JP2003063991A - Cytokine inducing agent for use in oral administration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cytokine inducing agent for use in oral administration which is useful in treating various diseases such as infectious diseases, autoimmunity diseases, inflammations, tumors by which production of cytokines such as IFN-γ, IL-8 and the like in the living body can be promoted by oral administration thereof. SOLUTION: A microorganism cell body, on the surface of which there is expressed cytokine inducing substances as to fusion protein, is used as cytokine inducing agent for use in oral administration. As to the above-mentioned microorganism, a lactobacillus such as Lactobacillus casei and the like may be used. The above-mentioned fusion proteins are the expressed products of DNA being combined in order with signal sequences, genes of cytokine inducing substances, and membrane anchor sequences. When a listeria hemolysin is used as to the gene of cytokine inducing substance, then IFN-γ can be induced, and when a flagellar protein flagellin of Slmonella is used as to the gene of cytokine inducing substance, then IL-8 can be induced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oral cytokine inducer such as an oral interferon-γ (IFN-γ) inducer, and more specifically, it carries a cytokine inducer such as Listeria hemolysin on its surface. The present invention relates to an oral cytokine inducer containing microbial cells such as lactic acid bacteria.

[0002]

BACKGROUND OF THE INVENTION Interferon (IFN) was originally discovered as a substance produced by virus-infected cells to make virus-infected cells resistant, and a therapeutic agent for hepatitis B and C,
Alternatively, it has been put to practical use as a cancer immunotherapy agent. IFN
Shows an antiviral effect against various viruses to varying degrees, but it is effective only in cells of the same species in which it is produced, and that long-term treatment is required depending on the target disease. Since it is a glycoprotein having a molecular weight of about 20,000 and is hardly absorbed by oral administration or gastric administration, an IFN inducer which can be orally administered has been demanded. Hitherto, as an IFN inducer, high molecular weight polymers such as double-stranded nucleic acid, poly I: poly C and polycarboxylate, and low molecular weight compounds such as fluoresins and pyrimidinones have been reported. However, they are not used for treatment because they have safety problems and have drawbacks such as low IFN-inducing effect by oral administration.

Further, in order to immobilize various useful proteins on the cell surface, Lactococcus lactis (La
ctococcus lactis) A step of obtaining a fusion gene sequence in which a gene sequence encoding an anchor sequence of 763 protease from NCDO763 strain and a gene sequence encoding a target protein are connected, and the fusion gene sequence is introduced into a host bacterium. And a step of culturing a host bacterium into which the fusion gene sequence has been introduced to immobilize the target protein on the surface of the microbial cell. No. 117783), and in order to effectively enhance the immune reaction to an antigen, 1) collagen, gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose or gum arabic, 2) Polymers of glycolic acid, lactic acid or amino acids or copolymers of two or more of these And 3) Carrying an antigen or an antigen-inducing substance on a carrier made of a biocompatible material selected from the group consisting of hydroxyapatite, polymethylmethacrylate, polydimethylsiloxane, polytetrafluoroethylene, polypropylene, polyethylene or polyvinyl chloride. And an immunopotentiating composition (JP-A-11-193246) comprising a composition further containing an immunomodulator or other additives as desired, ascorbic acid, lactic acid bacteria and 3-O-α-D-glucopyranosyl-D. -A drug, a quasi drug, a food, a feed, or a preparation for cosmetics that synergistically enhances the immunostimulatory effect by using together with another immunostimulant such as a saccharide containing glucose as a constitutional unit (JP 2001-64174) is known.

[0004]

[Problems to be Solved by the Invention] Cytokines, which are released from cells and are known as proteinaceous factors that mediate the interaction between cells, control their immune response, antitumor effect, antiviral effect, cell proliferation / differentiation. It is used for the treatment of various diseases due to its regulatory action. In many cases, a treatment method in which cytokine itself is administered is carried out, but it is accompanied by side effects such as bone marrow suppression, gastrointestinal disorder, cardiac dysfunction and hair loss, and it is a heavy burden on the patient to be treated. For example, IFN has attracted attention as a dream antiviral drug, but purified IFN-γ and its inducer could not be expected to have a sufficient effect by oral administration. An object of the present invention is to administer IFN-γ to an organism by oral administration.
It is an object of the present invention to provide an oral cytokine inducer which can promote the production of cytokines such as, and is useful for the treatment of various diseases such as infectious diseases, autoimmune diseases, inflammations and tumors.

