JP2002526116A - Therapeutically active proteins in plants - Google Patents

Abstract

  (57) [Summary] The present invention discloses transgenic plants that express a therapeutically active protein, preferably targeted from the plastid genome or to the vacuole. The present invention also describes the administration of the above transgenic plants to a host in need thereof for the prevention or treatment of a disease. In a preferred embodiment, the plant or a component derived from the plant is administered orally to a host.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to transgenic plants that express therapeutically active proteins. In particular, the invention relates to plants that express therapeutically active proteins in intracellular organelles, preferably in vacuoles or more preferably in plant plastids. The present invention also relates to therapeutic uses of the above transgenic plants.

[0002] A wide variety of diseases afflict animals, including humans, pets and livestock. These illnesses not only cause tremendous damage, but also cause significant economic losses. If a worker is ill, they may be unable to work or have reduced productivity, and diseases affecting livestock may dramatically reduce agricultural yields. All human civilizations deal with illnesses with their own medications, and current medicine has struggled with and continues to be successful, but many other illnesses It is still resistant to treatment or can only be partially treated.

[0003] Problems that adversely affect the treatment of disease include the cost of many drugs or their limited supply. That is, in some cases, especially in developing countries, only a small number of patients receive effective treatment, and expensive treatments are burdening global health care systems. In addition, in the agricultural sector, diseases that cause low yields may be inadequately treated because the costs far outweigh the benefits obtained from effective treatments. An important factor in the high cost of some drugs is the lack of economical production methods, especially for drugs based on proteins or peptides. Another problem that is sometimes overlooked but significant in current medicine resides in administering a therapeutically effective amount of a drug to a target host organ or body part. In fact, currently available delivery methods may not always be satisfactory, for example in the treatment of allergies or autoimmune diseases. An additional problem with drug therapy is their transport, especially in hot climates, which is even more expensive due to the need for expensive cooling equipment and expensive formulations. It would be highly advantageous to have a wide variety of drugs available that would avoid expensive transportation or administration costs. Because plants are edible and can have high biomass yield, they are attractive candidates for therapeutically active protein expression.
Expression of therapeutically active proteins in cellular compartments sequestered from cytoplasmic proteases is a particularly attractive way to produce therapeutically active compounds in plants. Thus, on the whole, the need for new drugs that can be administered to patients or hosts through a wide variety of methods, are readily available anywhere in the world, and are available in large quantities and at low cost remains unmet.

[0004] The present invention addresses the need for large and inexpensive supplies of drugs to prevent or treat diseases, especially protein-based drugs. Thus, the present invention
There is provided a transgenic plant capable of expressing the therapeutically active protein, especially a transgenic plant capable of expressing the therapeutically active protein in an intracellular organelle, preferably a vacuole or more preferably a plant plastid. The plants of the present invention are particularly useful for suppressing or reducing unwanted immune responses. The protein expressed in the transgenic plant can be administered to the host, preferably orally, by a wide variety of delivery methods. For example, and optionally and as described below, a plant or plant material containing a therapeutically active protein can be taken orally without prior extraction or purification or with minor extraction or purification. Such prevention or treatment of dietary illness is advantageous and significantly reduces the cost of the drug. Alternatively, proteins expressed in transgenic plants can be extracted and administered using methods well known in the medical arts.

[0005] In particular, the present invention relates to transgenic plants that express therapeutically active proteins from the plastid genome. The expression level in the plastid according to the present invention,
Constantly exceeds the level of nuclear expressed transgene. Such high levels of expression result in high protein concentrations per gram of plant tissue, which has been a long-awaited, but unprecedented, opportunity for the use of plants or plant materials derived from such plants in a wide range of therapeutic applications. The desire for achievement is achieved. In addition, transgene expression levels are stable for long periods of time due to the absence of gene silencing, and position effects may occur due to the insertion of the transgene at the precise targeted location in the plastid genome by homologous recombination. Change does not matter. Also,
Because proteins expressed in plant plastids remain sequestered in organelles, they are advantageously protected from degradation, especially in the digestive system when plants are ingested orally. Also, sequestration in organelles prevents the interaction of the plastid-expressed transgene with the cytoplasmic environment. This feature is also essential if the protein expressed in the plant exhibits activity against certain components of the plant. Also, an inducible plastid expression system is available (WO 98/1123).
5), which limits transgene expression to the desired time point, thus limiting the potentially deleterious effects of high transgene expression levels over time. Similarly, proteins expressed from nuclear genomes targeted to other intracellular compartments, such as the vacuole, are also protected from degradation or from deleterious interactions with the cytoplasmic environment. The present invention also provides a therapeutic composition comprising a plant substance derived from the transgenic plant, a novel method for expressing a therapeutically active protein, and a human,
A novel method for improving the condition of a wide range of hosts, including livestock, pets and other animals, is provided. Also provided is a method for administering a plant-derived therapeutic agent.

[0006] Accordingly, the present invention provides a plastid comprising at least one DNA molecule encoding at least one protein that exhibits therapeutic activity when administered to a host, preferably a host in need of treatment, in a therapeutically effective amount. Provided is a plant that is contained in the genome and that can express the protein (s). In a preferred embodiment, the therapeutically active protein is accumulated in the plastids of the plant. In another preferred embodiment, the protein is expressed in an edible part of the plant. In another preferred embodiment, the protein is administered orally to the host. In another preferred embodiment, the host is a vertebrate, preferably a mammal, more preferably a human, cow, sheep, pig, dog or cat. In a further preferred embodiment, the therapeutically active protein is an antigen, preferably an immunoactive antigen. Thus, the present invention relates to a DN encoding an antigen, preferably an immunoreactive antigen.
Provided is a plant comprising an A molecule in its plastid genome, wherein the plant can express an antigen. Preferably, the antigen is expressed in the plant, and more preferably, the immune response to the antigen is reduced after ingestion by the host of the plant. In another preferred embodiment,
The antigen may suppress or reduce the immune response of the animal to the antigen, preferably by inducing host tolerance to the antigen, for example, by contact or ingestion by the intestinal mucosa. Preferred antigens are allergens, preferably airborne allergens, preferably pollen allergens. Examples of preferred allergens are Der f I
, Der f II, Der p I and Der p II, Can f II, Lol p
V, Sorh I, Amb a I, preferably Amb a I.1, Amb a II,
Alng I, Cor a I, Bet VI, Feld I, or rAed a
1 and rAed a 2. For example, the allergen expressed in the plants of the present invention is not glycosylated. Another preferred antigen is an autoantigen, such as collagen, preferably type I or type III collagen, type II collagen, myelin basic protein, myelin proteolipid protein, interphotoreceptor binding protein, acetylcholine receptor, S-antigen, insulin , Glutamate dehydrogenase, islet cell-specific antigen or thyroglobulin, or transplantation antigens, such as allo- or xenotransplantation antigens, such as MHC proteins, preferably MHC
Class II proteins, preferably a or b chains. In another preferred aspect, the immunoreactive antigen is capable of inducing immunization of an animal against the antigen. In yet another preferred embodiment, the therapeutically active protein is a blood protein, hormone, growth factor, cytokine, enzyme, receptor, binding protein, immune system protein, translation or transcription factor, oncoprotein or protooncoprotein (proto-oncoprotein). -oncoprote
in), milk protein, muscle protein, myeloprotein, neurostimulatory peptide or tumor growth inhibitory protein or peptide, such as angiostatin or endostatin, both of which inhibit angiogenesis. In another preferred embodiment of the invention, the therapeutically active protein is a preservative peptide, for example a BPI (bactericidal permeability enhancing protein). In yet another embodiment, the immune system protein is an antibody. A DNA molecule according to the present invention is operably linked to a promoter capable of expressing the DNA molecule in plant plastids. In a preferred embodiment,
The promoter is a clpP promoter, and in another preferred embodiment 16S r
-An RNA gene promoter. In a particularly preferred embodiment of the invention, said promoter is a transactivator transduction promoter, in particular the T7 gene 10 promoter. Further, the present invention provides a transactivator-encoding D
There is provided a plant further comprising a heterologous nuclear expression cassette comprising an NA sequence. In a preferred embodiment, the transactivator is T7 polymerase. The invention further provides a plant comprising in its nuclear genome a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host in need of treatment in a therapeutically effective amount, wherein the therapeutically active protein is a mosquito. Allergens rAed a1 and rAed a2, bactericidal permeability enhancing protein (BPI) and pollen allergens Amb a I, Amb a I.
1, Amb a II, Amb t V, Alng I, Cor a I, Lol p V,
Sor h and Bet VI.

The present invention also provides a plant comprising in its nuclear genome a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host in need of treatment in a therapeutically effective amount, wherein the therapeutically active protein Is targeted to the intracellular organelles of the plant. In a preferred embodiment, the therapeutically active protein is targeted to the vacuole. The therapeutically active protein targeted to the vacuole is preferably an allergen, such as Der f I, Der f II, Der p
I and Der p II, Can f II, Lol p V, Sor h I, Amb
aI, preferably Amb aI.1, Amb a II, Amb t V, Alng I
, Cor a I, Bet VI, Feld I or rAed a 1 and rAe
da2, autoantigens such as collagen, preferably type I or type III collagen, type II collagen, myelin basic protein, myelin proteolipid protein, interphotoreceptor binding protein, acetylcholine receptor, S
An antigen, insulin, glutamate dehydrogenase, islet cell-specific antigen or thyroglobulin, or a transplantation antigen, such as an allo- or xenotransplantation antigen, such as an MHC protein, preferably an MHCII class protein, preferably a or b chain, or blood. Proteins, hormones, growth factors, cytokines, enzymes, receptors, binding proteins, immune system proteins, translation or transcription factors, oncoproteins or proto-oncoproteins, milk proteins, muscle proteins, bone marrow proteins ( myeloprotein), a neurostimulatory peptide or a tumor growth inhibitory protein or peptide, eg, selected from the group consisting of angiostatin or endostatin, both of which inhibit angiogenesis. In another preferred embodiment, the therapeutically active protein targeted to the vacuole is an antiseptic peptide, for example (a bactericidal permeability enhancing protein).

In a further preferred embodiment, the plant according to the invention is an edible plant. In another preferred embodiment, the plant is a dicot, preferably tobacco, tomato, soy or spinach. In another aspect, the plant is a monocotyledonous plant, preferably corn or rice.

In a further preferred embodiment, the expression of the protein in the plant is regulatable, preferably chemically regulatable. Alternatively, the expression of the protein is constitutive, tissue specific or developmentally regulated.

[0010] This also includes seeds of the above plants, wherein the seeds are optionally treated (eg,
Priming or coating) and / or packaged, eg, in a bag or other container, along with instructions for use. A host according to the invention is a vertebrate, especially a mammal, more particularly a human, cow, sheep, pig, dog or cat.

The invention further relates to a composition comprising a plant according to the invention or a plant material derived from said plant, wherein said composition comprises a therapeutically effective amount of a protein and said plant is processed prior to administration to a host. Provided compositions.

Further, the present invention provides that a composition of the present invention is administered to a host in need thereof in an amount effective to improve the condition of the host, wherein the composition is administered orally to the host, and the protein is an antigen. In some cases, the immune response of the host to the antigen is suppressed or reduced,
There is provided a method wherein the antigen is in particular an allergen, an autoantigen or a transplantation antigen.

[0013] The present invention also provides a method for treating or preventing a disease comprising administering to a host in need of a therapeutically effective amount of a plant of the present invention or a plant material derived from the plant. In one embodiment, the disease is an allergy, an autoimmune disease or a transplant rejection. In yet another embodiment, a therapeutically effective amount is administered orally to a host.

Furthermore, the present invention provides a method for treating a disease, eg, an allergy, in a patient in need of treatment or prevention, eg, by inducing tolerance, eg, oral tolerance.
Provided is a plant of the present invention for use as a medicament for treating or preventing an autoimmune disease or transplant, or for immunizing a host.

The invention further provides for the treatment of a patient in need of treatment or prophylaxis, eg, by induction of tolerance, eg, oral tolerance, preferably by illness, eg, allergy,
Provided is a plant of the present invention used as a pharmaceutical food for treating or preventing an autoimmune disease or transplantation, or for immunizing a host.

[0016] The invention further provides a plastid transformation vector comprising a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host in need thereof in a therapeutically effective amount, wherein the DNA molecule is in a plant plastid. A vector operably linked to a promoter capable of directing the expression of a DNA molecule is provided. In a preferred embodiment, the protein is accumulated in the plastids of the plant. In another preferred embodiment, the promoter in the plastid transformation vector is a clpP promoter, 16S r-R
An NA gene promoter, a psbA promoter, an rbcL promoter, or a transactivator transduction promoter regulated by a nuclear transactivator (eg, a T7 gene 10 promoter when the transactivator is T7).

The present invention provides a plastid containing the above-mentioned transformation vector. The present invention further provides a plant cell containing the plastid, which is capable of producing a protein.

Furthermore, the invention relates to a transactivator (preferably a transactivator not naturally occurring in plants, preferably an RNA polymerase or a DNA binding protein such as T7 RNA polymerase), but optionally a plastid targeting sequence such as a leaf A promoter, eg, an inducible promoter, operably linked to a DNA sequence encoding a transactivator that may be fused to a chloroplast targeting sequence (eg, a plant-expressable expression cassette);
For example, a wound- or chemo-inducible promoter, such as the tobacco PR-1a promoter, or a heterologous nuclear expression cassette containing a tissue or organ-specific or developmentally regulated promoter, and a transactivator (eg, the transactivator is T7 RNA). DNA that is regulated by the T7 gene 10 promoter when it is a polymerase and encodes a therapeutically active protein of the invention
Provided is a plant comprising a heterologous plastid expression cassette comprising a transactivator transduction promoter operably linked to the molecule, and the seed of such a plant, optionally treated (eg, primed or coated) Also included are seeds that may be packaged, for example, in a bag or other container with instructions for use.

The invention further includes a composition comprising the above plant or plant material, wherein the composition comprises a therapeutically effective amount of the protein when administered to a host in need thereof. In a preferred embodiment, the plant material is processed before being administered to the host. The processing method is preferably a processing method of the type commonly used in the food or feed industry. In another preferred embodiment, the composition of the invention comprises an immunologically effective amount of the antigen.

The present invention further provides a pharmaceutical composition comprising the plant or plant substance of the present invention and a pharmaceutical food composition comprising the plant or plant substance of the present invention.

The present invention further provides a method comprising transforming a plastid genome of a plant with the above-mentioned transformation vector, and preferably further comprising expressing a therapeutically active protein in the above-mentioned plant. In a preferred embodiment, the therapeutically active protein is an antigen, preferably an immunoreactive antigen.

Further, the present invention also provides a method comprising administering to a host in need thereof the above composition in an amount effective to improve the condition of the host. Preferably, the method comprises oral administration of the composition to a host.

Further, the present invention is a method of treating or preventing a disease, such as an allergy, an autoimmune disease or transplant rejection, or immunizing a host, eg, by inducing tolerance in the host in need of treatment or prevention, comprising the steps of: And a therapeutically effective amount of the plant of the present invention or a plant substance derived therefrom.

Further, the invention is directed to the treatment or prevention of disease, eg, by treating tolerance or induction of tolerance in a host in need thereof, eg, for the treatment of allergy, autoimmune disease or transplantation, or immunization of a host. It is intended to provide a use of the plant of the present invention in production of a desired medicine.

Further, the invention is directed to the treatment or prevention of a disease, for example, the treatment of an allergy, an autoimmune disease or a transplant, or the immunization of a host, for example by inducing tolerance in the host in need of the treatment or prevention. It is intended to provide a use of the plant of the present invention in production of a target pharmaceutical food.

The present invention further provides a use of the plant of the present invention for producing an antigen for measuring an immunological activity.

Further, the present invention provides an antibody specific to an antigen expressed in the plant of the present invention. Also provided is an antibody that prevents binding of an antigen expressed in the plant of the present invention and an antibody specific to the expressed antigen.

The present invention further provides a food product comprising an edible part of a plant comprising a DNA molecule according to the present invention, the food product having a therapeutic activity when administered to a host in need thereof in a therapeutically effective amount. I do.

The present invention further provides an agricultural product derived from a plant or plant part comprising a DNA molecule of the present invention, wherein the agricultural product has a therapeutic activity when administered to a host in need thereof in a therapeutically effective amount.

Furthermore, the present invention also provides all the novel products, processes and uses described herein.

Definitions In a broad sense, a "therapeutically active protein" contributes positively to a host's condition when administered to the host in a therapeutically effective amount. Therapeutically active proteins have curative, therapeutic or palliative properties against the disease and may be administered to ameliorate, alleviate, reduce, reverse or reduce the severity of the disease. "Therapeutically active proteins" also have prophylactic properties,
It is used to prevent the onset of the disease or to reduce the severity of the disease or condition when it appears. The term "therapeutically active protein" encompasses the entire protein or peptide or a therapeutically active fragment thereof. It also includes a therapeutically active analog of a protein or peptide, or an analog of a protein or peptide fragment. The term "therapeutically active protein" also encompasses a plurality of proteins or peptides that act therapeutically or synergistically to provide a therapeutic benefit.

“Analogs” of a therapeutically active protein include proteins that are so structurally related to a protein that they have the same biological activity as the protein.

“Immune response” means a physiological response that results from the activation of the immune system by an antigen. In the present invention, the immune response can be suppressed through induction of tolerance based on exposure to the antigen, especially when the antigen is administered orally.

As used herein, “immunologically active” means that the immune system can be modulated, eg, by stimulating an immune response or suppressing or reducing an immune response or inflammatory condition.

An “antigen” is a substance that stimulates an “immune response” by interacting with the immune system, preferably with products having specific humoral or cellular immunity. The antigen is
Preferably it is a polypeptide and a "therapeutically active protein". An antigen includes the entire polypeptide or a portion of the polypeptide. Said part of the polypeptide is, for example, an epitope of an antigen or an antigenic determinant. An antigen may include one or more epitopes or antigenic determinants. Antigens also include "analogs" of antigens that contain molecules that are so closely related to the antigen that they have the same biological activity, ie, the same immunological activity, as the antigen. In the context of the present invention, antigens include, for example, allergens, autoantigens and transplantation antigens.

An “epitope” is that portion of an antigen that determines its ability to bind to the specific binding site of the corresponding antibody in an antigen-antibody interaction.

An “antigenic determinant” is that part of an antigen that determines the specificity of the immune response stimulated by the antigen.

An “adjuvant” is mixed with an antigen, and when administered, in particular, mixed with the antigen;
It is a substance that, when injected, nonspecifically enhances the immune response to an antigen. Representative adjuvants are complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (Lando et al. (1981) JPI Immunol. 126: 1526).

“Autoantigens” are usually found within the body of an animal, but are unusual conditions, such as in the case of an autoimmune disease, because they are no longer recognized by the animal's immune system as part of the animal itself. Includes all substances attacked by the immune system as if they were.

“Allergen” is an antigen that induces an allergic reaction in a host.

“Immunization” of a host against a pathogen or toxin is one aspect of stimulating an immune response, and here refers to protecting the host from the pathogen or toxin by inducing the immune system. Preferably, both an immediate immune response and an immune memory are elicited, preferably providing immediate and long term protection of the host. The antigen of the host of the toxin is used for immunization. Vaccination is also included in the term immunization.

“Administration” of a therapeutically active protein to a host in need thereof is accomplished in a manner that retains the therapeutic efficacy of the protein for a period of time sufficient to provide the desired beneficial effect in the host. The host should be provided with a therapeutically active protein.

“Oral administration” of a therapeutically active protein means administration primarily through the mouth, preferably by eating, but is intended to include all dosage forms that provide the protein to the host stomach or gastrointestinal tract. . In a preferred embodiment, oral administration results in contact between the therapeutically active protein and the intestinal mucosa.

A “host” is an animal to which a therapeutically active protein of the invention is administered. "animal"
The term encompasses all living forms with an immune system, including humans, cows, sheep, pigs, dogs or cats.

“Food” or “food product” means here a liquid or solid food or foodstuff or feed, and is a plant, plant part or plant derived therefrom that is consumed by humans and other animals. Substance. The term encompasses all raw plants or plant materials that can be fed directly to humans and other animals or plant substances processed together with nutritional carriers that are fed to humans and other animals. And Plant derived material shall include all plant components that are consequently consumed by humans or other animals.

“Pharmaceutical food” includes compositions that are eaten and consumed by the host and have a therapeutic effect on the host. The pharmaceutical food contains, for example, the plant of the present invention or a plant substance derived therefrom. Pharmaceutical foods can be taken alone or in combination with pharmaceutical compositions well known in the pharmaceutical arts. Pharmaceutical foods also include equal-content feeds for non-human animals.

As used herein, “produce” includes edible parts of plants that are eaten in whole, such as alfalfa shoots, radish shoots, wheat shoots, etc. or plants that are consumed by humans in raw or cooked form. . Edible portions include roots, such as rutabaga, beet, carrots and sweet potatoes, tubers or storage stems, such as potatoes, jerusalem artichokes and taro, stems such as asparagus and kohlrabi, buds such as brussels sprouts, corms such as onions and garlic, stalks. Or petiole, such as celery and rhubarb, leaves, such as cabbage, lettuce, parsley and spinach,
Flower buds can be, for example, cauliflower, broccoli and artichokes, seeds, immature fruits such as eggplant, cucumber and sweet corn (corn), or mature fruits such as tomatoes, peppers, apples, pears, bananas, oranges, berries and the like.

“Plant” includes any plant, especially a seed plant.

“Plant cell” encompasses the structural and physiological units of a plant, including protoplasts and cell walls. Plant cells can be isolated single cell or cultured cell morphology, or a high tissue unit, such as a part of a plant tissue or plant organ.

“Plant material” includes leaves, stems, roots, seeds, flowers or flower parts, fruits, pollen, pollen tubes,
The ovule, embryo sac, egg cell, zygote, embryo, seed, cutting, cell or tissue culture, or other parts or products of the plant, if any, are also included.

“Vegetable material” includes any part of a plant at any stage of development, preferably that part that can be administered orally. Plant substances include edible parts of plants,
For example, leaves, seeds, fruits, tubes, or other plant parts that can be consumed in a raw or raw state. Plant substances also include isolated fractions of plants, such as intracellular organelles, such as plastids or vacuoles. Vegetable substances also include parts of the plant that have been subjected to various types of processing steps, especially processing steps commonly used in the food or feed industry. The above steps include, for example, the formation or formation of pellets by concentration or condensation of plant solids, the production of pastes, the drying or freeze-drying, or the fractionation of plants to varying degrees by cutting or grinding, or the extraction of liquid parts of plants by soup. , Syrup or juice production, but is not limited to these. The processing step can also include cooking of the plant or plant material.

“Expression” refers to transcription and / or transcription of an endogenous gene or transgene in a plant.
Or mean translation. For example, in the case of an antisense construct, expression can mean transcription of only the antisense DNA.

