JP5641232B2 - Ogonori-derived cyclooxygenase gene and method for producing prostaglandins using the gene - Google Patents

Description

  The present invention relates to a cyclooxygenase gene, which is a gene involved in the biosynthesis of prostaglandins (hereinafter also abbreviated as “PG”) derived from tiger beetle (Gracilaria vermiculophylla) and its use. The present invention also relates to a method for producing prostaglandins using E. coli (Escherichia coli, also simply referred to as E. coli), plant transformants and the like.

  Prostaglandins, which are one class of eicosanoids, have peripheral vasodilatory action, platelet aggregation inhibitory action, cytoprotective action, angiogenic action, etc., and are used as these therapeutic agents as so-called lipoPG agents.

  Polyunsaturated fatty acids such as arachidonic acid (hereinafter sometimes abbreviated as “AA”) and eicosapentaenoic acid (hereinafter sometimes abbreviated as “EPA”) are lipids in the cell membrane mainly in the nervous system in humans and the like. Is included in many. From these highly unsaturated fatty acids, pharmacologically very important physiologically active substances such as prostaglandins are biosynthesized by the reaction of cyclooxygenase. These biosynthetic metabolic pathways are called arachidonic acid cascades (FIG. 1).

  As described above, it has been reported that prostaglandins and the like which are physiologically active substances are biosynthesized in animal cells. Prostaglandins in blood have been prepared for quantitative analysis (Patent Document 1).

  Currently, prostaglandins are produced by total chemical synthesis from lactones by chemical synthesis. This raises the problem of high production costs. Prostaglandins are considered to be biosynthesized by reaction of cyclooxygenase from AA or EPA, for example, in the human body. In these reactions, AA or EPA is first released from cell membrane lipids into cells by phospholipase, and the AA or EPA is oxidized by two molecules of oxygen by cyclooxygenase and converted into prostaglandins.

  The seaweed Ogonori belongs to the Protozoic kingdom, Rhodophyta, and is classified into the order Ogonori, Ogonoriaceae, Ogonori. In addition to ogonori, birch, shiramo, and tsurushiramo are known. Accumulation of prostaglandins has been reported in Ogonori (Non-patent Document 1 and Patent Document 2). Although the presence of prostaglandin-like substances has been reported in onions, poplars, larch, evening primrose, morning glory, bean diatom and certain Thai species, details are unknown (Non-patent Document 2).

Published JP 2007-278750 Japanese Patent Laid-Open No. 3-206895

Bull. Of the Jpa. Society of Science Fisheries 50 (3), p465-469, 1984 Botanical Review 63, p199, 1997

  The present invention has been made in view of the above-mentioned conventional problems, and its object is to use cyclooxygenase derived from the order of organisms, etc., that can produce prostaglandins using microorganisms such as E. coli, plants, and the like. It is to provide genes as well as their usage.

  As a result of intensive studies to solve the above problems, the present inventors have identified a gene encoding cyclooxygenase from a cDNA clone derived from Gracilaria vermiculophylla. In addition, the present inventors have found that prostaglandins can be produced in E. coli by introducing the gene into E. coli and expressing it, and transformants obtained by introducing the gene into microorganisms or plants As a result, it was conceived that prostaglandins could be produced efficiently. For example, when an E. coli transformant into which the gene encoding cyclooxygenase is introduced so as to be expressed is cultured in the presence of AA and / or EPA, prostaglandins using AA or EPA in the cells (cells) as a substrate The prostaglandins biosynthesized intracellularly are secreted into the culture medium. For this reason, the E. coli transformant into which the gene and the gene can be expressed can be used to supply raw materials such as drugs and reagents in the production of prostaglandins in the pharmaceutical industry and various material industries. Very useful.

  In addition, the transformant of the genus Coleoptera that has been introduced so that the gene encoding the cyclooxygenase can be expressed (according to the order of the genus Coleoptera) accumulates prostaglandins in the cell as it grows or grows. Since the genus Coleoptera biosynthesizes arachidonic acid and eicosapentaenoic acid, which are prostaglandin synthesis substrates, the use of such plant transformants eliminates the need to add these substrates to the medium. Play.

That is, the present invention includes the following inventions [1] to [16].
[1] A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 154th to 1842th base sequences in the base sequence shown in SEQ ID NO: 1
(B) hybridizes under stringent conditions with DNA consisting of the 154th to 1842th base sequences of the base sequence shown in SEQ ID NO: 1 or DNA complementary to the DNA, and arachidonic acid or A DNA encoding a protein derived from the order of the genus Pleurotus with cyclooxygenase activity using eicosapentaenoic acid as a substrate.
[2] A gene containing the DNA of any one of (a) to (c) below.
(A) DNA comprising the base sequence shown in SEQ ID NO: 1
(B) a DNA derived from the organism of the order Ogonori that encodes a protein having a cyclooxygenase activity comprising a part of the base sequence represented by SEQ ID NO: 1 and using arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Cyclooxygenase activity which hybridizes under stringent conditions with all or part of DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and which uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the order of the order of the organism that encodes the protein
[3] A gene encoding the following protein (A) or (B).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B) From the amino acid sequence shown in SEQ ID NO: 2 in which one to several amino acids are substituted, deleted, inserted and / or added A protein derived from the order of the genus Ogonori having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate

[4] A protein encoded by the gene according to any one of [1] to [3].
[5] A recombinant expression vector comprising the gene according to any one of [1] to [3].
[6] An E. coli transformant obtained by introducing at least the gene according to any one of [1] to [3] into E. coli which is a host cell.
[7] A genus Coriolis transformant obtained by introducing at least the gene according to any one of the above [1] to [3] into a genus Coriolis, or the cranigo having the same properties as the transformant Or a tissue of the genus C. elegans, which is a descendant of the organism transformant.
[8] A reproductive material obtained from the genus Geometridae transformant according to [7] above or the tissue of the genus Geometridae organism transformant.

[9] A prosta using arachidonic acid or eicosapentaenoic acid as a substrate, comprising the step of culturing the E. coli transformant according to [6] in the presence of arachidonic acid and / or eicosapentaenoic acid How to produce grangeins.
[10] A method for producing prostaglandins using arachidonic acid or eicosapentaenoic acid as a substrate, the method comprising a step of proliferating or growing the genus Geometridae or the tissue thereof according to [7].
[11] A step of preparing a protein fraction from the E. coli transformant according to [6] above or the genus Geometridae transformant according to [7] above. A method for producing prostaglandins using icosapentaenoic acid as a substrate.
[12] A method for altering the prostaglandin composition of a genus Coleoptera or a tissue thereof, which comprises the step of introducing the gene according to any one of [1] to [3] above into the genus Amanita .
[13] An antibody that recognizes the protein encoded by the gene according to any one of [1] to [3].
[14] A gene detection instrument using at least a part of the base sequence of the gene according to any one of [1] to [3] or a complementary sequence thereof as a probe.

[15] A method for screening for a substance that regulates the protein using the protein encoded by the gene according to any one of [1] to [3].
[16] (I) a step of contacting arachidonic acid and / or eicosapentaenoic acid and a candidate substance in vitro with the protein encoded by the gene according to any one of [1] to [3], (II) a step of monitoring the amount of prostaglandins produced using arachidonic acid or eicosapentaenoic acid as a substrate, and (III) the amount of prostaglandins produced in step (II) as compared with the case where no candidate substance is contacted. The method for screening according to [15] above, further comprising the step of selecting a candidate substance in which an increase or decrease is detected as a substance that regulates the protein.

  Prostaglandins based on arachidonic acid or eicosapentaenoic acid mean prostaglandins biosynthesized from arachidonic acid or eicosapentaenoic acid.

  One of the preferred embodiments of the gene according to the present invention is a gene encoding a cyclooxygenase gene isolated from ogonori, and a protein having an activity equivalent to the protein encoded by these genes. For example, when the cyclooxygenase gene is expressed in a host cell such as E. coli or a plant, genes related to prostaglandin biosynthesis function well in the host cell, and as a result, prostaglandins are biosynthesized. The effect that it is done.

  In addition, the E. coli transformant according to the present invention can produce prostaglandins and has the effect of secreting prostaglandins synthesized intracellularly. For this reason, prostaglandins can be produced efficiently. By using such an E. coli transformant according to the present invention, it is possible to significantly reduce the production cost of prostaglandins useful as an active ingredient of pharmaceuticals, and at the same time, to realize a production process that is easier to the environment. it can. Further, in the plant transformant according to the present invention, prostaglandins are generated and accumulated in the cells with the growth or growth thereof. That is, according to the present invention, it is possible to produce prostaglandins at low cost by a low-cost and environmentally friendly production process. Furthermore, since an E. coli transformant can be mass-produced by a microorganism culture method, it is possible to industrially supply cheaper prostaglandins efficiently. The prostaglandins thus obtained have an effect that they can be used as pharmacologically active substances, raw materials for health foods, and the like.

FIG. 1 is a diagram showing a reaction in which prostaglandins are produced from arachidonic acid or eicosapentaenoic acid by cyclooxygenase in the arachidonic acid cascade. FIG. 2 is a diagram showing an outline of the flow of prostaglandin production by the E. coli transformant of the present invention. FIG. 3A to FIG. 3B are diagrams showing an example of a procedure for producing a phenotypic expression construct (FIG. 3A: E. coli, FIG. 3B: yeast) for introducing a cyclonigenase gene derived from ogonori. FIG. 4C to FIG. 4D are diagrams showing an example of a procedure for producing a trait expression construct (plant trait expression recombinant vector) for introducing a cyclogonogenase gene derived from ogonori. 5A to 5D respectively show an E. coli expression recombinant vector (FIG. 5A), a yeast expression vector (FIG. 5B), and a plant expression expression recombinant vector for introducing a cyclooxygenase gene derived from the order of the genus Pleurotus. It is a schematic diagram of (FIG. 5C and FIG. 5D).

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

  Hereinafter, the biosynthetic pathway of prostaglandins, the gene according to the present invention, the protein according to the present invention, the protein according to the present invention and the method for obtaining the gene, and the method for producing the gene and protein according to the present invention (utility, ie, The present invention will be described in detail in the order of production of prostaglandins.

(1) Prostaglandin biosynthetic pathway (Figure 1)
In the cell, prostaglandins start from arachidonic acid and eicosapentaenoic acid (substrate), and the substrate is oxidized by two molecules of oxygen and biosynthesized into prostaglandins. These reactions are catalyzed by cyclooxygenase (COX) as shown in FIG. These reaction pathways are called arachidonic acid cascades.

(2) Gene according to the present invention The gene of the present invention is a cyclooxygenase gene. The gene of the present invention can be suitably used for, for example, preparation of recombinant proteins, production of prostaglandins, production of E. coli, yeast and plant transformants with modified prostaglandin composition. It can be done.

