JPH0940520A - Production of plant component by animal, preparation of stress resistant animal and use of plant component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enabling to produce a plant component useful for an animal (especially a domestic animal) by the animal and relating techniques. SOLUTION: In this method for producing a plant component, a gene relating to a synthesis of the plant component (e.g. a carotenoid such as phytoene, etc.) having antioxidation activity, lipid antioxidative ability or cancer preventive ability is brought into an animal and manifested in the animal and the plant component having such bioactivities is produced by the animal. A method for preparing an oxidative stress resistant animal, lipid-oxidative stress resistant animal or an animal having cancer protective ability is claimed in this patent. An anticarcinogenesis promoter agent and a cancer cell-proliferation inhibitor containing phytoene as an effective ingredient and a beverage and a cosmetic product containing phytoene are also claimed.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

TECHNICAL FIELD The present invention relates to a method for producing plant components in animals (particularly domestic animals), a method for producing stress-tolerant animals, and uses of plant components. The plant-derived ingredients here do not contain proteins or carbohydrates and cannot be biosynthesized by animals.
It usually says something that must depend on the plant. Examples of botanical components include the carotenoid phytoene. The industrial field of use of the present invention is wide, and, for example, if a plant component can be produced in animal cells, it is not necessary for the animal to ingest the plant component from the outside world. Therefore, it can be used, for example, for producing transgenic livestock that does not depend on food. Further, if the botanical component is phytoene, it can be used for foods and drinks, cosmetics and the like having a cancer-preventing action and the like.

[0002]

2. Description of the Related Art Animal is a protein,
In addition to carbohydrates, it is essential to get botanicals from plants. That is, the incorporated plant components have various and important physiological functions in animals. For example, various vitamins or their precursors are included in the botanical composition. In addition, there are many plant components that function as antioxidants against active oxygen and free radicals that cause various diseases such as cancer and aging (Masayasu Inoue, Maiko Yamaguchi, “Active oxygen metabolism and food antioxidants”). ", Bioindustry, 12, 27-33, 1995.). However, its supply amount is not constant and is unstable because it depends on the intake from the outside world, and it was often insufficient.

[0003]

If a plant component having an important function such as antioxidative ability can be produced in the cells of an animal (particularly for domestic animals), the component must depend on the intake from the outside world. Disappears. The development of such a technique is expected to have a wide range of applications and great merits, but such a technical idea has never existed, and the development of such a technique has hitherto been impossible. There are several possible causes. For example, since an animal does not have a gene for biosynthesizing a plant component, it needs to be introduced as a foreign gene. However, usually the gene is not one, but the introduction of multiple genes,
Although it is possible to produce it for the first time by expression, the gene itself is often unknown. Also, even if it is known, it would be technically difficult to introduce, express and control multiple genes. Also,
Even if the above is successful, it cannot be denied that the newly synthesized plant component may disrupt the normal functions of animal cells or cause the cells to die because of their diverse physiological functions. Ah It is an object of the present invention to provide a method for producing an animal (particularly a domestic animal) a plant component useful for sustaining the life of the animal, and a related technique therefor.

[0004]

Means for Solving the Problems The present inventors have found that all animal cells have the ability to isolate and analyze genes necessary for biosynthesis of plant components such as phytoene and other carotenoids. We succeeded in obtaining the genes necessary for synthesizing plant components from precursor substances such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) (Norihiko Misawa, "Elucidation of carotenoid biosynthetic pathway at the gene level" And metabolic engineering ", Pharmacia, 31, 369-37
2, 1995.). Therefore, it may be possible to synthesize the plant component in the animal by introducing and expressing the synthetic gene for the plant component in the animal. However, since the newly synthesized plant component has various physiological functions, there is a possibility that the normal functions of animal cells may be disturbed or the cells may die. Therefore, the present inventors have selected a plant component which is known to be accumulated in animal cells and tissues, and whose excess disorder is not known even when it is overdose. Furthermore, since it is meaningless that the newly synthesized plant component is not effective for animal cells such as having an antioxidant action, a plant component confirmed by the present inventors to exhibit various efficacy was selected. . By making full use of the above means, we succeeded in producing useful plant components in animals, and completed the present invention based on this finding.

That is, the present invention provides the following methods and applications. (1) A method for producing a plant component in an animal, which comprises introducing a gene involved in the synthesis of the plant component into the animal and expressing the gene in the animal. (2) The method according to (1), wherein the botanical component is a carotenoid (preferably phytoene). (3) A method for producing an oxidative stress-tolerant animal, which comprises introducing a gene involved in the synthesis of a plant component having an antioxidant ability into an animal and expressing the gene in the animal. (4) A method for producing a lipid oxidative stress-tolerant animal, which comprises introducing a gene involved in the synthesis of a plant component having an anti-lipid oxidation ability into an animal and expressing the gene in the animal. (5) A method for producing a carcinogenic stress-resistant animal, which comprises introducing a gene involved in the synthesis of a plant component having a cancer-preventing ability into an animal and expressing the gene in the animal. (6) A method for producing a carcinogenic stress-resistant animal, which comprises introducing a gene involved in the synthesis of a plant component capable of suppressing cancer growth into an animal and expressing the gene in the animal. (7) The production method according to (3) to (6) in which the plant component is phytoene, and the animal is a domestic animal (1) to
Method (6). Further, in the present invention, it is possible to provide a use of phytoene utilizing these effects by first revealing that phytoene, which is a plant component, has antipromotion activity and cancer growth inhibitory activity. That is, (8) An anti-carcinogenic promoter agent or cancer cell growth inhibitor, which contains phytoene as an active ingredient, and a cancer-preventing cosmetic material comprising phytoene, and a food or drink or cosmetic product containing phytoene.