As used herein, an “expression cassette” refers to a 3 ′ sequence, eg, a 3 ′ regulatory sequence or a nucleotide sequence of interest that may be operably linked to a termination signal. A DNA sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, including an operably linked promoter. It also typically contains sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest,
It may also encode a functional RNA of interest, such as an antisense RNA or an untranslated RNA that inhibits the expression of a particular gene in the sense or antisense orientation, such as an antisense RNA. Since the expression cassette containing the nucleotide sequence of interest can be chimeric, the nucleotide sequence is a recombinant DNA
The technique consists of a plurality of DNA sequences of different origin fused together by the technique, resulting in a nucleotide sequence which is not found in nature and especially not found in transformed plants. The expression cassette may also be naturally occurring, but obtained in a recombinant form useful for heterologous expression.

However, in general, the expression cassette is heterologous with respect to the host, ie, the particular DNA sequence of the expression cassette is not naturally present in the host cell, and is introduced into the host cell or host cell ancestor by a transformation event. Must have been done.
Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter may also be specific for a particular tissue or organ or stage of development. A nuclear expression cassette is usually inserted into the nuclear genome of a plant and can direct the expression of a particular nucleotide sequence from the nuclear genome of the plant. The plastid expression cassette is usually inserted into the plastid genome of a plant and can direct the expression of a particular nucleotide sequence from the plastid genome of the plant. In the case of the plastid expression cassette, expression of the nucleotide sequence from the plastid genome requires an additional element, a ribosome binding site, or a 3 'stem-loop structure that prevents plastid RNA polyadenylation and subsequent degradation. obtain.

“Gene” refers to a coding sequence and associated regulatory sequences, wherein the coding sequence is R
It is transcribed into NA, eg, mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences include promoter sequences, 5 '
And 3 'untranslated and termination sequences. Yet another element that may be present is, for example, an intron.

As used herein, “heterologous” means of different natural or synthetic origin. For example, if a host cell is transformed with a nucleic acid sequence not found in the untransformed host cell, the nucleic acid sequence is said to be heterologous with respect to the host cell. The transforming nucleic acid can include a heterologous promoter, a heterologous coding sequence or a heterologous termination sequence. Alternatively, the transforming nucleic acid can be completely heterologous or can comprise any possible combination of heterologous and endogenous nucleic acid sequences. Similarly, heterologous is used for nucleotide sequences that are derived and inserted from the same natural progenitor cell type, but that are in a non-native state, eg, at a different copy number or under the control of different regulatory elements.

A “marker gene” is a gene that encodes a selectable or screenable property.

A regulatory DNA sequence is a DNA encoding RNA or protein when two sequences are positioned such that the regulatory DNA sequence affects the expression of the coding DNA sequence.
It is said to be "operably linked" or "associated with" the NA sequence.

“Regulatory elements” include sequences involved in conferring expression of a nucleotide sequence. Regulatory elements include a promoter operably linked to the nucleotide sequence of interest and optionally a 3 'sequence, such as a 3' regulatory sequence or termination signal. They also typically include sequences required for proper translation of the nucleotide sequence.

“Intracellular organelle” encompasses intracellular organelles that have a characteristic structure and function. An intracellular organelle is, for example, a vacuole, plastid, mitochondria, cell nucleus, endoplasmic reticulum or plasma membrane.

“Homoplasmic” is used on plants, plant tissues or plant cells, in which all plastids are genetically identical. This is the normal state in a plant if the plastids have not been transformed, mutated or otherwise genetically modified. At different tissues or stages of development, plastids can take different forms, such as chloroplasts, proplastids (primoplastids), ethioplasts, amyloplasts, chromosomes, and the like.

“Promoter” means a DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression, such as activators, enhancers and / or repressors.

An “inducible promoter” is a promoter that, unlike a constitutive promoter or a promoter specific to a specific tissue or organ or developmental stage, initiates transcription only when a plant is exposed to some particular external stimulus. is there. Particularly preferred for the present invention are chemically inducible promoters and wound inducible promoters. Chemically inducible promoters include plant-derived promoters, such as promoters in the systemic acquired resistance pathway, such as the PR promoter, such as PR
There are -1, PR-2, PR-3, PR-4 and PR-5 promoters, especially the tobacco PR-1a promoter and the Arabidopsis PR-1 promoter, which plants are exposed to BTH and related chemicals. Transfer is started when See U.S. Pat. No. 5,614,395, which is hereby incorporated by reference, and WO 98/03536, which is hereby incorporated by reference. Chemically inducible promoters can also be used in receptor-mediated systems, such as those from other organisms, such as steroid-dependent gene expression, such as the glucocorticoid, progesterone and estrogen receptor systems, copper-dependent gene expression, such as ACE1
, Tetracycline-dependent gene expression, Lac repressor system and Drosopyla mediated by juvenile growth hormone and its agonists (Drosop
hila) using the USP receptor (described in WO 97/13864, which is hereby incorporated by reference), and, for example, described in WO 96/27673 (which is incorporated herein by reference) ) And systems using combinations of receptors. Additional chemically inducible promoters include elicitor inducible promoters, safener inducible promoters and the alcA / alcR gene activation system which can be induced by certain alcohols and ketones (WO 93/21334).
(1998) Nat Biotechnol 16: 177-180, the contents of which are hereby incorporated by reference. Wound-inducible promoters include promoters for proteinase inhibitors, such as the proteinase inhibitor II promoter from potato, and other plant-derived promoters involved in the wound response pathway, such as the promoters for polyphenyloxidase, LAP and TD. In general, see C. Gatz, `` Chemical Control of Gene Expressio
n ", Annu. Rev. Plant Physiol. Plant Mol. Biol. (1997) 48: 89-1.
08 (incorporated herein by reference).

A “transactivator” is a protein that, alone or in combination with one or more additional proteins, can trigger transcription of a coding region under the control of a corresponding transactivator-mediated promoter. An example of a transactivator system is the bacteriophage T7 gene 10 promoter, the transcriptional activation of which is dependent on a specific RNA polymerase, such as phage T7 RNA polymerase. Transactivators typically interact with a particular promoter to activate transcription by directly activating the promoter or inactivating a repressor gene, for example, by suppressing expression or accumulation of a repressor protein. RNA polymerase or DNA binding protein that can be initiated. The DNA binding protein can be a chimeric protein that includes a binding region (eg, a GAL4 binding region) linked to a suitable transcriptional activator domain.
Some transactivator systems may have multiple transactivators, such as promoters that require not only a polymerase but also a specific subunit (sigma factor) for promoter recognition, DNA binding or transcriptional activation. . The transactivator is preferably heterologous with respect to the intracellular organelles or components of the plant or plant cell from which the induction is performed.

“Minimal promoters” include promoter elements, especially TATA elements, which are inactive or have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable upstream activation sequence fused to the minimal promoter and the corresponding transcription factor, transcription can be effected by the function of the minimal promoter.

“Recombinant DNA technology” includes, for example, the method used to ligate DNA sequences together as described by Sambrook et al. (1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). I do.

A “screenable marker gene” refers to a gene whose expression does not confer a selective advantage on transformed cells, but whose expression phenotypes the transformed cells from non-transformed cells. Include.

“Selectable marker gene” means a gene whose expression in a plant cell confers a selective advantage on the cell. The selective advantage of cells transformed with a selectable marker gene may be due to the ability to grow in the presence of a negative selection agent, such as an antibiotic or herbicide, as compared to the growth of non-transformed cells. The selective advantage that transformed cells have over non-transformed cells is also due to their enhanced or novel ability to utilize compounds added as nutrients, growth factors or energy sources. obtain. Selectable marker genes also include genes or combinations of genes whose expression in a plant cell confers on the cell both negative and positive selective advantages.

“Synthetic” is used for nucleotide sequences that contain structural features not present in the native sequence. For example, G + C of dicot and / or monocot genes
Artificial sequences that closely resemble content and normal codon distribution are said to be synthetic.

“Transformation” means introducing a nucleic acid into a cell. In particular, it refers to the stable integration of DNA molecules into the genome of the organism of interest.

(Brief description of sequence in sequence list) 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 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 Oligo Nucleotides 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 E spovL SEQ ID NO: 40 oligonucleotide AmboeU

The present invention discloses transgenic plants that express a therapeutically active protein, especially an antigen. The DNA molecule encoding the protein is expressed from the plastid genome of the plant or expressed in the cell nucleus, and the protein is targeted to the cytosol or intracellular organelle, eg, vacuole, of the cell. The plants of the present invention can express proteins in a cost-effective manner and can be easily obtained. The plant can express the protein in large amounts in a cost-effective manner. further,
The therapeutically active protein expressed in plastids is conveniently packaged, which particularly simplifies purification and processing. Such compartmentalization is also advantageous for oral administration as the therapeutic molecule can be protected during digestion. The therapeutically active proteins expressed in the transgenic plants according to the present invention can be administered to a host, including humans, pets or livestock, by a wide range of methods to prevent or treat a variety of diseases, thereby altering the condition of the treated host. Can be improved. In particular, the transgenic plants of the invention or plant material derived from said plants can be taken orally by the host, preferably by inducing host tolerance to antigens, for example for the treatment of allergies, autoimmune diseases or the prevention of transplant rejection. Can be used. The present invention also relates to a composition comprising the plant or a plant substance derived from the plant, for example, a pharmaceutical composition,
Also disclosed is a method of improving the condition of a host by administering the composition of the present invention.

Therapeutically Active Proteins Expressed in Transgenic Plants The proteins of the present invention can preferably modulate the immune response of the host, examples are provided below. Thus, they can be used for treating or preventing an unwanted immune response, for example by inducing tolerance, for example for suppressing or reducing the immune response of the host. Alternatively, they may also contribute to or induce immunization of the host against a disease, such as a bacterial, parasite or viral disease, by stimulating the host's immune system. In a preferred embodiment, a single protein is expressed in a transgenic plant. In this case, if some proteins are desired for a particular treatment,
A mixture of plants, each expressing a different protein, is used. In another preferred embodiment, several different proteins are expressed in the same transgenic plant.
In this case, the DNA molecules encoding the different proteins are contained in different expression cassettes and are transformed into plants or, alternatively, the DNA molecules are contained in the same expression cassette. For example, when expressed from the plastid genome, DNA molecules encoding different therapeutically active proteins can be engineered into expression cassettes to form a single polycistronic messenger RNA. For purposes of simplicity and clarity, "a" (one) therapeutically active protein as described throughout this specification means "at least one" therapeutically active protein and includes one or more proteins Shall be. Also, a therapeutically effective amount of a therapeutically active protein of the present invention can be obtained from a single plant or a plant material derived from a single plant, or can be obtained from a plurality of plants, such as sister cells of a plant.

The plants transformed according to the present invention are monocotyledonous or dicotyledonous, and may be corn, wheat, barley, rye, sweet potato, beans, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish , Spinach, asparagus, onion, garlic, pepper, celery, pumpkin, peppermint, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, Pineapple, avocado, papaya, mango, banana, soy, tomato,
Sorghum, sugar cane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, turfgrass, ornamental and woody plants, such as conifers and deciduous trees, may be included. However, it is not limited to these. Once the gene of interest has been transformed into a particular plant species, it can be propagated in that species or transferred to other varieties of the same species, including commercial varieties, using traditional breeding techniques. In addition, the present invention,
Edible algae such as unicellular green algae (eg, Chlamydomonas), multicellular green algae (eg, Ulva), unicellular red algae (eg, Porphyridium)
) And multicellular red algae (eg, Porphyra), which contain a plastid genome substantially similar to that of higher plants that can be transformed in a similar manner.

(Expression of Therapeutically Active Protein in Plant Plastids) The present invention particularly relates to the expression of therapeutically active proteins from the plastid genome of plants. In this case, some or all of the thousands of copies of the cyclic plastid genome present in each plant cell will be transformed by a DNA molecule encoding a therapeutically active protein of the invention. The huge plastid-specific transgene copy number allows for expression levels that typically exceed expression levels typically obtained from nuclear expressed genes. This high level of expression further reduces the cost of producing the therapeutically active protein in the plant, and as in the case of treating allergies, autoimmune diseases or preventing transplant rejection, for example, by ingesting the plant or plant material orally. The transgenic plants can be used in applications requiring high levels of protein in the plant material. Plastid gene expression also has some additional advantages. For example, due to the absence of gene silencing and position effect variations, transgene expression levels are stable over time, and the polycistronic operon can be expressed synchronously from a single promoter or regulatory sequence, resulting in some Can be produced in equimolar amounts and the genetic traits of the parthenogenetic plastid gene prevent pollen transmission of foreign DNA in very economically important crops, thus migrating laterally to wild or cultivated plants. The possibility of doing so is reduced. In addition, since plastid transgene integration is performed by a homologous recombination method, it means that accurate targeting genetic engineering technology and gene replacement can be easily performed.
In addition, proteins expressed in plastids remain sequestered in organelles, preventing their interaction with the cytoplasmic environment. This feature is also essential if the protein expressed in the plant exhibits activity or interacts detrimentally with certain components of the plant cytosol.

The plastid transformation technique is described in US Pat. No. 5,451,513,
545818 and 5576198, PCT Application Nos. WO95 / 16783 and WO97 / 32977, and McBride et al., Proc. Natl. Acad. Sci. USA 91.
: 7301-7305 (1994), which is hereby incorporated by reference. Plastid transformation by biolistics is described in Chlamydomonas reinhardtii, a unicellular green alga (Boynton et al. (1988) S
cience, 240: 1534-1537, incorporated herein by reference.)
This method, which uses the selectivity for the cis-acting antibiotic resistance locus (spectinomycin / streptomycin resistance) or the complementation of the non-photosynthetic mutant phenotype, was immediately used in Nicotiana tabacum ( Nicotiana tabacum
(Svab et al. (1990) Proc. Natl. Acad. Sci. USA. 87: 8526).
-8530, incorporated herein by reference).

The basic technique for tobacco plastid transformation involves PEG-mediated incorporation of plasmid DNA in protoplasts with a particle bombardment of leaf or callus tissue or a cloned plastid DNA region flanked by selectable antibiotic resistance markers. . The 0.5-1.5 kb flanking region, called the targeting sequence, is replaced or modified with a specific region of the 156 kb tobacco plastid genome to facilitate homologous recombination with the plastid genome. First, chloroplast 16S rDNA conferring resistance to spectinomycin and / or streptomycin
And point mutations in the rps12 gene were used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Proc.
Natl. Acad. Sci. USA 87, 8526-8530, Staub, JM and Maliga, P. (
1992) Plant Cell 4, 39-45, incorporated herein by reference.
. As a result, stable homoplasmic transformants were obtained at a frequency of about once per 100 bombardies of the target leaf. The presence of a cloning site between these markers created plastid targeting vectors for foreign gene transfer (Staub, JM. And Maliga, P., EMBO J. 12: 601-606 (1993).
) Incorporated herein by reference). Substituting the recessive rRNA or ribosomal protein antibiotic resistance gene with the bacterial aadA gene encoding the dominant selectable marker, spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase, resulted in a substantial increase in transformation frequency. (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913).
−917, incorporated herein by reference.) Previously, by using this marker, the green alga Chlamydomonas reinharti
dtii) has achieved high frequency transformation of the plastid genome (Goldschmidt-C
lermont, M. (1991) Nucl. Acids Res. 19, 4083-4089, which is hereby incorporated by reference. Recently, plastid transformation of protoplasts from tobacco and moss Physcomitrella patens has been achieved using polyethylene glycol (PEG) -mediated DNA incorporation (O ').
Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996) Planta.
199: 193-201, both of which are hereby incorporated by reference.)
Particle bombardment and protoplast transformation are both suitable in the present case. A DNA molecule encoding a therapeutically active protein of the invention is inserted into a plastid expression cassette that includes a promoter capable of expressing the DNA molecule in plant plastids. Preferred promoters that allow expression in plant plastids are those that are isolated from the 5 ′ flanking region upstream of the coding region of the plastid gene, which can be from the same or a different species, and that the natural product is generally It is found in most plastid types, including those present in non-green tissues. Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (see Stern et al. (1997) Trends in Plant Sciences 2: 308-315, herein incorporated by reference). Part of the book). Plastid promoters generally contain -35 and -10 elements typical of prokaryotic promoters, and some of the plastid promoters are primarily E. coli encoded by the plastid genome. Are recognized by RNA polymerases, which are so-called PEP (plastid-encoded RNA polymerase) promoters, while other plastid promoters are recognized by nuclear-encoded RNA polymerase (NE
P promoter). Both types of plastid promoters are suitable for the present invention.
Examples of plastid promoters include the promoter of the clpP gene, eg, tobacco cl
pP gene promoter (WO97 / 06250, incorporated herein by reference) and Arabidopsis clpP gene promoter (
Positions 71882 and 723 in the Arabidopsis plastid genome
Contained between position 71, the sequence is located at the URL at Kazusa DNA Research Institute, Takakaz Kaneko and Satoshi Tabata, ftp: //genome-ftp.stanf
ord.edu/pub/arabidopsis/chloroplast/). Another promoter capable of expressing DNA molecules in plant plastids is derived from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., Microbiol. Rev. 58: 700).
-754 (1994); Shinozaki et al., EMBO J. 5: 2043-2049 (198
6), both of which are hereby incorporated by reference). Other examples of promoters capable of expressing DNA molecules in plant plastids include the psbA promoter or rbc
L promoter. The plastid expression cassette also preferably includes a plastid gene 3 'untranslated sequence (3'UT) operably linked to a DNA molecule of the present invention.
R). The role of the untranslated sequence is preferably not transcription termination, but rather 3 of the transcribed RNA.
'To direct processing. Preferably, the 3′UTR is a plastid rp
3 'untranslated sequence of s16 gene or Arabidopsis plastid psbA
Gene 3 'untranslated sequence. In a further preferred embodiment, the plastid expression cassette comprises a poly-G region rather than a 3 'untranslated sequence. The plastid expression cassette also preferably further comprises a 5 'untranslated sequence (5'UTR) functional in the plant plastid operably linked to the DNA molecule of the present invention.

The plastid expression cassette is contained in a plastid transformation vector, which preferably further comprises a flanking region for integration into the plastid genome by homologous recombination. The plastid transformation vector can optionally include at least one chloroplast origin of replication. The invention also encompasses plant plastids transformed with such plastid transformation vectors, wherein the DNA molecule can be expressed in the plant plastid. The invention also includes plants or plant cells, including the plant plastids, including progeny thereof. In a preferred embodiment, the plant or plant cell, including its progeny, is homoplasmic for the transgenic plastid. Other promoters capable of expressing DNA molecules in plant plastids are transactivator-regulated promoters, which are preferably heterologous with respect to the intracellular organelle or component of the plant or plant cell in which expression is performed. In these cases, the DNA molecule encoding the transactivator is inserted into a suitable nuclear expression cassette and transformed into plant nuclear DNA. Transactivators are targeted to plastids using plastid transit peptides. The transactivator and the transactivator-driven DNA molecule comprise a DN encoding the transactivator operably linked to a plastid targeting sequence and operably linked to a nuclear promoter.
Crosses the transgenic system containing the A molecule into a selected plastid transformation system, or comprises a DNA molecule encoding a transactivator operably linked to a plastid targeting sequence and operably linked to a nuclear promoter. Ligation is achieved by directly transforming the plastid transformation vector containing the DNA molecule of interest into the transgenic system. If the nuclear promoter is an inducible promoter, in particular a chemically inducible promoter, the expression of the DNA molecule in the plant plastid is activated by the foliar application of a chemically inducible substance. Such an inducible transactivator-mediated plastid expression system can preferably be tightly regulated, with no detectable expression before induction, with very high expression and accumulation of proteins after induction. Preferred transactivators are, for example, viral RNA polymerases. A preferred promoter of this type is a promoter that is recognized by a single subunit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by a bacteriophage T7 DNA-dependent RNA polymerase. The gene encoding T7 polymerase is preferably transformed into the nuclear genome, and T7 polymerase is targeted to the plastid using a plastid transit peptide.
Suitable promoters for nuclear expression of a gene, eg, a gene encoding a viral RNA polymerase, eg, T7 polymerase, are described below. Expression of DNA molecules in plastids can be constitutive or inducible. Expression of DNA molecules in plastids can also be organ or tissue specific. These various aspects are fully described in WO 98/11235 and are hereby incorporated by reference.

Nuclear Expression of Transactivators or Therapeutically Active Proteins For expression in certain transgenic plants, nucleotide sequences according to the present invention may require modification and optimization. Low expression in transgenic plants may be due to heterologous nucleotide sequences having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention have been modified to be compatible with plant preferences, thereby encoding The amino acids used can be maintained. Furthermore, high expression in plant nuclei is most efficiently achieved from coding sequences having a GC content of at least 35%, and preferably greater than 45%. In addition, nucleotide sequences with low GC content may cause ATTT to destabilize the message.
It is poorly expressed in plant nuclei due to the presence of the A motif and the AATAAA motif, which can induce inappropriate polyadenylation. Preferred gene sequences can be adequately expressed in both monocotyledonous and dicotyledonous plant species, but by modifying the sequences monocotyledonous or dicotyledonous plants that have already been shown to differ in preference Plant specific codon preference and GC content preference may be accounted for (Murray et al., Nucl. A
cids Res. 17: 477-498 (1989)). DNA molecules are also screened for the presence of illegitimate splice sites that trigger message truncation. All changes that need to be made within a DNA molecule, such as those described above, are for example site-directed mutagenesis, PCR, and published patent application EP 0385962.
, EP 0359472 and WO 93/07278.

For efficient translation initiation, sequences adjacent to the initiation methionine may require modification. For example, sequences known to be effective in plants can be included. Jo
shi reports a consensus sequence suitable for plant nuclei (NAR 15: 6643-6653).
(1987)), Clontech suggests yet another common translation initiator (catalog 1993/1994, page 210). These consensus sequences correspond to the DN of the present invention.
Suitable for use with the A molecule. These sequences up to and including ATG (including ATG)
The second amino acid remains unmodified) or, alternatively, is incorporated into constructs containing DNA molecules up to and including the GTC following the ATG (with the potential to modify the second amino acid of the transgene).

The expression of a nucleotide sequence encoding a transactivator or therapeutically active protein in transgenic plants is driven by a promoter that has been shown to be functional in plants. The choice of promoter will depend on the temporal and local requirements for expression, and will also vary with the host species.
Expression of a DNA molecule of the present invention in a plant may be constitutive or inducible, eg, chemo-inducible, or expression of the DNA molecule may be organ or tissue specific. Although many promoters from dicots have also been shown to be functional in monocots, and vice versa, ideally, dicot promoters are suitable for expression in dicots. A monocot promoter is selected for expression in a monocot plant. However, there are no restrictions on the origin of the selected promoter. It is sufficient if they are functional in driving the expression of the nucleotide sequence in the target cell. Preferred promoters that are constitutively expressed include the CaMV 35S and 19S promoters, and promoters from genes encoding actin or ubiquitin. The DNA molecules of the invention can also be expressed under the control of a chemically regulated promoter. Preferred techniques for chemically inducing gene expression are detailed in patent application EP 0 332 104 and US Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter. A preferred class of promoters is wound inducible. Various promoters have been reported that are expressed at wound sites and at sites of plant pathogen infection. Ideally, such promoters are only active locally at the site of infection. Preferred promoters of this type include Stanford et al., Mol. Gen. Genet. 215: 200.
-208 (1989); Xu et al., Plant Molec. Biol. 22: 573-588 (19
93), Logemann et al., Plant Cell 1: 151-158 (1989), Rohrmeier
& Lehle, Plant Molec. Biol. 22: 783-792 (1993); Firek et al., Pl.
ant Molec. Biol. 22: 129-142 (1993), and Warner et al., Plant.
J. 3: 191-201 (1993). Preferred tissue-specific expression patterns include green tissue-specific, root-specific, stem-specific, fruit-specific and flower-specific. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis, many of which have been cloned from both monocots and dicots. A preferred promoter is maize PEPC from the phosphophenol carboxylase gene.
Promoter (Hudspeth & Grula, Plant Molec. Biol. 12: 579-5).
89 (1989)). Preferred promoters for root-specific expression are de Framond (FE
BS 290: 103-106 (1991), EP 0452269), and yet another preferred root-specific promoter is from the T-1 gene (de Framond et al., FEBS 290: 103-106 ( 1991), E
P0452269). Preferred stem-specific promoters are described in US Pat.
6, which drives the expression of the maize trpA gene. A preferred embodiment of the present invention is a transgenic plant expressing a DNA molecule in a root-specific manner. Yet another preferred embodiment is a transgenic plant that expresses a DNA molecule in a wound- or pathogen-inducing manner. Additional promoters are synthetic promoters, such as the Gervin Super MAS promoter (Ni
(1995) Plant J. 7: 661-676).