[Cyclooxygenase gene according to the present invention]
The cyclooxygenase gene according to the present invention is a gene encoding a protein having cyclooxygenase activity that uses arachidonic acid or eicosapentaenoic acid as a substrate and oxidizes the substrate with two molecules of oxygen. ~ 7. It is a gene described in.
1. A gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 1.
2. A gene comprising a part of the base sequence shown in SEQ ID NO: 1 and containing a DNA derived from the order of the organism of the order of the organism that encodes a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate.
3. A protein having a cyclooxygenase activity that hybridizes under stringent conditions with all or part of a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and uses arachidonic acid or eicosapentaenoic acid as a substrate A gene containing DNA derived from the order of the genus Gonoid.

4). A gene comprising DNA consisting of the 154th to 1842th base sequences in the base sequence shown in SEQ ID NO: 1.
5. It hybridizes under stringent conditions with DNA comprising the nucleotide sequence 154 to 1842 of the nucleotide sequence shown in SEQ ID NO: 1 or DNA complementary to the DNA, and arachidonic acid or eicosapentaene A gene comprising a DNA encoding a protein derived from the order of the genus Lepidoptera having a cyclooxygenase activity using an acid as a substrate.

6). A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
7). In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one to several amino acids are substituted, deleted, inserted and / or added, and has cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate A gene that encodes a protein derived from the order of the genus Lepidoptera.
In the present invention, the term “several” is usually about 20, preferably about 10, more preferably about 7, more preferably about 5, particularly preferably about 3, and most preferably about 2. is there.
In the present invention, the protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate may use arachidonic acid and eicosapentaenoic acid as substrates.
The cyclooxygenase gene according to the present invention is preferably derived from the order of the genus Lepidoptera. More preferably, it is derived from Gracilaria vermiculophylla.

  The DNA consisting of the base sequence shown in SEQ ID NO: 1 is a gene derived from Gracilaria vermiculophylla. Of the base sequence shown in SEQ ID NO: 1, the 154th to 1842th base sequences are regions that are translated into a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a cyclooxygenase that oxidizes arachidonic acid or eicosapentaenoic acid with two molecules of oxygen. The 1840th to 1842th base sequence (TAG) of SEQ ID NO: 1 is the start codon. Of the base sequence shown in SEQ ID NO: 1, the 154th to 1842th base sequence is shown in SEQ ID NO: 3.

Examples of the DNA derived from the order of the organism of the order Ogonori which consists of a part of the base sequence shown in SEQ ID NO: 1 and encodes a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate include, for example, SEQ ID NO: 1 Of the nucleotide sequences shown, DNA having the 154th to 1842th nucleotide sequence (that is, DNA having the nucleotide sequence shown in SEQ ID NO: 3) is preferable. The DNA comprising a part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1 is, for example, a base complementary to the DNA having the 154th to 1842th base sequences in the base sequence of SEQ ID NO: 1. Sequence DNA is preferred.
In addition, as a gene containing DNA consisting of the 154th to 1842th base sequences (DNA of SEQ ID NO: 3) among the base sequences shown in SEQ ID NO: 1, for example, the base sequence shown in SEQ ID NO: 1 is preferably used. It is done.

  It should be noted that the above “stringent conditions” means that hybridization occurs only when at least about 90% identity, preferably at least about 95% identity, and most preferably about 97% identity exists between sequences. Means what happens.

The hybridization, J. Sambrook et al. Molecular Cloning, A lLaboratory Manual. 2 nd Ed., Like the method described in Cold Spring Harbor Laboratory (1989), can be performed by a conventionally known method. Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous gene can be obtained. As hybridization conditions, conventionally known conditions can be preferably used, and are not particularly limited. For example, a commercially available DIG (digoxigenin) hybridization buffer (Roche Diagnostics) is used. And so on.

The cyclooxygenase gene of the present invention can be obtained from the order of the genus Pleurotus.
The above-mentioned `` Orchidaceae organism '', that is, the seaweed Ogonori belongs to the Protozoic kingdom, Rhodophyta, and is classified as Ogonori, Ogonoriaceae, Ogonori genus, in addition to Ogonori, birch, Shiramo and Tsurusilamo are known. Accumulation of prostaglandins has been reported in organisms of the order of Gogonidae (Bull. Of the Jpa. Society of Science Fisheries 50 (3), p465-469, 1984).

  Obtaining the cyclooxygenase gene from these organisms is easy with the current state of the art. For example, it is generally known that genes encoding enzymes with similar functions in related organisms cross-hybridize.

  The gene of the present invention includes not only double-stranded DNA but also each single-stranded DNA and RNA called sense strand and antisense strand. The antisense strand can be used as a probe or as an antisense compound. DNA includes, for example, cDNA and genomic DNA obtained by cloning or chemical synthesis techniques, or a combination thereof. Furthermore, the gene of the present invention comprises a promoter region, an untranslated region (a 5′UTR sequence, a terminator sequence including 3′UTR, etc.), a vector sequence (for example, an E. coli expression expression recombinant vector sequence described later, etc.). It may contain a sequence such as a replacement expression vector sequence).

(3) Protein according to the present invention The protein according to the present invention is a cyclooxygenase protein.

[Cyclooxygenase protein according to the present invention]
The cyclooxygenase protein according to the present invention may be any protein as long as it exhibits substrate specificity for the above-described cyclooxygenase activity, that is, AA or EPA, and has an action of converting these into prostaglandins. More specifically, the following proteins are preferred. Preferably, it is a protein having cyclooxygenase activity derived from the order of the genus Lepidoptera. More preferably, it is a protein having cyclooxygenase activity derived from Gracilaria vermiculophylla.
1. 1. a protein encoded by the cyclooxygenase gene according to the present invention described in (2) above; 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Cyclooxygenase activity comprising an amino acid sequence in which one to several amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted and / or added, and using arachidonic acid or eicosapentaenoic acid as a substrate Protein with Protein The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a cyclooxygenase derived from Gracilaria vermiculophylla.

  The above "one to several amino acids are substituted, deleted, inserted, and / or added" means substitution, deletion, insertion by a known mutant protein production method such as site-directed mutagenesis. And / or a number that can be added (usually 20 or less, preferably 10 or less, more preferably 7 or less, still more preferably 5 or less, even more preferably 1 to 3, particularly preferably 1 to 2). Or most preferably about 1 amino acid) is substituted, deleted, inserted and / or added.

(4) Gene and protein acquisition method according to the present invention The gene and protein acquisition method (production method) according to the present invention is not particularly limited, but typical methods include the following methods. it can.

[Method of obtaining genes]
The method for obtaining the gene of the present invention is not particularly limited, and examples thereof include a method using differential screening (subtraction cloning). In this method, according to a known technique, direct hybridization in a test tube is repeated to concentrate the target cDNA (the gene of the present invention).

  Each step in the differential screening may be performed under conditions that are normally used. The clone thus obtained can be analyzed in more detail by preparing a restriction enzyme map and determining its nucleotide sequence (sequencing). From these analyses, it can be easily confirmed whether a DNA fragment containing the gene sequence of the present invention has been obtained.

  Moreover, as a method for obtaining the gene of the present invention, a method of isolating and cloning a DNA fragment containing the gene of the present invention by a known technique can be mentioned. For example, a probe that specifically hybridizes with a part of the base sequence of the gene of the present invention may be prepared and a genomic DNA library or cDNA library may be screened. As such a probe, any probe of any sequence / length may be used as long as it specifically hybridizes to at least part of the base sequence of the gene of the present invention or its complementary sequence.

  Further, for example, if the sequence of the probe is selected from among the regions that are well conserved among the above-mentioned organisms, and a genomic DNA (or cDNA) library of other organisms is screened, A gene encoding a homologous molecule or a similar molecule having a function similar to that of a protein can be isolated and cloned.

  Alternatively, as a method for obtaining the gene of the present invention, a method using amplification means such as PCR can be mentioned. For example, among the cDNA sequences of the gene of the present invention, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and genomic DNA (or cDNA) is used as a template using these primers. By performing PCR or the like and amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing the gene of the present invention can be obtained. Operations such as PCR and primer preparation can be performed by a known genetic engineering technique (gene manipulation technique). The gene of the present invention can also be synthesized by a DNA synthesizer.

The protein encoded by the gene obtained by the above method has cyclooxygenase activity.For example, the protein is produced by introducing the gene into E. coli, yeast, etc., and using the prepared protein fraction, The enzyme activity of can be confirmed by in vitro analysis.
Cyclooxygenase activity is usually measured by Hashimoto et al, J. MoI. Biol. Chem. 254, 829-836, (1979).

[Protein acquisition method]
The method (production method) for obtaining the protein of the present invention is not particularly limited as described above. First, the protein is isolated from microorganisms such as E. coli and plants that express the protein of the present invention. A purification method can be mentioned. The purification method is not particularly limited, and an extract is prepared from an E. coli transformant or the like by a known method, and the extract may be purified using a known method such as a column.

  Moreover, as a method for obtaining the protein of the present invention, a method using a gene recombination technique or the like can also be mentioned. In this case, for example, after the gene of the present invention is incorporated into a recombinant vector or the like, it is introduced into a host cell so that it can be expressed by a known method, and the resulting transformed cell (transformant) is cultured. For example, a method of purifying the above-mentioned protein obtained by translation in step 1 can be employed. The host cell may be a plant, microorganism, or animal cell.

  For the purpose of obtaining the protein of the present invention, for example, when a foreign gene is introduced into E. coli as a host cell, an E into which a promoter that functions in E. coli is incorporated for the expression of the foreign gene. Since there are various E. coli expression vectors, it is sufficient to select one according to the purpose. The method for purifying the protein produced in the host cell differs depending on the host used and the nature of the protein, but the protein is recovered from the host cell or its culture supernatant by a method known to those skilled in the art. Is possible. For example, the target protein can be purified relatively easily by using tags or the like.

  The method for producing the mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto et al. Gene 152, 271-275 (1995), a method of introducing a point mutation into a nucleotide sequence using PCR method, or creating a mutant protein, or by inserting a transposon A well-known mutant protein production method such as a mutant strain production method can be used, and a commercially available kit may be used for the production of the mutant protein.

The method for obtaining the protein of the present invention is not limited to the method described above, and may be, for example, chemically synthesized. For example, the protein of the present invention may be synthesized from the gene of the present invention using a cell-free protein synthesis solution. Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. 1985 Development of pharmaceuticals Continued Vol. 14, peptide synthesis, Hirokawa Shoten, 1991, etc., can also be synthesized by peptide synthesis methods.
It can be confirmed that the protein has cyclooxygenase activity by measuring the activity by the method described above.