[Detailed Description of the Invention] The present invention is described in detail below. 1. Prevention and proliferation of cancer (definition) There is an idea of chemoprevention of cancer. The idea is to identify and eliminate the factors that cause carcinogenesis, and to prevent cancer by ingesting tumor suppressors, which is a very popular idea in the United States. Carcinogenesis is known to occur in multiple stages and is generally initiated (initiati
on), promotion, and progression (Toshihiko Osawa, “Development Trends of Designer Foods” Bioindustry, 12, p16-26, 1995). What is an initiation?
This is the stage where the incorporated carcinogen precursor is transformed into the ultimate carcinogen in vivo and acts on DNA to fix the mutation on the DNA. A substance that has such an action is called an initiator, and DM
BA (dimethylbenzanthracene) and the like are known. Promotion is the stage at which the growth of fixed latent tumor cells described above is promoted. A substance with such an action is called a promoter, and TPA
(12-O-tetradecanoylphorbol-13-acetate) (diterpene contained in plants of Euphorbiaceae), glycoxal (glycoxal: contained in coffee etc.), NaCl, bile acid,
High-fat foods are known. It is known that the action of the promoter is reversible, and if promotion can be suppressed at this stage, cancer will not occur unless the initiator is very strong. Therefore, if this promotion can be suppressed, it will lead to the prevention of cancer. Those having such an action are called anti-carcinogenic promoters. Progression is the stage at which a formed tumor grows, and it is at this stage that it is discovered that it is a cancer.
It is an anticancer drug that suppresses and stops the growth of this tumor.

[0007] 2. Botanical ingredients The phytochemicals referred to here are those that do not contain proteins and carbohydrates and cannot be biosynthesized by animals, and animals usually have to rely on plants for their intake. Examples of such botanical ingredients include:
For example, various vitamins or their precursors, carotenoids, etc. can be mentioned. In addition, in order to synthesize a plant component in an animal, a gene for synthesizing the plant component from a precursor substance commonly possessed by animal cells must be clarified. For carotenoids such as phytoene, lycopene and β-carotene, the genes for their synthesis have already been clarified (Norihiko Misawa, “Clarification of carotenoid biosynthetic pathways at the gene level and metabolic engineering”, Pharmacia, 31, 369-372, 1995
). The present inventors have selected phytoene as a preferred example of the plant component as described above, but even in plant components other than phytoene, if the gene is clear, the plant component (for example, lycopene, β- It is possible to synthesize carotenoids such as carotene, etc. in animals using the gene transfer system of animals (edited by Isao Ishida and Tamsuke Ando, "Gene Expression Experiment Manual", 1994.). However,
In this case, it must be a plant component that is not toxic even when accumulated in animals, and the number of genes that must be introduced to make a plant component is large (for example, 5
Since the above is difficult with the current technology of gene manipulation,
It should be synthesizable with the introduction of few genes.

[0008] 3. Carotenoids and phytoene Carotenoids are widespread pigments in nature, and up to now about 600 species have been isolated (H.
Pfander, "Key to Carotenoids", Birkhauser, Ba
sel, 1987). Carotenoids are synthesized by the isoprenoid biosynthetic pathway starting from mevalonic acid. C15 farnesyl pyrophosphate (FPP) produced by the isoprene-based biosynthesis system is condensed with C5 isopentenyl pyrophosphate (IPP) to form C20 geranylgeranyl pyrophosphate (GGPP). This GGPP
Is a substrate of geranylgeranylated protein that is ubiquitously present in animal cells, and can be considered to be possessed by all animal cells. Subsequent carotenoid genes do not exist in animals. In plants and certain microorganisms, two molecules of GGPP are condensed to synthesize the first carotenoid, colorless phytoene. Phytoene is converted into red lycopene by a series of unsaturated reactions, and lycopene is orange to yellow β-carotene (β-carot) due to cyclization reaction.
ene)). Then, it is thought that various oxidized carotenoids (xanthophyll) are synthesized by introducing a hydroxyl group or a keto group into β-carotene (G. Britton,
"Biosynthesis of carotenooids". Plant Pigments. A
academic Press, London, 1988, p.133-182 (TW Good
win))). Carotenoids such as phytoene, lycopene and β-carotene are plant components that are commonly observed to accumulate in various animal organs (M.
M. Mathews-Roth, "Recent progress in the medical a
pplication of carotenoids ", Pure & Appl. Chem., 63,
147-156, 1991). The present inventors have cloned the carotenoid biosynthesis gene group of the plant resident non-photosynthetic bacterium Erwinia uredovora into Escherichia coli using the yellow color tone as an index,
By expressing various combinations of these genes in microorganisms such as Escherichia coli, it has become possible to cause microorganisms such as Escherichia coli to produce carotenoids such as phytoene, lycopene and β-carotene (Norihiko Misawa, “At the gene level Of Carotenoid Biosynthetic Pathway and Metabolic Engineering in ", Pharmacia, 31, 369-372, 1995). In order to synthesize phytoene in animal cells, it is considered that a phytoene synthase gene that catalyzes the synthesis of phytoene from GGPP should be introduced. The present inventors have introduced the crtB gene Erwini a encoding synthase phytoene from GGPP, by expressing, and succeeded in the production of phytoene in animal cells such as NIH3T3. There has been no report that animal cells have produced plant components such as phytoene.