In addition to selecting a suitable promoter, the construct for expressing a DNA molecule in the plant nucleus preferably contains a suitable 3 ′ sequence, such as a 3 ′ regulatory sequence or transcription terminator, and can function downstream of a heterologous nucleotide sequence. Are combined. Several such terminators are available and known in the art (eg, tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the present invention. A variety of other sequences can be incorporated into the expression cassette for the DNA molecules described in this invention. These also include sequences that have been shown to enhance expression, such as intron sequences (eg, from Adh1 and bronze1) and viral leader sequences (eg, from TMV, MCMV and AMV).

In another preferred embodiment, the nuclear-expressed DNA molecule of the present invention is targeted to an intracellular location (s) in a plant. For example, the therapeutically active protein is secreted from the cell into the apoplast or the therapeutically active protein is targeted to a particular intracellular organelle, such as the vacuole or endoplasmic reticulum. This can be achieved, for example, by fusing the DNA molecule of the present invention with an appropriate targeting sequence by using techniques known in the art. That is, since the protein is targeted toward apoplasts or vacuoles, the protein preferably includes an appropriate signal peptide, preferably at its N-terminus, to target the protein to organelles. In a preferred embodiment, the proteins of the invention are targeted to vacuoles. Because the protein is targeted to the vacuole in addition to the N-terminal signal peptide, the protein also preferably further comprises a vacuolar targeting sequence, such as from the tobacco chitinase gene, preferably at its C-terminus (Neuhaus et al. (1991) Proc. Natl
Acad. Sci. USA 88, 10362-10366). Expression of the protein encoded by the DNA molecule of the present invention can also be targeted to plastids using a suitable plastid transit peptide, preferably contained at the N-terminus of the protein detailed below. The proteins of the present invention may also include, for example, mitochondrial targeting sequences, such as N. plumbaginifolia.
olia) by fusion with the F1-adenosine triphosphatase β-subunit,
Can be targeted to mitochondria (Chaumont et al. (1994) Pl
ant Molecular Biology 24: 631-641).

Suitable vectors for plant transformation are described elsewhere in this specification.
In the case of Agrobacterium-mediated transformation, at least one T
-A binary vector (s) carrying a DNA border sequence is preferred, and for direct gene transfer any vector is suitable, and linear DNA containing only the construct of interest may be preferred. Single DNA for direct gene transfer
Species transformation or co-transformation can be used (Schocher et al., Biotechnolog
y 4: 1093-1096 (1986)). For both direct and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) performed with an antibiotic (eg, kanamycin, hygromycin or methotrexate) or a herbicide (eg, vasta / phosphino). (A tricine or an inhibitor of protoporphyrinogen oxidase). However, selection of the selectable marker is not critical to the present invention.
Examples of expression cassettes and transformation vectors are detailed below.

[0085] The DNA molecules of the present invention are introduced into plant cells by several known methods. It will be appreciated by those skilled in the art that the choice of method may vary depending on the type of plant targeted for transformation, ie, monocot or dicot. Suitable transformation methods for plant cells include microinjection (Crossway et al., Bio Techniques 4:
320-334 (1986)), electroporation (Riggs and Bates, Proc. Natl. Ac.
ad.Sci.USA 83: 5602-5606 (1986)), Agrobacterium (Ag
robacteriumu) -mediated transformation method (Hinchee et al., Biotechnology, 6: 915-92).
1 (1988)), direct gene transfer method (Paszkowski et al., EMBO J. 3: 2717-).
2722 (1984)) and ballistic particle acceleration methods using equipment available from Agra Citas, Inc. (Madison, Wis.) And Dupont, Inc. (Wilmington, Del.) (Eg, US Pat. No. 4,945,050, and McCabe). Et al., Biotechnology 6: 923-9.
26 (1988), and Weissinger et al., Annual Rev. Genet. 22: 421-.
477 (1988), Sanford et al., Particulate Science and Technology 5: 2.
7-37 (1987) (onion), Christou et al., Plant Physiol. 87: 671.
-674 (1988) (soybean), McCabe et al., Bio / Technology 6: 923-92.
6 (1988) (soy), Datta et al., Bio / Technology 8: 736-740 (19).
90) (rice), Klein et al., Proc. Natl. Acad. Sci. USA, 85: 4305-430.
9 (1988) (maize), Klein et al., Bio / Technology 6: 559-56.
3 (1988) (maize), Klein et al., Plant Physiol. 91: 440-4.
44 (1988) (maize), Fromm et al., Bio / Technology 8: 833-8.
39 (1990), and Gordon-Kamm et al., Plant Cell 2: 603-618 (
1990) (maize), Koziel et al. (Biotechnology 11: 194-200).
(1993)) (maize), Shimamoto et al., Nature 338: 274-27.
7 (1989) (rice), Christou et al., Biotechnology 9: 957-962 (1
991) (rice), European Patent Application EP 0 323 581 (Camogaya and other Pooideae), Vasil et al. (Biotechnology 11: 1553-155).
8 (1993) (wheat)), Weeks et al. (Plant Physiol. 102: 1077-10).
84 (1993) (wheat)), Wan et al. (Plant Physiol. 104: 37-48 (1)
994) (barley)), Umbeck et al., (Bio / Technology 5: 263-266 (198).
7) (Cotton))).

(Administration of Therapeutically Active Protein to Host) An advantage of the present invention is that the therapeutically active protein expressed in the transgenic plant can be administered to the host by a wide variety of methods, including oral, enteral, Methods include nasal, parenteral, especially intramuscular or intravenous, rectal, topical, ocular, pulmonary routes or by contact application. In a preferred embodiment, the allergen expressed in the transgenic plant is administered orally to the host. In a preferred embodiment, the therapeutically active protein expressed in the transgenic plant is extracted, purified and used for the manufacture of a pharmaceutical composition. The localization of the expressed therapeutically active protein in plastids greatly simplifies the extraction and purification. For example, initial separation of intact plastids by centrifugation facilitates extraction and purification of the therapeutically active protein. In another preferred embodiment, conventional conditions and techniques known in the art that have been previously used for the separation of said proteins, such as extraction, precipitation, chromatography,
The protein is separated and purified by affinity chromatography, electrophoresis and the like. The compositions typically contain an effective amount of a therapeutically active protein together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials. The therapeutically active protein produced according to the present invention is used in dosage forms such as tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, powder pills or liquid solutions, in which case the protein The biological activity shall not be destroyed by the above dosage forms. For example, the protein may be provided as a pharmaceutical composition by conventional mixing, granulating, dragee-making, dissolving, lyophilizing or similar methods. The dose of the protein will depend on the patient's weight, age and physical and pharmacokinetic status, and will also depend on the method of delivery.

When the protein is to be administered enterally, it can be introduced in solid, semi-solid, suspension or emulsion form, formulated with any pharmaceutically acceptable carrier, including water, suspensions, emulsifiers. Can be done. The protein of the present invention may also be administered by pump means or sustained release forms, particularly when administered as a precautionary measure to prevent the onset of disease in a subject, or when administered to ameliorate or delay an already established disease. obtain.

The therapeutically active proteins produced according to the present invention are particularly suitable for oral administration as pharmaceutical compositions. Compositions for oral administration comprise the protein provided as a dry powder, foodstuff, aqueous or non-aqueous solvent, suspension or emulsion. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, such as saline and buffered pharmaceutical parenteral vehicles, such as sodium chloride solution, Ringer's dextrose solution, dextrose + sodium chloride solution, lactose-containing Ringer's solution or the like. There is a fixed oil. In a preferred embodiment, such compositions are taken orally alone or with food or feed or beverages.

In another preferred embodiment, the transgenic plant expressing the therapeutically active protein of the present invention or a plant substance derived from said plant is administered orally as a pharmaceutical food. The edible composition is consumed by eating in solid form or by drinking in liquid form. In a preferred embodiment, the transgenic botanical is taken directly without prior processing steps or after minimal cooking preparation. For example, therapeutically active proteins are expressed in directly edible plant parts, such as fruits or vegetables. Preferably, the protein is expressed in plastids of edible plant parts. All types of plastids, regardless of the plant, are suitable for the present invention. For example, the protein is expressed in spinach or lettuce chloroplasts, tomato chromoplasts or potato amiloplasts. In another preferred embodiment, the plants are processed and the plant material recovered after the processing step is consumed. The processing method preferably used in the present invention is a method commonly used in the food or feed industry. The final product of the above method still contains significant amounts of protein and can be conveniently eaten and consumed. The final product may also be in other food or feed forms, such as salt,
It can be mixed with carriers, chemical seasonings, antibiotics and the like and consumed in solid, semi-solid, suspension or emulsion form. In another preferred embodiment, the method comprises the step of storing
This includes, for example, pasteurization, cooking or storage and the addition of preservatives. In the present invention, all plants are used and processed to produce edible or drinkable plant material. For example, if tobacco plants are used according to the present invention, it may be necessary to treat the plants or plant matter to remove harmful substances, such as nicotine. In a preferred embodiment,
Low alkaloid tobacco plants are used for expression of the proteins of the invention. In a preferred embodiment, the amount of the therapeutically active protein in the edible or drinkable plant material according to the invention is tested. Such an amount can be determined, for example, by ELISA using an antibody specific for the protein.
Measured by SA or Western blot analysis. The results of this measurement are used to normalize the amount of protein consumed. For example, the amount of therapeutically active protein in a juice, such as tomato juice, can be measured and adjusted, for example, by mixing batches of products with different levels of protein, so that the amount of juice consumed per dose can be standardized. It is clear that the present invention provides a new and effective method of producing and administering plant-expressed therapeutic actives as pharmaceutical foods.

The therapeutically active proteins produced in plants and eaten by the host are absorbed by the digestive system. One advantage of consuming a plant or plant material, especially an intact plant or plant material, or a plant that has been lightly processed, is that the protein is encapsulated or sequestered in the plant cells. That is, a high proportion of active protein is available and ingested because the protein can be protected, at least in part, from digestion in the upper gastrointestinal tract before reaching the gastrointestinal tract or intestine.

If the plant used for the expression of the therapeutically active protein according to the invention is tobacco, the raw, so-called “F2 soluble protein fraction” can be used, for example, for therapeutic administration. An additional fraction, F1, is produced from the raw tobacco extract by precipitation of the macromolecular protein complex. In non-transgenic plants, this F
One fraction is almost exclusively constituted by ribulose bisphosphate carboxylase as disclosed in US Pat. No. 4,347,324. Some transgenic therapeutically active proteins produced by the method of the present invention may be able to be split into F1 fractions. In this case, the F1 fraction can also be used for therapeutic administration.

[0092] Preferred hosts or receptors for the therapeutically active proteins of the present invention are animals. Animals preferably include vertebrates, preferably mammals. Preferred mammals are humans
Pets or companion animals such as cats, dogs, rodents, ferrets, primates,
Fish or birds, or agricultural animals such as cows, pigs, poultry, and the like.

Immunization Some current vaccines are not readily available in developing countries because they require costly refrigeration. Also, vaccines that are effective against certain pathogens are not yet produced in large quantities or are still not safe enough to be widely distributed. In an attempt to ameliorate this situation, hepatitis B virus surface antigens are expressed in plants and administered through edible plants with the goal of vaccination against hepatitis B (see, eg, US Pat. Nos. 5,484,719 and 5,612,487). However, efficient production of edible vaccines was ruled out because the vaccine-encoding genes were expressed from the plant nuclear genome and vaccines were obtained only in relatively low yields. Immunization against pathogenic microorganisms has also been attempted by expressing antigenic determinants of the pathogen in transgenic plants and administering the plants orally to the host (
U.S. Pat. No. 5,654,184, 56798880, 5686079). However,
In these cases, too, the relatively low levels of expression of the transgene from the plant nuclear genome have limited the commercial success of the method. Thus, a preferred embodiment of the invention is to express an antigen capable of inducing immunization of a host against a pathogen, such as a bacterial, parasite or viral pathogen, or in a transgenic plant, in particular an intracellular organelle, preferably a fluid. Immunization of the host against toxins in vesicles, more preferably in plant plastids. The plant of the present invention includes, for example, polio, measles, mumps, rubella, smallpox, yellow fever, viral hepatitis B, influenza, rabies, adenovirus infection, Japanese encephalitis, varicella, diarrhea, acute respiration Used for immunization of humans against organ infections, malaria, whooping cough, diphtheria, tetanus or neonatal tetanus. The protein may also be used in diseases such as horse infections,
Canine distensa, rabies, canine hepatitis, parvovirus enteritis and feline leukemia, Newcastle disease, rinderpest, swine cholera, blue tongue and foot-and-mouth disease, brucellosis, poultry cholera, anthrax and blackleg, and protozoan and helminthic infections It is also advantageously used in immunizing animals to prevent disease. The plants of the present invention are also used for immunizing animals, including humans, with various toxins or stimulants, such as snake or bee venom, mosquito saliva, and urushi. The host is immunized after the challenge, preferably by oral ingestion of the plant or plant material derived from the plant. As is well known in the immunology art, an adjuvant can be added to a composition in an amount that improves the immunological activity of the antigen-containing composition of the invention and thereby the therapeutic efficacy of the composition. Information about the immune system and immunological responses is also described in Hood et al. (1984) Immunology (The Benjamin / Cummings Publishing Company, Inc., Menlo Park, Calif.).

Tolerization The medical arts relating to the suppression or reduction of unwanted immune responses also need improvement. The animal's immune system is a complex network of differentiated cells and organs that act on a variety of specific signals. One of its main functions is “self”
And "non-self." This basic property allows the host to be protected from invasive pathogens without eliciting a deleterious immune response against the host itself. Typically, the immune response is characterized by a cellular response provided by certain cells of the lymphatic system and a humoral response provided by the antibody, initiated by the encounter of non-self-determining factors by the immune system, and Self is destroyed. However, in some cases, the discrimination between self and non-self is incomplete, causing an immune response to certain parts of the self, leading to the development of an autoimmune disease. In other cases, an immune response to specific non-self antigens may also elicit undesirable effects, such as rejection of non-autologous tissue or organ transplants, or the development of an allergic response to environmental determinants. In these cases, effective suppression or reduction of the immune response is desirable. Several approaches have been tried to achieve this goal. For example, certain autoimmune diseases have been prevented by aerosol administration of self antigens (US Patents 5,641,473 and 5,641,474). Other therapeutic attempts are based on oral tolerization of the host to the antigen, by which the ingested protein triggers a suppression or reduction of the normal immune response to specific foreign substances or self-antigens (
For example, Weiner (1994) Proc. Natl. Acad. Sci. 91, 10762-10765.
, Friedman et al. (1994), Grandstein RD (eds.): Mechanisms of Immune Regu
lation, Chem. Immunol. Basel, Kalgar, vol. 58, 259-290).
There are multiple mechanisms involved in oral tolerance, two of which are active cell suppression or clonal anergy. This is thought to be due to absorption by the small intestinal mucosa. The antigen is absorbed in the gastrointestinal tract and directed to differentiated mucosal tissues, such as Peyer's patch cells, which are entry points into the gastrointestinal tract-associated immune system. This induced immunological hypo- or non-responsiveness is associated with food allergy suppression usually performed in most individuals and is the primary means of tolerizing various potential antigens that enter the gastrointestinal tract as food components. is there. In the therapeutic area, oral administration of insulin has shown some relief in the treatment of type I diabetes (U.S. Pat. No. 5,763,396), and oral administration of type I or type III collagen has shown some success in the treatment of autoimmune arthritis. (US Pat. No. 5,733,547). In addition, attempts have been made to suppress transplant rejection by oral administration of polymorphic type II MHC allopeptide (US Pat. No. 5,559,369).
8). In these earlier cases, the antigen was purified from natural sources. However,
Large amounts of antigen must be taken in order for oral tolerization to be effective. Many antigens, especially airborne antigens, are difficult or expensive to obtain in large quantities and cannot be easily manufactured in orally ingestible compositions. That is, many of the successes of the above methods have been hampered by the lack of sufficient amounts of protein available for oral ingestion. Recently, the expression of certain transplantation and autoantigens in transgenic plants and their enteral or oral administration has been attempted (WO 95/08347). However, again, the relative levels of nuclear transgene expression are relatively low and these methods may not yield favorable results.

Thus, in a preferred embodiment, the antigen capable of suppressing or reducing the immune response or inflammatory state of the host is expressed in plants, especially plant plastids. Preferably, the antigen is administered orally to the host. Transgenic plants expressing the above antigens are particularly useful for treating, preventing or ameliorating diseases such as allergies, autoimmune diseases or transplant rejections, preferably by inducing host tolerance to one or more antigens. In the case of oral ingestion, plants expressing the antigen or plant substances derived from said plants are eaten raw or eaten or processed after the processing step, for example after the processing step. In a preferred embodiment, oral tolerance of the plant or plant material results in induction of host tolerance to the antigen. The present invention provides, for the first time, a rich and inexpensive source of antigens that can be used for oral tolerization by expressing the antigens from the plastid genome of plants. In a preferred embodiment, edible plants that have been genetically engineered to overexpress the specific peptide antigens described in this invention are ideal vehicles for pharmaceutical food-based oral tolerization therapies. . The use of transgenic plants for oral tolerance has some potential advantages over current methods (biochemical purification or recombinant expression in cell culture systems and protein expression in transgenic milk). These advantages include low production costs per dose, low risk of transmission of mammalian pathogens, or the need for purification, processing or encapsulation when antigen-containing plants or plant materials are consumed directly as food. May be missing or limited.

[0096] In another preferred embodiment, the antigen of the present invention is co-administered to a host with a tolerizing adjuvant. Such adjuvants are, for example, toxins, such as the cholera toxin B subunit (eg, Arakawa et al. (1998) Nat Biotechnol 16: 934-8.
). Such toxin subunit molecules are known in the art to promote oral tolerance as opposed to mucosal immunity (Sun et al., PNAS (1996) 93: 7196).
-201). Another tolerizing adjuvant used in the present invention is Escherichia coli labile enterotoxin B. In a preferred embodiment, a DNA molecule encoding the fusion protein is produced by fusing a nucleic acid sequence encoding a toxin to a nucleic acid sequence encoding an antigen. Plants containing the DNA molecule are produced as described in the present invention, and the fusion protein is administered to a host as described in the present invention. In another preferred embodiment, the toxin and antigen are expressed in the plant as separate polypeptides and administered to a host as described in the present invention. Alternatively, the toxin and antigen are expressed in different plants and combined prior to administration to the host. Alternatively, the toxin is obtained from a different organism, preferably purified, and then combined with the antigen before administration to the host.

Allergens Allergy causes significant distress in most of the population and is suspected to have deleterious effects on animals. The magnitude of the allergic response in the human population ranges from mild signs to heavy and dramatic aspects, such as anaphylactic shock, and common pathological aspects include food allergies, skin reactions (urticaria or atopic Dermatitis), upper respiratory allergic reactions (eg, hay fever or allergic rhinitis) or lower respiratory system allergic reactions that are a major cause of asthma (for review, see O'Hehir et al. (1991) Annu. Rev. Immunol. 9: 67-95). Allergic responses are typically accompanied by an inflammatory response due to excessive IgE responses to various types of allergens (eg, pollen, animal dander, insect dung components or dust and food allergens). The pathology of the IgE-antigen interaction is due to mast cell degranulation, which in particular results in the release of histamine, heparin and leukotriene. The agents of the invention are primarily non-specific and take a form of treatment with antagonists of mast cell degranulation, eg, antihistamine or epinephrine. More specific treatments have also been developed, based on the periodic injection of allergens at lower than allergenic doses, leading to desensitization of the host to the allergen. However, the injections are burdensome and desensitization is poorly or not directed at the T cell response normally associated with allergies. Therefore, injections for desensitization are often of limited efficacy. The present invention provides a novel and specific method for treating allergy by expressing a DNA molecule encoding an allergen in a plant, and administering the plant or a plant substance derived from the plant to a host. Such a plant or botanical material is preferably administered orally to the host, where it produces tolerance to the allergen. With regard to the treatment of infants or young individuals who may be at risk of developing an allergic disease in the future based on their pedigree, taking preventive measures can also prevent the development of allergies. Alternatively, the allergen expressed in the transgenic plants of the present invention may be extracted and purified according to methods well known in the art, and the host may be desensitized to the allergen by periodic injection of a sub-allergenic dose of the allergen. Used for cropping.

[0098] Allergens suitable for use in the context of the present invention include food allergens, drug allergens, venom allergens, plant allergens, fungal allergens, bacterial allergens, animal allergens, other allergens from natural or synthetic substances and extracts thereof. There is. Food allergens include, for example, seafood, strawberries, fresh fruits and vegetables, peanuts and milk. Drug allergens include, for example, penicillin and insulin. Venom allergens include, for example, bee, wasp and mosquito venom. A preferred allergen is the honey bee venom peptide PLA-2. Plant allergens include, for example, pollen, such as tree pollen, grass pollen and weed pollen allergens. Fungal allergens include, for example, mold spores. Animal allergens include, for example, scales or saliva from dogs, cats, horses, and the like. Preferred allergens of the present invention are house dust mite allergens, such as allergens from Dermatophagoides farinae and Dermatophagoies pteronyssinus, preferably Der f I, Der f II, Der f II I and Der p II allergens (US Pat. Nos. 5,552,142, 5,770,202 and 5798099). Another preferred allergen is ryegrass pollen, e.g.
lium perenne) derived and contains the isolated peptide of Lol p V (US Pat. No. 571)
0126). Still other preferred allergens include allergens derived from Johnsongrass pollen, such as Sor h I, Sorghum Halpense (So
rghum halepense) (US Patent No. 54809)
72 and 5691167). Further preferred allergens are ragweed pollen allergens, such as Amb a I and Amb a II allergens (US Pat. No. 5,698,204). Further, more preferred allergens include alder (Alnus)
(Alnus species) Alng I allergen, Hazel (Coryus species) C
There are the or a I allergen and the Bet VI allergen of birch (Betula sp.) (described in US Pat. No. 5,693,495). Still other allergens include cat antigens, such as Fel d I allergen (US Pat. No. 5,329,891), and canine scale allergens, such as Can f II (US Pat. No. 5,939,283). Still other allergens include rAed a 1 and rAed a 2 allergens from the mosquito Aedes aegypty (WO 98/04274) and bee venom antigens (Lomnitzer and Rabson (1986) J. Allergy Clin. Immuu).
no. 78: 25-30) or murine urine protein (Gurka et al. (1989) J. All.
ergy Clin. Immunol. 83: 945-954).