(5) Gene and protein utilization method according to the present invention (usefulness, prostaglandin productivity)
(5-1) Recombinant Expression Vector A recombinant expression vector containing the gene of the present invention is also encompassed by the present invention.
The recombinant expression vector of the present invention is, for example, a transformant, prostagland, for producing the above-described protein (cyclooxygenase protein) according to the present invention in a host cell or having increased prostaglandin production ability described later. It can be suitably used for the production of a transformant that has acquired the ability to produce gins and a transformant in which the intracellular composition of prostaglandins is modified.

  The recombinant expression vector according to the present invention is not particularly limited as long as it contains the cyclooxygenase gene according to the present invention described in (2) above. For example, a recombinant expression vector into which the cDNA of the gene according to the present invention is inserted can be mentioned. The recombinant expression vector of the present invention preferably further contains regulatory sequences such as a promoter necessary for the expression of the gene in order to express the gene according to the present invention in a host cell. The method for producing the recombinant expression vector of the present invention may be performed using a known method.

  The recombinant expression vector containing the gene according to the present invention usually contains the gene, promoter, terminator and the like according to the present invention at the multicloning site of the underlying vector (pET, for convenience in the following description, referred to as the basic vector). What is necessary is just to build and build.

  The specific type of the basic vector is not particularly limited, and a vector that can be expressed in the host cell may be appropriately selected. Here, plasmids, phages, cosmids, and the like can be used as the basic vector, but are not particularly limited. That is, according to the type of host cell, a promoter sequence or the like is appropriately selected in order to reliably express the gene, and a gene obtained by incorporating this and the gene of the present invention into a basic vector such as various plasmids can be used as a recombinant expression vector. That's fine.

  Examples of the host cells include plants, animals other than humans, insects, bacteria such as E. coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), and Xenopus laevis oocytes A cell such as a cell can be mentioned, but is not particularly limited. As a bacterium, E. coli is preferable. Among them, the host cell for preparing the protein according to the present invention is preferably a plant, E. coli, yeast or the like, more preferably a plant, E. coli or the like, and even more preferably E. coli.

  As E. coli, for example, E. coli BL21 (DE3) derived from E. coli B strain is preferable.

  One of the preferred embodiments of the recombinant expression vector of the present invention is an E. coli expression recombinant vector capable of expressing the gene of the present invention in E. coli cells. The recombinant expression vector (E. coli expression expression recombinant vector) is introduced into E. coli to produce an E. coli transformant (E. coli expression body), or E. coli AA or EPA is When modifying the composition of prostaglandins used as a substrate, it is preferable to use a vector for E. coli described later as a basic vector.

  A vector for E. coli such as pET is suitable as a basic vector used for the production of a recombinant vector expressing E. coli expression. In addition, a promoter that constantly expresses a gene in an E. coli cell, for example, a vector having an rrn promoter or a vector having a promoter inducibly activated by an external stimulating IPTG can be used.

Any promoter that can be expressed in E. coli cells may be used as a promoter for expressing the above-described gene according to the present invention contained in the recombinant expression vector in E. coli cells. For example, the T7 promoter is preferable. Furthermore, an artificially designed and modified promoter such as a tac promoter may be used. Various promoters derived from E. coli genes can also be used. Among them, since it is preferable that the introduced gene is constantly expressed in E. coli, a constitutive promoter is preferably used, and an rrn promoter or the like is more preferable.
The terminator can be appropriately selected, and examples thereof include a tT7 terminator.

When the recombinant expression vector of the present invention is introduced into yeast to produce a transformant, or prostaglandins using AA or EPA as a substrate are produced by yeast, a pPICZA vector or the like may be used as a basic vector. preferable. Any promoter may be used as long as it can be expressed in yeast as a promoter for expressing the gene of the present invention described above in yeast cells. For example, the pAOX1 promoter is preferable.
The terminator can be appropriately selected, and examples thereof include a tAOX1 terminator.

  For example, when the recombinant expression vector of the present invention is introduced into a plant to produce a plant transformant, or when prostaglandins using AA or EPA as a substrate are produced by plant cells, the plant described later as a basic vector It is preferable to use a cell vector.

  The recombinant expression vector used for plant transformation is not particularly limited as long as it can express the inserted gene in the plant cell. Examples of basic vectors used for the production of recombinant expression vectors include pUC plasmids such as pUC18 and pUC19; plasmid DNA for plant host cells such as pBI221, or pWTT23132 (manufactured by DNAP), Gateway (manufactured by Invitrogen), and the like. Plant cell vectors such as binary vectors and PCS31 vectors (for chloroplasts) are preferred. In addition, as described above, a recombinant expression vector having a promoter that constantly expresses a gene in a plant cell (for example, the 35S promoter of the cauliflower mosaic virus described above) or an inducibly activated by an external stimulus. A recombinant expression vector having a promoter can be used. The plant cells into which the recombinant expression vector is introduced include various types of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus. The gene of the present invention may be introduced into intracellular organelles other than the nucleus such as chloroplasts in plant cells by a recombinant expression vector.

Any promoter may be used as long as it can be expressed in plant cells as a promoter for expressing the gene according to the present invention contained in the recombinant expression vector in plant cells. For example, a plant-derived promoter such as a cauliflower mosaic virus 35S promoter, rd29A gene promoter or rbcS promoter; or a cauliflower mosaic virus 35S promoter enhancer sequence added to the 5 ′ side of an Agrobacterium-derived mannopine synthase promoter sequence. A constitutive promoter such as -1 promoter is preferred. Furthermore, an artificially designed and modified promoter such as a tac promoter may be used. Various promoters derived from plant genes can also be used. Among them, a constitutive promoter is preferably used because the gene encoding the introduced cyclooxygenase protein is constitutively expressed in plants, and the cauliflower mosaic virus 35S promoter and the like are more preferable.
The terminator can be appropriately selected. Examples thereof include AG7 terminator, NOS terminator, 35S terminator, rps16 terminator, CaMV35S terminator and the like.

  In order to confirm whether or not the gene of the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell, various markers may be used. The marker is not particularly limited, and a marker known per se may be used. For example, a gene deleted in the host cell is used as a marker, and a plasmid containing this marker and the gene of the present invention is introduced into the host cell as an expression vector. Thus, the introduction of the gene of the present invention can be confirmed from the expression of the marker gene. Examples of such marker genes include various drug resistance genes and genes that complement plant auxotrophy. More specifically, examples include hygromycin, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, tetracycline resistance gene or herbicide chlorsulfuron resistance gene, spectinomycin resistance gene and the like. It is done. Alternatively, the protein of the present invention may be expressed as a fusion protein. For example, the protein of the present invention may be expressed as a GFP fusion protein using the green fluorescent protein (GFP) derived from Aequorea jellyfish as a marker. For example, when the host cell is a plant cell, the marker is preferably a hygromycin resistance gene. When the host cell is E. coli, an ampicillin resistance gene, a kanamycin resistance gene and the like are preferable. Moreover, it is preferable to have a promoter and a terminator for recognizing the gene upstream and downstream of the marker gene.

(5-2) Transformant The transformant according to the present invention is not particularly limited as long as it is a transformant into which the cyclooxygenase gene of the present invention described in (2) is introduced. Preferably, it is a transformant in which the cyclooxygenase gene of the present invention described in (2) above is introduced into a host cell so that it can be expressed. Such a transformant is capable of producing (expressing) the protein (cyclooxygenase protein) according to the present invention. A transformant in which the cyclooxygenase gene of the present invention is introduced into a host cell so that it can be expressed is usually enhanced in the ability to produce prostaglandins using AA or EPA as a substrate, or obtains the ability to produce the prostaglandins. Transformant. The transformant of the present invention is preferably a transformant in which the intracellular content of prostaglandins using AA or EPA as a substrate is increased as compared with that before transformation.

  Here, “a gene has been introduced into a host cell so that it can be expressed” means that the gene is transferred into the target cell (eg, E. coli, yeast, plant cell, etc.) by a known genetic engineering technique (gene manipulation technique). It is introduced so that it can be expressed.

  The production method (production method) of the transformant of the present invention is not particularly limited. For example, a method of transforming by introducing the above-described recombinant expression vector of the present invention into a host cell is suitable.

  Examples of the host cell include the above-mentioned plants, animals other than humans, insects, bacteria such as E. coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis Examples of such cells include, but are not limited to, oocytes. As a bacterium, E. coli is preferable.

  Transformants obtained by introducing the recombinant expression vector of the present invention into a host cell and having enhanced the ability to produce prostaglandins using the transformant, preferably AA or EPA as a substrate, or have obtained the ability to produce the prostaglandins. When producing a transformant, the host cell is preferably a microorganism or a plant cell. More preferred host cells are plants, E. coli, yeast, etc., more preferably plants, E. coli, etc., with E. coli being particularly preferred. For example, when the recombinant expression vector of the present invention is introduced into a host cell to produce a transformant in which the intracellular composition of prostaglandins using AA or EPA as a substrate is modified, the host cell may be a plant Cells are preferred.

Plant cells that can be used as host cells are preferably those capable of biosynthesizing AA and / or EPA in the cells. For example, cells such as algae, Physcomitrella patens and genus Coleoptera are preferred, and organisms belonging to the family Asteraceae are more preferred. Even a plant that cannot biosynthesize AA and EPA can be suitably used as a host cell in the present invention by introducing a gene related to AA and / or EPA biosynthesis. Genes relating to the biosynthesis of AA and / or EPA and methods for introducing them into plant cells are described in, for example, JP2011-000123A, JP2010-279387A, International Publication WO2005 / 061713, and the like. . When a recombinant expression vector containing the gene of the present invention is introduced into a plant cell to produce a plant transformant, preferably a plant transformant having a modified prostaglandin composition, Of these, among others, Marchantia polymorpha is preferable.
The above-mentioned “Amanitaceae organisms” are not limited to (Marchantia polymorpha), but include organisms belonging to the Amanitaceae (Marchantales). Examples of organisms belonging to the order of the genus Mantiantiaceae (Marchantiales) include plants such as the clam sphagnum family, sphagnum genus department, jago department, azuma genus department, genus moss department, genus moss department, genus genus department, genus department, etc. Can be mentioned.
Here, the “plant transformant” means not only plant cells, tissues and organs but also individual plants (plants).

  In the case of producing prostaglandins using AA or EPA as a substrate by the plant transformant of the present invention, the host plant is preferably a genus Pleurotus. Among them, Zenigoke (Marchantia polymorpha) is more preferable.

  In producing the transformant of the present invention, a method for introducing a recombinant expression vector into a host cell, that is, a transformation method may be a known method, and may be appropriately selected depending on the host. For example, conventionally known methods such as an electroporation method, a calcium phosphate method, a liposome method, a DEAE dextran method, an Agrobacterium method, and a particle gun method can be suitably used. In addition, for example, when the protein of the present invention is transferred and expressed in insects, an expression system using baculovirus can be employed.

