power/permeability strengthen albumen (BPI) and pollen allergen Amb a I, Amb a I.1, Amb t V, Amb a II, Alng I, Cor a I, Lol p V, Sor h and Bet v I.
29. a plant that contains a kind of dna molecular in its nuclear gene group, this dna molecule encode have the protein of therapeutic activity when the host to needs uses with the treatment significant quantity, wherein this therapeutic activity protein is directed in the subcellular organelle of this plant.
30. according to the plant of claim 29, wherein said subcellular organelle is a vacuole.
31. according to each plant of claim 1 or 28-30, wherein this plant is a kind of dicotyledons.
32. according to the plant of claim 31, wherein this plant is tobacco, tomato, soybean or spinach.
33. according to the plant of claim 1 or 28-30, wherein this plant is a kind of monocotyledons.
34. according to the plant of claim 33, wherein this plant is corn or rice.
35. according to claim 31 or 33 each plants, wherein this plant is a kind of edible plants.
36. according to each plant of claim 1 or 28-30, but the expression of wherein said protein in this plant is Chemical Regulation.
37. according to each plant of claim 1 or 28-30, the expression of wherein said protein in this plant is composing type.
38. according to each plant of claim 1 or 28-30, the expression of wherein said protein in this plant is tissue-specific.
39. according to each plant of claim 1 or 28-30, wherein said host is a kind of vertebrates.
40. according to the plant of claim 39, wherein said vertebrates is a kind of Mammals.
41. according to the plant of claim 40, wherein said Mammals is people, ox, sheep, pig, dog or cat.
42. one kind contain with good grounds claim 1 or 28-30 each plant or derive from the composition of the plant material of this plant, wherein said composition contains this protein for the treatment of significant quantity.
43., wherein before the host is used, process described plant according to the composition of claim 42.
44. a method comprises that the host to needs effectively measures the composition of using according to claim 42 to improve host's state of an illness.
45. according to the method for claim 42, wherein to the described composition of host's dosage forms for oral administration.
46. according to the method for claim 45, wherein said protein is a kind of antigen, the host is suppressed this antigenic immunne response or reduces thus.
47. according to the method for claim 46, wherein said antigen is a kind of allergen, autoantigen or transplantation antigen.
48. a treatment or prophylactic method, it comprise to host's administering therapeutic significant quantity of needs according to claim 1 or 28-30 each plant or derive from the plant material of this plant.
49. according to the method for claim 48, wherein said disease is transformation reactions, autoimmune disease or transplant rejection.
50. according to the method for claim 49, wherein to the described treatment significant quantity of host's dosage forms for oral administration.
51. a plastid conversion carrier, it contains a kind of coding has therapeutic activity when the host is used protein DNA molecule, and wherein this dna molecular effectively is connected with guiding this dna molecular expression promoter in plant plastid.
52. plastid that contains the conversion carrier of with good grounds claim 51.
53. a vegetable cell that contains the plastid of claim 52, wherein this vegetable cell can produce described protein.
54. plant that contains the vegetable cell of with good grounds claim 53.
55. a method comprises and uses the plastom that transforms plant according to the plastid conversion carrier of claim 51.
56. according to the method for claim 55, it further is included in the described protein of expression treatment significant quantity in the described plant.
57. a food, it contains each the edible part of plant of dna molecular according to claim 1 or 28-30, and wherein when the host to needs used with the treatment significant quantity, this food had therapeutic activity.
58. agricultural-food, it derives from and comprises each the plant or the plant part of dna molecular according to claim 1 or 28-30, and wherein when the host to needs used with the treatment significant quantity, these agricultural-food had therapeutic activity.
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