Furthermore, the anaphylactic reaction and the IgE-mediated reaction are separable, suggesting that disulfide bridges are involved in anaphylaxis.
Dermatophagoides farinae Der f I
Removal of cysteine residues involved in disulfide bridge formation in the I allergen has been shown to greatly reduce the anaphylactic reaction without altering the allergen epitope (Takai et al. (1997) Nature Biotechnology 15:75).
4-758). The invention also encompasses the expression of allergens modified in this way to lack disulfide bridges. The DNA molecule encoding the allergen is cloned into a plastid transformation vector using techniques well known in the art to produce a transgenic plant. The plant or plant material of the present invention can be administered alone to the host or combined with other therapies for allergy.

(Antigen Expressed in Transgenic Plant) A preferred embodiment of the present invention is to express the antigen in a transgenic plant, especially in a plant plastid. Preferably, the antigen achieves the desired therapeutic effect by being able to modulate the immune system of the host in need of the modulation. Transgenic plants expressing the above antigens are particularly useful in methods of treating, preventing or ameliorating, or immunizing a disease, such as an allergy, an autoimmune disease or transplant rejection, preferably by inducing host tolerance to one or more antigens. is there. The transgenic plants are administered by various methods known in the art, preferably orally, preferably by eating or drinking a plant of the present invention or a plant material derived from the plant.

Antigens or Autoantigens in Autoimmune Diseases Autoimmune diseases are dysfunctions of the immune system in animals, including humans, where the immune system is composed of foreign substances in the animal and part of the normal makeup of the animal. Cannot distinguish from certain substances.
As a dysfunction of immune tolerance, T-cells or B-cells or both appear with receptors that can make them recognize and attack self components. As a result, an autoimmune disease is induced in the host having the T cells or B cells. A variety of diseases have been diagnosed, at least in part, caused by autoantigens. For example, blood cells are the most common type of cells that undergo this effect, for example, idiopathic thrombocytopenic purpura, where antibodies to platelets are formed, or autoantibodies to polymorphonuclear leukocytes. It leads to diseases such as granulocytopenia. It has also been reported that hemolysis and anemia may be due to autoantibodies directed against the surface of red blood cells. Antibodies to cell surface receptors can also trigger disease by interfering with receptor function, for example in the case of myasthenia gravis, the formation of antibodies against the acetylcholine receptor interferes with neuromuscular transmission . An opposite effect, i.e., receptor stimulation by anti-receptor autoantibodies, has been found in Graves' disease or hyperthyroidism. Still other examples of diseases in which immune tolerance dysfunction is suspected or known are, for example, multiple sclerosis, diabetes mellitus, systemic lupus erythematosus, multiple chondritis, systemic sclerosis, Wegener's granulomatosis, Dermatomyositis, chronic active hepatitis, psoriasis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (eg, including ulcerative colitis and Crohn's disease), sarcoidosis, primary biliary cirrhosis, uveitis (Anterior and posterior), keratoconjunctivitis sicca and spring, stromal pulmonary fibrosis, psoriatic arthritis and glomerulonephritis (eg, with and without nephrotic syndrome, including idiopathic nephrotic syndrome or micronephrotic nephropathy) Cases), and arthritis (eg, rheumatoid arthritis, chronic progressive arthritis and osteoarthritis). In the case of allergic autoimmune diseases, current drugs are largely lacking in specificity and are often associated with unpleasant side effects, such as global immunosuppression of the host. Accordingly, a preferred embodiment of the present invention is to provide a novel treatment for autoimmune diseases by expressing a self-antigen targeted by autoantibodies in plant plastids. The plant or plant material derived from the plant is preferably administered orally, preferably to a host in need thereof by eating or drinking by the host. The host's tolerance for specific autoantibodies then manifests without affecting the host's overall immune response capacity. The plant or plant material of the present invention is administered to a host alone or in combination with immunosuppressive or anti-inflammatory agents such as cyclosporine, rapamycin, FK506 and steroids.

Preferred autoantigens according to the invention are eg collagen for treating or preventing arthritis, preferably rheumatoid arthritis, preferably type I or type III collagen (see US Pat. No. 5,733,547), treating multiple sclerosis. Or myelin basic protein for preventing, S-antigen for treating or preventing autoimmune uveoretinitis, insulin for treating or preventing type I diabetes (see US Pat. No. 5,763,396). Glutamate decarboxylase or islet cell-specific antigen to treat or prevent diabetes, thyroglobulin to treat or prevent autoimmune thyroiditis, acetylcholine receptor to treat or prevent myasthenia gravis is there.

A DNA molecule encoding an autoantigen is cloned into plastid or nuclear transformation vectors using techniques well known in the art to produce transgenic plants.

Transplantation Antigens There is a great need for transplantation or transplant surgery to replace severely damaged tissues and organs. Transplantation from one individual to itself (autologous transplantation) is generally successful, but transplantation from a homologous, genetically dissimilar individual (allogeneic transplant) or transplantation between xenogeneic individuals (xenotransplantation) is without immunosuppressant Does not usually succeed. Without continued immunosuppressive therapy, the organ is rejected. Thus, general methods that induce tolerance to transplanted organs and increase the likelihood of successful allogeneic or xenotransplantation have great potential and are addressed in the present invention.

Transplant rejection is initiated by an immune response to a cell surface antigen that distinguishes a host from a donor. The cell surface belongs mainly to histocompatibility antigens, in particular the major histocompatibility complex (MHC). Class I and II MHC gene products are involved in antigen presentation. Therefore, they are particularly important in the recognition of non-self antigens by T cells and play an important role in transplant rejection. Class I MHC complexes comprise a transmembrane glycoprotein homodimer (heavy chain, HC) bound to the b2 microglobulin moiety (
Bjorkman et al. (1987) Nature 329: 506). Coding the HC part 3
Class Ia loci (HLA-A, B and C in humans, H-A in mice)
2K, H-2D and H-2L) and several class Ib loci have been identified (York and Rock (1996) Annu Rev Immunol 14: 369-396). Class II MHC is a heterodimer consisting of two glycoprotein a and b chains (B
rown et al. (1993) Nature 364: 33). The MHC locus is conserved among vertebrates, especially mammals (Klein (1986) Nature History of the Major H
DNA molecules encoding the istocompatibility Complex, Oxford: Blackwell Sci.), MHC antigens, can also be isolated from various mammals, including pigs, which are preferred donors for xenotransplantation.

The importance of MHC antigens in transplant rejection was determined by monoclonal antibody class II
It was further established by showing that molecular targeting attenuates transplant rejection. Thus, MHC antigens are good candidates for treatments that involve tolerance of the transplant recipient to specific MHC determinants of the donor. Recently, the expression of certain transplantation and autoantigens in transgenic plants and their enteral or oral administration has been attempted (WO 95/08347). However, again, these methods may not be successful due to the relatively low level of nuclear transgene expression.

In the present invention, the MHC gene, in particular the gene encoding the class I HC part and the class II MHC gene encoding the a or b chain are expressed from the plastid or nuclear genome of the plant. Transgenic plants containing the above plastids are preferably administered to the host before and / or after tissue or organ transplantation (allogeneic or xenotransplant) to induce host tolerization. Preferably, the plant or plant material derived from the plant is administered orally to a host to induce oral tolerization of the host. DNA molecules encoding different known allotypes of MHC class I HC genes and class II MHC genes encoding a or b chains
Introduced in transgenic plants. For use in specific transplants, the allotype of both the donor and the recipient is determined, and the recipient is preferably orally administered a botanical material containing the selected MHC antigen of the donor. The plant or plant material of the present invention is administered to a host alone or in combination with an immunosuppressant, including, for example, cyclosporine.

Other Therapeutically Active Proteins Other therapeutically active proteins expressed in plants in the context of the present invention include, for example, blood proteins (eg, coagulation factors VIII and IX, complement factors and complement, hemoglobin or other Blood proteins, serum albumin, etc., hormones (eg, insulin, growth hormone, thyroid hormone, catecholamine gonadotropin, PMS
G, stimulating hormone, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc., growth factors (eg, EGF, PDGF, NGF, IGF, etc.), cytokines (eg, interleukin, CSF, G-CSF, GMCSF,
EPO, TNF, TGFa, TGFb, interferon, etc.), enzymes (eg, tissue plasminogen activator, streptokinase, cholesterol biosynthesis or degradability, steroid synthase, kinase, phosphodiesterase, methylase, demethylase, dehydrogenase, cellulase, Proteases, lipases,
Phospholipase, aromatase, cytochrome, adenylate or guanylate cyclase, neuraminidase, etc., hormones or other receptors (eg, steroid proteins, peptides, lipids or prostaglandins, etc.), binding proteins (eg, steroid binding proteins, growth hormones) Or growth factor binding proteins), immune system proteins (eg, antibodies, antibody fragments, chimeric antibodies, variable regions, etc., or MHC genes), antigens (eg, bacteria, parasites, viruses, allergens, antigens in autoimmune diseases, Transplantation antigens), translation or transcription factors, oncoproteins or proto-oncoproteins, milk proteins (eg, casein, lactalbumin, whey, etc.), muscle proteins (eg, myosin, tropomyosin, etc.), bone marrow proteins Protein, neurostimulatory peptide (eg, enkephalin), collagen, preservative peptide (eg, BPI (bactericidal permeability enhancing protein))
Or a tumor growth inhibitory protein or peptide, such as, but not limited to, angiostatin or endostatin, both of which block angiogenesis.

Expression of Bactericidal Permeability Enhancement (BPI) Protein in Plants The present invention also relates to the transduction of BPI or a fragment thereof in transgenic plants, especially intracellular organelles, preferably vacuoles or more preferably plant plastids. It is about expression. BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs), which are blood cells essential for defense against invasive microorganisms in mammals (U.S. Pat. No. 5,641,874; Wilde et al. (1994) J. Biol. Ch.
em. 269: 17411-17416). BPI is a potent bactericide that is active against a wide variety of Gram-negative bacteria (eg, US Pat.
27816 and 5763567, which are incorporated herein by reference.) It exhibits a high degree of specificity in its cytotoxic effect, ie 10
At -40 nanomolar (0.5-2.0 micrograms) 90 against 107 susceptible bacteria
%, But 100 times higher concentration of BPI is non-toxic to other microorganisms and eukaryotic cells. BPI isolated from both human and rabbit PMNs has been purified to homogeneity. The molecular weight of human BPI is about 58,000 daltons (58 kDa) and that of rabbit BPI is about 50 kDa. Since the selectivity is sharp and lacks cytotoxicity against cells other than Gram-negative bacteria,
PI fragments are particularly useful as specific therapeutic agents. Common gram-negative bacterial infections such as Escherichia coli, various types of Salmonella, Klebsiella or Pseudomonas
domonas) are treated with antibiotics such as penicillin derivatives, aminoglycosides and chloramphenicol. Gram-negative bacteria exhibit significant endogenous resistance to many commonly available antibiotics, and the fact that resistance factor plasmids are acquired and thus tend to readily develop additional resistance results in the efficacy of antibiotics. Is limited. However, the production of BPI in recombinant systems is problematic, and purification from mammalian cells is not cost-effective given that doses in the gram range are preferred for therapeutic efficacy. One reason for the need for high doses is the high rate of clearance of BPI from the bloodstream. Therefore, expressing a DNA molecule encoding a BPI or BPI fragment from the plastid genome of a plant is a preferred embodiment of the present invention. Expression in plastids in high yield is particularly advantageous in this case.

The BPI or BPI produced according to the present invention when used in the treatment of bacteremia (ie, the presence of bacteria in the bloodstream) or sepsis (bacterial contamination of body fluids) induced by Gram-negative bacteria. The fragments are preferably administered parenterally, and most preferably intravenously, in amounts ranging between about 1 and 1000 micrograms and preferably between 10 and about 250 micrograms per treatment. The duration and number of treatments can vary between individuals depending on the severity of the disease. A typical treatment may include intravenous administration of about 100 micrograms of the BPI fragment three times a day. To help avoid rapid inactivation, the BPI or BPI fragment may be conjugated to a physiologically acceptable carrier, such as a commonly found serum protein (eg, albumin or lysozyme). BP of the present invention
I or BPI fragments can also be used topically to treat mammals with skin infections caused by infectious gram-negative bacteria found in bedridden or burn patients who suffer from floor rubs (pressure ulcers). In a preferred embodiment, B of the present invention
PI or BPI fragment is 1 microgram per dose and 100 micrograms per dose.
An amount in the range between 0 micrograms may be mixed with the antibiotic. In another preferred embodiment of the invention, the pharmaceutical formulation for treating a mammal afflicted with a Gram-negative bacterial infection comprises a standard amount (known in the art) of an antibiotic such as penicillin-G (E.R. And Sons, available from Incorporated, Princeton,
New Jersey) or cephalosporins (available from Eli Lilly and Company, Indianapolis, Indiana) plus the BP of the present invention
I fragment. In a particularly preferred embodiment, the BPI fragments of the invention can be mixed with hydrophobic antibiotics, such as rifampicin and hydrophobic penicillins, such as penicillin-V benzathine (Redale Labs, Pearl River, NY). The enhanced permeability of Gram-negative bacteria after BPI treatment is expected to increase the efficacy of the antibiotic, which is not readily accessible to non-permeable bacteria. The BPI or BPI fragment of the present invention is effective against Gram-negative bacteria,
It is expected that it would be ideally suited for co-treatment with any antibiotics, immune system cells or factors, such as T cells or interleukin 2, cytotoxic agents and the like. The BPI fragments of the present invention are expected to reduce the bacterial resistance to the above factors due to the increased susceptibility of the virulent, smooth Gram-negative bacteria to the fragments of the present invention. Substantially simultaneous administration of the fragment of the invention and the selected antibiotic is preferred. In another preferred embodiment, the BPI or BPI fragment is orally ingested according to the above embodiments, preferably for treating bacterial infections of the gastrointestinal tract. The protein is administered as a pharmaceutical composition or taken as a pharmaceutical food.

BPI has also been shown to be active in the treatment of medical conditions, including neutralizing the anticoagulant activity of heparin, inhibiting angiogenesis, tumor and endothelial cell proliferation, and treating chronic inflammatory diseases (US See US Pat. No. 5,807,818; incorporated herein by reference.)

Hereinafter, the present invention will be further described with reference to detailed examples. These examples are for illustrative purposes only and are not intended to be limiting unless otherwise specified.

EXAMPLES Standard recombinant DNA and molecular cloning techniques used herein are known in the art and are described in J. Sambrook, EFFritsch and T. Maniatis, Molecular C
loning: A Laboratory manual, Cold Spring Harbor laboratory, Cold Spring H
arbor, NY (1989) and TJSilhavy, MLBerman and LWEnquist, Experim
ents with Gene Fusions, Cold Spring Harbor laboratory, Cold Spring Harbo
r, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology
, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).

Example 1: Isolation of Amb a I.1 cDNA from short ragweed (Ambrosia artemisiifolia) pollen by RT-PCR amplification
Separation by DS method (Current Protocols in Molecular Biology). All RN
First strand cDNA from 0.5 mg A is synthesized with the Advantage RT for PCR kit (Clontech) using oligo (dT) ₁₈ primer. Single-stranded DNA
Using a Pfu turbo DNA polymerase kit using the following oligonucleotides (
(Rafnar et al. (1991) J. Biol. Chem. 266: 1229-1236). The “top strand” primer is
A NcoI site was added to the translation initiation site of Amb a I.1 (5′-GCG GCC
ATG GGG ATC AAA CAC TGT TGT TA-3 ', SEQ ID NO: 1)
, The "bottom strand" primer has an Xba after the stop codon in the 3 'untranslated region.
Add an I site (5′-GCG GTC TAG ATC ATT ATA AGT
GCT TAG T-3 ', SEQ ID NO: 2). The 1.2 kb band was digested with NcoI and XbaI to subclone, the entire cDNA was sequenced, and Genebank
Compare with accession number M80558. Although a 7 bp difference is observed, none of the differences alters the amino acid composition.

Example 2 Construction of Vector for Homologous Recombination into Tobacco Plastid Genome By adding modifications to the trnV and rps12 / 7 intergenic regions of the tobacco plastid genome, a chimeric gene was inserted by homologous recombination. Is done. 1.78kb
Region (positions 139255 to 141036; Shinozaki et al. (1986) EMBO J 5: 2
043-2049) is PCR-amplified from the tobacco plastid genome, and the PstI site is inserted after position 140169 to obtain flanking plastid DNAs 5 ′ and 3 ′ of 915 bp and 867 bp of the PstI insertion site. PCR amplification (Pfu Turbo DNA polymerase, Stratagene, La Jolla, CA)
Shows a BsiEI site (5′-TAA CGG CCG CGC CGC before position 139255)
CCA ATC ATT CCG GAT A-3 ', SEQ ID NO: 3) and 1401
After position 69, a PstI site (5′-TAA CTG CAG AAA GAA GGC
Performed by a primer pair that inserts CCG GCT CCA A-3 ', SEQ ID NO: 4). PCR amplification also requires a PstI site (5′-C
GC CTG CAG TCG CAC TAT TAC GGA TAT G-3 ', SEQ ID NO: 5) and a BsiWI site (position 5'-CGC CGT) after position 141036.
ACG AAA TCC TTC CCG ATA CCT C-3 ', SEQ ID NO: 6). The PstI-BsiEI fragment was inserted into the PstI-SacII site of pBluescript SK + (Stratagene) to obtain pAT216, and the PstI-BsiWI fragment was converted to pB
By insertion into the PstI-Acc65I site of luescript SK +, pA
T215 is obtained. PAT218 contains a 1.78 kb plastid DNA with a PstI site for insertion of the chimeric gene and a selectable marker, and 2.0 kb of pAT215.
PstI-ScaI fragment and 2.7 kb PstI-Sc of pAT216
Constructed by ligation of the aI band.

Example 3 Amplification of Tobacco 16S rRNA Gene Promoter and rbcL
The rbs16S rRNA gene promoter of the gene is derived from tobacco DNA (N. tabacum cv. Xanthi).
) And fused to the synthetic ribosome binding site (rbs) of the tobacco plastid rbcL gene. The "top strand" primer has 1 before position 102568
Insert an EcoRI site at the 5 'end of the 6S rRNA gene promoter (
5'-GCC AGA ATT CGC CGT CGT TCA ATG AGA AT
G-3 ′, SEQ ID NO: 7). The "bottom strand" primer amplifies up to position 102675 of the 16S rRNA gene promoter and removes the two upstream ATGs by changing positions 102661 (from A to C) and 102670 (from A to C). Rbs of the rbcL gene (57569-575)
No. 84) and insert a BSphI site at the 3 ′ end of rbs (5′-GCC
TTC ATG ATC CCT CCC TAC AAC TAT CCA GGC
GCT TCA GAT TCG-3 ', SEQ ID NO: 8). The 142 bp amplification product was gel purified and cleaved with EcoRI and BSphI to give the rbs gene rbs
A 128 bp fragment containing the tobacco 16S rRNA gene promoter fused to is obtained.

Example 4: Isolation of Arabidopsis 16S rRNA Gene Promoter Region Isolation of the Arabidopsis 16S rRNA gene promoter region was carried out in a plant in which the gene order in the Arabidopsis plastid genome is known and the whole plastid genome is known. tabacum), which is easy to do because it is likely to be preserved. In the case of Sinapis alba, a closely related species to Arabidopsis, the 16S rRNA gene and valine tRNA are in the same orientation as in tobacco (Genebank accession number CHSARRN1).
The Arabidopsis 16S rRNA gene promoter region is used as a template for both the entire Arabidopsis thaliana (A. thaliana) (variety “Landsberg erecta”) and both Nicotiana (Nicotiana) and Sinapis alba. Primers stored in: "top strand"
Primer (5'-CAG TTC GAG CCT GAT TAT CC-3 ', SEQ ID NO: 9) and "bottom strand" primer (5'-GTT CTT ACG CGT
TAC TCA CC-3 ', SEQ ID NO: 10) (Pfu turbo DNA polymerase, Stratagene, La Jolla, CA). The putative 379 bp amplification product containing the Arabidopsis 16S rRNA gene promoter region corresponding to nucleotides 102508-102873 of the tobacco plastid genome (Shinozaki et al. (1986) EMBO J 5: 2043-2049) was purified by pGE.
M5Zf (-) (Promega) is blunt-ended to the EcoRV site, sequenced and compared to the tobacco 16S rRNA gene promoter.

Example 5: Tobacco plastid rps16 gene 3 ′ untranslated RNA sequence (3′UTR)
The tobacco plastid rps16 3'UTR is PCR amplified from tobacco DNA (N. tabacum cv. Xanthi) using the following oligonucleotide pairs. That is, SpeI
The site is the “top strand” primer (5′-CGC GAC TAG TTC AAC
CGA AAT TCA AT-3 ') added immediately after the stop codon of the plastid rps16 gene encoding ribosomal protein S16, and a PstI site was added to the "bottom strand" primer (5'-CGC TCT GCA GTT CAA TGG).
AAG CAA TG-3 ', SEQ ID NO: 12) 3' of rps16 3 'UTR
Appended to the end. The amplification product was gel-purified and digested with SpeI and PstI. Tobacco rps16 3′UTR flanked by 5 ′ by the SpeI site and 3 ′ by the PstI site (positions 4941 to 5093 of the tobacco plastid genome, Sh
(1986) EMBO J 5: 2043-2049).

Example 6: 16S rRNA gene promoter for plastid transformation selection :: aa
Construction of dA gene :: rps16 3 'UTR cassette Bacterial gene encoding aminoglycoside 3 "adenyltransferase, which confers resistance to spectinomycin and streptomycin, a
The coding sequence of the adA gene is pRL277 (Black et al. (1993) Molecular M
icrobiology 9: 77-84 and Prentki et al. (1991) Gene 103: 17-2.
3). The 5 'major portion of the aadA coding sequence was isolated as a 724 bp BSphI-BssHII fragment from pRL277 (start codon is at the BSphI site) and the 3' remaining portion of the aadA gene was used as a template for pR27.
L277 and the following oligonucleotide pair: "top strand" primer (5'-A
CC GTA AGG CTT GAT GAA-3 ′, SEQ ID NO: 13) and Spe
"Bottom strand" primer (5'-CCC ACT AGT TTG
AAC GAA TTG TTA GAC-3 ', SEQ ID NO: 14), modified by adding a 20 bp SpeI site after the stop codon by PCR amplification. The 658 bp amplification product was gel purified, digested with BssHII and SpeI, and the 89 bp fragment was converted to an aad carried by a 724 bp BSphI-BssHII fragment.
The 5 'portion of the A gene, the 16S rRNA gene promoter carried on the 128 bp EcoRI-BSphI PCR amplified fragment and the rbcL rbs and EcoRI-SpeI digested pLITMUS28 vector (New England Biolab
Ligation to s) gives pAT223. The three-way ligation reaction is 16S rRN
EcoR of pAT223 containing A gene promoter-rbs driven aadA gene
I-SpeI 0.94 kb fragment, 163 bp including rps16 3 'UTR
Performed on pUC19 (New England Biolabs) cut with SpeI, PstI digested PCR fragment and EcoRI, PstI, rps1
PAT229 containing the 16S rRNA gene promoter driving the aadA gene with 6 3 'UTR was obtained.

