KR101483473B1 - Composition comprising cystatin like protein isolated from Trichinella spiralis for treating or preventing inflammatory bowel disease or autoimmune desease - Google Patents

Abstract

The present invention relates to an inflammatory bowel disease (IBD) or an autoimmune disease comprising cystatin-like protein (rTs-CLP) isolated from a trichinella spiralis parasitized in a mammalian muscle as an active ingredient and a composition for preventing or treating autoimmune disease. The cystatin-like protein isolated from the tymobacteria according to the present invention inhibits the activity of papain, a kind of cysteine protease, activates innate immune receptors of intestinal epithelial cytokines, inhibits enteritis, By modulating cytokines, inflammation and immune response by modulating the symptoms of inflammatory bowel disease and autoimmune disease is superior to suppress the ability to inhibit.

Description

[0001] The present invention relates to a composition for preventing or treating an inflammatory bowel disease or autoimmune disease, which comprises a cystatin-like protein isolated from a tilapia, and a composition for preventing or treating autoimmune desease,

The present invention relates to a composition for the prophylaxis or treatment of inflammatory bowel disease or autoimmune disease comprising, as an active ingredient, a cystatin-like protein (rTs-CLP) isolated from a trichinella spiralis parasitizing in the muscles of mammals .

"Parasite" refers to an invertebrate animal that lives parasitically depending on its prey and environment in the body of another organism. Parasitic infections are a major cause of mortality and mortality in rabbits and humans. In general, parasites are parasitic or non-parasitic species belonging to flat animals and linear animals. The parasite species enters the host's body in the form of, for example, eggs or invasive larvae, which is the result of food ingestion by the host, including the egg or oil layer. The parasite then develops and travels slowly through other tissues, blood and / or lymph. Eventually reaching "desirable" organs to grow and mature. Thus, the parasitic organ of which they are affected is harmful.

One type of parasite, " Trichinella spiralis ", is a nematode of the Trichinella genus, and is one of the common parasites that infest many mammals and human fields such as pigs, dogs, and mice. Adult male and female adult parasites in the small intestine mucosa of an animal,

The parasite is parasitized in muscle fibers distributed throughout the body's rhabdomyosus through the blood vessels and lymph nodes of the barrier. It does not come out during development.

Parasites must enter the parasitic site to infect the host. To do this, parasites secrete proteolytic enzymes. Because these proteolytic enzymes induce an inflammatory immune response in the host, the parasite secretes proteolytic enzymes and protease inhibitors together.

"Cystatin", a cysteine protease inhibitor, is known to control inflammation and immune responses to immune diseases such as allergic asthma and inflammatory bowel disease. In addition, there is a report that when a host infects a parasite, hypersensitivity and immune response are suppressed.

Recently, various studies on immunoregulatory factors have been published, but a clear mechanism has not been elucidated. In developed countries, autoimmune diseases are classified as autoimmune diseases, and inflammatory bowel disease (IBD) is classified as autoimmune disease. In the world, inflammatory bowel disease patients are continuously increasing. As a treatment method for this disease, a method of directly ingesting parasitic eggs in the western world has been introduced, but a clear immune mechanism has not been elucidated.

The present inventors determined that the cystatin-like protein (CLP) isolated from the tiger bug was able to regulate the host immune response and determined that the cystatin-like protein Ts (Ts) isolated from Trichinella spiralis 3 ^rd stage larva - CLP) was used to verify the inhibitory activity of the host inflammatory reaction, thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the prophylaxis or treatment of inflammatory bowel disease or autoimmune disease which comprises cystatin-like protein isolated from a tiger bug as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating an inflammatory bowel disease or autoimmune disease, which comprises cystatin-like protein isolated from a tiger bug as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease or autoimmune disease, comprising cystatin-like protein isolated from a tiger bug as an active ingredient.

The present invention provides a food composition for preventing or ameliorating an inflammatory bowel disease or autoimmune disease comprising, as an active ingredient, a cystatin-like protein isolated from a tiger bug.

The cystatin-like protein isolated from the tymobacteria according to the present invention inhibits the activity of papain, a kind of cysteine protease, activates innate immune receptors of intestinal epithelial cytokines, inhibits enteritis, By modulating the cytokine, the inflammation and immune response by modulating the symptoms of inflammatory bowel disease or autoimmune disease is excellent in suppressing ability.