[0009] Carotenoids are not only precursors of vitamin A in animals, but are also attracting attention as antioxidants for removing active oxygen and free radicals (Takao Matsuno, Mikiwa, “Carotenoids in animals”). Physiology and Biological Activity ", Chemistry and Biology, 28, 219-227, 1990
). In addition, carotenoids have recently been known to have a function of preventing various cancers (Sai Nishino,
"Suppression of carcinogenesis by carotenoids in foods", Journal of Japan Society of Agricultural Chemistry, 67, 39-41, 1993). In phytoene, it was also reported that this suppresses the development of UV-induced skin cancer (MM Mathews-Roth et al., "Antitumor ac
tivity of β-carotene, canthaxanthin and phytoen
e ", Oncology, 39, 33-37, 1982). However, according to their research, phytoene only suppressed the initiation of UV-induced carcinogenesis, and did not induce DMBA-induced carcinogenesis or non-UV-induced carcinogenesis. Not suppressed (MMMathews-Roth et al., "Antitumor activit
y of β-carotene, canthaxanthin and phytoene ", Onc
ology, 39, 33-37, 1982 and MM Mathews-Roth,
"Carotenoids and cancer prevention-experimental
and epidemiological studies ", Pure & Appl. Chem.,
57, 717-722, 1985). This means that, unlike other carotenoids, phytoene does not have long conjugated double bonds that cause absorption of active oxygen and free radicals,
Further, since it has a strong ultraviolet ray absorbing action, it is convincing that phytoene absorbs harmful ultraviolet rays and consequently suppresses the development of skin cancer. However,
The present inventors have heretofore shown that phytoene has an antitumor promoter activity that leads to the prevention of cancer, and that it exhibits an antitumor effect such as having an effect of suppressing the growth of cancer cells. It was the first time in the world to reveal it. The present inventors have further found that phytoene-producing animal cells are not only resistant to canceration but also resistant to peroxidation by active oxygen or free radicals. confirmed. Therefore, the inventors of the present invention, rather than ingesting plant components such as phytoene from the outside world,
He discovered that the method of making cells directly produce is effective against stress such as peroxidation and canceration.

The genes involved in the synthesis of phytoene are known, for example, the above-mentioned Erwinia crtB gene or tomato psy gene (GHBartle).
y etal, “A Tomato gene expressed during fruit rip
ening encodes an enzyme of the carotenoid biosynthe
sis pathway ”J. Biol. Chem, 267, p5036-5039, 1992).
For details on the crtB gene and its acquisition method, see (N. Misaw
a et al., "Elucidation of the Erwinia uredovora ca
rotenoid biosynthetic pathway by functional analys
is of gene products expressed in Escherichia col
i ", J. Bacteriol., 172, 6704-6712, 1990, and
G. Sandmannn and N. Misawa, "New functional assign
ment of the carotenogenic genes crtB and crtE with
constructs of these genes from Erwinia species ",
FEMS Microbiol. Lett., 90, 253-258, 1990) and can be isolated from Erwinia according to the method (the DNA sequence of the crtB gene is shown in FIG. 1) with the corresponding amino acid sequence). The crt gene is a gene involved in the synthesis of carotenoids (phytoene, lycopene, β-carotene, etc.) containing the crt gene in the Institute of Microbial Technology, Ministry of International Trade and Industry (now the Institute of Biotechnology and Industrial Technology). It has been deposited as a deposit number (BP-2377). As a phytoene synthetic gene to be introduced into an animal, a known gene as described above or a partial modification of the encoded amino acid (substitution, deletion, insertion, addition, etc.)
Genes corresponding to the above sequences and their degenerate genes can be used.

4. Animal cells and method for introducing and expressing plant component synthesizing gene into animal cells The animals of interest in the present invention are mainly domestic animals (eg, bovines, pigs, etc.). Currently, various animal cells can be distributed from several research institutes. For example, it can be ordered from the JCRB cell bank. In addition, in the present invention, HGC-27 cells (gastric cancer), GOTO cells (neuroblasts), PANC-1 cells (pancreatic cancer), OS for phytoene cancer prevention test or cancer growth suppression test.
Using T-KYO-1 cells (osteosarcoma), HeLa cells (uterine cancer),
NIH3T3 as a host for gene transfer of plant component synthesis gene
Cells (mouse fibroblasts) were used. SV40, bovine papillomavirus (BPV) as a vector for introducing animal cells
A variety of vectors that utilize the replication initiation region of the viral genome, such as a retrovirus and a retrovirus, are known (J. Sambroo
k et al., "Molecular Cloning Second Edition", Cold
Spring Harbor Laboratory Press, New York, 1989
). Various promoters are known as promoters that enable the expression of foreign genes in animal cells, such as promoters of various viruses such as SV40 and β-actin promoter of animal origin (J. Sambroo
k et al., "Molecular Cloning Second Edition", Cold
Spring Harbor Laboratory Press, New York, 1989
). The inventors of the present invention, pCAGGS (H. N, which is a vector utilizing the replication initiation region of SV40 and the β-actin promoter.
iwa et al., "Efficient selection for high-expressi
on transfectants with a nevel eucaryotic vector ",
Gene, 108, 193-200, 1991) and the retrovirus vector LXSH (Gene Bank, Accession number M7723).
Although 9) was used, various other vectors described above can be used. As a method for introducing a vector containing a foreign gene into animal cells, various methods such as calcium phosphate coprecipitation method, microinjection method and electroporation method are known (J. Sambrook et al.,
"Molecular Cloning Second Edition", Cold Spring H
arbor Laboratory Press, New York, 1989, and Isao Ishida, Minsuke Ando, "Gene Expression Experiment Manual",
1994). The inventors used the electroporation method, but other various methods or methods described above can be used.