Example 7: Amplification of the bacteriophage T7 gene 10 promoter The bacteriophage T7 gene 10 promoter is PCR amplified from pET-3d (Stratagene) using the following oligonucleotide pair. That is, the "top strand" primer inserts an EcoRI site at the 5 'end of the T7 promoter (5'-CCC GAA TTC ATC CCG CGA AAT TAA TA-3'
, SEQ ID NO: 15), the “bottom strand” primer inserts an NcoI site at the 3 ′ end (5′-CGG CCA TGG GTA TAT CTC CTT CTT AA
A GTT AAA-3 ′, SEQ ID NO: 16). The amplification product is gel-purified and EcoRI
Cleavage with NcoI produces a 96 bp fragment containing the T7 promoter.

Example 8 Amplification of the Bacteriophage T7 Gene 10 Terminator The bacteriophage T7 gene 10 terminator is PCR amplified from pET-3d (Stratagene) using the following oligonucleotide pair. That is, "
The “top strand” primer inserts a HindIII site at the 5 ′ end of the terminator (5′-GCG AAG CTT GCT GAG CAA TAA CTA GC
A TAA-3 ′, SEQ ID NO: 17), “bottom strand” primer inserts a PstI site at the 3 ′ end of the terminator (5′-GCG CTG CAG TCC GG
A TAT AGT TCC TCC T-3 ', SEQ ID NO: 18). The amplification product is gel purified and cleaved with HindIII-PstI to produce an 86 bp fragment containing the T7 terminator.

Example 9: Arabidopsis thaliana plastid psbA 3 ′ untranslated RNA sequence (UT
R) Amplification The A. thaliana plastid psbA3'UTR is PCR amplified from Arabidopsis thaliana (A. thaliana) DNA (Ecotype Landsberg erecta) using the following oligonucleotide pairs: That is, the “top strand” primer adds a SpeI site to the 5 ′ end of the 3′UTR and eliminates the XbaI site in the native sequence by G to A mutagenesis (underlined) (5′-GCG ACT AGT TAG TGT
TAG TCT A A A TCT AGT T -3 ', SEQ ID NO: 19), "bottom strand"
The primer adds a HindIII site at the 3 ′ end of the UTR (5′-CCG
CAA GCT TCT AAT AAA AAA TAT ATA GTA-3 ', SEQ ID NO: 20). The amplification region is 1350 to Genebankaccession number X79898.
Extends to 1552. The 218 bp PCR product was gel purified, SpeI and Hi
ndIII, digested with HindIII-PstI digested PCR fragment carrying T7 terminator, SpeI-P of pBluescriptSK- (Stratagene)
Ligation to the stI site generates pPH171. Genebankaccession numb
Sequence analysis of the psbA3 'UTR region of pPH171 compared to erX79898 shows a deletion of adenine nucleotides at positions 1440 and 1452.

Example 10: Construction of a vector using the polyguanosine domain as a surrogate for the 3'UTR The polyguanosine domain is functionally functional in vivo in the plastid atpB gene 3'UTR.
(Drager et al. (1996) RNA 2: 6).
52-663). A poly-G region containing 18 consecutive guanosine residues flanked by SpeI and HindIII cohesive ends at the 5 'and 3' ends, respectively, contains two kinased oligonucleotides: (5'-CTA GTG GGG GGG GGG GGG GGG
GGG GGA-3 ', SEQ ID NO: 21) and (5'-AGC TTC CCC CC
Assembled by annealing C CCC CCC CCC CCA-3 ′, SEQ ID NO: 22). SpeI, poly _{G 18} zone containing a HindIII sticky end, SpeI of pBluescriptSK + (Stratagene) HindIII, the PstI digested PCR fragment containing the T7 terminator, was ligated into the PstI site, PAT222 is generated.

Example 11: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Ragweed pollen allergen Am fused to promoter and terminator
ba I.1 coding sequence and Arabidopsis plastid psbA3 ′
Preparation of chimeric gene containing UTR Bacteriophage T7 gene 10 promoter :: Amb aI.1 :: A. Thaliana psbA3′UTR :: T7 terminator cassette is a 96 bp EcoRI, NcoI PCR fragment containing T7 promoter 1.2 kb NcoI, XbaI PCR fragment containing the Amb a I.1 cDNA and the 295 bp XbaI, PstI fragment of pPH171 containing the (A. thaliana) psbA3′UTR and the T7 terminator were pGEM-3.
It is constructed by a four-way ligation reaction to the EcoRI and PstI sites of Z (Stratagene) to generate plasmid pAT230. Am driven by T7 promoter
The ba I.1 gene cassette contains the 16S rRNA gene promoter-rbs ::
1.1 kb Hin of pAT229 containing aadA :: rps16 3'UTR cassette
dIII, EcoRI fragment and T7 promoter :: Amb a 1.1:
: psbA3'UTR: 1.6kb EcoRI, which carries the T7 terminator cassette,
The PstI pAT230 fragment was converted to pBluescript SK + (Stratagene)
Is ligated into the aadA selectable marker cassette by cloning into the HindIII and PstI sites, generating plasmid pAT234. The plastid transformation vector pAT238 ligates the 2.7 kb PstI band from pAT234 containing Amb a I.1 and the selectable marker cassette to the PstI site of pAT218, with the Amb a I.1 gene in the same orientation as the rps12 / 7ORF. It is constructed by screening for the transcribed insertion orientation.

Example 12: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Ragweed pollen allergen Am fused to promoter 0 and terminator
Preparation of chimeric gene containing baI.1 coding sequence and polyguanosine region T7 gene 10 promoter :: Amb aI.1 :: polyguanosine region :: T7 terminator and 16S rRNA gene promoter-rbs :: aadA ::
The plastid transformation vector pAT239 containing the rps16 3'UTR cassette is:
96 bp EcoRI, NcoI PCR fragment containing T7 promoter, A
mba I.1 1.2 kb NcoI containing cDNA, 30 bp XbaI of pAT222 carrying the XbaI PCR fragment and the poly _{G 18} region, the HindIII fragment, a selectable marker, 5 of pAT238 containing the T7 terminator and flanking homologous plastid regions. A 4-kb ligation reaction was performed with a 9 kb EcoRI and HindIII vector fragment, and the Amb a 1.1 gene was rps12 / 7O
It is constructed by screening for an insertion orientation that is transcribed in the same direction as the RF.

Example 13: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Dermatophagoides fused to a promoter and terminator (Derm
atophagoides) allergen Der f I coding sequence and Arabidopsis (Arab)
Preparation of a chimeric gene containing the plastid psbA3′UTR. The coding sequence of the Der f I gene is described in US Pat. No. 5,770,202. Using this sequence, Dermatophagoides farinae (Dermatophagoides
farinae) PCR primers are designed for amplification of the Der f I coding sequence from cDNA. The cDNA was reverse transcribed from total RNA extracted from D. farinae dust mite product (Glia Laboratories).
Amb a I.1). Bacteriophage T7 gene 10 promoter :: Der f I :: Arabidopsis thaliana
The psbA 3'UTR :: T7 terminator cassette is constructed in the same way as for pAT230. The T7 promoter-driven Der f I gene cassette comprises a
linked to the adA selectable marker cassette, pBluescript SK + (Stratagene)
At the HindIII and PstI sites. The plastid transformation vector is then constructed as described in Example 11.

Example 14: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Dermatophagoides fused to a promoter and terminator (Derm
atophagoides) Allergen Der f II coding sequence and Arabidopsis (Ara
Bidopsis) Preparation of a Chimeric Gene Containing the Plastid psbA3'UTR The coding sequence of the Der f II gene is described in US Pat. No. 5,770,202. Using this sequence, Dermatophagoid farinae (Dermatophagoid
es farinae) PCR primers are designed for amplification of the Der f II coding sequence from the cDNA. cDNA is D. farinae (D. farinae)
Obtained by reverse transcription (as for Amb a I.1) from total RNA extracted from a dust mite product (Glia Laboratories). Bacteriophage T7
Gene 10 promoter :: Der f II :: Arabidopsis thaliana (A. thalia
na) The psbA 3'UTR :: T7 terminator cassette is constructed in the same way as for pAT230. The T7 promoter driven Der f II gene cassette is ligated to the aadA selectable marker cassette and pBluescript SK + (Stratag
ene) into the HindIII, PstI sites. The plastid transformation vector is then constructed as described in Example 11.

Example 15: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Dermatophagoides fused to a promoter and terminator (Derm
atophagoides) allergen Der p I coding sequence and Arabidopsis (Arab)
Preparation of a Chimeric Gene Containing the Plastid psbA3′UTR The coding sequence of the Der p I gene is described in US Pat. No. 5,770,202. Using this sequence, Dermatophagoides pteronisinus (Dermatophago
PCR primers are designed for amplifying the Der pI coding sequence from C. ides pteronyssinus) cDNA. The cDNA was obtained from Dermatophagoides pteronicinus (D
.pteronyssinus) obtained by reverse transcription (as for Amb a I.1) from total RNA extracted from a dust mite product (Glia Laboratories). The bacteriophage T7 gene 10 promoter :: Der p I :: A. Thaliana psbA 3'UTR :: T7 terminator cassette is pA
It is constructed in the same manner as in the case of T230. The T7 promoter driven Der p I gene cassette is ligated to the aadA selectable marker cassette and pBluescriptS
Inserted into HindIII and PstI sites of K + (Stratagene). The plastid transformation vector is then constructed as described in Example 11.

Example 16: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Dermatophagoides fused to a promoter and terminator (Derm
atophagoides) allergen Der p II coding sequence and Arabidopsis (Arab)
Preparation of a chimeric gene containing the plastid psbA3′UTR. The coding sequence of the Der p II gene is described in US Pat. No. 5,770,202. Using this sequence, Dermatophagoides pteronisinus (Dermatophago
PCR primers are designed for amplification of the Der p II coding sequence from C. ides pteronyssinus) cDNA. The cDNA was obtained from Dermatophagoides pteronicinus (D
.pteronyssinus) obtained by reverse transcription (as for Amb a I.1) from total RNA extracted from a dust mite product (Glia Laboratories). The bacteriophage T7 gene 10 promoter :: Der p II :: A. Thaliana psbA 3'UTR :: T7 terminator cassette contains p
It is constructed in the same manner as in the case of AT230. T7 promoter driven Der p II
The gene cassette is linked to the aadA selectable marker cassette and pBluescript
It is inserted into HindIII and PstI sites of SK + (Stratagene). Then
The plastid transformation vector is constructed as described in Example 11.

Example 17: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Johnson grass pollen allergen Sorhl coding sequence and Arabidopsis plastid fused to promoter 0 and terminator
Preparation of Chimeric Gene Containing bA3'UTR The coding sequence of the Sor h I gene is described in US Pat. No. 5,480,972. A bacteriophage T7 gene 10 promoter :: Sorhl :: A. Thaliana psbA 3'UTR :: T7 terminator cassette is constructed. The T7 promoter driven Sor h I gene cassette is aad
ASelected marker cassette, linked to pBluescript SK + (Stratagene)
indIII, inserted into PstI site. The plastid transformation vector is then constructed as described in Example 11.

Example 18: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Birch pollen allergen Bet VI coding sequence and Arabidopsis plastid ps fused to promoter 0 and terminator
Production of chimeric gene containing bA3'UTR The coding sequence of the Bet VI gene was determined by Breiteneder et al. (1989) EMBO J.8
1935-1938. Bacteriophage T7 gene 10 promoter :: Bet VI :: Arabidopsis thaliana psbA3
'UTR :: T7 terminator cassette is constructed. T7 promoter driven B
The et VI gene cassette is ligated to the aadA selectable marker cassette and the pB
Inserted into the HindIII and PstI sites of luescript SK + (Stratagene). The plastid transformation vector is then constructed as described in Example 11.

Example 19: Bacteriophage T7 gene 1 in tobacco plastid transformation vector
Mosquito saliva allergen rAed fused to 0 promoter and terminator
a1 coding sequence and Arabidopsis plastid psbA3'UT
Production of Chimeric Gene Containing R The coding sequence of the rAed a1 gene is described in WO98 / 04274. A bacteriophage T7 gene 10 promoter :: rAed a1 :: A. Thaliana psbA 3'UTR :: T7 terminator cassette is constructed. The T7 promoter-driven rAed a1 gene cassette contains a
linked to the adA selectable marker cassette, pBluescript SK + (Stratagene)
At the HindIII and PstI sites. The plastid transformation vector is then constructed as described in Example 11.

Example 20: Preparation of a chimeric gene containing the glutamate decarboxylase (GAD) coding sequence fused to the bacteriophage T7 gene 10 promoter and terminator in a tobacco plastid transformation vector and the Arabidopsis plastid psbA3'UTR The human GAD gene Is obtained from Genebanl (accession number L16888). Bacteriophage T7 gene 10 promoter :: GAD :: A.thalianaps
Construct the bA3'UTR :: T7 terminator cassette. The GAD gene cassette driven by the T7 promoter is ligated to the aadA selectable marker cassette and inserted into HindIII and PstI sites of pBluescript SK + (Strategene). A plastid transformation vector is then constructed as described in Example 11.

Example 21: Preparation of a thyroglobulin coding sequence fused to the bacteriophage T7 gene 10 promoter and terminator in a tobacco plastid transformation vector and a chimeric gene containing the Arabidopsis plastid psbA3'UTR. Obtained from Genebank (accession number U93033). Bacteriophage T7 gene 10 promoter :: thyroglobulin :: A.th
Construct the aliana psbA3′UTR :: T7 terminator cassette. The thyroglobulin gene cassette driven by the T7 promoter is ligated to the aadA selectable marker cassette, and HiBlue of pBluescript SK + (stratagene) is used.
ndIII, inserted into PatI site. And plastid transformation vector
Construct as described in 11.

Example 22: Biolistic Transformation of Tobacco Plastid Genome 7 seeds of Nicotiana tabacum cv'Xanthi nc 'per plate in a 1 "cicular array were germinated on T agar and seeded 12-14. Days later, Svab, Z. and Maliga, P. (1
993) Bombard with 1 μm tungsten particles (M10, Biorad, Hercules, Calif.) Coated with plasmid DNA as described in PNAS 90,913-917. Bombarded seedlings after excision of leaves were incubated on T medium for 2 days, and 500 μg / ml spectinomycin dihydrochloride (500-500 μmol photon / m ² / s) with the hypocotyl side up in bright light (350-500 μmol photon / m ² / s). RMOP medium (Sigma, St. Louis, MO) (Svab, Z., Hajdukiewicz)
, P. and Maliga, P. (1990) PANS 87, 8526-8530).

[0136] Three to eight weeks after bombardment, resistant shoots exhibiting whitened leaves at the bottom are subcloned on the same selection medium to allow callus formation, and secondary shoots are isolated and subcloned. Complete isolation of the transformed plastid genomic copies (homoplasmicity) in independent subclones was confirmed by Southern blot (Sambrook et al.).
al., (1989) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Labor
atory Cold Sprong Harbor). BamHI / Ec
oRI-digested whole cell DNA (Metteler, IJ (1987) Plant Mol Biol Reporter 5,346
-349) was separated on a Tris-borate (TBE) agarose gel of 1 and a nylon membrane (Amer
sham) and transfer from pC8 containing part of the rps7 / 12 plastid targeting sequence.
The probe is a ³² P-labeled random-primed DNA sequence corresponding to a 7 kb BamHI / HindIII DNA fragment (see patent application WO 98/11235).
Homoplasmic shoots are transferred to MS / IBA medium containing spectinomycin (McBride
, KE et al. (1994) PNAS 91, 7301-7305), aseptically rooted and transferred to a greenhouse.

Example 23: Construction of plastid-targeted bacteriophage T7 RNA polymerase gene fused to tobacco PR-1a promoter NcoI restriction site and ATG start codon followed by rbcS gene Synthetic oligonucleotide linker (top strand: 5'-CAT GGC TTC C) containing the first seven plastid transit peptide codons from the subunit encoding) and an endogenous PstI restriction site.
TC AGT TCT TTC CTC TGC A-3 ', SEQ ID NO: 23; Bottom chain: 5'-GAG GAA AGA ACT GAG GAA GC-3', SEQ ID NO: 24)
Containing the bacteriophage T7 RNA polymerase gene (T7 Pol) driven in reading frame at the 3 'portion of the rbcS gene transit peptide coding sequence
2.8 kb PstI of GN4205 (McBride, KE et al. (1994) PNAS 91,7301-7305)
/ SacI DNA fragment, 0.9 kb NcoI / KpnI DN of pCIB296 containing tobacco PR-1a promoter with NcoI restriction site introduced at start codon
A fragment (Uknes et al. (1993) Plant Cell 5, 159-169) as well as the binary Agrobacterium transformation vector pSGCGC1 (pGPEM4 (Promega, Madison Wis.) Containing the polylinker and cloned into the SacI / HindIII site.
The 4.9 kb SfiI / KpnI and 6.6 kb SacI / SfiI fragments of TV-Hyg derivative) are ligated to construct pPH110.

Example 24: Chimeric PR by Agrobacterium-mediated leaf disk transformation
Introduction of the 1a / T7 Pol Gene into the Tobacco Nuclear Genome Hygromycin-resistant NT-pPH110 tobacco plants were isolated from N. tabacum'X as described.
Regenerate from shoots obtained following co-culture of anthi 'and "NahG" (Friedrich et al. (1995) Plant Mol Biol 29: 959-68) leaf discs with the pV110 binary vector carried by the GV3101 Agrobacterium. .

For each transgenic line, repeat about 2-3 cm ² leaf punches ca.300
Incubate for 2 days in 3 ml BTH (5.6 mg / 10 ml) or sterile distilled water under μmol / m ² / s radiant flux density. Collect leaf material, flash freeze in liquid nitrogen,
Grind. Total RNA was extracted (Verwoerd et al. (1989) NAR 17,2362) and using a radiolabeled T7 RNA polymerase gene probe (Ward et al. (1991)
) Northern blot analysis is performed as described in The plant Cell 3,1085-1094). Transferring 19 NT-110X (Xanthi gene background) and 7 NT-110N (NahG gene background) T1 plants showing a range of T7 Pol expression to the greenhouse,
Self-pollinate. Progeny that segregate 3: 1 for linked hygromycin resistance markers are selfed and homozygous T2 lines are selected.

Example 25: Induction of ragweed allergen expression in plastids of transgenic plants Homozygous NT-110X and NT- containing PR-1a-T7 RNA Pol construct
The 110N plant is used to pollinate a homoplasmic plastid transformed line with the pAT238 and pAT239 constructs inherited from the maternal line. Nt
pAT238 × NT-110X or NT 110N, and NT pAT239 × NT-110X or NT 110N F1 progeny (they are heterozygous for the PR-1 / T7 polymerase nuclear expression cassette and have T7 / Amb aI.1 plastid expression Germinate on the soil (which was homoplasmic for the cassette).

When reaching a height of 20-40 cm, the T7 of the Amb a.1 gene, which is localized in plastids
The inducer compound BTH, which induces Pol-regulated expression, is sprayed. Immediately prior to induction and 8 hours and 1, 2, 3, 7 and 14 or 28 days after induction, the plants are harvested and flash frozen in liquid nitrogen. Der f I, Sor
Applies to transgenic plants containing the hI, Bet VI and rAed a1 allergen genes, and the GAD and thyroglobulin genes.

Example 26: Quantification of antigen expression and content of transgenic plants Total RNA was extracted (Verwoerd et al. (1989) NAR 17,2362) and radiolabeled specific for the Amb a I.1 gene Northern blot analysis is performed as described using the probe (Ward et al. (1991) The Plant Cell 3, 1085-1094). To determine the amount of Amb a I.1 present in the tissue of the transgenic plant, chemiluminescence (
(Amersham) Western blot analysis was performed according to manufacturer's instructions and Harlow and Lane (198
8) Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spri
Antisera against Amb a I.1 protein and purified according to ng Harbor
Performed using Amb a I.1 protein standard. A similar procedure is followed by Der f I, S
Applies to or h I and Bet VI, rAed a 1 allergen, and GAD and thyroglobulin.

Example 27: Induction of oral tolerance in rats Female Lewis or Wistar Furth rats weighing 150 to 220 g (6-8 weeks of age) are used.
Rats are immunized in both posterior footpads with 50 μg of Guinea pig antigen emulsified in Complete Freund's Adjuvant (CFA). In one experiment, 50 μg of ovalbumin (Sigma) is added to the emulsified antigen and injected as well.

For the induction of oral tolerance, the transgenic plants of the invention are administered to rats.
Feed 5 times at 3 day intervals. Nine days after immunization, rats are sacrificed and popliteal lymph nodes are removed. A single cell suspension is prepared by pressing the lymph nodes through a stainless steel mesh. All of the 105 lymph node cells (LNC) were harvested from irradiated (2000 Rad) or untreated LNC and round bottoms from the indicated number of fed rats.
Culture in duplicate in 96-well plates (Costar). Antigen (50 μg / ml) is added to the culture in a volume of 20 μl. Incubate the culture for 80 hours and allow for the last 16 hours of culture.
Pulse with 1 μCi [3H] TdR / well. The culture is then collected on an automatic cell harvester and counted on a standard liquid scintillation counter. The percentage of primed LNC (PLNC) proliferation is then calculated. Growth medium (Proliferation
Media) RPMI (Gibco) is used for all experiments. The medium is filtered and sterilized after adding 2 × 10 −5 M 2-mercaptoethanol, 1 sodium pyruvate, 1 penicillin and streptomycin, 1 non-essential amino acid, and 1 autologous serum.

Example 28: Measurement of Antibodies A. Serum Levels of Antibodies A solid-phase enzyme-linked immunosorbent assay (ELISA) is used to quantitate antibody titers to antigen. Incubate the microtiter plate with 0.1 ml per well of 10 μg antigen / ml in double distilled water. Incubate the plate for 18 hours at 25 ° C. 3 times
After washing with PBS / Tween-20 (Bio-Rad) pH 7.5, the plate was washed with 3 BSA / PBS for 2 hours 37
Incubate at 0 ° C., wash twice, and add 100 μl of diluted serum in 4 replicates. Incubate the plate for 2 hours at 37 ° C. After three rinses with PBS / Tween-20, plates were diluted with peroxidase-conjugated goose anti-rat IgG antibody (Tago, USA) diluted 1: 1000 in 100 μl / well of 1 BSA / PBS. Incubate together at 25 ° C for 1 hour.
D-phenylenediamine (0.4mg / ml phosphate) citrate buffer containing 30H2O2 for color reaction
, pH 5.0). The reaction was stopped by adding 0.4N H2 SO _4, read the OD492nm ELISA reader.