[0005]

[Means for Solving the Problems] The present inventors have fixed the expression of Listeria hemolysin or flagella protein of Salmonella flagellin on the surface layer of lactic acid bacteria by fixing it with a membrane anchor sequence to express M cells or epithelial cells in the intestinal tract. Through, it becomes easier to be recognized by the immune system, and effective IFN-γ
It was found that an inducing ability and an interleukin-8 (IL-8) inducing ability are obtained, and the present invention has been completed.

That is, the present invention provides an oral cytokine inducer characterized by containing a microbial cell carrying a cytokine-inducing substance on its surface (claim 1) and a microbial cell carrying a cytokine-inducing substance on its surface. Is a microorganism having a cytokine-inducing substance expressed as a fusion protein on the surface thereof, and the oral cytokine-inducing agent (claim 2) or the fusion protein according to claim 1,
The oral cytokine inducer (claim 3) or the fusion protein according to claim 2, which is an expression product of a DNA in which a signal sequence, a cytokine inducer gene and a membrane anchor sequence are sequentially bound. 4. Oral cytokine induction according to claim 3, which is a fusion protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence, and which is capable of regulating the cytokine inducing ability. The agent (Claim 4) or the cytokine-inducing substance is Listeria hemolysin, and the oral cytokine-inducing agent (Claim 5) according to any one of claims 1 to 4 and the microorganism are lactic acid bacteria. An oral cytokine inducer according to any one of claims 1 to 5 (claim 6),
The oral cytokine inducer according to claim 6, wherein the lactic acid bacterium is a Th1-induced lactic acid bacterium strain (claim 7).
Or, the Th1-induced lactic acid bacterium strain is a Th1-induced lactic acid bacterium strain of Lactobacillus casei, wherein the oral cytokine inducer (claim 8) or the cytokine is interferon-γ. The present invention relates to the oral cytokine inducer according to any one of claims 1 to 8 (claim 9).

Further, the present invention comprises a prophylactic / therapeutic agent for a disease requiring the production of an endogenous cytokine, which comprises the oral cytokine inducer according to any one of claims 1 to 9 as an active ingredient ( (10) or an oral cytokine inducer is an oral interferon-γ inducer, and the preventive / therapeutic agent for a disease requiring production of an endogenous cytokine according to (10). 11) and prevention of diseases requiring the production of endogenous cytokine, which comprises the oral cytokine inducer according to any one of claims 1 to 9 as an active ingredient
The present invention relates to a therapeutic functional food material or food (claim 12), and a method for inducing cytokine (claim 13), which comprises orally administering the oral cytokine inducer according to any one of claims 1 to 9. .

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The oral cytokine inducer of the present invention may be any one as long as it contains microbial cells carrying a cytokine inducer on its surface. Examples of cytokines that are induced in vivo by oral administration of an inducer include IF
Interferons such as N-γ, IFN-β and IFN-α, IL-1, IL-2, IL-3, IL-4 and IL
-5, IL-6, IL-7, IL-8, IL-9, IL
-10, IL-11, IL-12, IL-13, IL-
14, IL-15, IL-16, IL-17, IL-1
Interleukins such as 8 and TNF-β, TNF-α
Necrosis factors such as GM-CSF, G-CSF, M
-Colony stimulating factors such as CSF and epidermal growth factor (E
GF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), insulin-like growth factor (IGF), nerve growth factor family (NGF), brain-derived neurot
rophic factor (BDNF), ciliary neurotrophic fa
In addition to growth factors such as ctor (CNTF), oncostatin M (OSM) and hepatocyte growth factor (IIGF), erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF), leukemia inhibitory factor (LI)
F), prolactin hormone and the like can be exemplified. It is considered that the induction of these cytokines occurs when microbial cells carrying (immobilizing) the cytokine-inducing substance are orally administered to the intestinal tract and act on M cells or epithelial cells of the intestinal tract.

The cytokine-inducing substance is not particularly limited as long as it is a substance capable of inducing the endogenous release or production of cytokines.
Listeria monocytogenes-derived Listeria hemolysin, endotoxins such as LPS (lipopolysaccharide), flagellin of Salmonella flagellin, pertussis vaccine, diphtheria vaccine, bacterial components such as lipoteichoic acid, concanavalin A, lentil lectin, T lymphocyte stimulating lectins such as pokeweed lectin, MDP (muramyl dipeptide), a human cell-derived protein that induces the production of IFN-γ in immunocompetent cells (see JP-A-09-289896), 4-
Amino-2-ethoxymethyl-α, α-dimethyl-1H
Examples thereof include compounds such as imidazo [4,5-c] quinoline-1-ethanol.