5. Use of Phytoene As described above, it was confirmed that phytoene has an anti-tumor promoter activity and a cancer cell growth inhibitory activity (see Examples for details). Therefore, phytoene can be used as an anti-tumor promoter and a cancer cell growth inhibitor. However, since the cancer cell growth inhibitory activity of phytoene is not sufficiently strong as compared with the anticancer agents currently used clinically (for example, mitomycin C), the use of phytoene is more than that of pharmaceuticals, such as foods and drinks for cancer prevention and cosmetics. It is considered to be more suitable as a material for. That is, phytoene can be applied to various foods and drinks for use as a health food. In order to obtain foods and drinks containing phytoene, for example, oils or powders for processed foods and oils or liquids for foods such as beverages (health drinks, etc.) may be added depending on the type of foods and drinks. Phytoene has not been reported to cause any physical damage due to overdose, is contained in green-yellow vegetables, and is colorless and transparent and almost odorless, so there is no need to worry about overdose. Therefore, the amount of intake is not particularly limited, but in order to obtain more effect, intake of several mg / day or more is preferable. Further, since it has the above-mentioned properties, phytoene can be used as a cosmetic material for cancer prevention. Therefore, in the present invention, this phytoene is used as a cosmetic material for cancer prevention, and the phytoene is used as a cosmetic material for cancer prevention by blending it with an ordinary cosmetic base material (for example, paraben, dibutylhydroxytoluene) in an appropriate ratio. You can In addition, it goes without saying that phytoene can be used as a material for sunscreen cream and the like because it absorbs ultraviolet rays well.

[0013]

The present invention will be described below in more detail with reference to examples. The scope of the invention is not limited by these examples. [Example 1] Anti-carcinogenic promoter activity of phytoene As an index for measuring the cancer promotion activity of TPA, Epst
A system of ein Barr (EB) virus activation exists (Y. Ito
et al., "A short-term in vitro assay for promoter
substances using human lymphoblastoid cells latent
ly infected with Epstein-Barr virus ", Cancer Lette
rs, 13, 29-37, 1981). In other words, when the TPA promoter is applied to B lymphocytes (Raji cells) containing the EB virus genome, the EB virus is activated.
The promoter activity can be measured by using a fluorescence-labeled antibody against the early antigen of EB virus that has emerged with this. Using this system, it was examined whether phytoene has anti-carcinogenic promoter activity. That is, EB
It was investigated whether phytoene suppressed the activation of the virus. The result is shown in FIG. As is clear from FIG. 1, phytoene exhibited antipromoter activity in a concentration-dependent manner. The effect became clear when it was applied at a concentration of 500 times (molar ratio) that of TPA, and at a concentration of 2000 times, a 90% or more inhibitory effect was observed. As another assay method for anti-carcinogenic promoters, human uterine cancer cells HeLa cells were treated with phytoene (50 μg / ml), and 1
After time, TPA (50 nM) and ³² Pi were added. After further culturing for 4 hours, the radioactivity incorporated in the phospholipid fraction was measured. As a result, it was confirmed that phytoene has an inhibitory effect on TPA-induced enhancement of phospholipid synthesis. This revealed that phytoene exhibited antipromoter activity even in another system. These are the first reports showing that phytoene has anti-carcinogenic promoter activity.

Example 2 Cancer Cell Proliferation Inhibitory Effect Various human cancer cells were used to examine whether phytoene has a cancer cell proliferation inhibitory effect. First, Eagle's minimum medium (H. Eagle, Sc) containing 10% fetal bovine serum is used.
ience, 130, 432-437, 1959) with various concentrations of phytoene added to the gastric cancer cells HGC-27 cells,
The cells were cultured in the presence of 5% CO ₂ at 37 ° C, and the effect of phytoene on the cell growth inhibition was examined. The result is shown in FIG. As is clear from FIG. 2, phytoene was found to suppress the growth of cancer cells in a concentration-dependent manner. The ID _{50 on the} 4th day after the treatment was 20 to 50 μM. next,
Using 50 μM phytoene, in addition to HGC-27 cells (gastric cancer), GOTO cells (neuroblasts), PANC-1 cells (pancreatic cancer), OST-
The inhibitory effect on the growth of KYO-1 cells (osteosarcoma) and HeLa cells (uterine cancer) was examined. The culture conditions for various cancer cells were the same as those for the HGC-27 cells described above.
The number of cells on the day was counted. The results are shown in Table 1. As can be seen from Table 1, it was revealed that phytoene suppresses the growth of various human tumor cells other than HGC-27 cells. In particular, the proliferative effect on the neuroblastoma GOTO cells was strong, and its potency was stronger than that on HGC-27 cells. This is the first report showing that phytoene has a cancer cell growth inhibitory effect.