B. In Vitro Measurement of Antibody Production Popliteal and spleen LNCs were obtained from fed, untreated and challenged rats and were given alone or irradiated (2000 rad) at a concentration of 107 cells per ml Petri dish. Sow with other RLNC, as directed. Culture the antigen in growth medium (20 μg / ml)
Maintain and collect in the incubator for 3 days, with or without. The diluted supernatant is used to test the in vitro production and secretion of IgG antibodies, and antibody production is measured using an ELISA test as described above.

Example 29: Induction of oral tolerance in mice Female Balb / c mice, 6 to 8 weeks of age, are used. To induce oral tolerance, mice are fed an extract from tobacco plants expressing recombinant ragweed from the T7 / Amb a I.1 plastid expression cassette according to Example 22.

More specifically, mice were treated with fractions 1 or 2 of tobacco plants transformed with the T7 / Amb a I.1 plastid expression cassette, or fraction 1 from control tobacco plants not transformed as control. Or feed fraction 2. Untreated animals are divided into three recombinant ragweed-fed groups (0.1, 1, and 10 mg recombinant ragweed / day) and one non-recombinant tobacco-fed group (as controls). Each group contains 8 animals. Oral administration of antigen to animals is initiated after the first 10 days of acclimatization to the housing facility. Animals are fed the corresponding tobacco fraction or phosphate buffered saline prepared with H ₂ O to a final volume of 0.2-0.5 ml once daily for 5-8 days.

[0149] Induction of oral tolerization was achieved with the last 4 oral feedings of tobacco-expressed allergen.
Evaluate after days. All four groups of mice are sensitized three times at two week intervals by intraperitoneal injection of recombinant ragweed (10 μg / animal). To demonstrate that mice can mount an immune response against other antigens, each mouse was also challenged with ovalbumin (10 μg / animal; Sigma Chemical, St. Louis, MO, USA) in addition to recombinant ragweed. Sensitize. The antigen was dissolved in 0.9 saline and mixed with aluminum hydroxide (2: 1; Alu Gel S Serva, Fein
biochemica GmbH, Heidelberg, Germany), the desired concentration in a final volume of 200 μl / mouse.

Serum samples are collected four times for quantification of antigen-specific IgE, IgG1 and IgG2a levels: once prior to the start of oral administration of antigen and at appropriate time points for each of the three immunizations. Aliquot the serum and store at -20 ° C until assay.

All serum samples are adsorbed and a solid-phase enzyme-linked immunosorbent assay (ELIZA) is performed for quantification of recombinant ragweed and ovalbumin-specific serum IgE, IgG1, and IgG2a levels. 96-well microtiter plates (Immunoplates M
axisorp brand Surface, Nunc, Roskilde, Denmark) with 0.1 ml per well of the corresponding antigen (1-10 μg / ml) in 0.015 M sodium carbonate bicarbonate buffer (pH 9) for 2 hours
Incubate at 37 ° C followed by overnight at 4 ° C.

All washes are performed with 0.8 saline containing 0.05 Tween-solution (pH 7.4; wash buffer; Sigma Chemical). Serum samples, labeled antibodies and reagents are diluted in wash buffer containing 1% heat-inactivated fetal bovine serum unless otherwise specified, all incubations are performed in moist air, and reaction volumes are 100 μl / well .
After coating, wash the plate three times with wash buffer. Serum samples are processed into antigen-coated wells in 3-fold serial dilutions and incubated at 37 ° C for 2 hours. After three washes in wash buffer, the corresponding biotin-labeled labeled antibody (So.Biotechno
logy Assoc., Birmingham, AL.USA; Binding Site, Birmingham, UK)
Treat for 1 hour at ° C.

After washing, horseradish peroxidase-labeled avidin D is treated to detect immunoreactivity (1: 3000, 60 minutes, 30 ° C .; Vector Lab., Burlingame, Calif.,
USA). After rewashing, the plate is washed three more times with citrate buffer (substrate buffer).
, pH 5). Enzyme activity o-phenylenediamine (Sigma Chemical)
Detection in substrate buffer (0.5 mg / ml) and hydrogen peroxide (1 μl / ml) at 4
Stop with NH ₂ SO ₄ (50 μl / well; Merck KgaA, Darmstadt, Germany). The optimal density is quantified spectrophotometrically at 492 nm by an ELISA reader. Final results are expressed in relative ELISA units (REU) compared to a standard serum pool (arbitrarily assigned 100 REU) or in absolute protein concentrations (μg / ml).

A similar procedure for testing oral tolerance was described in Der f I, Sorh I, Be.
Applies to transgenic plants containing the tVI and rAed a1 allergen genes, and the GAD and thyroglobulin genes or any other antigen of the invention.

The induction of oral tolerance for house dust mite extract was
to et al., Immunology 95: 193-199, 1998. The house dust mite test in humans is described by Passalacqua et al., Lancet, 351: 629-632, 1998.

Example 30: Chemically regulated expression of the human BPI gene in plant plastids Construction of a Plastid Transformation Vector Containing the BPI Gene Under the Control of a Bacteriophage T7 RNA Polymerase-Responsive Promoter Element The coding sequence for the human BPI gene (as described in US Pat. It is cloned as an EcoRI restriction fragment (containing the 5 'portion of the cDNA and incorporating an initiation codon at the NcoI site) and as a 1288 bp EcoRI / XbaI restriction fragment (including the 3' portion of the cDNA sequence and a termination codon). These fragments are gel-purified and ligated in a three step reaction to a 7693 bp NcoI / XbaI fragment from plasmid pT7 GUS. About the obtained plasmid (pT7 BPI), the sequence 5'-GGG AGA CCA CAA CGG TTT CCC TCT AG-
Plasmid pET3a with a chimeric 5 'untranslated leader containing 3' (SEQ ID NO: 25) (
Novagen), BPI gene downstream of the T7 gene 10 promoter, plasmid pC
The linker sequence 5'-CGAGG-3 'from the StuI linker of 8 and the 5' leader of the tobacco plastid psbA gene modified to include an NcoI restriction site at the initiation codon (underlined) Sequence 5'-GGG AGT CCC
TGA TGA TTA AAT AAA CCA AGA TTT TA C CAT G
The inclusion of G- 3 '(SEQ ID NO: 26) is confirmed by DNA sequencing and restriction analysis.

The pT7 GUS was dephosphorylated with a 386 bp gel purified EcoRI / NcoI fragment of the plasmid pPH142 containing the chimeric T7 promoter / psbA5 ′ leader, using the GUS plasmid transformation vector pC8 (International Patent Application WO 98/11235). It is constructed by ligating to a gel purified 9241 bp NcoI / EcoRI fragment. Plasmid pPH142 was digested with BsaBI to plasmid pPH136 (2 hours at 60 ° C.), and the gel purified and linearized plasmid DNA was re-digested with BsmFI (1
Time 37 ° C), treatment with alkaline phosphatase (1 hour 37 ° C followed by phenol extraction) and the resulting gel-purified 3398 bp fragment was phosphorylated with T4 polynucleotide kinase (1 hour 37 ° C). Oligonucleotide T73a U
(5′-CGA TCC CGC GAA ATT AAT ACG ACT CACT
AT AGG GAG ACC ACA ACG GTT TCC C-3 ', SEQ ID NO: 2
7) and T73a L (5'-TAG AGG GAA ACC GTT GTG GT
C TCC CTA TAG TGA GTC GTA TTA ATT TCG CGG
GAT CG-3 ′, SEQ ID NO: 28) is constructed by ligating to a synthetic double-stranded oligonucleotide created by annealing.

The plasmid pPH136 DNA is amplified in a strain of E. coli (DM-1, Strategene, La Jolla, California) lacking dam methylase activity to allow digestion with BsaBI. Next, pPH136 was converted to a 3428 bp gel-purified StuI / Nc from plasmid pPH120.
The oI fragment was ligated to the oligonucleotide minpsb U (5'-GGG AGT CC
C TGA TGA TTA AAT AAA CCA AGA TTT TAC-3 ',
SEQ ID NO: 29) and minpsb L (5′-CAT GGT AAA ATC TT
G GTT TAT TTA ATC ATC AGG GAC TCC C-3 ′, SEQ ID NO: 30), constructed by ligating to a synthetic double-stranded oligonucleotide created by annealing. This oligonucleotide contains a 38 nt portion of the 5 'untranslated leader sequence of the tobacco psbA gene which is implicated in the up-regulation of the psbA gene product in an in vitro plastid translation system (Hirose and Sugiur
a, EMBO J. 15: 1687-1695.1996).

PPH120 contains a modified portion of the aadA gene driven by the psbA promoter and the divergent T7-lac promoter of plasmid pC8 (derived from the chimeric ribosome binding site from Novagen plasmid pET21d and the tobacco plastid rbcL gene). Into which a fragment of yeast DNA is inserted to inhibit the low-level bidirectional transcriptional activity of the psbA promoter, which is characteristic of vectors derived from plasmid pPRV111A (Allison et al., EMBO J. 1996 Jun 3; 15 (11): 2802-9) .PPH1
20 is a 256 bp EcoRI / HincII fragment of the Saccharomyces cerevisiae LEU2 gene, 2645 bp EcoRI / Dralll of pPH119, and 569 bp Dralll / pPH119 of pPH119.
Constructed by ligating the Klenow-blunted EcoRI fragment. Next, PPH119 is constructed by digesting plasmid pPH118 with StuI and ligating to remove the two StuI linkers present in the pC8-derived chimeric T7 promoter of pPH118. PPH118 contains the 235 bp SpeI / NcoI fragment of pC8, which was isolated and gel purified and the cloning vector pGEM
Ligation to 5Zf (+) (Promega, Medison WI), which is digested with NcoI and SpeI.

II: Biolistic Transformation of the Tobacco Plastid Genome with the Plasmid pT7 BPI Transformation of the plastid genome is performed as described in Example 22. Resistant shoots that exhibit whitened leaves at the bottom are subcloned 3 to 8 weeks after bombardment on a similar selective medium to allow callus formation, and the secondary shoots are isolated and subcloned. Complete isolation of transformed plastid genomic copies (homoplasmicity) in independent subclones was confirmed by Southern blot (Sambrook et al., (1)
989) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory,
(Cold Spring Harbor).

BamHI / EcoRI-digested whole cell DNA (Metteler, IJ (1987) Plant Mol Bi
ol Reporter 5,346-349) was separated on one Tris-borate (TBE) agarose gel, transferred to a nylon membrane (Amersham) and 0.7 kb BamHI / pB from pC8 containing part of the rps7 / 12 plastid targeting sequence. The probe is a ³² P-labeled random-primed DNA sequence corresponding to the HindIII DNA fragment. desired
A homoplasmic shoot containing a 1.25 kb fragment and lacking the wild-type 3.3 kb fragment was transformed into an MS / IBA medium containing spectinomycin (McBride, KE et al.
1994) Root aseptically on PNAS 91,7301-7305) and move to greenhouse.

III: B in tobacco plants transformed with the plasmid pT7 BPI and carrying the bacteriophage T7 RNA polymerase gene with a plastid targeting sequence in the nuclear genome under the control of the BTH-responsive PR-1a promoter
Chemical induction of PI expression A homoplasmic Nt pT7 BPI plant of line C35BPI-5B-4
Pollinated by 10X6b-5 and seeds are tested for maternal inheritance of the spectinomycin resistance marker carried by the transformation plasmid pT7 BPI. These F1
Additional seeds of progeny are germinated in 6-inch clay pot soil. Eight weeks after sowing, the plants are sprayed with 1.0 mM BTH foliage until dripping.

Tissues are harvested at 0, 1, 2, 3, 7, 14, 21, 28 and 35 day intervals after BTH application for analysis of BPI accumulation. Simultaneously germinate control plants sprayed with water or inert components of the same strain and C35BPI-5B-4 plants that have only the plastid BPI gene and no chemically inducible transactivator Using a similar time schedule. BPI accumulation is assayed by standard techniques of Northern hybridization (mRNA) and Western blot (protein). In addition, plant-expressed BPI can be used for gram negative bacteria (e.g., E.col.
To assess the ability to inhibit exponential exponential growth in liquid medium of i), an activity assay is performed using leaf extracts.

Example 31: Construction of an expression cassette for nuclear expression of a transgene in plants A gene sequence intended for expression in a transgenic plant is first placed behind a suitable promoter and preferably with a suitable transcription terminator Upstream in the expression cassette. All the requirements for the construction of a plant expression cassette apply to DNA molecules of the invention, in particular to DNA molecules encoding transactivators or therapeutically active proteins, using techniques well known in the art. Implemented using

Selection of Promoter The spatial and temporal expression pattern of the transgene in the transgenic plant is determined by the choice of promoter used for the expression cassette. The selected promoter causes the transgene to be expressed in a particular cell type (leaf epidermal cells, mesophyll cells, root cortex cells) or in a particular tissue or organ (e.g., root, leaf or flower), and this selection involves transgene biosynthesis. Affects the desired localization of Alternatively, the selected promoter may drive the expression of the transgene under a light-induced or other temporally regulated promoter. Alternatively or alternatively, the selected promoter can be chemically regulated. From this, desired,
Only when triggered by treatment with a chemical inducer does the possibility of inducing the expression of the transgene be offered.

3 ′ Regulatory Sequences Various 3 ′ regulatory sequences are available for use in expression cassettes. In the plant nucleus, these sequences are responsible for, for example, termination of transcription across the transgene and its correct polyadenylation. Suitable transcription terminators and those known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants.

Sequences for Enhancement or Regulation of Expression A number of sequences enhance gene expression from within the transcription unit, and these sequences bind to the transgene to increase its expression in transgenic plants. It has been discovered that it can be used.

A variety of intron sequences have been shown to enhance expression, especially in monocot plant cells. For example, it has been discovered that the intron of the maize Adh1 gene significantly enhances the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective in enhancing the expression of fusion constructs made with the chloramphenicol acetyltransferase gene (Callis et al., Genes Dvelop 1:
1183-1200 (1987)). In a similar experimental system, introns from the maize bronze1 gene had a similar effect in enhancing expression (Callis et a
l. supra). Intron sequences have been routinely incorporated into plant transformation vectors, typically into untranslated leaders.

Many non-translated leader sequences from the virus are known to enhance expression and are particularly effective in dicotyledonous plant cells. In particular, tobacco mosaic virus (TMV Ω-sequence), maize repressed mottle virus (Maize Chlorotic M
ottle Virus, MCMV), and leader sequences from alfalfa mosaic virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al. Nucl. Acids Res. 15: 8693-8711 ( 1987); Skuzeski et al. Plant
Molec. Biol. 15; 65-79 (1990)).

Example 32: Targeting of gene products in cells Various mechanisms for the targeting of gene products are known to exist in plants, and the sequences that control the functioning of these mechanisms are described in some detail. Has been characterized. For example, the targeting of gene products to plastids, e.g., chloroplasts, is controlled by signal sequences found at the amino terminus of various proteins, which are cleaved during import into the chloroplast to yield the mature protein (e.g., Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products and affect the transfer of the heterologous product into the chloroplast (van den Broeck et al. Nature 313: 358-363 (1985)). . The DNA encoding the appropriate signal sequence is the 5 'end of cDNA encoding RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein and many other proteins known to be localized in chloroplasts. Can be isolated from The signal sequence chosen should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site required for cleavage.

In some cases, this requirement is met by adding a small number of amino acids between the cleavage site and the transgene ATG, or replacing certain amino acids in the transgene sequence. Fusions constructed for transfer to chloroplasts are obtained by in vitro translation of the in vitro transcribed construct, followed by in vitro chloroplast uptake.
(Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular
Biology, Elsevier.pp 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205: 446
-453 (1986)), the efficiency of incorporation into chloroplasts can be tested. Thus, any of these signal sequences can be fused to the nucleotide sequence encoding the transactivator, targeting the transactivator to the chloroplast. The mechanism described above for intracellular targeting uses the cognate promoter with its cognate promoter to provide the goal of targeting a particular cell under the transcriptional control of a promoter having an expression pattern different from that of the promoter from which the targeting signal is derived. It is used not only for binding to a heterologous promoter but also for binding to a heterologous promoter.

Other gene products are localized to other subcellular organelles, such as mitochondria and peroxisomes (eg, Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can be engineered to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATP-ase and the specific aspartate aminotransferase isoform of mitochondria. Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-651 for targeting to cellular protein granules).
6 (1985)). In addition, sequences have been characterized that cause the targeting of the gene product to other cellular compartments. The amino terminal sequence is ER,
It is responsible for targeting to apoplasts and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, the amino terminal sequence, in conjunction with the carboxy terminal sequence, is responsible for targeting the gene product to the vacuole (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

Example 33: Example of construction of an expression cassette The present invention encompasses expression of a DNA molecule of the present invention under the control of any promoter that can be expressed in plants, regardless of the origin of the promoter. The DNA molecule of the present invention is then inserted into any expression cassette using techniques well known in the art. These expression cassettes can then be easily delivered to the plant transformation vectors described below. The invention further encompasses the use of any plant-expressible promoter associated with any additional sequences required or selected for expression of the transgene. Such sequences include transcription terminators, exogenous sequences that enhance expression (such as introns [eg, Adh intron 1], viral sequences [eg, TMV-Ω]), and targeting of gene products to specific organelles and cellular compartments. Including but not limited to the sequences contemplated for

Constitutive expression: CaMV 35S promoter The construction of plasmid pCGN1761 is described in published patent application EP0392225. pCGN1761 contains a "double" 35S promoter and a tml transcription terminator, has a specific EcoRI site between the promoter and the terminator, and has a pUC-type backbone. A derivative of pCGN1761 is constructed that has a modified polylinker containing NotI and XhoI sites in addition to the existing EcoRI site. This derivative is named pCGN1761ENX. pCGN1761ENX is useful for cloning cDNA or gene sequences (including microbial ORF sequences) within its polylinker for its expression under the control of the 35S promoter in transgenic plants.

The complete 35S promoter-gene sequence-tml terminator cassette of such a construct was constructed with a 5 'site to the promoter by HindIII, SphI, SalI, and XbaI and a 3' site to the terminator by XbaI, BamHI and Bgll. It can be excised for delivery to a transformation vector. In addition, the double 35S promoter fragment contains HindIII, SphI,
5 ′ excision with SalI, XbaI or PstI and 3 ′ excision at any polylinker restriction site (EcoRI, NotI or XhoI) can be removed to replace other promoters.

Expression Under Chemically Regulatable Promoter (1) PR1-a Promoter This section describes the replacement of the double 35S promoter with any selected promoter in pCGN1761ENX; PR-1a promoter is described. The selected promoter is preferably excised from its source with a restriction enzyme, but can alternatively be PCR-amplified using primers having appropriate terminal restriction sites. If PCR-amplification is performed, the promoter should be resequenced after cloning of the amplified promoter in the target vector to confirm amplification errors.

A chemically regulatable tobacco PR-1a promoter was added to plasmid pCIB1004 (EP0
332104) and delivered to the plasmid pCGN1761ENX. Nco for pCIB1004
The 3 ′ overhang of the linear fragment obtained by digestion with I is blunt-ended by treating with T4 DNA polymerase. And the fragment is HindII
The resulting fragment containing the PR-1a promoter is gel-purified and cloned into pCGN1761ENX from which the double 35S promoter has been removed.
It was cut with XhoI, blunt-ended with T4 polymerase, and subsequently
This is accomplished by cutting and isolating the larger fragment containing the vector-terminator, into which the pCIB1004 promoter fragment is to be cloned, with HindIII.

This has the PR-1a promoter and tml terminator, and an intervening polylinker with unique EcoRI and NotI sites.
This gives rise to the pCGN1761ENX derivative. The nucleotide sequence encoding the DNA molecule of the present invention is inserted into this vector and the fusion product (i.e., promoter-gene-terminator) is then delivered to any selected transformation vector, including those described in the present application. And provides for chemically inducible expression of the transactivator.

(2) Ethanol-Inducible Promoters Promoters inducible by certain alcohols or ketones, such as ethanol, are also used to enable inducible expression of the transgenes of the invention. Such a promoter is, for example, the alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat Biotechnol 16: 177-180). A.nid
In ulans, the alcA gene encodes alcohol dehydrogenase I and its expression is regulated by the AlcR transcription factor in the presence of a chemical inducer. For the purposes of the present invention, the CAT gene sequence in the plasmid palkA: CAT containing the alcA gene promoter sequence fused to the minimal 35S promoter (Caddick et al.
1998) Nat Biotechnol 16: 177-180) to replace the nucleotide sequence encoding the transgene to form an expression cassette having the nucleotide sequence encoding the transgene under the control of the alcA gene promoter. .

This is performed using methods well known in the art. In a preferred embodiment, the minimal minimal 35S promoter is replaced with any convenient minimal promoter, such as, for example, the maize Bronze-1 minimal promoter (Roth et al. (1991) The Plant Cell 3: 317-325). The termination signal in palcA: CAT can also be replaced with other termination signals well known in the art.
The resulting construct is transformed into the desired plant species. The alcR gene is also included in plants comprising a nucleotide sequence encoding a DNA molecule of the present invention fused to the alcA gene promoter, and expression of the alcR gene is determined by a plant known in the art or described herein. It can be controlled by any promoter suitable for expression in. The tissue- or organ-specificity of the alcR gene product is then achieved, leading to the inducible tissue- or organ-specificity of the transgene. Any termination signal in the art is suitable for the alcR expression cassette.

(3) Glucocorticoid-inducible promoter Induction of expression of the DNA molecules of the invention using steroid hormone-based systems is also contemplated. For example, using the glucocorticoid-mediated induction system (Aoya
ma and Chua (1997) The Plant JouRNAl 11: 605-612), gene expression, for example, synthetic glucocorticoids, preferably dexamethasone, preferably 0.1 mM to 1 mM
, More preferably by application of glucocorticoid at a concentration ranging from 10 mM to 100 mM. For the purposes of the present invention, the transgene under the control of six copies of the GAL4 upstream activating sequence, replacing the luciferase gene sequence with the nucleotide sequence encoding the transgene and fused to a 35S minimal promoter. An expression cassette having the encoding nucleotide sequence is formed. This is performed using methods well known in the art. In a preferred embodiment, the minimal 35S promoter is, for example, a maize Bronze-1 minimal promoter (Roth et al. (1991) Th.
e Replace with any convenient minimal promoter, such as Plant Cell 3: 317-325).

The termination signal can also be replaced with other termination signals known in the art. The resulting construct is transformed into the desired plant species. The trans-activation factor is fused to the transactivation domain of the herpes virus protein VP16 (Triezenberg et al. (1998) Genes Devel. 2: 718-7
29), fused to the hormone-binding domain of rat glucocorticoid receptor
(Picard et al. (1988) Cell 54: 1073-1080), GAL-4 DNA-binding domain (Keegan
et al. (1986) Science 231: 699-704).

[0183] Expression of the fusion protein is controlled by any suitable promoter known in the art or described herein for expression in plants. This expression cassette is also included in plants containing a nucleotide sequence encoding the transgene fused to the 6xGAL4 / minimal promoter. The tissue- or organ-specificity of the fusion protein is then achieved, leading to the inducible tissue- or organ-specificity of the transgene. Any termination signals known in the art are also suitable for expression cassettes containing the fusion protein.