In the present invention, the method for supporting (immobilizing) the cytokine-inducing substance on the surface of the microbial cell is not particularly limited. For example, a method for expressing the cytokine-inducing substance on the surface of the microbial cell as a fusion protein, or a bacterium Examples of the method include a method of binding a cytokine-inducing substance to a substance having a specific affinity for various types of receptors existing on the body surface, a method of immobilizing it on the cell surface using a synthetic polymer, and the like. When the inducer is composed of a protein such as Listeria hemolysin or flagella protein flagellin, the method of expressing the cytokine inducer as a fusion protein on the surface of the fungus body, in particular, the proteinaceous cytokine inducer It is preferable because it can be stably supported (fixed) on the surface.

As a method for expressing a cytokine-inducing substance as a fusion protein on the surface of the above-mentioned microbial cells, the above-mentioned JP-A-10-117783 and JP-A-10-10783 are available.
Although it is not particularly limited including the known method described in JP-A-508207, the promoter controlling the expression level in the bacterial cell and the produced proteinaceous cytokine inducer pass through the bacterial cell membrane. A signal sequence that helps
DNA (fusion gene sequence) in which a membrane anchor sequence encoding a hydrophobic linker capable of binding to a cell membrane is sequentially bound
A method of transforming a microorganism with a vector incorporating Then, the construction of a vector incorporating the fusion gene sequence, the introduction of such a vector into a microbial cell, the culturing of a transformed microorganism and the like can be carried out by a conventional method in this field.

In the present invention, a mutant cytokine inducer gene is used in which a part of the nucleotide sequence constituting the gene DNA of the above-mentioned proteinaceous cytokine inducer is subjected to mutation treatment such as deletion, substitution or addition, A cytokine-inducing substance gene that is genetically mutated can be used to regulate the cytokine-inducing ability, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in its constituent amino acid sequence. Fusion proteins can also be used. By using such a fusion protein, it becomes possible to regulate the cytokine inducing ability in animals including humans.

Microorganisms used for the preparation of the oral cytokine inducer of the present invention are not particularly limited, but microorganisms that act on M cells or epithelial cells of the intestinal tract to efficiently induce cytokine induction, A microorganism that is harmless to humans and animals is preferable, and among them, a microorganism that can express a cytokine-inducing substance as a fusion protein on the surface of the bacterial cell is preferable. As such microorganisms, lactic acid bacteria, baker's yeast (Saccharomyces cerevisiae), natto bacteria (Bacillus subtilis), which have been conventionally eaten as food ingredients and whose safety has been confirmed.
Lactic acid bacteria are preferred in view of their ability to induce cytokines and the ability of related bacteria to form a normal flora in the intestinal tract.

The above-mentioned lactic acid bacteria include Lactobacillus,
Lactococcus genus, Bifidobacterium genus, Streptococcus genus, Pediococcus genus, can be mentioned lactic acid bacteria belonging to the genus Leuconostoc, such as Lactobacillus casei (Lactobacillus casei) Th1-induced lactic acid bacterial strains, It is preferable because of its ability to induce cytokines and long-term safe eating experience in humans. The Th1-induced lactic acid bacterium strain is a helper T cell clone Th1.
-Induced IL-2 and IF secreted by Th1
It can be advantageously used for inducing cytokines such as N-γ and TNF-β. Further, Th2-induced lactic acid bacterial strains such as Lactobacillus reuteri and Lactobacillus brevis can be advantageously used in inducing cytokines such as IL-4 secreted by Th2.

As a prophylactic / therapeutic agent for diseases requiring the production of endogenous cytokine of the present invention, oral IFN-γ
As long as it contains the oral cytokine inducer of the present invention such as an inducer as an active ingredient, any drug such as a drug or a quasi drug may be used, and a disease requiring the production of an endogenous cytokine is C Examples include infections caused by viruses such as hepatitis B and influenza, microorganisms such as intracellular parasitic bacteria such as Listeria monocytogenes, various malignant tumors such as epithelial malignant tumors, autoimmune diseases, and inflammation. The prophylactic / therapeutic agent for diseases that require the production of endogenous cytokine of the present invention includes tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, etc. It can be used in the form of various pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrators, solubilizers, etc. Compounding ingredients for dispensing can be added. The dose of the prophylactic / therapeutic drug of the present invention can be appropriately determined according to the age, sex, other conditions of the patient, the degree of disease and the like.