[Example 3] Construction of plasmid for introduction into animal cells The crtB gene of Erwinia uredovora, a plant-resident bacterium, encodes a phytoene synthase that catalyzes the synthesis of phytoene from geranylgeranyl pyrophosphate (GGPP) (N. Misawa). et al., "Elucidation of the Erwinia ur
edovora carotenoid biosynthetic pathway by functio
nal analysis of gene products expressed in Escheri
chia coli ", J. Bacteriol., 172, 6704-6712, 1990,
And G. Sandmannn and N. Misawa, "New functional
assignment of the carotenogenic genes crtB and cr
tE with constructs of these genes from Erwinia spe
cies ", FEMS Microbiol. Lett., 90, 253-258, 199
0). 1.1 kb Eco52I (49 with only this crtB gene
26)-EcoRI (6009) DNA fragment (N. Misawa et al., "El
ucidation of the Erwinia uredovora carotenoid bios
ynthetic pathway by functional analysis of gene pr
oducts expressed in Escherichia coli ", J. Bacterio
PCR using l., 172, 6704-6712, 1990) as a template
Due to this, the DNA sequence near the N-terminus and C-terminus of CrtB was partially modified. That is, the TTG (5057) upstream of the crtB gene was modified to ATG of the start codon, and the Kozak sequence and the XhoI sequence were introduced immediately upstream (Fig. 3). Furthermore, an XhoI sequence was introduced at the EcoRI site downstream of the crtB gene. 1.1 kb XhoI containing the resulting crtB gene
The fragment was cut out and the vector for animals pCAGGS (H. Niwa et a
l., "Efficient selection for high-expression transf
ectants with a nevel eucaryotic vector ", Gene, 10
8, 193-200, 1991) by introducing it into the XhoI site,
A plasmid pCAcrtB was prepared (Fig. 3). In addition, the above 1.1 kb XhoI fragment was used as an animal vector LXSH (Gene Ban
k, Accession number M77239) was introduced into the XhoI site to prepare the plasmid LcrtBSH (FIG. 4).

[Example 4] Introduction of plasmid into animal cells NIH3T3 cells were prepared from Dulbecco's MEM containing 4 mM L-glutamine, 10% bovine serum, penicillin and streptomycin.
(DMEM) Medium (HJ Morton, In Vitro, 6, 93-94, 19
70 and LP Rutzky, RW Pumper, In Vito, 9, 4
68-469, 1974) and cultured at 37 ° C in the presence of 5% CO2. The plasmid prepared in Example 3 was prepared by electroporation (J. Sam) in the presence of the plasmid pKOneo.
brook etal., "Molecular Cloning Second Edition", C
old Spring Harbor LaboratoryPress, New York, 1989
) Was introduced into NIH3T3 cells.

[Example 5] Northern analysis of transformed cells NIH3 using various plasmid DNAs obtained in Example 4
Total RNA from T3 transformed cells was analyzed by the guanidine thiocyanate method (J. Sambrook et al., "Molecular CloningSecond E
dition ", Cold Spring Harbor Laboratory Press, New
York, 1989). 20 μg of total RNA for 1.2
Electrophoresed on a% formaldehyde agarose gel and transferred to a nitrocellulose filter. Northern blot analysis was performed using the 1.1 kb XhoI fragment containing the crtB gene shown in Example 3 labeled by the random primer labeling method (Amersham) as the probe DNA. FIG. 6 shows the result. As is clear from this figure, it was confirmed that the crtB gene was expressed in the NIH3T3 transformed cells with the plasmid pCAcrtB or LcrtBSH containing the crtB gene.

[Example 6] Examination of phytoene synthesizing ability of transformed cells Plasmid pC containing vector pCAGGS and crtB gene
Lipids were extracted from AcrtB-transformed NIH3T3 cells (10 ⁷ to 10 ⁸ ) with 50 ml of acetone, and then re-extracted with 50 ml of hexane. After washing with water several times, the upper hexane layer was concentrated and dried. After dissolving in a small amount of methanol, a Waters Nova-pak HR column (6 μC18,
High-performance liquid chromatography (HPLC) using 3.9 x 300 mm), and elution was performed with acetonitrile / methanol / 2-propanol (90/6/4) at a flow rate of 1 ml / min and 286 nm with a JASCO 875 UV detector. Was monitored. As a result, plasmid pCAc containing the crtB gene
In lipids prepared from NIH3T3 cells by rtB , Erwi
A standard phytoene (15,15'-cis-phytoene purified from Escherichia coli containing the crtE and crtB genes of nia uredovora.
A peak not detected in NIH3T3 cells having only the vector was detected at the same position as () (holding time 59 minutes) (Fig. 7 (a), (b)). Therefore, in addition, HP
Purified by preparative method from LC. The UV absorption spectrum was measured by Shimadzu diode array detector SPD-M10A, and the spectrum was in agreement with that of phytoene. In addition, the FD-MS spectrum of the JEOL SX-102
As measured by a mass spectrometer, the molecular ion peak (m / m) was found at the position corresponding to the molecular weight of phytoene.
z 545) occurred (Fig. 8). From the above results, this substance was identified as phytoene (15,15'-cis-phytoene).