For example, when producing prostaglandins using AA or EPA as a substrate by the transformant of the present invention, the host cell is preferably a microorganism, a plant, or the like. Among these, E. coli is more preferable as the microorganism.
One of the preferred embodiments of the transformant of the present invention is an E. coli transformant produced by introducing the cyclooxygenase gene of the present invention described in (2) above into E. coli. Such an E. coli transformant obtained by introducing the gene of the present invention into E. coli can be suitably used for the production of prostaglandins using AA or EPA as a substrate as described later. It is. Here, the “transformant” is an E. coli cell. Prostaglandins produced by an E. coli transformant into which the gene of the present invention has been introduced may be secreted outside the E. coli and accumulated in the cell.

  As a transformant according to the present invention, the cyclosoxygenase gene according to the present invention is introduced into E. coli so that it can be expressed, the prostaglandins producing ability using AA or EPA as a substrate is enhanced, or the prostaglandins More preferred is an E. coli transformant that has acquired a similar productivity. Such a transformant can be suitably used for production of prostaglandins. Such an E. coli transformant with increased prostaglandin-producing ability or acquired prostaglandin-producing ability can be obtained, for example, by using the cyclooxygenase gene according to the present invention (preferably the E. coli trait described above). It can be produced by introducing an expression vector into E. coli (using an expression recombinant vector). That the prostaglandin production ability is enhanced means that the prostaglandin production ability is higher than that before the transformation with the gene of the present invention.

  The method for introducing an E. coli expression recombinant vector into E. coli, that is, the E. coli expression method is not particularly limited as described above, and may be appropriately selected according to the type of E. coli. . In addition to the conventional E. coli transformation method, polyethylene glycol (PEG-calcium phosphate) method, electroporation method (electroporation method) (for example, Heiei, Y. et al., Plant J., 6,271-282, 1994, Takaiwa) F. et al., Plant Sci. 111, 39-49, 1995. Axelos, M. et al. Plant Physiol. Biochem. 30, 123-128, 1992), etc., can be used.

  The E. coli transformant produced by the above method is capable of expressing the introduced cyclooxygenase gene, but in order to efficiently express the gene, it is possible to induce expression of the gene by adding IPTG or the like. preferable. For example, when prostaglandins are produced using E. coli transformants as described later, for example, IPTG may be added to the medium before culturing in the presence of AA and / or EPA. preferable. It is preferable that IPTG is usually added to the medium in an amount of about 0.05 to 0.2 mM. After the addition of IPTG, the introduced cyclooxygenase gene is efficiently expressed by culturing usually at about 15 to 30 ° C., preferably about 20 to 25 ° C. for about 2 to 24 hours.

  A yeast transformant in which the cyclooxygenase gene according to the present invention is introduced into yeast so that the prostaglandins can be produced using AA or EPA as a substrate, or the prostaglandins can be produced is also obtained. This is one of the preferred embodiments of the present invention.

  Another preferred embodiment of the transformant of the present invention is a plant transformant produced by introducing the cyclooxygenase gene of the present invention described in (2) above into a plant cell. Preferably, a plant trait in which prostaglandin production ability using AA or EPA as a substrate is enhanced or the prostaglandin production ability is obtained and / or the intracellular composition of the prostaglandin is modified It is a conversion body. Such a plant transformant can also be suitably used for production of prostaglandins using AA or EPA as a substrate. Such a plant transformant can be produced, for example, by introducing the cyclooxygenase gene of the present invention into a plant so that the gene can be expressed. In addition, a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant, or a tissue of the plant transformant is also a preferred embodiment of the present invention.

As another preferred embodiment of the present invention, a method for producing a plant transformant in which the cyclooxygenase gene of the present invention described in (2) above is introduced into a plant so as to be expressed is described below.
A plant transformant is produced by introducing, for example, a recombinant expression vector produced using the aforementioned plant basic vector into a plant cell.

  For introduction of a recombinant expression vector into a plant cell, a polyethylene glycol method, an electroporation method (electroporation method), an Agrobacterium-mediated method (for example, Heiei, Y. et al., Plant J., 6, 271-282, 1994, Takaiwa, F. et al., Plant Sci. 111, 39-49, 1995), and various methods known to those skilled in the art, such as a particle gun method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell.

  For example, there are already several techniques for creating plant transformants of the genus Amanita, such as a method of directly regenerating plants by introducing a gene directly into cells by the particle gun method, and a method using Agrobacterium. Has been established. In the present invention, these known methods can be suitably used. A technique for producing such a transformant of the genus Coleoptera is described in, for example, Plant Cell Physiol., 49, p1048 2008.

  Once a plant transformant in which the gene of the present invention has been introduced into the genome is obtained, offspring can be obtained from the plant body of the plant transformant by sexual reproduction or asexual reproduction. Further, from the plant transformant, its progeny, or clone, a propagation material such as a leaf, embryo, spore, temporary root, strain, callus, protoplast, etc. is obtained and based on them It is possible to mass-produce plant transformants. Therefore, the present invention includes a plant transformant into which the gene of the present invention has been introduced, a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant, and the plant trait This includes the organization of converters. Furthermore, the present invention also includes a propagation material obtained from the plant transformant or the tissue of the plant transformant. As a method for obtaining a propagation material from the plant transformant or the tissue of the plant transformant, a known method can be used. There are no particular restrictions on the method for growing or growing a plant or plant tissue into which a gene has been introduced by the above recombinant expression vector, the method for regenerating a plant from plant cells, the method for cultivating, etc. The conditions according to the above can be used as appropriate.

  A plant transformant having a modified prostaglandin composition using AA or EPA as a substrate, wherein the gene according to the present invention is introduced so as to be expressed in the plant, and the plant having the same properties as the plant transformant A plant transformant that is a descendant of the transformant, a tissue of the plant transformant, and a propagation material obtained from the plant transformant or the tissue of the plant transformant are also included in the present invention. “Prostaglandin composition has been altered” means that the composition of prostaglandins in the plant before transformation is different from the composition of prostaglandins in the plant transformant or its tissue after transformation. To do. For example, (1) a plant transformant produced by transforming a plant that originally did not contain prostaglandins with AA or EPA as a substrate into the composition according to the present invention. When the prostaglandin composition contains prostaglandins with AA or EPA as substrate, (2) the content of prostaglandins with AA or EPA as substrate in the prostaglandins composition compared to before transformation And the like can be mentioned.

  The plant heterogeneous transformant of the present invention is preferably a Paramecium organism transformant obtained by introducing the cyclooxygenase gene of the present invention described in the above (2) into the Paramecium organism. A preferred embodiment of the present invention is also a genus genus biotransformation, or a tissue of the genus genus biotransformation, which is a descendant of the genus genus L. This is one aspect. More preferred is a genus moss transformant obtained by introducing the cyclooxygenase gene of the present invention described in the above (2) into genus genus. A more preferred embodiment of the present invention is a Zenigoke transformant that is a descendant of the Zenithoke transformant having the same properties as the Zenigoke transformant, or a tissue of the Zenithoke transformant. The propagation material obtained from the tissue of the genus Amanita or the organism of the genus Amanita is also included in the present invention.

  Prostaglandins and the like can be produced by a low-cost and environment-friendly production process using a transformant obtained by introducing such a gene according to the present invention into host cells such as E. coli and plants. In the present invention, the plant may be, for example, the whole plant or a part of the plant, or may be a plant cell such as protoplast or callus.

(5-3) Prostaglandin production method In the present invention, a prostaglandin is produced using a transformant transformed with the gene of the present invention or a fraction containing a protein prepared from the transformant. A method of producing a variety is included. For example, when a protein fraction is prepared from the transformant of the present invention and a substrate such as AA or EPA is added to the protein fraction, prostaglandins are produced by cyclooxygenase contained in the protein fraction. The protein fraction from the cells can be prepared by a known extraction method.

  For example, when the transformant is a transformant in which the gene according to the present invention is introduced so that it can be expressed in a microorganism or plant such as E. coli or yeast, an extract of the transformant (preferably a protein) Fractions) can be used to produce prostaglandins in vitro.

  In the method for producing prostaglandins using an E. coli transformant in which the gene according to the present invention is introduced so as to be expressed in E. coli, for example, the E. coli transformant is converted into a prostaglandin. By culturing in the presence of AA and / or EPA, which are precursors, prostaglandins using AA or EPA as a substrate can be produced. Such a method for producing prostaglandins using AA and / or EPA as a substrate, which includes the step of culturing the E. coli transformant in the presence of AA and / or EPA, is also encompassed in the present invention. Prostaglandins produced by E. coli transformants may be secreted outside the E. coli transformants or accumulated intracellularly.

An example of production of prostaglandins using E. coli transformants is outlined in FIG. First, the gene of the present invention (GvCOX gene (SEQ ID NO: 1) obtained in Example 1 is shown as an example in FIG. 2) is introduced into E. coli by a recombinant expression vector or the like (FIG. 2). (1)), it is expressed in cells. The obtained E. coli transformant is cultured in the presence of substrates AA and / or EPA ((2) in FIG. 2). From AA or EPA incorporated into cells, induced by IPTG, and expressed by the introduced gene, cyclooxygenase (COX) causes AA to prostaglandin H ₂ (PGH ₂ ), prostaglandin G ₂ (PGG ₂ ), etc. Prostaglandins are produced. Prostaglandins such as prostaglandin H ₃ (PGH ₃ ) and prostaglandin G ₃ (PGG ₃ ) are produced from EPA. The produced prostaglandins are accumulated in the cells or secreted outside the cells ((3) in FIG. 2).

  The culture conditions of the E. coli transformant are not particularly limited. For example, the E. coli transformant is first cultured under aerobic conditions to increase the cell concentration, and then the E. coli transformant is transformed into AA and / or Or it is preferable to culture in the presence of EPA. By culturing under aerobic conditions, it is possible to cultivate to a high microorganism concentration in a short time. A known method is used as a means for culturing. The culture is performed under aerobic conditions, for example, shaking culture, liquid culture such as jar fermenter culture or tank culture, or solid culture. The medium may be any medium that is usually used for culturing E. coli. The culture temperature can be appropriately selected within the range in which the E. coli transformant can grow, but is usually about 15 to 37 ° C, preferably about 20 to 25 ° C. The pH of the medium is preferably in the range of about 6-8. Although the culture time varies depending on the culture conditions, it is usually preferably about 12 to 48 hours, more preferably about 18 to 36 hours, further preferably about 20 to 30 hours, and particularly preferably about 24 hours.

  In E. coli transformants, when induced cyclooxygenase gene expression is induced, it is usually preferable to increase the cell concentration and then add IPTG or the like to induce expression. Inducible expression may be performed simultaneously with the culture in the presence of AA and / or EPA.