As a functional food material or food for the prevention / treatment of diseases requiring production of endogenous cytokine of the present invention, the oral cytokine inducer of the present invention such as oral IFN-γ inducer is effective. Any food or food material contained as an ingredient may be used, and it can be prepared by adding or blending the oral cytokine inducer of the present invention to a food material or food. As the food material or food, cheese, yogurt, drink yogurt, juice, milk, soy milk, alcoholic beverages, coffee, tea, sencha, oolong tea, various drinks such as sports drinks, pudding, cookies, bread, cakes, jelly, rice crackers Such as baked confectionery, Japanese sweets such as yokan, frozen desserts, bread and confectionery such as chewing gum, noodles such as udon and buckwheat, fish paste products such as kamaboko, ham and fish sausage, miso, soy sauce, dressing, mayonnaise, sweetness Seasonings such as ingredients,
Tofu, konjac, other gyoza, gyoza, croquettes, salads and other various side dishes can be mentioned.

The method for inducing cytokines of the present invention may be a method of orally administering the oral cytokine inducer of the present invention to animals such as humans, horses, cows, pigs and chickens to induce cytokines in vivo. In particular, although not particularly limited, when the production of endogenous IFN-γ is induced by the method for inducing cytokine of the present invention, the function of helper T cell is inclined to Th1 type by such IFN-γ, It is effective in ameliorating a decrease in Th1 type immune function, and includes, for example, recovery from tumor-induced immunosuppressive state or immune function decrease induced by anticancer drug treatment,
Prevention of AIDS onset, prevention and treatment of type I allergy,
It is effective in controlling various biological functions such as suppressing stress and deterioration of immune function due to aging, and improving resistance to various diseases.

[0018]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. Example 1 (Induction of blood IFN-γ by oral administration) 1-1 (Preparation of Listeria hemolysin gene and its related gene) Listeria hemolysin, listeriolysin O (LLO), was used as an IFN-γ inducer. Using. As a source of LLO gene, Listeria monocytogenes Y in which produced LLO has hemolytic activity
The LLO gene and its related genes were prepared using Listeria monocytogenes BUG strain in which 7 strains and produced LLO are mutated and do not show hemolytic activity. First, a chromosomal DNA was prepared from Listeria monocytogenes Y7 strain by a conventional method, and using this chromosomal DNA as a template, a sense primer comprising the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2. PCR is carried out using an antisense primer consisting of
The LLO full-length gene (Y7) shown in SEQ ID NO: 3 of p was prepared. Similarly, Listeria monocytogenes BU
From the chromosomal DNA of the G strain, a mutation represented by SEQ ID NO: 4 having a total length of about 1500 bp using a sense primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 and an antisense primer consisting of the nucleotide sequence represented by SEQ ID NO: 2 The LLO gene (BUG) was prepared. In addition, PCR is 94 ℃ 60
Seconds, 45 ℃ 60 seconds, 72 ℃ 120 seconds as one cycle,
This was repeated 30 times.

1-2 (Construction of membrane surface anchoring expression vector and secretory expression vector) The above LLO full-length gene (Y7), mutant LLO gene (B
UG) was incorporated into the membrane surface anchoring plasmid vector pLP series developed by Dr. Leah et al. Of Netherlands TNO to construct a membrane surface anchoring expression vector. This membrane surface anchoring plasmid vector p
LPM11 is Lactobacillus amylophorus (Lact
obacillus amylovorus) α-amylase gene-derived promoter (Pamy) and signal sequence (SS),
An anchor sequence (Anchor) derived from protease P of Lactobacillus casei (Anchor),
Lactobacillus planta
rum) cbh-derived terminator sequence (Tcbh). The LLO-related gene was inserted into the restriction enzyme BamHI and SalI cleavage sites at the junction between SS and Anchor using the restriction enzymes BamHI and SalI. A schematic diagram of a fusion protein expression unit of a membrane surface anchoring expression vector in which such an LLO-related gene is incorporated is shown in FIG. Similarly, a secretory plasmid vector obtained by removing the anchor sequence (Anchor) from the membrane surface anchoring plasmid vector pLPM11 was used in the same manner as L
LO full-length gene (Y7), mutant LLO gene (BUG)
Was incorporated to construct a secretory expression vector.

1-3 (Preparation of oral IFN-γ inducer) Lactobacillus casei ATCC3 as a host lactic acid bacterium
Using the 93 strain, the membrane surface anchoring expression vector and secretory expression vector constructed above were introduced by electroporation. Then, by Western blot, hemolysis test and flow cytometry analysis, the transformants into which the target membrane surface anchoring expression vector and secretory expression vector were introduced were selected, and oral IF
Various transformed lactic acid bacteria containing an N-γ inducer were prepared.