[Example 7] Resistance test of transformed cells to oxidative stress NIH3T3 transformed cells confirmed to synthesize phytoene were cultured to 80-90% "confluent" state (culture method was carried out. Example 4), with 374 mM iron (III) chloride
Oxidative stress was applied by culturing in DMEM medium containing 10 mM ADP for 4 hours. Then, wash with PBS (-) 3 times, scrape the cells with a cell scraper, wash the collected cells again with PBS (-), and finally with 1 mM PBS (-).
After suspending in, the cells were freeze-thawed once. The cell suspension was diluted with 6 ml of chloroform / methanol (2/1) mixture to 2
The chloroform layer was extracted twice and concentrated to dryness. Next, after dissolving in a small amount of an HPLC developing solvent, it was subjected to chemiluminescence HPLC to quantify lipids and lipid peroxides. The lipid component is a JASCO Finepack SIL NH2-5 column (250 mm x
4.6 mm id), 2-propanol / hexane / methanol / water (7/5/1/1), flow rate 1 ml / min, 3
Develop and elute at 5 ° C and 210 nm with TOSHO UV-8011 detector.
Was detected by monitoring. The lipid peroxide in the separated lipids was
Chemiluminescence was generated by mixing with g / ml cytochrome C and borate buffer solution (pH 10) containing 2 μg / ml luminol, and the resulting chemiluminescence was produced by Tohoku Denshi CLD-110.
Detection was performed with a detector at a flow rate of 1.1 ml / min. As a result, as shown in Table 2, NIH3 that produces phytoene
The amount of lipid peroxide in T3 transformed cells was found to be down to 55% of NIH3T3 transformed cells carrying the vector alone. This means that NI producing phytoene
It is shown that the H3T3 transformed cells have acquired resistance to oxidative stress.

Example 8 Cancer Prevention Test of Transformed Cells NIH3T3 transformed cells into which pCAcrtB and pCAGGS were introduced were added to 1.5 × 10 ⁵ cells each and cultured for 2 days (the culture method was Example 4). reference). After 2 days of culture, plasmids pNCO102 and pNCO602 containing the oncogene hst-1 were respectively subjected to the calcium phosphate method (J. Sambrook et al., "M.
olecular Cloning Second Edition ", ColdSpring Harbo
r Laboratory Press, New York, 1989). At the same time, for comparison of transfection efficiency, a plasmid containing a hygromycin resistance gene was used.
Also introduced LXSH. After culturing in a medium containing DNA and calcium phosphate for 8 hours, the medium was exchanged and further culturing was continued for 24 hours. Then, the cells were divided into 4 plates, and 2 plates were cultured in DMEM medium containing 5% bovine serum to examine the formation of foci. The other two were cultured in DMEM medium containing 10% bovine serum and hygromycin B to examine the transfection efficiency. The medium was changed every 2-3 days, and the number of foci was counted after 17 days and 32 days. As a result, Table 3
As shown in Fig. 7, after 17 days, the focus number of the NIH3T3 transformed cells introduced with pCAcrtB having the crtB gene was about 1/2 or lower than the focus number of the NIH3T3 transformed cells introduced with the vector pCAGGS. It was below. It was also found that the number of foci of NIH3T3 transformed cells into which pCAcrtB had been introduced did not increase even after 32 days. Therefore, it was demonstrated that the focus formation of NIH3T3 cells by the oncogene was suppressed by the introduction of pCAcrtB. No significant difference was observed in transfection efficiency. From the above results, it was shown that NIH3T3 transformed cells producing phytoene are effective in preventing cancer and suppressing the growth of cancer cells.

Table 1 Effect of phytoene on proliferation of various cancer cells Number of viable cells (x 10 ^-4 / dish) Spore control + Phytoene (50 μM) inhibition% GOTO 71.5 0 100 HGC-27 65.0 9.0 86.2 PANC-1 18.5 7.5 59.5 OST-KYO-1 58.0 31 0.0 46.6 HeLa 50.0 33.0 34.0

Table 2 Protective effect against phospholipid peroxidation in NIH3T3 cells carrying plasmid pCAcrtB Plasmid PCOOH + PEOOH / PC + PE Lipid peroxide amount ratio pCAGGS 4.56 E-4 pmol / area 1 pCAcrtB 2.50 E-4 pmol / area 0.55 The data represent the average value of 6 replicate experiments.

[0023]Table 3 Focus formation assay using NIH3T3 transformed cells Oncogene \ Plasmid pCAGGS pCAcrtB pCAcrtB17 days later 17 days later 32 days later − 44.5 35 110 hst-1 (pKOHST1) 135 (90.5) 89.5 (54.5) 147 (37) ras-1 (pNCO102) 91 (46.5) 57 (22) 122.8 (12.8)ras-2 (pNCO602) 124 (79.5) 50 (15) 138.5 (28.5)  The data shows the average value of the focus numbers of two dishes. The numbers in parentheses show the converted values based on the number of control focus points.