Constitutive Expression: Actin Promoters Several isoforms of actin are expressed in most cell types, and consequently the actin promoter is known to be a good choice for a constitutive promoter. In particular, the promoter from the rice Act1 gene has been cloned and its properties have been characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). A 1.3 kb fragment of the promoter has been found to contain all the regulatory elements required for expression in rice protoplasts. In addition, a number of expression vectors based on the Act1 promoter have been constructed, especially for use in monocots (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These include the Act-intron 1, the Adh1 5 'flanking sequence, and the sequences from the Adh1-intron 1 (from the corn alcohol dehydrogenase gene) and the CaMV 35S promoter.

Vectors that exhibit the highest expression are 35S and the Act1 intron or a fusion of the Act1 5 'flanking sequence and the Act1 intron. Optimization of sequences around the initiating ATG (of the GUS receptor gene) also enhances expression. McElroy et al. (Mol.Gen.Genet.2
31: 150-160 (1991)) can be easily modified for expression of the nucleotide sequences of the present invention and are particularly suitable for use in monocotyledonous hosts. For example, a fragment containing a promoter
It can be removed from the roy construct and used to replace the double 35S promoter in pCGN1761ENX, which is available for insertion or specific gene sequences.

The fusion gene thus constructed can be subsequently delivered to a suitable transformation vector. In another report, the rice Act1 promoter with the first intron was also found to direct high expression in cultured barley cells (Chibbar
et al. Plant Cell Rwp. 12: 506-509 (1993)). A nucleotide sequence encoding a DNA molecule of the present invention is inserted downstream of such a promoter and a fusion product (i.e., promoter-gene-terminator)
Is then delivered to any selected transformation vector, including those described herein.

Constitutive Expression: Ubiquitin Promoter Ubiquitin is another gene product known to accumulate in many cell types, the promoter of which has been cloned from several species for use in transgenic plants. (Eg, sunflower-Binet et al. Plant Sci.
ence 79: 87-94 (1991), corn-Christensen et al. Plant Molec. Biol. 12
: 619-632 (1989)). The corn ubiquitin promoter has been developed in a transgenic monocot system, and its sequences and vectors constructed for the transformation of monocot plants have been published in patent application EP0342926. Furthermore, Taylor et a
l. (Plant Cell Rep. 12: 491-495 (1993)) is a vector containing the corn ubiquitin promoter and the first intron (pAHC25), and the cell suspension of many monocots when introduced by microprojectile bombardment. It describes its high activity in turbidity.

The ubiquitin promoter is particularly suitable for the expression of nucleotide sequences in transgenic plants, especially monocots. Suitable vectors are pA modified by the introduction of a suitable ubiquitin promoter and / or intron sequence.
A derivative of HC25 or any of the transformation vectors described herein. Arabidopsis ubiquitin 3 (UBQ3) gene promoter (Norris et al. (1993
) Plant Mol Biol21: 895-906) are also suitable for the expression of the transgenes of the invention in plants. The nucleotide sequence encoding the transgene is inserted into any of these vectors, and the fusion product (ie, promoter-gene-terminator) is used to transform a plant and the transgene is constitutively expressed.

Specific Expression in Roots The preferred pattern of expression for the transgenes of the present invention is expression in roots. Suitable root promoters are as described by de Framond (FEBS 290: 103-106 (1991)) and in published patent application EP 0 452 269. The promoter is delivered to a suitable vector, such as pCGN1761ENX, and the nucleotide sequence encoding the transgene is inserted into such a vector. The complete promoter-gene-terminator cassette is then delivered to a transformation vector of interest.

Wound-Inducible Promoters Wound-inducible promoters are particularly suitable for transgene expression. Many such promoters have been described (e.g., Xu et al. Plant Molec. Biol. 22:57
3-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989). Rohmeier & Lehle, P
lant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-14.
2 (1993), Warner et al. Plant J. 3: 191-201 (1993)), all suitable for use in the present invention.

Logemann et al. (Supra) describe the 5 ′ upstream sequence of the dicotyledonous potato wun1 gene. Xu et al. (Supra) describe that a wound-inducible promoter from the dicotyledon potato (pin2) is also active in monocot rice. Further Rohr
mier & Lehle (supra) describes the cloning of maize Wipl cDNA which is wound induced and can be used to isolate the cognate promoter using standard techniques. Similarly, Firek et al. (Supra) and Warner (supra) describe a wound-inducing gene from the monocotyledonous plant Asparagus officinalis, which is expressed at the site of a local wound or pathogen invasion. Using cloning techniques well known in the art, these promoters can be delivered to a suitable vector, fused to the nucleotide sequence encoding the transgene, and expressed in order to express these genes at the site of the pest infection. Can be used.

Preferred expression in the marrow Patent application WO 93/07278 describes the isolation of a maize trpA gene that is selectively expressed in marrow cells. Using standard molecular biology techniques, the promoter or a portion thereof can be delivered to a vector such as pCGN1761;
The 35S promoter can be replaced and used to drive expression of the transgenes of the invention as preferred in the marrow. Indeed, fragments containing a pith-preferred promoter or portion thereof can be delivered to any vector and modified for convenience in transgenic plants. Preferred expression in the pith of the nucleotide sequence encoding the transgene is achieved by inserting the nucleotide sequence encoding the transgene into such a vector.

Pollen-Specific Expression Patent application WO 93/97278 describes maize calcium expressed in pollen cells.
The isolation of the dependent protein kinase (CDPK) gene is further described. Its gene sequence and promoter span 1400 bp from the start of transcription. Using standard molecular biology techniques to deliver the promoter, or a portion thereof, to a vector such as pCGN1761, displace the 35S promoter and drive expression of the transgene of the invention in a pollen-specific manner. Can be used for In fact, a pollen-specific promoter or a fragment containing a portion thereof can be delivered to any vector and modified for convenience in transgenic plants.

Leaf-Specific Expression A maize gene encoding phosphoenol carboxylase (PEPC) was described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biology techniques, the promoter for this gene is used to drive expression of the transgene of the invention in a leaf-specific manner in the transgenic plant.

Expression of chloroplast targeting Chen & Jagendorf (J. Biol. Chem. 268: 2363-2367 (1993) described the successful use of chloroplast transit peptides for the transfer of heterologous transgenes. This peptide used is a transit peptide from the rbcS gene from Nicotiana plumbaginifolia (Poulsen et al. Mol. Gen. Genet. 205: 193-200 (1986
)). Using restriction enzymes DraI and SphI, or Tsp5091 and SphI,
The DNA sequence encoding this transit peptide is
It can be excised from S-8B (Poulsen et al., Supra) and manipulated for use with any of the constructs described above. The DraI-SphI fragment is the starting rbcS AT
G from position -58 to the first amino acid (methionine) of the mature peptide immediately after the import cleavage site (including it), while the Tsp5091-SphI fragment is the first amino acid of the mature peptide from position -8 relative to the starting rbcSATG. Spans amino acids (including it).

[0196] These fragments are then replaced with a selected promoter (eg, 35S, PR-
1a, actin, ubiquitin, etc.) can be suitably inserted into the polylinker of any selected expression cassette, resulting in a transcriptional fusion to the untranslated leader.
Insertion in a precise fusion of the nucleotide sequence encoding the transgene downstream of the transit peptide is possible. This type of construction is routine in the art. For example, while the Dral end is already blunt, the 5 'Tsp5091 site
4 can be blunt-ended by polymerase treatment, or alternatively can be ligated to a linker or adapter sequence to facilitate fusion to the selected promoter. The 3 'SphI site may be maintained as such, or alternatively, its selection may be made in such a way as to make suitable restriction sites available for subsequent insertion of the nucleotide sequence encoding the transactivator. Can be ligated to an adapter of a linker sequence to facilitate insertion into the vector.

The ATG at the SphI site is maintained and ideally contains the first ATG of the DNA molecule. Chen & Jagendorf (supra) provide a consensus sequence for ideal cleavage for chloroplast transfer, in which case the first part of the mature protein is preferably methionine. Subsequent parts are more diverse, and amino acids cannot be so critical. In any case, Bartlett et al. (Edelmann et al (Eds.) Me
thods in Chloroplast Molecular Biology, Elesevier.pp 1081-1091 (1982) :) and in vitro using methods described by Wasmann et al. (Mol.Gen. Genet. 205: 446-453 (1986)). The efficiency of the transfer can be evaluated. Typically, the best approach is to use DNA containing modifications only when it is clear that such fusions are not transferred to the chloroplast with high efficiency without modification at the amino terminus
Is to create a fusion using the molecule, in which case the modification is in the established literature (Che
n & Jagendorf, supra; Wasman et al. supra; Ko & Ko, J. Biol. Chem. 267: 13910-13916 (19
92)).

A preferred vector is the Dral-Sph encoding fragment from prbSS-8B.
Constructed by delivering the transit peptide into the cloning vector pCGN1761ENX / Sph-. This plasmid was cut with EcoRI, and the end was T4 DNA.
It is made blunt by treatment with a polymerase. Plasmid prbcS-8B is cut with SphI and ligated to the annealed molecular adapter.
The resulting product is phosphorylated by treating the 5 'end with T4 kinase. The blunt-end-ex-Ec of the modified vector was subsequently cut by DraI.
Release the transit peptide encoding the fragment ligated to the oRI site. Clones originating at the 5 'end of the insert adjacent to the 3' end of the 35S promoter are identified by sequencing.

These clones contain the mature protein A from position -58 for rbcS ATG.
The SphI site, which spans the TG and is unique at that position, and the newly created EcoRI site, and the RNotI and XhoI where pCGN1761ENX is present
It has a DNA fusion of the 35S leader sequence to the rbcS-8A promoter-transit peptide sequence containing the site. This new vector is named pCGN1761 / CT.
The DNA sequence is amplified using PCR technology and SphI, NSphI, or NlaIII
The site is incorporated into the amplified ATG and, after restriction enzyme digestion with the appropriate enzyme, is delivered to pCGN1761 / CT in frame by ligating it to SphI-cut pCGN1761 / CT. To facilitate construction, it may be required to change the second amino acid of the cloned gene; however, in most cases, the cleavage site and site can be reduced by using PCR with standard site-specific induction. The construction of any desired sequence around the first methionine of the mature protein is possible.

A further preferred vector is the double 35S promoter of pCGN1761ENX,
It is constructed by replacing the BamHI-SphI fragment of prbS-8A containing the full-length light-regulated rbc-8A promoter from the position (relative to the transcription start site) to the first methionine of the mature protein. Modified pCGN1761 with a disrupted SphI site is cut with PstI and EcoRI and blunt-ended with T4 DNA polymerase. PrbcS-8A is cleaved with SphI and ligated to an annealed molecular adapter of the sequence described above. The resulting product is phosphorylated at the 5 'end by treatment with T4 kinase. Subsequent cleavage at BmHI results in T4
The promoter-transit peptide containing the fragment treated with DNA polymerase and blunted at the BamHI end is released.

The thus-produced promoter-transit peptide fragment was cloned into the prepared pCGN1761ENX vector to construct a construct comprising the rbc-8A promoter and a transit peptide having an SphI site located at a cleavage site for insertion of a heterologous gene. Generate. It also has EcoRI (regenerated), NotI, and XhoI cloning sites downstream of the SphI site. This construct is named pCGN1761rbcS / CT. Similar manipulations contemplate the use of other GS2 chloroplast transit peptides encoding sequences from other sources (monocotyledonous and dicotyledonous) and from other genes.

Modification of pCGN1761ENX by Optimizing Translation Start Site For any of the constructs described in this section, modifications around the cloning site can be made by introducing sequences that can enhance translation. This is particularly useful when introducing genes from micro-organs into plant expression cassettes, since these genes may not contain sequences flanking the initiation methionine which may be suitable for initiating translation in plants. If a gene from a micro-organ is to be cloned in its ATG into a plant expression cassette, it may be useful to modify its insertion site to optimize its expression. The modification of pCGN1761ENX is described as an example that includes one of several optimized sequences for plant expression (e.g., Josh
i, supra). pCGN1761ENX is cut with SphI, treated with T4 DNA polymerase, religated, and the SphI site located 5 'of the double 35S promoter is destroyed.

This generates the vector pCGN1761ENX / Sph-. EcoRI with pCGN1761ENX / Sph-
And ligate to the annealed molecular adapter. This allows
Sph suitable for cloning of a heterologous gene in the initiation methionine itself
This gives rise to the vector pCGNSENX, which contains the so-called optimized plant translation initiation sequence TAAA-C adjacent to the ATG, which is part of the I site. Downstream of the SphI site, Eco
Has RI, NotI, and XhoI sites. An alternative vector utilizing the Ncol site in the starting ATG is constructed. This vector, designated pCGN1761NENX, is created by inserting the annealed molecular adapter into the pCGN1761ENX EcoRI site. This vector thus contains the start A site within the NcoI site.
Contains the so-called optimized sequence TAAACC adjacent to TG. The downstream site is EcoR
I, NotI, and XhoI.

However, prior to this operation, two NcoI sites in the pCGN1761ENX vector
(At the position upstream of the 5'35S promoter unit) using the same technique as described above for SphI or alternatively, the "inside-outside" P
CR (Innes et al. PCR Protocols: A guide to methods and applications.Academi
c Press, New York (1990)). This operation can be performed on any plant cDNA.
Alternatively, all possible deleterious effects on expression due to insertion of the reporter gene sequence into the cloning site can be assayed for subsequent analysis of routine expression in plants. As a result, the nucleotide sequence encoding the transgene was converted to pCGN1761ENX.
Insert for constitutive expression under the control of the CaMV 35S promoter with a translation initiation site optimized for

Example 34: Construction of Plant Transformation Vectors Many transformation vectors are available for plant transformation, and a nucleotide sequence encoding a DNA molecule of the present invention can be inserted into any of the expression cassettes described above. , They can express the transgene in the desired cell under appropriate conditions. Then, the obtained expression cassette is incorporated into any appropriate transformation vector described below. The choice of vector for use depends on the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred.

The selection marker routinely used for transformation is the nptII gene (Vieria and Messing, Gene 19) which confers resistance to kanamycin and related antibiotics.
: 259-268 (1982); Bevan et al., Nature 304: 183-187 (1983)), hph gene that confers resistance to the antibiotic hygromycin (Blochliger & Diggelmann, Mol Cell B
iol 4: 2929-2931), confer resistance to methatrexate
dhfr gene (Bourouis and Jarry., EMBO J. 2 (7): 1099-1104 (1983)), and aminoglycoside 3'-adenylyltransferase. aadA gene
(Goldschmidt-Clermont, 1991, Nucl. Asids Res. 19: 4083-4089). Another marker used is the bar gene (which confers resistance to the herbicide phosphinothricin).
White et al., Nucl Acids Res 18: 1062 (1990), Spencer et al. Theor Appl Gene
t 79: 625-631 (1990)), conferring a mutant EPSP synthase gene encoding glyphosate resistance (Hinchee et al., 1988, Bio / Technology 6: 915-922), imidazolion or sulfonylurea resistance Mutant acetolactate synthase (ALS)
Gene (Lee et al., 1988, EMBO J. 7; 1241-1248), a mutant psbA gene that confers resistance to atrazine (Smeda et al., 1993, Plant Physiol. 103: 911-917),
Or a mutant protoporphyrinogen oxidase gene as described in EP079059.

[0207] Selectable markers that result in positive selection, such as the phosphomannose isomerase gene as described in patent application WO 93/05163, are also used. Identification of the transformed cells will result in phenotypically distinct characterization of chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), luciferase, and green fluorescent protein (GEP) or transformed cells. It can also be achieved by expression of a screenable marker gene, such as any other protein conferred.

(1) Construction of Vector Suitable for Agrobacterium Transformation Many vectors can be used for transformation using Agrobacterium tumefasiens. Typically, they have at least one T-DNA border sequence and have pBIN19 (
Vectors such as Bevan, Nucl. Acids Res. (1984). The following describes the construction of two typical vectors. Construction of pCIB200 and pCIB2001 The binary vectors pCIB200 and pCIB2001 are used to construct a recombinant vector for use in Agrobacterium and are constructed in the following manner. pTJS75ka
n is made by digestion of pTJS75 (Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)) with NarI, excising the tetracycline-resistance gene, followed by insertion of the AccI fragment from pUC4K with NPTII (Vieira and Messing, Gene 19: 2
59-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant
Molecular Biology 14: 266-276 (1990)). Ligating the XhoI linker to the EcoRV fragment of pCIB7 containing the left and right T-DNA borders, the plant selectable nos / nptII chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)); XhoI-digested fragment was converted to SalI-digested pT
It is cloned into JS75kan to create pCIB200 (see EP0332104). pCIB200 has the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII.
, XbaI, and SalI.

PCIB2001 was created by insertion of an additional restriction site into the polylinker.
0 derivative. The unique restriction site in the polylinker of pCIB2001 is Eco
RI, SstI, KpnI, BglII, XbaI, SalI, MluI, Bc
II, AvrII, ApaI, HpaI, and StuI. pCIB2001 is
In addition to containing these unique restriction sites, plant and bacterial kanamycin selection, left and right T-D for Agrobacterium-mediated transformation
It has a trfA function for transfer between the NA border, RK-2-derived E. coli and other hosts, and an OriT and OriV function from RK2. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing these own regulatory signals. Any one of the aforementioned plant expression cassettes is inserted into pCIB2001, preferably using a polylinker.

Construction of pCIB10 and Hygromycin Selection of Derivatives The binary vector pCIB10 contains a gene encoding kanamycin resistance, T-DNA right and left border sequences for selection in plants, and has a broad host-range. It incorporates sequences from plasmid pRK252 and is capable of replication in both E. coli and Agrobacterium. The construction is Rothstein et a
l. (Gene53: 153-161 (1987)). Gritz et al. (Gene25: 179-
188 (1983)), various derivatives of pCIB10 have been constructed which incorporate the gene for hygromycin B phosphotransferase. These derivatives allow selection of transgenic plant cells on hygromycin alone (pCIB743) or on hygromycin and kanamycin (pCIB715, pCIB717). This vector is used for transforming the expression cassette of the present invention.

(2) Construction of Suitable Vectors for Non-Agrobacterium Transformation Transformation without Agrobacterium tumefaciens circumvents the need for T-DNA sequences in the selected transformation vector, and consequently Can be used in addition to vectors such as those described above containing a T-DNA sequence. Agrobacterium-free transformation techniques include particle bombardment, protoplast uptake (eg, PEG and electroporation), microinjection or pollen transformation (US Pat. No. 5,629,183). The choice of the vector will often depend on the selection preferred for the species to be transformed. The construction of a typical vector is described below.

Construction of pCIB3064 pCIB3064 is a pUC-derived vector suitable for direct gene delivery combined with selection with the herbicide vasta (or phosphinothricin). Plasmid pCIB246 is E
35S promoter and CaMV operably fused to the .coli GUS gene
It contains the 35S translation terminator and is described in published PCT application WO93 / 07278. The 35S promoter of this vector contains two ATG sequences at the 5 'position of the start site. These sites are mutated using standard PCR techniques, such as removing ATG and creating restriction sites SspI and PvuII. New restriction site is unique Sa
96 and 37 bp away from the I1 site and 101 and 42 bp away from the true start site. The resulting derivative of pCIB246 is named pCIB3025. And S from pCIB3025
The GUS gene is excised by digestion with all and SacI, blunted and ligated to create plasmid pCIB3060.

Plasmid pJIT82 was obtained from John Innes Centre, Norwich and used for Streptomyces vi
The 400 bp SmaI fragment containing the bar gene from ridochromogenes was excised and pCIB3
060 (Thompson et al. EMBO J6: 2519-2523 (1987)). The generated pCIB3064 contains the bar gene under control of the CaMV 35S promoter and terminator for herbicide selection, the gene for ampicillin resistance (for selection in E. coli) and unique sites SphI, PstI, HindIII. , And B
Includes a polylinker with amHI. This vector is suitable for cloning a plant expression cassette that contains its own regulatory signals that govern the expression of the transgene of the invention.

Construction of pSOG19 and pSOG35 pSOG35 is E. coli as a selectable marker that confers resistance to methotrexate.
It is a transformation vector using the dihydrofolate reductase (DHER) of the coli gene. 35S promoter (~ 800 bp), intron 6 (~ from corn Adh gene)
PCR is used to amplify the GUS untranslated leader sequence from pSOG10 at 550 bp) and 18 bp. 25 encoding the E. coli dihydrofolate reductase type II gene
The 0 bp fragment was amplified by PCR and these two PCR fragments were
9 pBI221 (including vector backbone and nopaline synthase terminator)
Clontech) with the SacI-PstI fragment. Construction of these fragments results in pSOG19 containing the 35S promoter fused to the intron 6 sequence, GUS leader, DHFR gene and nopaline synthase terminator. pSOG1
Replacement of the GUS leader in 9 with the leader sequence from maize repressed mottle virus (MCMV) yields the vector pSOG35. pSOG19 and pSOG35 have the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites which can be used for cloning foreign sequences, especially the transgenes of the present invention.

Plastid Transformation Vector For the constitutive expression of a nucleotide sequence of the present invention in plant plastids under the control of a clpP gene promoter element, the plastid transformation vector pPH143 (WO97 / 32
Example 36) of Example 011 is used. The nucleotide sequence is inserted into pPH143, thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of spectinomycin resistant transformants. Alternatively, the nucleotide sequence is inserted into pPH143, replacing the aadA gene under the control of the psbA gene promoter. In this case, transformants are selected for their resistance to the PROTOX inhibitor. In an alternative embodiment, the plastid 16S ribosomal RNA of tobacco or Arabidopsis
A promoter from the gene is used to express the PROTOX coding sequence.

Example 35: Plastid transformation of maize Type 1 embryogenic callus culture (Green et al. (1983) in A. Fazelahmad, K. Downey, J. et al.
Schultz, RW Voellmy, eds. Advances in Gene Technology: Molecular Genetics o
f Plants and Animals.Miami Winter Symposoium Series, Vol.20.Academic Pres
s, NY) is initiated from a 1.5-2.5 mm long immature corn embryo from greenhouse growing material. Embryos are aseptically removed from surface-sterilized ears about 14 days after pollination. Embryos were harvested with 2% sucrose and 5 mg / L chlorambene (Duncan et al. (1985) Planta 165: 322
-332) containing D-callus initiation medium or 3% sucrose and 0.75 mg / L
KM callus initiation medium containing 2,4-d (Kao and Michayluk (1975) Planta
126, 105-110). The embryo and embryogenic culture are subsequently cultured in the dark. Embryogenesis reactions are removed from explants after 1414 days.