1-4 (Hemolytic test using sheep red blood cells) Lactic acid bacterium (Anchor Y7) in which the LLO full-length gene (Y7) was expressed on the cell surface, and mutant LLO gene (BU
G) on the surface of lactic acid bacteria (AnchorBU
G), a lactic acid bacterium (Y7) of a type that secretes LLO having hemolytic activity from cells, and a mutant LL that does not exhibit hemolytic activity
A hemolytic assay using sheep red blood cells was performed using the culture supernatant and the washed bacterial cell suspension of lactic acid bacteria (BUG) of the type that secretes O from the bacterial cells. For the hemolytic assay, 100 μl of the test sample was placed on a 96-well V-shaped plate, sheep blood cells were added so that the final concentration was 1%, and hemolysis was observed 20 minutes later. The results are shown in Figure 2. The lysis picture shown in FIG.
A secretory type in which Y7 having hemolytic activity was incorporated as LO, a bacterial cell surface-bound recombinant, a negative control culture supernatant containing only an anchor, and a washed bacterial cell suspension were prepared from above,
Samples diluted 2-fold, 4-fold, 8-fold and 16-fold are shown. No bacteriolytic activity was found in any of those incorporating BUG.

1-5 (Induction of IFN-γ in Blood by Oral Administration) The above-mentioned various transformed lactic acid bacteria were treated with 8-week-old male C57BL / 6C.
r mice (n = 3 to 5 each) were intragastrically administered 5 × 10 ⁸ CFU / mouse three times each week using a sonde. In addition, as a control, lactic acid bacteria expressing only anchor (NC)
For comparison, a considerable amount of purified LLO having hemolytic activity was also intragastrically administered. Two weeks after the final administration, whole blood was collected and IFN-γ induced in the blood was analyzed by mouse IFN-γ ELISA kit EM-.
Quantification was performed using 1001 (ENDOGEN). Figure 3 shows part of the result
Shown in. Induction of IFN-γ in blood was significantly observed in mice of the lactic acid bacterium (Y7 + A) administration group in which the LLO full-length gene (Y7) having hemolytic activity was expressed on the cell surface, but a mutant LLO gene having no hemolytic activity was observed. Only a slight increase in blood IFN-γ was observed in the mice of the lactic acid bacterium (BUG + A) -administered group in which (BUG) was expressed on the cell surface, and as a control, the lactic acid bacterium expressing only the membrane surface anchor (NC ) Induction of blood IFN-γ was not observed in the mice of the administration group. In the mouse of the purified LLO administration group having hemolytic activity, weak induction of IFN-γ was observed in only one of the five mice.

Example 2 (Induction of IL-8 in intestinal model) 2-1 (Preparation of Salmonella flagella protein flagellin gene) IL-8 was monocyte-derived neutrophil chemotaxis by Matsushima et al. In 1987. Purified as factor (MDNCF) (Proc.N
atl.Acad.Sci.USA, 84, 9233, 1987), a cytokine produced locally in inflammation during an inflammatory reaction, and such IL
As a inducer of -8, Salmonella flagella protein flagellin was used. The source of the flagellin gene is Enteritidis (Salmonella enter
Itidis) # 40 strain was used. First, a chromosomal DNA is prepared from the above-mentioned Enteritidis bacterium by a conventional method, and using this chromosomal DNA as a template, it comprises a sense primer consisting of the nucleotide sequence represented by SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6. PCR was carried out using an antisense primer to amplify the DNA to prepare the flagellin gene represented by SEQ ID NO: 7. The PCR is 94 ° C for 30 seconds,
One cycle of 55 ° C. for 60 seconds and 72 ° C. for 120 seconds was repeated 25 times.

2-2 (Preparation of Transformed Lactic Acid Bacteria) The flagellin gene prepared above was used at the junction between SS and Anchor of the membrane surface anchoring plasmid vector described in Example 1 using the restriction enzymes BamHI and SalI. It was inserted into the existing restriction enzyme BamHI and SalI cleavage sites.
The membrane surface anchoring plasmid vector pLPM11 as a negative control was introduced into Lactobacillus casei ATCC393 strain as a host lactic acid bacterium by electroporation. Expression of the flagellin protein on the surface layer of the bacterial cells was confirmed by Western blot and flow cytometric analysis, and a transformed lactic acid bacterium as an oral IL-8 inducer was prepared.