  Cultivation of E. coli transformants in the presence of AA and / or EPA is preferably performed under aerobic conditions. Cultivation in the presence of AA and / or EPA can be performed using a normal nutrient medium containing a carbon source, a nitrogen source, a mineral source and the like used for culturing E. coli. Examples of the carbon source include glucose, fructose, galactose, mannose, lactose, sucrose, cellulose, molasses, glycerol and the like, and examples of the nitrogen source include inorganic nitrogen such as ammonia, ammonium salt, nitrate and the like, organic nitrogen As the source, for example, urea, amino acids, proteins, etc. can be used alone or in admixture of two or more. Further, as the mineral source, for example, potassium monohydrogen phosphate, magnesium sulfate, etc., mainly containing K, P, Mg, S and the like can be used. In addition to these, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin and thiamine can be added to the medium as necessary. Usually, the carbon source concentration at the start of culture is preferably about 0.1 to 20% (W / V), more preferably about 1 to 5% (W / V).

As a culture condition, the temperature is usually preferably about 15 to 30 ° C, more preferably about 20 to 25 ° C. The pH range is preferably about 6 to 10, more preferably about 6 to 8. It is preferable to control the pH at the same time, and it is also possible to adjust the pH using an acid or alkali. The initial concentration of the microorganism in the culture in the presence of AA and / or EPA is usually about OD ₆₀₀ = 0.6 to 0.8, but is not particularly limited.
Although the culture time varies depending on the culture conditions, it is usually preferably about 12 to 48 hours, more preferably about 18 to 36 hours, further preferably about 20 to 30 hours, and particularly preferably about 24 hours. The culture is preferably stirred culture.

  A method for supplying AA and / or EPA in culture in the presence of AA and / or EPA is not particularly limited. For example, AA and / or EPA as a substrate can be added to a medium (culture solution) of an E. coli transformant. Can be added. The amount of AA and / or EPA to be added is not particularly limited as long as the effects of the present invention are exhibited. For example, the amount is preferably about 0.01 to 10 mM, and preferably about 0.05 to 1 mM in the medium. More preferably, it is more preferably about 1 mM. Such a method for producing prostaglandins including the step of adding AA or EPA to the medium of an E. coli transformant is one of the preferred embodiments of the present invention. The culture in the presence of AA and / or EPA can also be performed, for example, by co-culturing a microorganism producing AA and / or EPA and an E. coli transformant. Examples of microorganisms that produce AA and / or EPA include Mortierella microorganisms.

In the production of prostaglandins using AA or EPA as a substrate, by culturing E. coli transformants in the presence of AA and / or EPA, prostaglandins are efficiently produced in the E. coli transformants. Produced well. Prostaglandins produced are accumulated in E. coli transformants or secreted into the culture medium.
Prostaglandins accumulated in the E. coli transformant or secreted into the medium can be separated and purified by a known technique. Further, an E. coli transformant containing prostaglandins in cells and a medium containing secreted prostaglandins can be used as they are for pharmaceuticals, foods, veterinary drugs, feeds and the like.

A method for producing prostaglandins using AA or EPA as a substrate, which comprises the step of preparing a protein fraction from an E. coli transformant, is also encompassed in the present invention. When a protein fraction is prepared from an E. coli transformant and AA and / or EPA is added to the protein fraction, prostaglandins are produced by cyclooxygenase contained in the protein fraction. The protein fraction from the cells can be prepared by a known extraction method.
In addition, cyclooxygenase can be purified from the protein fraction by a known method, and the cyclooxygenase can be used for production of prostaglandins. The reaction between the protein fraction or cyclooxygenase and the substrate AA or EPA is, for example, at a temperature of about 5 to 37 ° C. (more preferably about 5 to 25 ° C., more preferably about 15 to 25 ° C.) and a pH of about It is preferably carried out in a solution of 6 to 8 (preferably pH about 7 to 8). The solution for performing the reaction is not particularly limited. For example, a buffer solution such as Tris-HCl buffer is suitable. Moreover, it is preferable to add about 1-10 mM EDTA to the solution which reacts. The protein concentration and substrate concentration in the reaction solution can be appropriately set. In the present invention, it is also preferable to carry out the reaction by immobilizing an enzyme. A known method can be adopted as the immobilization method.

  In the above method for producing prostaglandins using E. coli transformants, prostaglandins can also be produced by using yeast transformants instead of E. coli transformants. As the culture conditions of the yeast transformant, conditions usually used for yeast culture can be employed.

  Using the plant transformant into which the gene of the present invention is introduced (preferably a plant transformant having a modified prostaglandin composition using AA or EPA as a substrate) or the tissue of the plant transformant, AA or A method for producing prostaglandins using EPA as a substrate is also included in the present invention. For example, the plant transformant according to the present invention in which the content of prostaglandins in cells is increased becomes a valuable product as a raw material having high pharmaceutical and pharmacological activity. Such plant transformants are also useful as raw materials for animal drugs, functional foods, feeds, and the like.

  In the production of prostaglandins using the plant body of the plant transformant or the tissue of the plant transformant, the prostaglandin is produced in the cells of the plant body or the tissue as the plant body or the tissue grows or grows. Grangeins are produced efficiently. The produced prostaglandins accumulate in the cells of the plant transformant or its tissue. A method for producing prostaglandins using a plant transformant or a tissue of the plant transformant comprises a step of growing or growing the plant transformant or the tissue (preferably by asexual reproduction (vegetative reproduction)). It is preferable to include. In order to proliferate or grow the plant transformant or tissue thereof, the plant transformant or tissue thereof may be appropriately cultivated or cultured. The conditions and time for growing or growing (cultivating or cultivating) the plant transformant or its tissue may be appropriately selected depending on the type of plant. For example, when using the genus Coleoptera transformed with the gene according to the present invention, it is preferable to use asexual reproduction (vegetative reproduction) as the breeding form of the genus Coleoptera. That is, for the genus Coleoptera, it is preferable to grow or grow the transformant or the tissue thereof by asexual reproduction (vegetative reproduction) because the growth speed is high and mass production is possible even as a plant factory system. The method for asexual reproduction of the genus Amanita is described in "Aseptic culture and transformation method of Amanita" (Plant Cell Engineering Series 14-Plant Genome Research Protocol, Shujunsha, pp155-162., 2001) Has been. A well-known method can be used about the plant cultivation floor and the plant cultivation method. In addition, for example, a method described in JP 2010-088314 A (cultivation apparatus using a vertical multi-stage cultivation shelf and a mat system) can be used. For example, a transformant (transformed cell) in which the gene of the present invention has been introduced into a cell of the genus Coleoptera is usually cultured for 20 to 49 days, preferably 31 to 49 days, so that prostaglandins can be obtained. Accumulate in cells. As the medium or the like, a medium usually used for culturing or cultivating the genus Coleoptera can be used. In the case of the genus Coleoptera, the culture or cultivation temperature is usually about 20-30 ° C, preferably about 25-30 ° C.

A method for producing prostaglandins using AA or EPA as a substrate, which comprises the step of preparing a protein fraction from a plant transformant, is also encompassed in the present invention. When a protein fraction is prepared from a plant transformant and a substrate such as AA or EPA is added to the protein fraction, prostaglandins are produced by cyclooxygenase contained in the protein fraction. The protein fraction from the cells can be prepared by a known extraction method.
In addition, cyclooxygenase can be purified from the protein fraction by a known method, and the cyclooxygenase can be used for production of prostaglandins. The reaction conditions of the protein fraction or cyclooxygenase and the substrate AA or EPA are the same as the reaction conditions of the protein fraction or cyclooxygenase prepared from the E. coli transformant described above and AA or EPA. is there. In the present invention, it is also preferable to carry out the reaction by immobilizing an enzyme. A known method can be adopted as the immobilization method.

An E. coli transformant, a yeast transformant, a plant transformant, or a transformant such as a tissue thereof that produced prostaglandins according to the present invention can be directly used as a raw material containing prostaglandins, Although it can be used for food, veterinary medicine, feed, etc., it may further comprise a step of purifying the produced prostaglandins from the transformant or tissue thereof. The method for purifying prostaglandins is not particularly limited, and can usually be performed by an extraction method using an organic solvent or a high-pressure high-temperature steam distillation method.
As described above, the E. coli transformant, yeast transformant, plant transformant or transformant thereof according to the present invention that produces prostaglandins has a high prostaglandin content, It becomes a high value as a raw material with high pharmacological activity. Such a transformant is also useful as a raw material for animal drugs, functional foods, feeds and the like.

(5-4) Material The present invention includes a material obtained by the above-described prostaglandin production method and a material containing at least one prostaglandin. This “raw material” means all materials that can be used for the above-mentioned pharmaceutical raw materials, food raw materials, veterinary pharmaceutical raw materials, feeds, and the like.

  The prostaglandins of the above materials have unique physical properties such as having a plurality of double bonds in the molecule. Therefore, it is currently synthesized by total chemical synthesis using lactone as a raw material. Therefore, for example, these prostaglandins are produced by producing prostaglandins with the transformant of the present invention (preferably, E. coli transformant, yeast transformant, plant transformant or tissue thereof). The production cost of grangeins can be significantly reduced. In addition, according to the present invention, an environmentally friendly prostaglandin production process can be realized.

Examples of the prostaglandins produced by the present invention include prostaglandins G ₂ (PGG ₂ ) and prostaglandins H ₂ (prostaglandins produced from arachidonic acid (using arachidonic acid as a substrate). And prostaglandins (PG) such as PGH ₂ ), prostaglandin E ₂ (PGE ₂ ), and prostaglandin F _2α (PGF _2α ). Examples of prostaglandins produced from eicosapentaenoic acid (using eicosapentaenoic acid as a substrate) include prostaglandins such as prostaglandin H ₃ (PGH ₃ ) and prostaglandin G ₃ (PGG ₃ ). It is done. Among them, the method of the present invention includes prostaglandin H ₂ (PGH ₂ ), prostaglandin G ₂ (PGG ₂ ), prostaglandin E ₂ (PGE ₂ ), prostaglandin F _2α (PGF _2α ), prostaglandin. It is suitable for production of prostaglandins (PG) such as H ₃ (PGH ₃ ) and prostaglandin G ₃ (PGG ₃ ).

(5-5) Prostaglandin composition modification method The present invention includes a method of modifying the prostaglandin composition of a host using the gene according to the present invention. For example, the prostaglandin composition of the host cell can be altered by preparing a transformant introduced with the gene of the present invention as described above. The target for modifying the prostaglandin composition is not particularly limited, and any organism such as a plant, an animal, a bacterium, and a yeast can be targeted.

  A method for modifying the composition of prostaglandins using AA or EPA as a substrate in the host cell, which comprises the step of introducing the gene according to the present invention described above into the host cell, is also one aspect of the present invention. Preferably, the cyclooxygenase gene according to the present invention is introduced into a plant cell or the like. For example, the composition of prostaglandins using AA or EPA as a substrate of a plant transformant comprising a step of introducing a gene according to the present invention into a plant cell (preferably, Amanita, more preferably, Amanita) is modified. The method is one of the preferred embodiments of the present invention.