The embryogenic reaction from the D-callus initiation medium was determined using 2 sucrose and 0.5 mg / L
In a D callus maintenance medium containing 2,4-d, while those from the KM callus initiation medium are placed in a KM callus maintenance medium containing 2 sucrose and 5 mg / L Dicamba. Three
After selective subculture on fresh maintenance medium for 8 weeks to 8 weeks, good and compact embryogenic calli are produced. Active growing callus pieces are selected as target tissues for gene delivery. Approximately 4 hours prior to gene delivery, callus fragments are plated on target plates containing maintenance medium containing 12 sucrose. Callus fragments are arranged in a circle with a radius of 8 to 10 mm from the center of the target plate. Plasmid DN
A is precipitated on a gold microcarrier as described in the DuPont bombardment manual. Two to three μg of each plasmid is used to prepare each six shots of microcarrier. The gene is delivered to target tissue cells using a PDS-1000He biolistic device.

The settings for the bombardment apparatus are as follows: 8 mm between the rupture disc and the macrocarrier, 10 mm between the macrocarrier and the stopping screen and 7 cm between the stopping screen and the target. Shoot each target plate twice using a 650psi bursting disc. A 200 × 200 stainless steel mesh (McMaster-Carr, New Brunswick, NJ) is placed between the stopping screen and the target tissue. After 5 days, callus fragments bombed with 2 sucrose and 0.
Transfer to maintenance medium containing 5 mg / L 2,4-d but no amino acids and containing 750 or 1000 nM of Formula XVII. Callus fragments are placed on a light shelf 4-5 hours after transfer or the next day for 1 hour and stored at 27 ° C. for 5-6 weeks in the dark. Following the first selection stage of 5-6 weeks, the yellow to white tissue is transferred to a new plate containing the same medium supplemented with 500 or 750 nM of Formula XVII. Tissues are placed on light shelves 4-5 hours after transfer or the next day for 1 hour and stored at 27 ° C. in the dark for 3-4 weeks.

Following a 3-4 week secondary selection stage, transfer to plates containing the same medium supplemented with 500 nM of Formula XVII. Place the healthy growing tissue on a light shelf for 1 hour and
Store at 7 ° C. Subculture every two weeks until colonies are large enough for regeneration. At this point the colonies were modified to contain 3 sucrose (MS3S) and no selection reagent
Transfer to MS medium (Murashige and Skoog (1962) Physiol. Plant 15: 473-497) and place in light. 0.25 mg / L ancimidol and either 0.5 mg / L kinetin or 2 mg / L benzyladenine are added to this medium to induce embryo germination. Two weeks later, the regenerating colonies are transferred to MS3S medium without ancimidol and kinetin or benzyladenine, respectively. MS3S regenerated shoots with or without roots
Transfer to a box containing medium, the plantlets with roots are finally regenerated and transplanted to soil in a greenhouse.

Example 36 Preparation of a Chimeric Gene Containing Ragweed Pollen Allergen Amb a I.1 Coding Sequence Fused to the Constitutive Arabidopsis UBQ3 Promoter for Nuclear Expression in Plants The Arabidopsis UBQ3 promoter (Norris et al. 1993) Plant Mol. Biol. 21:89.
5-906), Amb as a 0.83 kb NcoI, SalI fragment from pAT230.
a methylated X at the 5 'half of a I.1 and at the 3' end of the Amb a I.1 gene
The ragweed pollen allergen Amb was inserted by inserting the 3 'end of Amb a I.1 as a PCR-generated fragment of 0.39 kb SalI, XbaI, intended to remove the baI site, into the NcoI, XbaI sites of pPH121. a Fusing the I.1 coding sequence. PCR amplification was performed using pAT230 as a template and the following primer set: "top strand" primer (5'-GTC GCT TTC AAC ACG TTC AC-3 ', SEQ ID NO: 31) and "bottom" excluding A before the XbaI site Strand '' primer (5'-GCG CTC TAG ACA TTA TAA GTG CTT A
GT-3 ', SEQ ID NO: 32). The 452 bp amplification product was gel purified and Sal
Digest with I and XbaI and ligate as described above to generate pAT240. Am
HindIII-Xb of pAT240 containing the UBQ3 promoter driving the ba I.1 gene
aI is ligated to the 11.3 kb HindIII, XbaI digested pPH108 fragment to create a binary vector with a constitutively expressed Amb I.1 gene.

Example 37 Preparation of a Chimeric Gene Containing the Putative Mature Ragweed Allergen Amb a I.1 Coding Sequence Fused to the Constitutive Arabidopsis UBQ3 Promoter for Nuclear Expression in Plants Amb a I.1 It has a putative signal peptide cleavage site with an alanine at position 26, predicted to be the group +1 (Rafnar et al. (1991) J. Biol. Chem. 266: 1229-123
6). The Amb a I.1 coding sequence is modified to remove the signal peptide by PCR amplification using pAT230 as a template and the following set of oligonucleotides: the `` top strand '' primer is the first of the mature Amb a I.1 protein ATG, the first 20 bp of the mature protein and the NcoI site in the newly created ATG before the codon (5'-GCA CCA TGG CCG AAG ATC TCC AGG AAA T-3 ', SEQ ID NO: 33),
And the "bottom strand" primer is (5'-CTA CCA GCC CAT CAA CAG ACT TAC-3 '
, SEQ ID NO: 34). Consists of the 5 'end of Amb I.1 gene without signal sequence
Create a 594 bp amplification product. The fragment was gel purified and NcoI and ClaI
The 0.35 kb fragment was ligated to the 0.81 kb ClaI, 3 ′ end of the Amb a I.1 gene as an XbaI fragment and the NcoI of pAT240 and the 4.4 kb fragment of XbaI, and fused to the Arabidopsis UBQ3 promoter. The Amb a l.1 gene lacking the signal peptide is prepared and cloned into a terminator sequence and a binary vector.

Example 38: Preparation of a chimeric gene containing the Dermatophagoides farinae major allergen Der fI coding sequence fused to the constitutive Arabidopsis UBQ3 promoter for nuclear expression in plants was cloned as described in Example 13. Dermatophagoides allergen Der
The fI coding sequence is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector as described in Example 34.

Example 39: Preparation of a chimeric gene containing the Dermatophagoides farinae major allergen Der fII coding sequence fused to the constitutive Arabidopsis UBQ3 promoter for nuclear expression in plants was cloned as described in Example 13. Dermatophagoides allergen Der
The fII coding sequence is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector as described in Example 34.

Example 40: Preparation of a chimeric gene containing the Dermatophagoides pteronyssinus major allergen Der pI coding sequence fused to the constitutive Arabidopsis UBQ3 promoter for nuclear expression in plants was cloned as described in Example 13. Dermatophagoides allergen Der
The pII coding sequence is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector as described in Example 34.

Example 41: Preparation of a chimeric gene containing the Dermatophagoides pteronyssinus major allergen Der pII coding sequence fused to the constitutive Arabidopsis UBQ3 promoter for nuclear expression in plants was cloned as described in Example 13. Dermatophagoides allergen Der
The pII coding sequence is fused to the Arabidopsis UBQ3 promoter and inserted into a binary vector as described in Example 34.

Example 42 Preparation of a Chimeric Gene Containing the Johnson Grass Allergen Sor hl Coding Sequence Fused with the Constitutive Arabidopsis UBQ3 Promoter for Nuclear Expression in Plants And inserted into a binary vector as described in Example 34.

Example 43 Preparation of a Chimeric Gene Containing a Birch Pollen Allergen Bet VI Coding Sequence Fused with the Constitutive Arabidopsis UBQ3 Promoter for Nuclear Expression in Plants Birch pollen allergen Bet VI coding sequence Fused with the Arabidopsis UBQ3 promoter and inserted into a binary vector as described in Example 34.

Example 44 Preparation of Chimeric Gene Containing Mosquito Saliva Allergen rAed a1 Coding Sequence Fused with Constitutive Arabidopsis UBQ3 Promoter for Nuclear Expression in Plants Fuse and insert into binary vector as described in Example 34.

Example 45: Preparation of a chimeric gene for vacuolar expression containing the ragweed pollen allergen Amb a I.1 coding sequence fused to the constitutive Arabidopsis UBQ3 promoter Plasmid containing the ragweed pollen allergen gene Amb a I.1 Using pAT240 as a template for PCR, a “top strand” primer (positions 5′-GCA ACG GTC GCT TTC AAC ACG TTC A-3 ′ sequence) from position 775 to position 799 in the Amb a I.1 gene for endogenous ATG Number 35
) And the sequence is homologous to the last 21 bp of Amb a I.1, and the tobacco chitinase gene
(Shinshi et al., (1990) Plant Mol. Biol. 14, 357-368, Neuhaus et al. (1991) Proc.
Natl.Acad.Sci.USA 88,10362-10366) 21b of the vacuolar targeting sequence
p, a “bottom strand” primer containing the last codon of the same tobacco chitinase gene and an XbaI restriction site (5′-CGC TCT AGA TTA CAT AGT ATC GAC TAA AAG TCC GC
A AGG TGC TCC GGG TTG GCA-3 ', SEQ ID NO: 36). PCR amplification yields a 447 bp product, which is gel purified and digested with SalI and XbaI.

The 383 bp SalI, XbaI fragment containing the 3 ′ end of AmbalI fused to the tobacco chitinase vacuolar targeting sequence was ligated with the 0.83 kb Nc from pAT240.
ligated to the 5 'end of Amb a I.1 as a
A 4.4 kb NcoI, XbaI fragment of pAT240 containing the Q3 promoter and vector is obtained. The Amb a I.1 coding sequence cassette fused to the Arabidopsis UBQ3 promoter :: tobacco chitinase vacuolar targeting sequence is cloned into a terminator sequence and a binary vector.

Plasmid pSCH10 (Shinshi et al., (1990) Plant Mol. Biol. 14, 357-368, Neuuhau
USA et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10362-10366) as a template for PCR amplification with the first 22 bp of the 23 amino acid N-terminal signal peptide of tobacco chitinase, A "top strand" primer (ErspU 5'-CGG TCA TGA GGC TTT GTA AAT TCA CAG-3 ', SEQ ID NO: 37) homologous to the BSphI restriction site in ATG and the sequence is the predicted mature 5' of the Amb a I.1 gene A `` bottom strand '' primer homologous to the last 17 bp of the tobacco chitinase N-terminal signal peptide fused to the first 14 bp of the terminus (ErspL 5′-TGG AGA TCT TCG GCT GCC GAG GCA GAA AGC A-3 ′, sequence Use with number 38). The 88 bp amplification product was gel purified, cleaved with BSphI and BglII, and 76 bp containing the 23 amino acid N-terminal signal peptide of tobacco chitinase fused to the 5 ′ end of the Amb a I.1 gene lacking the original signal peptide. Is ligated to the 3 ′ end of the Amb a.1 gene containing the tobacco chitinase vacuolar targeting sequence as a BglII and XbaI fragment to obtain a 4.4 kb NcoI, XbaI fragment of pAT240 containing the UBQ3 promoter and vector. .

Arabidopsis UBQ promoter :: Amb a with the original signal peptide removed
The I.1 gene, N-terminal tobacco chitinase signal peptide and C-terminal tobacco chitinase vacuole targeting sequence cassette are cloned into a terminator sequence and binary vector.

Plasmid pSCH10 was used as a template for PCR amplification, with the sequence consisting of the first 22 bp of the 23 amino acid N-terminal signal peptide of tobacco chitinase and Bsp in ATG.
A "top strand" primer homologous to the hI restriction site (ErspU), and the sequence is Amba I
.I a `` bottom strand '' primer homologous to the last 17 bp of the N-terminal signal peptide of tobacco chitinase fused to the first 13 bp 5 ′ extension (ErspovL 5′-AGT GTT TGA TCC TCC CT
G CCG AGG CAG AAA GCA -3 ', SEQ ID NO: 39). Plasmid pAT240
With the first 25 after the start codon of sequence Amb a.1 as a template for PCR amplification.
`` Top strand '' that is homologous to bp and anneals to 13 bp of primer ErspovL
Used with primers (AmboeU5'-GGG ATC AAA CAC TGT TGT TAC ATC T-3 ', SEQ ID NO: 40) and a "bottom strand" primer that extends from position 135 to position 158 relative to endogenous ATG in the Amball gene.

The two PCR amplification products are gel purified and a PCR overlap extension technique is performed to fuse the two products as follows: 2 ml of each purified amplification product is combined in a PCR reaction tube and the fusion products are annealed Perform 10 cycles in the absence of primer to allow for extension. 5 ml of the reaction is then used as a template for PCR amplification using the "outside" primers ErspU and AmboeL. The final amplification product is AT
155 bp downstream from the first amino acid after G, tobacco chitinase N-terminal 23 with a BSphI site at the start codon, fused in reading frame to the Amb a.1 gene.
Includes amino acid signal peptide.

The product was gel purified, digested with BSphI and BglII, BglII, Xba
Amb containing tobacco chitinase vacuolar targeting sequence as an I fragment
a pA ligated to the 3 'end of the l.1 gene and containing the UBQ promoter and vector
A 4.4 kb NcoI, XbaI fragment of T240 is obtained. Arabidopsis UBQ promoter :: Amb a l.1 gene, N-terminal tobacco chitinase signal peptide and C
-Cloning the terminal tobacco chitinase vacuole targeting sequence cassette into the terminator sequence and binary vector.

Example 46: Ragweed pollen allergen A fused to tobacco PR-1a promoter
Preparation of Chimeric Gene for Vacuolar Expression Containing the mba I.1 Coding Sequence PR-1a DXhoNco (Pk
The R-1a promoter was excised as a 903 bp XhoI, NcoI fragment, ligated to the NcoI, XbaI fragment containing the Amb a I.1 gene, the N-terminal tobacco chitinase signal peptide and the C-terminal tobacco chitinase vacuole targeting sequence, and pLITMUS28 (New England Biolabs) XhoI and XbaI sites are obtained.

The PR-1a promoter :: Amb a I.1 gene, N-terminal tobacco chitinase signal peptide and C-terminal tobacco chitinase vacuole targeting sequence cassette are cloned into a terminator sequence and a binary vector. XhoI, NsoI
The PR-1a promoter fragment was replaced with an NcoI, XbaI fragment containing the Amb a.1 gene with the original signal peptide removed, the N-terminal tobacco chitinase signal peptide and the C-terminal tobacco chitinase vacuole targeting sequence cassette, pLITMUS28 Ligation at the XhoI and XbaI sites.
PR-1a Promoter: The Amb a.1 gene with the original signal peptide removed, the N-terminal tobacco chitinase signal peptide and the C-terminal tobacco chitinase vacuole targeting sequence cassette are cloned into a terminator sequence and a binary vector. When reaching 20-40 cm high, transgenic plants containing the chimeric gene described above are sprayed with the inducer compound BTH to induce expression of the Amb a I.1 gene. Immediately before and 8 hours after induction and 1, 2
Harvest after 3, 7, and 14 or 28 days and flash freeze in liquid nitrogen.

The embodiments described above are exemplary. This description of the invention will provide those having ordinary skill in the art the possession of many different variations of the invention. All such obvious and foreseeable changes are intended to be covered by the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 38/22 A61P 29/00 4C085 38/28 37/00 38/43 37/06 39/00 37/08 39/35 C12R 1:91) A61P 29/00 C12N 15/00 ZNAA 37/00 5/00 C 37/06 A61K 37/02 37/08 37/26 C12N 5/10 37/48 15/09 ZNA 37 / 04 // (C12N 5/10 37/24 C12R 1:91) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, W), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Stephen Arthur Goff Encinitas, Calif. No. 1040 (72) Inventor Anmary Bull Mu Tuttle U.S.A. 27618 Benfield Court, Garner, North Carolina, USA (72) Inventor Monica Else Griot-Benck Zecher-3179 Kriechenville, Schönenbühl F-term (reference) 2B030 AA02 AB03 AD20 CA06 CA17 CA19 CB02 CD03 CD07 CD09 CD13 CD14 CD17 4B018 MD20 MD48 ME03 ME04 ME05 ME06 ME07 ME08 ME09 ME10 MF14 4B024 AA01 BA01 BA07 BA21 BA31 BA63 CA04 DA01 DA05 EA03 EA04 FA02 GA11 HA01 4B065 AA11X AA88X AB01 BA02 CA24 A02 CA14 DA01 DB01 DB34 DB52 DC01 MA52 ZB02 ZB08 ZB11 ZB13 4C085 AA02 BA99 BB03 BB04 BB06 BB11 BB12 BB17 BB22 BB31 CC40 DD62 GG08

Claims (58)

[Claims] 1. A plant comprising in its plastid genome a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host in need of treatment in a therapeutically effective amount, said plant being capable of expressing said protein. 2. The plant according to claim 1, wherein the protein is orally administered to a host. 3. The plant according to claim 1, wherein the protein is an antigen. 4. The plant of claim 3, wherein the antigen is capable of suppressing or reducing a host immune response or inflammatory condition to the antigen. 5. The plant according to claim 4, wherein the antigen is an allergen. 6. The plant according to claim 5, wherein the allergen is an airborne allergen. 7. The plant according to claim 5, wherein the allergen is a pollen allergen. 8. The pollen allergen is ragweed allergen Amb a I, Am
ba I.1, Amb t V and Amb a II, Alnu allergen Alng
The plant according to claim 7, wherein the plant is selected from the group consisting of I, hazel allergen Cor a I, ryegrass allergen Lol p V, Johnson grass allergen Sor h and birch allergen Bet v I. 9. The antigen is a cat antigen Fel d I, canine allergen Can f
II, selected from the group consisting of mosquito allergens rAed a1 and rAed a2, mite allergens Der f I, Der f II, Der p I and Der p II, honey bee venom allergen peptide PLA-2 and murine urine protein. Item 5. The plant according to Item 5. 10. The plant according to claim 3, wherein the antigen is a self antigen. 11. The autoantigen may be collagen, myelin basic protein, myelin proteolipid protein, interphotoreceptor binding protein,
The plant according to claim 10, which is selected from the group consisting of acetylcholine receptor, S-antigen, insulin, glutamate decarboxylase, islet cell-specific antigen, and thyroglobulin. 12. The plant according to claim 3, wherein the antigen is a transplantation antigen. 13. The plant according to claim 12, wherein the transplantation antigen is an MHC protein. 14. The plant according to claim 12, wherein the transplantation antigen is an MHC II species protein. 15. The plant according to claim 12, wherein the transplantation antigen is an MHC II species a or b chain. 16. The plant of claim 3, which is an antigen derived from a pathogen, wherein the antigen is capable of immunizing a host against said pathogen. 17. The protein may be a blood protein, hormone, growth factor, cytokine, enzyme, receptor, binding protein, immune system protein, translation or transcription factor, oncoprotein or proto-onc protein.
o plant), a milk protein, a muscle protein, a bone marrow protein (myeloprotein), a neurostimulatory peptide or a tumor growth inhibitory protein. 18. The plant according to claim 1, wherein the protein is a preservative peptide. 19. The plant according to claim 18, wherein the preservative protein is a bactericidal permeability-enhancing protein. 20. The plant according to claim 1, wherein the immune system protein is an antibody. 21. The plant of claim 1, wherein the DNA molecule is operably linked to a promoter capable of expressing the DNA molecule in the plastid of the plant. 22. The promoter of claim 21, wherein the promoter is a clpP promoter.
The described plant. 23. The plant according to claim 21, wherein the promoter is a 16S r-RNA gene promoter. 24. The plant according to claim 21, wherein the promoter is a transactivator transmission promoter. 25. The transactivator transmission promoter is T7 gene 1.
25. The plant of claim 24, which is a 0 promoter. 26. The plant of claim 24, further comprising a heterologous nuclear expression cassette comprising a DNA sequence encoding a transactivator. 27. The plant according to claim 25, wherein the transactivator is T7 polymerase. 28. A plant comprising in the nuclear genome a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host in need of treatment in a therapeutically effective amount, wherein the therapeutically active protein is the mosquito allergen rAed a. 1 and rAed a 2,
Bactericidal permeability enhancing protein (BPI) and pollen allergen Amb a I, Am
A plant selected from the group consisting of ba I.1, Ambt V, Amb a II, Alng I, Cor a I, Lol p V, Sohr and Bet v I. 29. A plant comprising in the nuclear genome a DNA molecule encoding a protein that exhibits a therapeutic activity when administered to a host in need of treatment in a therapeutically effective amount, wherein the therapeutically active protein is intracellular in the plant. Plants targeted to organelles. 30. The plant according to claim 29, wherein the intracellular organelle is a vacuole. 31. The plant according to claim 1, wherein the plant is a dicotyledonous plant. 32. The plant according to claim 31, wherein the plant is tobacco, tomato, soybean or spinach. 33. The plant according to any one of claims 1 or 28 to 30, wherein the plant is a monocotyledonous plant. 34. The plant according to claim 33, wherein the plant is corn or rice. 35. The plant according to claim 31, wherein the plant is an edible plant. 36. The protein expression in the plant is chemically regulatable.
The plant according to any one of claims 1 or 28 to 30. 37. The plant according to any one of claims 1 or 28 to 30, wherein the protein expression in the plant is constitutive. 38. The plant according to any one of claims 1 or 28 to 30, wherein the protein expression in the plant is tissue-specific. 39. The plant according to any one of claims 1 or 28 to 30, wherein the host is a vertebrate. 40. The plant of claim 39, wherein the vertebrate is a mammal. 41. The plant according to claim 40, wherein the mammal is a human, cow, sheep, pig, dog or cat. 42. A composition comprising a plant according to any one of claims 1 or 28 to 30 or a plant substance derived from said plant, said composition comprising a therapeutically effective amount of a protein. 43. The composition of claim 42, wherein the plant has been processed prior to administration to the host. 44. A method comprising administering to a host in need thereof an amount of the composition of claim 42 in an amount effective to improve the condition of the host. 45. The method of claim 42, wherein said composition is administered orally to a host. 46. The method of claim 45, wherein the protein is an antigen, thereby suppressing or reducing a host immune response to the antigen. 47. The method of claim 46, wherein said antigen is an allergen, an autoantigen or a transplantation antigen. 48. The method according to claim 1 or 28 to a host in need of treatment or prevention.
30. A method for treating or preventing a disease, comprising administering a therapeutically effective amount of the plant according to any one of claims 30 or a plant substance derived from the plant. 49. The disease is an allergy, an autoimmune disease or a transplant rejection.
49. The method of claim 48. 50. The method of claim 49, wherein the therapeutically effective amount is administered orally to the host. 51. A plastid transformation vector comprising a DNA molecule encoding a protein that exhibits therapeutic activity when administered to a host, wherein the DNA molecule directs expression of the DNA molecule in plant plastids. A vector operably linked to a vector. 52. A plastid comprising the transformation vector according to claim 51. 53. A plant cell comprising the plastid of claim 52, wherein the plant cell is capable of producing a protein. 54. A plant comprising the plant cell according to claim 53. 55. A method comprising transforming the plastid genome of a plant with the plastid transformation vector of claim 51. 56. The method of claim 55, further comprising expressing a therapeutically effective amount of the protein in the plant. 57. A food product comprising an edible part of a plant comprising the DNA molecule of any one of claims 1 or 28-30, wherein the food product is administered to a host in need of treatment in a therapeutically effective amount. Sometimes food products that show therapeutic activity. 58. An agricultural product derived from a plant or plant part comprising a DNA molecule according to any one of claims 1 or 28 to 30, which is administered to a host in need of treatment in a therapeutically effective amount. An agricultural product sometimes showing therapeutic activity.