2-3 (Induction of IL-8 in intestinal model) 24-well Sumilon Celtite C- was prepared using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum.
Caco-2 cells (ATCC HTB-37) were 2 × 10 ⁵ cells / well in one plate (Sumitomo Bakelite).
Culture at 37 ° C. and 5% CO ₂ conditions to about 1
After culturing for 0 days, the cells were differentiated into a morphologically and functionally small intestine-like cell layer. The transformed lactic acid bacterium was treated with 2 × 10 ⁷ CFU / well (MO) of DMEM containing no antibiotics.
I 100) and let the bacteria attach to the cells at 4 ° C for 1 hour, and
After incubating at 7 ° C. overnight, the IL-8 produced in the culture medium was measured. IL-8 in the culture medium is Human In
It was quantified using terleukin-8 ELISA kit EH2-IL8 (ENDOGEN) (n = 3). The results are shown in Fig. 4. Lactic acid bacteria administration group (fliC +) in which the flagellin gene was expressed on the cell surface
Induction of IL-8 was significantly observed in the intestinal model of A), but in the lactic acid bacteria administration group (Anchor) and medium group (DMEM) into which only the membrane surface anchoring plasmid vector was introduced, IL-8 was induced. Production was low.

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, oral administration can promote in vivo production of cytokines such as IFN-γ and IL-8, which play important roles in immune responses, and can promote infections, autoimmunity. It becomes possible to facilitate the treatment and prevention of many diseases such as diseases, inflammations and tumors.