  For example, as described above, the prostaglandin composition of the host cell is modified by preparing a transformant such as an E. coli transformant, a yeast transformant, or a plant transformant introduced with the gene of the present invention. It becomes possible to do. In the present invention, a prostagland of the transformant is obtained by culturing the transformant such as the E. coli transformant or yeast transformant in the presence of a substrate of prostaglandins such as AA and EPA. Methods for modifying the gin composition are included. Culturing in the presence of a substrate for prostaglandins can be performed, for example, by adding AA, EPA, etc. to the medium of the transformant.

  By introducing the gene according to the present invention described above into a plant to produce a plant transformant, it becomes possible to modify the prostaglandin composition of the plant or its tissue. A method for modifying the prostaglandin composition using AA or EPA as a substrate of a plant transformant, which comprises the step of introducing the gene according to the present invention into a plant is also one aspect of the present invention. As the plant, a plant capable of biosynthesizing AA and / or EPA in a cell is preferable, and for example, the above-mentioned genus Coleoptera is preferable, and among them, the genus Magenta (Marchantia polymorpha) is more preferable. The present invention includes a method for modifying the prostaglandin composition of the transformant or tissue thereof by proliferating or growing the plant transformant or tissue thereof.

(5-6) Antibody An antibody that recognizes the protein encoded by the gene of the present invention is also encompassed by the present invention. The antibody according to the present invention is preferably an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein according to the present invention, or a partial protein or partial peptide thereof as an antigen. Known methods include, for example, literature (Harlow et al., “Antibodies: A laboratory manual” (Cold Spring Harbor Laboratory, New York (1988)), Iwasaki et al., “Monoclonal antibody hybridomas and ELISA, Mizodansha (1991). )). The antibody thus obtained can be used for detection and measurement of the protein of the present invention.

(5-7) Screening Method The screening method according to the present invention is a method for screening a gene that regulates the protein or a substance that regulates the protein, using the protein according to the present invention. As the screening method of the present invention, various conventionally known methods for examining the presence / absence of binding between substances and the presence / absence of dissociation can be applied and are not particularly limited. For example, the screening of the substance which accelerates | stimulates or inhibits the activity (cyclooxygenase activity) of the protein based on this invention can be mentioned.

  Examples of the screening method of the present invention include: (I) a step of contacting arachidonic acid and / or eicosapentaenoic acid and a candidate substance with a protein encoded by the gene of the present invention in vitro; (II) arachidonic acid or A step of monitoring the amount of prostaglandins produced using eicosapentaenoic acid as a substrate, and (III) detection of an increase or decrease in the amount of prostaglandins produced in step (II) compared to the case where no candidate substance is contacted A method including a step of selecting the candidate substance as a substance that regulates the protein is preferable.

  In step (I), the method of contacting AA and / or EPA and a candidate substance in vitro with the protein encoded by the gene of the present invention is not particularly limited. For example, the gene of the present invention is introduced. The candidate substance or the like may be brought into contact with the transformant or the protein isolated from the transformant. When a transformant is used, for example, a candidate substance or the like may be added to the medium in which the transformant is cultured.

  In step (II), the amount of prostaglandins using the produced AA or EPA as a substrate is monitored. For example, the amount of prostaglandins accumulated in the cells can be measured by analyzing the cell extract. The amount of prostaglandins secreted extracellularly or produced by an isolated protein (cyclooxygenase protein) can be measured, for example, by analyzing the composition of the culture medium or medium. Measurement of prostaglandins can be performed by a known method, for example, analysis by high performance liquid chromatography (HPLC) / mass spectrometer (LC-MS / MS).

  In the step (III), for example, compared to the case where the candidate substance is not contacted, the candidate substance in which the increase or decrease in the amount of prostaglandins produced using AA or EPA as the substrate is detected in the step (II) is as described above. Selected as a substance that regulates protein. For example, a candidate substance for which an increase in the production amount of the prostaglandins is detected is selected as an activator for the protein. In addition, the candidate substance in which a decrease in the amount of prostaglandins produced in the cell is detected in the step (II) as compared with the case where the candidate substance is not contacted is selected as an inhibitor of the protein.

Candidate substances are not particularly limited, and examples include substances such as genes such as nucleic acids, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, and the like. . The candidate substance may be a novel substance or a known substance. These candidate substances may form salts, and as salts of candidate substances, salts with physiologically acceptable acids and bases are used.
The present invention also includes genes or substances obtained by the above screening methods.

(5-8) Gene detection instrument The present invention also encompasses a gene detection instrument that uses at least a part of the base sequence of the gene of the present invention or a complementary sequence thereof as a probe.
Such a gene detection instrument can be used for detection / measurement of the expression pattern of the gene of the present invention under various conditions.

  Examples of the gene detection instrument of the present invention include a DNA chip in which the probe that specifically hybridizes with the gene of the present invention is immobilized on a substrate (carrier). Here, the “DNA chip” mainly means a synthetic DNA chip using a synthesized oligonucleotide as a probe, but also includes an affixed DNA microarray using a cDNA such as a PCR product as a probe.

  The sequence used as a probe can be determined by a conventionally known method for specifying a characteristic sequence from a cDNA sequence. Specifically, for example, SAGE: Serial Analysis of Gene Expression Method (Science 276: 1268, 1997; Cell 88: 243, 1997; Science 270: 484, 1995; Nature 389: 300, 1997; US Pat. No. 5,695) , No. 937).

  A known method may be employed for manufacturing the DNA chip. For example, when a synthetic oligonucleotide is used as the oligonucleotide, the oligonucleotide may be synthesized on a substrate by a combination of a photolithography technique and a solid phase DNA synthesis technique. On the other hand, when cDNA is used as an oligonucleotide, it may be pasted on a substrate using an array machine.

  Similarly to a general DNA chip, a perfect match probe (oligonucleotide) and a mismatch probe in which one perfect base substitution is performed in the perfect match probe may be arranged to further improve the accuracy of gene detection. Furthermore, in order to detect different genes in parallel, a plurality of types of oligonucleotides may be fixed on the same substrate to constitute a DNA chip.

  The gene detection instrument according to the present invention is not limited to the above-exemplified DNA chip, and any instrument using at least a partial base sequence of the gene according to the present invention or a complementary sequence thereof may be used.

  In the production of prostaglandins using AA or EPA as a substrate using the transformant of the present invention, for example, E. coli transformant, prostaglandins are efficiently produced in the E. coli transformant. The Prostaglandins produced are accumulated in the transformant or secreted extracellularly. When the plant transformant of the present invention is proliferated or grown, prostaglandins are efficiently produced in the cells of the transformant and accumulated in the cells. The present invention also includes the production of prostaglandins by adding the substrate AA or EPA to the extract of the transformant for reaction.

  The transformant produced by producing the prostaglandins according to the present invention can be used as it is as a raw material containing prostaglandins for pharmaceuticals, foods, veterinary medicines, feeds, etc. Purify prostaglandins produced by reaction by adding AA or EPA as a substrate to the extract of an E. coli transformant, yeast transformant, plant transformant or tissue thereof, for example. The process of carrying out may be included. The method for purifying prostaglandins is not particularly limited, and can be performed by the above-described known methods.

  Several examples will be shown below, and the embodiment of the present invention will be described in more detail. The present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Furthermore, the present invention is not limited to the technical embodiments, and various modifications shown in the claims can be made, and the embodiments of the present invention also apply to the embodiments obtained by appropriately combining the disclosed technical means. Included in the scope.

  In this example, the experimental procedure was the same as that described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989) unless otherwise specified.

Example 1
Ogonori-derived cyclooxygenase gene As a result of extensive comparison of the amino acid sequences of the cloned cyclooxygenase genes of other organisms, the amino acid sequences Tyr-Asn-Asp-Tyr-Arg-Glu-His (SEQ ID NO: 4) and Asp-Ile-Phe -Asn-Glu-Lys-Thr (SEQ ID NO: 5) was found to be conserved. Therefore, in order to isolate the cyclooxygenase gene of Gracilaria vermiculophylla, the following primers encoding the above amino acid sequences were used.
COX-F 5'-TAYAAYGAYTAYAGNGARCAY-3 '(SEQ ID NO: 6)
COX-R 5'-NGTYTTYTCRTTRAADATRTC-3 '(SEQ ID NO: 7)
In SEQ ID NOs: 6 and 7, “N” means that any of 4 bases (adenine, guanine, thymine / uracil, cytosine) may be used.

PCR was performed using the above primers (COX-F and COX-R), and the resulting DNA fragment was subcloned. The base sequence of the obtained clone was determined, and the partial sequence of the target cyclooxygenase gene was obtained. The experimental materials and methods will be described in detail.
Isolation of total RNA from gonogori used as a sample was performed according to the method recommended by the manufacturer using an RNA extraction kit RNeasy Mini kit (manufactured by QIAGEN). 5 μg of isolated total RNA was reverse-transcribed into cDNA using ReverTra Ace (manufactured by TOYOBO). PCR was performed by the method recommended by the manufacturer using 1 μL of cDNA as a template and the above primers (COX-F and COX-R) and enzyme (Takara Ex Taq, manufactured by Takara) 0.5 U. The reaction volume was 20 μL, and the PCR thermal cycler Dice (manufactured by Takara) was held at 95 ° C. for 2 minutes, and then the reaction at 95 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C. for 30 seconds was repeated 40 times. Then, it was cooled to 4 ° C.

The obtained PCR product was electrophoresed on a 0.7% (w / v) agarose gel, and an amplified fragment having a size expected from the base sequence of a conventional cyclooxygenase gene was used using a Gel Extraction kit (QIAGEN). And recovered from the gel. The recovered amplified fragment was ligated to pGEM-Teasy (Promega) and transformed into competent E. coli DH5α.
Use the 3130xl Genetic Analyzer (product name, manufactured by Applied Biosystems) to decode the partial nucleotide sequence of the candidate gene linked to pGEM-Teasy and use it for the RACE reaction based on the nucleotide sequence information Primer sequences were designed.
3'RACE (COX): 5'-CGACCGATGCGGCAATCTTGGCCAAGTTGAAGCAAGT-3 '(SEQ ID NO: 8)
5'RACE (COX): 5'-CGCCTATTGCTGCCAAGAAAGGTCCATGAACACCTCC-3 '(SEQ ID NO: 9)

  In order to clarify the full length sequence of the candidate gene, Rapid amplified cDNA ENDs (RACE) reaction was performed using SMART RACE cDNA Amplification Kit (Clontech). A 3'-RACE reaction was performed according to the method specified by the reagent manufacturer using 1 µg of total RNA extracted from ogonori and 3'-RACE (COX) primer (SEQ ID NO: 8). The 5′-RACE reaction was similarly performed using 1 μg of total RNA and 5′-RACE (COX) primer (SEQ ID NO: 9). The 3 ′ end sequence and 5 ′ end sequence of the candidate gene obtained by the RACE reaction were each ligated to pGEM-Teasy and transformed into competent E. coli DH5α.