[Example 9] Formulation example of food and drink containing phytoene When the crtE and crtB genes of Erwinia were introduced into Escherichia coli by an appropriate vector such as pUC19, the E. coli came to produce phytoene (N. Misawa et al., "Elu
cidation of the Erwinia uredovora carotenoid bio
synthetic pathway by functional analysis of gene p
roducts expressed in Escherichia coli ", J. Bacter
iol., 172, 6704-6712, 1990). This E. coli (for example, E. coli JM109 containing plasmid pCAR-AE) was
2 x with L suitable drug (eg ampicillin)
The cells were cultured in YT medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl) at 30 ° C for 24 hours. The cells collected by centrifugation were extracted with 500 ml of acetone and then re-extracted with 500 ml of hexane. After washing with water several times, the upper hexane layer was concentrated and dried. Silica gel column chromatography (30 cm x 3.
0 cm), developed and eluted with hexane: chloroform (1: 1), separated the band of phytoene having strong ultraviolet absorption, and further subjected to Sephadex LH-20 column chromatography (30 cm X 3.0 cm), Elution with chloroform: methanol (1: 1) elution, pure product 10 mg
I got 5 mg of phytoene dissolved in a small amount of ethanol was suspended in 50 ml of an emulsifier consisting of sucrose fatty acid ester (25%), glycerin fatty acid ester (5%) and glycerin (30%).
0.1 mg / ml). Add this to water or 70% orange juice (eg Kirin Hyper Orange) with 0.1%
%, 1% and 5% were added, and phytoene could be blended without separation of phytoene.

[Example 10] Blending example of cosmetics containing phytoene By the above-mentioned example, a pure phytoene product can be obtained. 5 mg phytoene in a small amount of cosmetic material (eg paraben or dibutyl hydroxytoluene)
And the final concentration of phytoene is 0.001%, 0.01%,
Add phytoene to skin cream (eg Mentholatum Medicated Hand Cream) or foundation (eg Corpcake (2-way) M23) at a concentration of 0.1% without phytoene separation when stirred. did it.

[0026]

INDUSTRIAL APPLICABILITY In the present invention, the production of plant components in animals (particularly domestic animals) was successful for the first time, which enabled the production of stress-resistant animals. This allows
The animal does not need to ingest useful botanical ingredients from the outside world, and can be used, for example, for production of feed-independent transgenic livestock. If the plant component is phytoene, it can be used as a cancer preventive agent or food.

[0027]

[Sequence list]

SEQ ID NO: 1 Sequence length: 930 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Organism name: Erwini a uredovora Strain name: 20D3 ATCC19321 Sequence ATG AAT AAT CCG TCG TTA CTC AAT CAT GCG GTC GAA ACG ATG GCA GTT 48 Met Asn Asn Pro Ser Leu Leu Asn His Ala Val Glu Thr Met Ala Val 1 5 10 15 GGC TCG AAA AGT TTT GCG ACA GCC TCA AAG TTA TTT GAT GCA AAA ACC 96 Gly Ser Lys Ser Phe Ala Thr Ala Ser Lys Leu Phe Asp Ala Lys Thr 20 25 30 CGG CGC AGC GTA CTG ATG CTC TAC GCC TGG TGC CGC CAT TGT GAC GAT 144 Arg Arg Ser Val Leu Met Leu Tyr Ala Trp Cys Arg His Cys Asp Asp 35 40 45 GTT ATT GAC GAT CAG ACG CTG GGC TTT CAG GCC CGG CAG CCT GCC TTA 192 Val Ile Asp Asp Gln Thr Leu Gly Phe Gln Ala Arg Gln Pro Ala Leu 50 55 60 CAA ACG CCC GAA CAA CGT CTG ATG CAA CTT GAG ATG AAA ACG CGC CAG 240 Gln Thr Pro Glu Gln Arg Leu Met Gln Leu Glu Met Lys Thr Arg Gln 65 70 75 80 GCC TAT GCA GGA TCG CAG ATG CAC GAA CCG GCG TTT GCG GCT TTT CAG 28 8 Ala Tyr Ala Gly Ser Gln Met His Glu Pro Ala Phe Ala Ala Phe Gln 85 90 95 GAA GTG GCT ATG GCT CAT GAT ATC GCC CCG GCT TAC GCG TTT GAT CAT 336 Glu Val Ala Met Ala His Asp Ile Ala Pro Ala Tyr Ala Phe Asp His 100 105 110 CTG GAA GGC TTC GCC ATG GAT GTA CGC GAA GCG CAA TAC AGC CAA CTG 384 Leu Glu Gly Phe Ala Met Asp Val Arg Glu Ala Gln Tyr Ser Gln Leu 115 120 125 GAT GAT ACG CTG CGC TAT TGC TAT CAC GTT GCA GGC GTT GTC GGC TTG 432 Asp Asp Thr Leu Arg Tyr Cys Tyr His Val Ala Gly Val Val Gly Leu 130 135 140 ATG ATG GCG CAA ATC ATG GGC GTG CGG GAT AAC GCC ACG CTG GAC CGC 480 Met Met Ala Gln Ile Met Gly Val Arg Asp Asn Ala Thr Leu Asp Arg 145 150 155 160 GCC TGT GAC CTT GGG CTG GCA TTT CAG TTG ACC AAT ATT GCT CGC GAT 528 Ala Cys Asp Leu Gly Leu Ala Phe Gln Leu Thr Asn Ile Ala Arg Asp 165 170 175 ATT GTG GAC GAT GCG CAT GCG GGC CGC TGT TAT CTG CCG GCA AGC TGG 576 Ile Val Asp Asp Ala His Ala Gly Arg Cys Tyr Leu Pro Ala Ser Trp 180 185 190 CTG GAG CAT GAA GGT CTG AAC AAA GAG AAT TAT GCG GCA CCT G AA AAC 624 Leu Glu His Glu Gly Leu Asn Lys Glu Asn Tyr Ala Ala Pro Glu Asn 195 200 205 CGT CAG GCG CTG AGC CGT ATC GCC CGT CGT TTG GTG CAG GAA GCA GAA 672 Arg Gln Ala Leu Ser Arg Ile Ala Arg Arg Leu Val Gln Glu Ala Glu 210 215 220 CCT TAC TAT TTG TCT GCC ACA GCC GGC CTG GCA GGG TTG CCC CTG CGT 720 Pro Tyr Tyr Leu Ser Ala Thr Ala Gly Leu Ala Gly Leu Pro Leu Arg 225 230 235 240 TCC GCC TGG GCA ATC GCT ACG GCG AAG CAG GTT TAC CGG AAA ATA GGT 768 Ser Ala Trp Ala Ile Ala Thr Ala Lys Gln Val Tyr Arg Lys Ile Gly 245 250 255 GTC AAA GTT GAA CAG GCC GGT CAG CAA GCC TGG GAT CAG CGG CAG TCA 816 Val Lys Val Glu Gln Ala Gly Gln Gln Ala Trp Asp Gln Arg Gln Ser 260 265 270 ACG ACC ACG CCC GAA AAA TTA ACG CTG CTG CTG GCC GCC TCT GGT CAG 864 Thr Thr Thr Pro Glu Lys Leu Thr Leu Leu Leu Ala Ala Ser Gly Gln 275 280 285 GCC CTT ACT TCC CGG ATG CGG GCT CAT CCT CCC CGC CCT GCG CAT CTC 912 Ala Leu Thr Ser Arg Met Arg Ala His Pro Pro Arg Pro Ala His Leu 290 295 300 TGG CAG CGC CCG CTC TAG 930 Trp Gln Arg Pro L eu *** 305