[0027]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION       National Institute of Infectious Diseases <120> Cytokine inducer for oral dose <130> 13-217 <140> <141> <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Sense primer <400> 1 aactgaggat ccggatgcat ctgcattcaa taaag 35 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense       primer <400> 2 attactgtcg acgttattcg attggattat ctac 34 <210> 3 <211> 1537 <212> DNA <213> Listeria monocytogenes <400> 3 actgaggatc cggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca 60 ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatgaaatc 120 gataagtata tacaaggatt ggactacaat aaaaacaatg tgttagtata ccacggagat 180 gcagtgacaa atgtgccgcc aagaaaaggt tacaaagatg gaaatgaata tattgttgtg 240 gagaaaaaga agaaatccat caatcaaaat aatgcagaca ttcaagttgt gaatgcaatt 300 tcgagcctaa cctatccagg tgctctcgta aaagcgaatt cggaattagt agaaaatcaa 360 ccagatgttc tccctgtaaa atgtgattca ttaacactca gcattgattt gccaggtatg 420 actaatcaag acaataaaat cgttgtaaaa aatgccacta aatcaaacgt taacaacgca 480 gtaaatacat tagtggaaag atggaatgaa aaatatgctc aagcttatcc caatgtaagt 540 gcaaaaattg attatgatga cgaaatggct tacagtgaat cacaattaat tgcgaaattt 600 ggtacagcat ttaaagctgt aaataatagc ttgaatgtaa acttcggcgc aatcagtgaa 660 gggaaaatgc aagaagaagt cattagtttt aaacaaattt actataacgt gaatgttaat 720 gaacctacaa gaccttccag atttttcggc aaagctgtta ctaaagagca gttgcaagcg 780 cttggagtga atgcagaaaa tcctcctgca tatatctcaa gtgtggcgta tggccgtcaa 840 gtttatttga aattatcaac taattcccat agtactaaag taaaagctgc ttttgatgct 900 gccgtaaacg gaaaatctgt ctcaggtgat gtagaactaa caaatatcat caaaaattct 960 tccttcaaag ccgtaattta cggaggttct gcaaaagatg aagttcaaat catcgacggc 1020 aacctcggag acttacgcga tattttgaaa aaaggcgcta cttttaatcg agaaacacca 1080 ggagttccca ttgcttatac aacaaacttc ctaaaagaca atgaattagc tgttattaaa 1140 aacaactcag aatatattga aacaacttca aaagcttata cagatggaaa aattaacatc 1200 gatcactctg gaggatacgt tgctcaattc aacatttctt gggatgaagt aaattatgat 1260 cctgaaggta acgaaattgt tcaacataaa aactggagcg aaaacaataa aagcaagcta 1320 gctcatttca catcgtccat ctatttgcct ggtaacgcga gaaatattaa tgtttacgct 1380 aaagaatgca ctggtttagc ttgggaatgg tggagaacgg taattgatga ccggaactta 1440 ccacttgtga aaaatagaaa tatctccatc tggggcacca cgctttatcc gaaatatagt 1500 aataaagtag ataatccaat cgaagtcgac taaaagt 1537 <210> 4 <211> 1537 <212> DNA <213> Listeria monocytogenes <400> 4 actgaggatc cggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca 60 ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatgaaatc 120 gataagtata tacaaggatt ggattacaat aaaaacaatg tattagtata ccacggagat 180 gcagtgacaa atgtgccgcc aagaaaaggt tacaaagatg gaaatgaata tattgttgtg 240 gagaaaaaga agaaatccat caatcaaaat aatgcagaca ttcaagttgt gaatgcaatt 300 tcgagcctaa cctatccagg tgctctcgta aaagcgaatt cggaattagt agaaaatcaa 360 ccagatgttc tccctgtaaa acgtgattca ttaacactca gcattgattt gccaggtatg 420 actaatcaag acaataaaat cgttgtaaaa aatgccacta aatcaaacgt taacaacgca 480 gtaaatacat tagtggaaag atggaatgaa aaatatgctc aagcttatcc aaatgtaagt 540 gcaaaaattg attatgatga cgaaatggct tacagtgaat cacaattaat tgcgaaattt 600 ggtacagcat ttaaagctgt aaataatagc ttgaatgtaa acttcggcgc aatcagtgaa 660 gggaaaatgc aagaagaagt cattagtttt aaacaaattt actataacgt gaatgttaat 720 gaacctacaa gaccttccag atttttcggc aaagctgtta ctaaagagca gttgcaagcg 780 cttggagtga atgcagaaaa tcctcctgca tatatctcaa gtgtggcgta tggccgtcaa 840 gtttatttga aattatcaac taattcccat agtactaaag taaaagctgc ttttgatgct 900 gccataagcg gaaaatctgt ctcaggtgat gtagaactaa caaatatcat caaaaattct 960 tccttcaaag ccgtaattta cggaggttcc gcaaaagatg aagttcaaat catcgacggc 1020 aacctcggag acttacgcga tattttgaaa aaaggcgcta cttttaatcg agaaacacca 1080 ggagttccca ttgcttatac aacaaacttc ctaaaagaca atgaattagc tgttattaaa 1140 aacaactcag aatatattaa aacaacttca aaagcttata cagatggaaa aattaacatc 1200 gatcactctg gaggatacgt tgctcaattc aacatttctt gggatgaagt aaattatgat 1260 cctgaaggta acgaaattgt tcaacataaa aactggagcg aaaacaataa aagcaagcta 1320 gctcatttca catcgtccat ctatttgcca ggtaacgcga gaaatattaa tgtttacgct 1380 aaagaatgca ctggtttagc ttgggaatgg gcgagaacgg taattgatga ccggaactta 1440 ccacttgtga aaaatagaaa tatctccatc tggggcacca cgctttatcc gaaatatagt 1500 aataaagtag ataatccaat cgaagtcgac taaaagt 1537 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Sense primer <400> 5 gaaaaggatc cggcacaagt cattaataca aacagcct 38 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense       primer <400> 6 tcgccgtcga cacgcagtaa agagaggacg tt 32 <210> 7 <211> 1535 <212> DNA <213> Salmonella enteritidis <400> 7 gaaaaggatc cggcacaagt cattaataca aacagcctgt cgctgttgac ccagaataac 60 ctgaacaaat ctcagtcctc actgagttcc gctattgagc gtctgtcctc tggtctgcgt 120 atcaacagcg cgaaagacga tgcggcaggc caggcgattg ctaaccgctt cacttctaat 180 atcaaaggtc tgactcaggc ttcccgtaac gctaacgacg gcatttctat tgcgcagacc 240 actgaaggtg cgctgaatga aatcaacaac aacctgcagc gtgtgcgtga gttgtctgtt 300 caggccacta acgggactaa ctctgattcc gatctgaaat ctatccagga tgaaattcag 360 caacgtctgg aagaaatcga tcgcgtttct aatcagactc aatttaacgg tgttaaagtc 420 ctgtctcagg acaaccagat gaaaatccag gttggtgcta acgatggtga aaccattacc 480 atcgatctgc aaaaaattga tgtgaaaagc cttggccttg atgggttcaa tgttaatggg 540 ccaaaagaag cgacagtggg tgatctgaaa tccagcttca agaatgttac gggttacgac 600 acctatgcag cgggtgccga taaatatcgt gtagatatta attccggtgc tgtagtgact 660 gatgcagcag caccggataa agtatatgta aatgcagcaa acggtcagtt aacaactgac 720 gatgcggaaa ataacactgc ggttgatctc tttaagacca ctaaatctac tgctggtacc 780 gctgaagcca aagcgatagc tggtgccatt aaaggtggta aggaaggaga tacctttgat 840 tataaaggcg tgacttttac tattgataca aaaactggtg atgacggtaa tggtaaggtt 900 tctactacca tcaatggtga aaaagttacg ttaactgtcg ctgatattgc cactggcgcg 960 acggatgtta atgctgctac cttacaatca agcaaaaatg tttatacatc tgtagtgaac 1020 ggtcagttta cttttgatga taaaaccaaa aacgagagtg cgaaactttc tgatttggaa 1080 gcaaacaatg ctgttaaggg cgaaagtaaa attacagtaa atggggctga atatactgct 1140 aacgccacgg gtgataagat caccttagct ggcaaaacca tgtttattga taaaacagct 1200 tctggcgtaa gtacattaat caatgaagac gctgccgcag ccaagaaaag taccgctaac 1260 ccactggctt caattgattc tgcattgtca aaagtggacg cagttcgttc ttctctgggg 1320 gcaattcaaa accgttttga ttcagccatt accaaccttg gcaatacggt aaccaatctg 1380 aactccgcgc gtagccgtat cgaagatgct gactatgcaa cggaagtttc taatatgtct 1440 aaagcgcaga ttctgcagca ggctggtact tccgttctgg cgcaggctaa ccaggttccg 1500 caaaacgtcc tctctttact gcgtgtcgac ggcga 1535