  As a result, one type of homolog gene candidate was isolated, and this gene was designated as GvCOX gene. The cDNA length of the GvCOX gene (excluding the poly A portion) was 2019 bp (SEQ ID NO: 1), and the deduced amino acid sequence was 562 residues. The base sequence is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.

  As a result of comparing the deduced amino acid sequence of GvCOX cDNA with the amino acid sequences of mouse and human cyclooxygenase, they were found to show 21.5% and 20.4% identity, respectively.

(Example 2)
Construction of recombinant expression vector to be introduced into E. coli and introduction of the vector into E. coli
In order to express the Ogonori cyclooxygenase gene (GvCOX gene) obtained in Example 1 in E. coli, an E. coli expression recombinant vector was constructed according to the following procedure. E. coli was used as the host cell.

  The PCR product obtained by PCR using the primer of SEQ ID NO: 6 and the primer of SEQ ID NO: 7 in Example 1 was electrophoresed on a 0.7% (w / v) agarose gel and predicted from the base sequence of the conventional cyclooxygenase gene. The amplified fragment having the size as described above was recovered from the gel using Gel Extraction kit (manufactured by QIAGEN). The collected ORF-amplified fragment of the sea cucumber cyclooxygenase gene (GvCOX gene) was ligated to pGEM-Teasy (Promega) to prepare a construct of a recombinant expression vector. This production procedure is shown in FIG. 3A.

  FIG. 3A shows an E. coli expression recombination vector from the basic vector pET-21a (+) (purchased from Merck) in order to express the cyclooxygenase gene (GvCOX) of Gracilaria vermiculophylla in E. coli. The produced method is shown. In FIG. 3A, pT7 means T7 promoter and tT7 means T7 terminator.

Specifically, a cDNA fragment containing the ORF of the Ogonori cyclooxygenase gene (GvCOX gene) was PCR amplified using the following primers to add a restriction enzyme site, introduced into pGEM-Teasy, and the 3130xl genetic analyzer (product name) (Applied Biosystems) was used to determine the base sequence.
Primers used for ORF amplification of the cyclogonogenase gene
COX-F5: 5'-CATATGGTGTTCAACAACTTCCG-3 '(SEQ ID NO: 10)
COX-R7: 5'-GCTCAGCTACACAGGGTTATTCTTCG-3 '(SEQ ID NO: 11)

  In PCR for amplifying the ogonyori cyclooxygenase gene (GvCOX gene), 0.5 U of PrimeSTAR DNA polymerase (manufactured by Takara) was used with ogonori cDNA as a template, maintained at 95 ° C. for 2 minutes, then at 98 ° C. for 10 seconds, 55 The reaction of 15 ° C. for 15 seconds and 68 ° C. for 2 minutes and 30 seconds was repeated 35 times, and then cooled to 4 ° C. The obtained fragment was treated with a restriction enzyme (NudI-Bpu1102I) to produce an insert. The resulting insert was ligated with pET21a (+) purified by restriction enzyme treatment with NdeI and Bpu1102I according to a conventional method according to a conventional method to obtain an E. coli expression recombinant vector (GvCOX / pET21a (+)). The structure of the E. coli phenotypic recombinant vector (GvCOX / pET21a (+)) is schematically shown in FIG.

Example 3
Transformation with E. coli phenotypic recombinant vector (GvCOX / pET21a (+)) and measurement of cyclooxygenase enzyme activity using the resulting extract of E. coli transformant The GvCOX gene prepared in Example 2 was transformed into E. coli. The vector (GvCOX / 21a (+)) introduced into the expression vector pET21a was introduced into E. coli BL21 (DE3) strain by electroporation. The Escherichia coli BL21 (DE3) strain is a bacterium that does not produce prostaglandins. E. coli colonies into which a GvCOX expression vector (GvCOX / 21a (+)) was introduced were inoculated into 3 mL of LB medium containing ampicillin (50 μg / mL), and cultured under shaking at 37 ° C. 100 μL of the culture solution was transferred to a fresh 10 mL LB medium, and further cultured with shaking at 37 ° C. until OD ₆₀₀ = 0.6. After reaching OD ₆₀₀ = 0.6, 0.1 mM IPTG (final concentration) was added to the medium, and then cultured with shaking at 20 ° C. for 2 hours.

  Only bacterial cells (E. coli transformants) were collected from the culture broth by centrifugation and suspended in 500 μL extraction buffer (100 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1 mM PMSF). After disrupting the cells using an ultrasonic disrupter, centrifugation was performed at 17,000 × G for 15 minutes, and the supernatant was collected.

250 μL of the collected supernatant and 250 μL of activity measurement buffer (100 mM Tris-HCl (pH 7.5), 5 mM EDTA, 2 μM Hematin) were mixed and allowed to stand at 25 ° C. for 5 minutes. Arachidonic acid was added to the mixed solution to a final concentration of 200 μM and reacted at 25 ° C. for 30 minutes. The final concentration of 5 mM SnCl ₂ was then added to stop the reaction.

  The reaction solution was adjusted to pH 3 with hydrochloric acid and extracted with 700 μL of ethyl acetate. The solvent was distilled off under reduced pressure using a rotary evaporator, and the residue was used as a crude fraction of prostaglandin. The crude fraction was dissolved in 200 μL ethanol, insoluble material was removed by centrifugation at 17,000 × G for 15 minutes, and the supernatant was used as a sample for measurement.

Prostaglandin included in the measurement sample was adjusted Jin _{F 2α (PGF 2α)} Liquid chromatography / mass spectrometry for (LC-MS / MS) (model number 3200QTRAP, Applied Biosystem Inc.) fragments from PGF ₂ alpha with Detection of ions (m / z = 309) and fragment pattern generated from molecular ion 353.2 [M−H] ^{− of} PGF _2α were performed. LC-MS / MS measurement conditions were as follows: Column: RP-18 GP 150 x 4.6 (5 mm) (manufactured by Kanto Chemical Industry Co., Ltd.), mobile phase: gradient elution with 2.5 mM ammonium acetate aqueous solution and 2.5 mM ammonium acetate methanol solution, flow rate : 400 mL / min, column temperature: 40 ° C., collision energy: 40 V, collision gas: nitrogen gas, electroion spray mode: negative.

Because the fragment pattern (Table 1) of the reaction product and the standard PGF _2α (manufactured by CAYMAN CHEMICAL) match, and the retention time of the liquid chromatography matches between the reaction product and the standard PGF _2α- d4 It was confirmed that PGF _2α was contained in the reaction product.

Example 4
Production of PGF _2α by E. coli culture AA was added to the E. coli transformant (in LB medium) prepared in Example 3 to the presence of IPTG to a concentration of 1 mM, and cultured in LB medium for 20 hours at 25 ° C. After culturing, the cells were removed by centrifugation (10,000 rpm, 15 minutes), the culture solution was collected, and prostaglandins were collected by the above prostaglandin extraction method. When the recovered prostaglandins were analyzed by the method described in Example 3, production of prostaglandin F _2α (PGF _2α ) was observed in the medium. From this result, it was confirmed that the Escherichia coli transformant prepared in Example 3 secreted prostaglandins using AA produced in cells as a substrate.

(Example 5)
Construction of Yeast Expression Vector to be Introduced into Yeast In order to express the ogonori cyclooxygenase gene (GvCOX gene) obtained in Example 1 in yeast, a yeast expression vector was constructed according to the following procedure.

  The PCR product obtained by PCR using the primer of SEQ ID NO: 6 and the primer of SEQ ID NO: 7 in Example 1 was electrophoresed on a 0.7% (w / v) agarose gel and predicted from the base sequence of the conventional cyclooxygenase gene. The amplified fragment having the size as described above was recovered from the gel using Gel Extraction kit (manufactured by QIAGEN). The ORF amplified fragment of the recovered ogonori cyclooxygenase gene (GvCOX gene) is digested with restriction enzymes KpnI and ApaI, and the yeast expression basic vector pPICZA (manufactured by Invitrogen) is digested with the restriction enzymes KpnI and ApaI. Ligated with a ligating enzyme. Thus, a yeast expression vector (GvCOX / pPICZA) was obtained. The outline of the production procedure of the yeast expression vector (GvCOX / pPICZA) is schematically shown in FIG.

The structure of the obtained yeast expression vector (GvCOX / pPICZA) is schematically shown in FIG. In FIG. 5B, pAOX1 represents the pAOX1 promoter. tAOX1 represents a tAOX1 terminator.
By introducing a yeast expression vector (GvCOX / pPICZA) into yeast and transforming it, a yeast transformant capable of producing prostaglandins using AA or EPA as a substrate can be produced.

(Example 6)
Construction of a vector for expression (Agrobacterium infection) to be introduced into the moss. In order to introduce and express the cyclooxygenase gene (GvCOX gene) of Ogonori obtained in Example 1 into genus moss using Agrobacterium, According to the procedure shown in the above, a vector for expression of A. niger (for Agrobacterium infection) was constructed.

  FIG. 4C schematically shows an outline of a procedure for producing a vector for expression of Amanita (for Agrobacterium infection). Plant expression basic vector pBin-Hyg-TX (Gatz C. et al., Plant J. 2, 397-404 (1992)) was digested with restriction enzymes KpnI and SalI. Next, the ORF-amplified fragment of the ogonori cyclooxygenase gene (GvCOX gene) obtained in Example 1 was digested with restriction enzymes KpnI and SalI. It was connected by. As a result, a vector for expression (for Agrobacterium infection) (GvCOX / pBin-Hyg-TX) was obtained.

  FIG. 5 (C) schematically shows the structure of the obtained vector for expression of Amanita (for Agrobacterium infection) (GvCOX / pBin-Hyg-TX). In FIG. 5C, p35S represents a cauliflower mosaic virus (CaMV) 35S promoter. tOCS represents an octapin synthase terminator.

(Example 7)
Transformation with Zenigo phenotype-expressing recombinant vector (GvCOX / pBin-Hyg-TX) and measurement of cyclooxygenase enzyme activity using the extract of the resulting Zenioke transformant Zenvoke expression of GvCOX gene prepared in Example 6 The vector (GvCOX / pBin-Hyg-TX) introduced into the vector (for Agrobacterium infection) vector pBin-Hyg-TX (Gatz C. et al., Plant J. 2, 397-404 (1992)) (Marchantia polymorpha) was introduced by the Agrobacterium infection method. The vector was introduced by the Agrobacterium infection method according to the method described in Ishizaki et al., Plant Cell Physiol., 49, 1084-1091. (2008). Zenigoke is a plant that does not produce prostaglandins. Zenigoke (transformed mushroom) introduced with a GvCOX expression vector (GvCOX / pBin-Hyg-TX) was grown on a 0M51C agar medium containing hygromycin (10 μg / mL) at 25 ° C. for 1 week.