[Brief description of drawings]

FIG. 1 is an explanatory view showing the effect of phytoene on the expression of TPA-induced EB virus early antigen in Raji cells.

FIG. 2 is an explanatory view showing the effect of phytoene on the growth of HGC-27 cells.

FIG. 3 is a structural diagram of plasmid pCAcrtB.

FIG. 4 is a structural diagram of plasmid LcrtBSH.

FIG. 5 is an explanatory view showing the nucleotide sequence of the crtB gene (chromosomal DNA) contained in the plasmids of FIGS. 3 and 4 and the corresponding amino acid sequence.

FIG. 6: Total RNA prepared from various NIH3T3 transformed cells
Electrophoresis photograph showing the result of Northern analysis of. The crtB gene was used as a probe.

FIG. 7: Spectra of HPLC analysis of lipids prepared from NIH3T3 cells carrying plasmid pCAcrtB. Phytoen (ph
The ytoene) peak (retention time 59 minutes) is shown in black.

FIG. 8: Mass (FD-MS) spectrum of phytoene purified from NIH3T3 cells carrying plasmid pCAcrtB.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 48/00 ADU A61K 48/00 ADU AER AER AGZ AGZ C12N 15/09 9162-4B C12N 15/00 A

Claims (14)

[Claims] 1. A method for producing a plant component in an animal, which comprises introducing a gene involved in the synthesis of the plant component into an animal and expressing the gene in the animal. 2. The method according to claim 1, wherein the botanical component is a carotenoid. 3. The method of claim 2, wherein the carotenoid is phytoene. 4. A method for producing an oxidative stress-tolerant animal, which comprises introducing a gene involved in the synthesis of a plant component having antioxidant ability into an animal and expressing the gene in the animal. 5. A method for producing a lipid oxidative stress-tolerant animal, which comprises introducing a gene involved in the synthesis of a plant component having an anti-lipid oxidation ability into an animal and expressing the gene in the animal. 6. A method for producing a carcinogenic stress-resistant animal, which comprises introducing a gene involved in the synthesis of a plant component capable of preventing cancer into an animal and expressing the gene in the animal. 7. A method for producing a carcinogenic stress-resistant animal, which comprises introducing a gene involved in the synthesis of a plant component capable of suppressing cancer growth into an animal and expressing the gene in the animal. 8. The plant-derived component is phytoene.
7. The method according to any one of items 7 to 7. 9. The method according to any one of claims 1 to 8, wherein the animal is a domestic animal. 10. An anti-carcinogenic promoter agent containing phytoene as an active ingredient. 11. A cancer cell growth inhibitor comprising phytoene as an active ingredient. 12. A food or drink containing phytoene. 13. A cosmetic material for cancer prevention comprising phytoene. 14. A cosmetic product containing phytoene.