[Brief description of drawings]

FIG. 1 is a schematic diagram of a fusion protein expression unit of a membrane surface anchoring expression vector incorporating the LLO gene of the present invention.

FIG. 2 is a schematic diagram showing the expression system of the oral IFN-γ inducer of the present invention and the result of the hemolytic test thereof.

FIG. 3 is a diagram showing the results of induction of IFN-γ in blood when the oral IFN-γ inducer of the present invention is intragastrically administered to mice.

FIG. 4 is a diagram showing the results of IL-8 induction when the oral IL-8 inducer of the present invention is administered to an intestinal tract model.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 31/00 A61P 31/04 31/04 31/12 31/12 31/18 31/18 35/00 35 / 00 37/02 37/02 37/04 37/04 37/08 37/08 43/00 111 43/00 111 117 117 117 A61K 37/02 F term (reference) 4B018 MD86 ME07 ME08 ME09 4C084 AA02 AA17 BA44 CA03 DA01 DA24 DC50 MA52 NA10 ZB032 ZB072 ZB092 ZB112 ZB132 ZB262 ZB322 ZB332 ZB352 ZC022 ZC552 4C087 AA01 AA02 BC56 BC57 CA09 MA52 NA10 ZB03 ZB07 ZB09 ZB11 ZB13 ZB26 ZB32 ZB33 ZB35 ZC02 ZC

Claims (13)

[Claims] 1. An oral cytokine inducer comprising a microbial cell carrying a cytokine-inducing substance on the surface thereof. 2. The oral cytokine-inducing agent according to claim 1, wherein the microbial cell carrying the cytokine-inducing substance on the surface is a microorganism in which the cytokine-inducing substance is expressed as a fusion protein on the surface. 3. The fusion protein is an expression product of DNA in which a signal sequence, a cytokine inducer gene and a membrane anchor sequence are sequentially linked.
The oral cytokine inducer described. 4. The fusion protein comprises an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the constituent amino acid sequence, and is a fusion protein capable of regulating cytokine inducing ability. The oral cytokine inducer according to claim 3. 5. The oral cytokine inducer according to any one of claims 1 to 4, wherein the cytokine inducer is Listeria hemolysin. 6. The oral cytokine inducer according to claim 1, wherein the microorganism is a lactic acid bacterium. 7. The oral cytokine inducer according to claim 6, wherein the lactic acid bacterium is a Th1-induced lactic acid bacterium strain. 8. The oral cytokine inducer according to claim 7, wherein the Th1-induced lactic acid bacterium strain is a Lactobacillus casei Th1-induced lactic acid bacterium strain. 9. The cytokine is interferon-γ
The oral cytokine inducer according to any one of claims 1 to 8, wherein 10. A preventive / therapeutic agent for a disease requiring the production of an endogenous cytokine, which comprises the oral cytokine inducer according to any one of claims 1 to 9 as an active ingredient. 11. The prophylactic / therapeutic agent for diseases requiring production of endogenous cytokine according to claim 10, wherein the oral cytokine inducer is an oral interferon-γ inducer. 12. A functional food for preventing / treating a disease requiring the production of an endogenous cytokine, comprising the oral cytokine inducer according to any one of claims 1 to 9 as an active ingredient. Material or food. 13. A method for inducing a cytokine, which comprises orally administering the oral cytokine inducer according to any one of claims 1 to 9.