  The transformed genus mushroom was frozen in liquid nitrogen, added with 400 μL of extraction buffer (100 mM Tris (pH 7.5), 5 mM EDTA, 0.5 mM PMSF, 1 μM hematin), and crushed with an ultrasonic crusher. Centrifugation was performed at 17,000 × G for 15 minutes, and the supernatant was collected.

The collected supernatant was allowed to stand at 20 ° C. for 5 minutes. Arachidonic acid was added to the mixed solution to a final concentration of 200 μM and reacted at 20 ° C. for 5 minutes. Next, the final concentration of 5 mM SnCl ₂ was added to stop the reaction.

  The reaction solution was adjusted to pH 3 with hydrochloric acid and extracted with 700 μL of ethyl acetate. The solvent was distilled off under reduced pressure using a rotary evaporator, and the residue was used as a crude fraction of prostaglandin. The crude fraction was dissolved in 200 μL ethanol, insoluble material was removed by centrifugation at 17,000 × G for 15 minutes, and the supernatant was used as a sample for measurement.

Prostaglandin F _2α (PGF _2α ) and prostaglandin E ₂ (PGE ₂ ) contained in the prepared measurement sample liquid chromatography / mass spectrometer (LC-MS / MS) (model number 3200QTRAP, manufactured by Applied Biosystem) ) To generate fragment patterns derived from PGF _2α- derived fragment ions (m / z = 309) and PGF _2α molecular ions 353.2 [M−H] ⁻ , and fragment ions derived from PGE ₂ (m / z = 271) ) And PGE ₂ molecular ion 351.2 [M−H] ⁻ . LC-MS / MS measurement conditions were as follows: Column: RP-18 GP 150 x 4.6 (5 mm) (manufactured by Kanto Chemical Industry Co., Ltd.), mobile phase: gradient elution with 2.5 mM ammonium acetate aqueous solution and 2.5 mM ammonium acetate methanol solution, flow rate : 400 mL / min, column temperature: 40 ° C., collision energy: 40 V, collision gas: nitrogen gas, electroion spray mode: negative.

The fragment pattern (Table 1 and Table 2) of the reaction product and the standard PGF _2α and PGE ₂ (manufactured by CAYMAN CHEMICAL) match, and the retention time of liquid chromatography is the same as the reaction product and the standard PGF _2α − Since it was in agreement with d4 and PGE ₂ , it was confirmed that PGF _2α and PGE ₂ were contained in the reaction product.

(Example 8)
Construction of a vector for expression of mushroom to be introduced into mushroom (for particle gun) In order to introduce and express the cyclooxygenase gene (GvCOX gene) of ogoryori obtained in Example 1 into mushroom by particle gun, A vector for expressing mushroom (for particle gun) was constructed.

  FIG. 4D schematically shows an outline of a procedure for producing a vector for expressing mushrooms (for particle gun). Plant expression basic vector pBI221 (manufactured by Takara) was digested with restriction enzymes SmaI and SacI. Next, the GvCOX fragment obtained by digesting the ORF amplified fragment of the ogonori cyclooxygenase gene (GvCOX gene) obtained in Example 1 with the restriction enzymes SmaI and SacI was ligated to the vector pBI221 treated with the restriction enzyme using a ligation enzyme. As a result, a vector for expressing mushroom (for particle gun) (GvCOX / pBI221) was obtained.

  FIG. 5 (D) schematically shows the structure of the obtained mushroom expression (particle gun) vector (GvCOX / pBI221). In FIG. 5D, p35S represents a cauliflower mosaic virus (CaMV) 35S promoter. tNOS represents the NOS gene terminator.

Example 9
Measurement of cyclooxygenase enzyme activity using an extract of the genus moss transformant (GvCOX / pBI221) and the resulting extract of the genus genus moss The GvCOX gene prepared in Example 8 is used for expression of genus The vector (GvCOX / pBI221) introduced into the vector pBI221 was introduced into the mushroom (Marchantia polymorpha) by the particle gun method. Introduction of the vector by the particle gun method was carried out according to the method described in Ishizaki et al., Plant Cell Physiol., 49, 1084-1091. (2008). Zenigoke is a plant that does not produce prostaglandins. Zenigoke into which the GvCOX expression vector (GvCOX / pBI221) was introduced was grown at 25 ° C. for 1 week on a 0M51C agar medium containing hygromycin (10 μg / mL).

  The transformed genus mushroom was frozen in liquid nitrogen, added with 400 μL of extraction buffer (100 mM Tris (pH 7.5), 5 mM EDTA, 0.5 mM PMSF, 1 μM hematin), and crushed with an ultrasonic crusher. Centrifugation was performed at 17,000 × G for 15 minutes, and the supernatant was collected.

The collected supernatant was allowed to stand at 20 ° C. for 5 minutes. Arachidonic acid was added to the mixed solution to a final concentration of 200 μM and reacted at 20 ° C. for 5 minutes. Next, the final concentration of 5 mM SnCl ₂ was added to stop the reaction.

  The reaction solution was adjusted to pH 3 with hydrochloric acid and extracted with 700 μL of ethyl acetate. The solvent was distilled off under reduced pressure using a rotary evaporator, and the residue was used as a crude fraction of prostaglandin. The crude fraction was dissolved in 200 μL ethanol, insoluble material was removed by centrifugation at 17,000 × G for 15 minutes, and the supernatant was used as a sample for measurement.

Prostaglandin F _2α (PGF _2α ) and prostaglandin E ₂ (PGE ₂ ) contained in the prepared measurement sample liquid chromatography / mass spectrometer (LC-MS / MS) (model number 3200QTRAP, manufactured by Applied Biosystem) ) To generate fragment patterns derived from PGF _2α- derived fragment ions (m / z = 309) and PGF _2α molecular ions 353.2 [M−H] ⁻ , and fragment ions derived from PGE ₂ (m / z = 271) ) And PGE ₂ molecular ion 351.2 [M−H] ⁻ . LC-MS / MS measurement conditions were as follows: Column: RP-18 GP 150 x 4.6 (5 mm) (manufactured by Kanto Chemical Industry Co., Ltd.), mobile phase: gradient elution with 2.5 mM ammonium acetate aqueous solution and 2.5 mM ammonium acetate methanol solution, flow rate : 400 mL / min, column temperature: 40 ° C., collision energy: 40 V, collision gas: nitrogen gas, electroion spray mode: negative.

The fragment pattern (Table 1 and Table 2) of the reaction product and the standard PGF _2α and PGE ₂ (manufactured by CAYMAN CHEMICAL) match, and the retention time of liquid chromatography is the reaction product and the standard PGF _2α. -It was confirmed that PGF _2α and PGE ₂ were contained in the reaction product because they matched with d4 and PGE ₂ .
Therefore, it was confirmed that prostaglandins can be produced by a transformant in which the cyclooxygenase gene is introduced into the genus Amanita.

  As described above, the gene and protein of the present invention are useful for producing prostaglandins. In addition, a transformant such as an E. coli transformant into which the gene of the present invention has been introduced is capable of producing prostaglandins in the pharmaceutical industry and various material industries. Very useful for reagent supply. In addition, in E. coli transformants and the like into which the gene of the present invention has been introduced, the content of prostaglandins in E. coli increases or is released into the culture medium. The Escherichia coli transformant is very useful in the pharmaceutical field and the like.

Claims (16)

A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 154th to 1842th base sequences in the base sequence shown in SEQ ID NO: 1
(B) hybridizes under stringent conditions with DNA consisting of the 154th to 1842th base sequences of the base sequence shown in SEQ ID NO: 1 or DNA complementary to the DNA, and arachidonic acid or A DNA encoding a protein derived from the order of the genus Pleurotus with cyclooxygenase activity using eicosapentaenoic acid as a substrate. A gene comprising any of the following DNAs (a) to (c):
(A) DNA comprising the base sequence shown in SEQ ID NO: 1
(B) a DNA derived from the organism of the order Ogonori that encodes a protein having a cyclooxygenase activity comprising a part of the base sequence represented by SEQ ID NO: 1 and using arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Cyclooxygenase activity which hybridizes under stringent conditions with all or part of DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and which uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the order of the order of the organism that encodes the protein A gene encoding the following protein (A) or (B).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (B) From the amino acid sequence shown in SEQ ID NO: 2 in which one to several amino acids are substituted, deleted, inserted and / or added A protein derived from the order of the genus Ogonori having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate   A protein encoded by the gene according to any one of claims 1 to 3.   A recombinant expression vector comprising the gene according to any one of claims 1 to 3.   An E. coli transformant obtained by introducing at least the gene according to any one of claims 1 to 3 into a host cell, E. coli.   At least the gene of any one of claims 1 to 3 and the progeny of the genus L. elegans having the same properties as that of the genus L. Or a tissue of the genus Coleoptera organism.   The propagation material obtained from the structure | tissue of the Paramecium organism transformant of Claim 7, or this Geniolia organism transformant.   Prostaglandin production using arachidonic acid or eicosapentaenoic acid as a substrate, comprising the step of culturing the E. coli transformant according to claim 6 in the presence of arachidonic acid and / or eicosapentaenoic acid Method.   A method for producing prostaglandins using arachidonic acid or eicosapentaenoic acid as a substrate, which comprises a step of proliferating or growing the transformant of the genus Coleoptera according to claim 7 or a tissue thereof.   A step of preparing a protein fraction from the E. coli transformant according to claim 6 or from the genus Coleoptera transformant according to claim 7, wherein arachidonic acid or eicosapentaenoic acid is used as a substrate Prostaglandin production method.   A method for altering the prostaglandin composition of a genus Coleoptera or a tissue thereof, which comprises the step of introducing the gene according to any one of claims 1 to 3 into the genus Amanita.   The antibody which recognizes the protein encoded by the gene of any one of Claims 1-3.   The gene detection instrument which uses the base sequence of at least one part of the gene of any one of Claims 1-3, or its complementary sequence as a probe.   A method for screening a substance that regulates the protein using the protein encoded by the gene according to any one of claims 1 to 3.   (I) a step of contacting arachidonic acid and / or eicosapentaenoic acid and a candidate substance with the protein encoded by the gene according to any one of claims 1 to 3 in vitro, (II) arachidonic acid or A step of monitoring the amount of prostaglandins produced using eicosapentaenoic acid as a substrate, and (III) detection of an increase or decrease in the amount of prostaglandins produced in step (II) compared to the case where no candidate substance is contacted The screening method according to claim 15, further comprising a step of selecting the candidate substance obtained as a substance that regulates the protein.

Images