FIG. 1 shows (A) secreted excretory protein and (B) cervical protein in a specific immunoglobulin in progeny mouse serum of a mouse infected with a tiger bug . [ T. spiralis (-) represents the progeny mouse serum of uninfected mouse , And T. spiralis (+) means offspring mouse sera of mice infected with the tiger bug ].
Fig. 2 is a diagram showing the result of analysis of 37.8 kDa protein of Ts-CLP obtained by MALDI-TOF.
3 is a diagram showing the result of amino acid sequence analysis of Ts-CLP using Signal P 3.0.
FIG. 4 is a graph showing the results of the statistical analysis of the amino acid sequence of Ts-CLP using MEGA 5. FIG.
T. Figure 5 is a diagram showing the level of expression of a developmental stage of the CLP-rTs spiralis [(A) NBL, the expression of Ts-CLP gene in 3L and adults, (B) rTs CLP-proteins (left: marker, right: rTs-CLP), (C) Results of rTs-CLP protein detection (left: secretory excretory protein, right: cervical protein) in the larval and larval excretory proteins of the third instar larva.
FIG. 6 is a graph showing the inhibitory effect of rTs-CLP on cysteine protease activity (papain inhibitory activity) [* P <0.05, ** P <0.01, 3 independent experiments, triplicate].
7 shows the expression of innate immune response by rTs-CLP in mouse mouse epithelial cell CT-26 cells [(A) Expression pattern of TLRs, IL-10 and TGF-β gene (B) (* P <0.05, ** P <0.01, *** P <0.001), respectively, indicating ERK, p38, JNK MAP kinases, NF-kb, Ikb and β-actin patterns.
FIG. 8 is a diagram showing the experimental schedule of a mouse model in which inflammatory bowel disease was induced for the purpose of the present invention. [The induction process of inflammatory bowel disease in the rTs-CLP-treated group and the rTs- Respectively. The number means elapsed time (per day).
FIG. 9 shows DAI, an index of length, weight, fecundity, and health status in a mouse model in which inflammatory bowel disease was induced by DSS [(A) weight change, (B) disease index scale ). (C and D) indicates the degree of shrinkage of the intestines. DW; Unprocessed, DSS; Enteritis induced group, DSS + rTs-cystatin; (* P <0.05, ** P <0.01, *** P <0.001, n = 5 mice / group) after treatment with rTs-CLP.
Fig. 10 Inflammatory Bowel Disease-Induced Mouse Model (A) Hematoxylin staining and eosine staining. (A) No-treatment group, (b) Enteral-induced group, and c) After rTs-CLP treatment, the group was divided into two groups. (B) was evaluated by scoring the degree of intestinal inflammation (** P <0.01, n = 5 mice / group).
11 shows cytokine changes by rTs-CLP treatment in the spleen and mesenteric lymph nodes of inflammatory bowel disease-induced mice [(A) spleen and (B) cytokine changes in the mesangial lymph nodes (* P <0.05, ** P <0.01, *** P <0.001, n = 5 mice / group).
12 shows the expression pattern of intracolonic cytokines [* P <0.05, ** P <0.01, *** P <0.001, n = 5 mice / group].

The present invention provides a composition for the prophylaxis or treatment of inflammatory bowel disease or autoimmune disease comprising cystatin-like protein isolated from the tiger bug as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in detail.

The cystatin-like protein, which is an active ingredient in the composition of the present invention, is a cystatin-like protein (cysteine protease inhibitor) isolated from the secretion of larval tincture larva and excretion protein and is produced from DNA isolated from the third stage larva It is a recombinant cystatin-like protein obtained by the cloning method, which consists of 714 bp nucleotides having 237 amino acids in total, and the original protein size is 27.563 KDa.

The cystatin-like protein is composed of the amino acid sequence shown in SEQ ID NO: 1, and the protein is encoded by the nucleotide sequence shown in SEQ ID NO: 2.

The cystatin-like protein isolated from the triticalum according to the present invention inhibits the activity of pispine, a kind of cysteine protease, activates the innate immune receptor of the intestinal epithelial cell cytokine, inhibits enteritis By modulating the cytokine in the model, it has excellent inhibitory properties that regulate inflammation and immune responses and relieve symptoms of inflammatory bowel disease and autoimmune diseases.

Thus, the cystatin-like protein isolated from the tiger bug of the present invention can be used as a medicament or a health food useful for the prevention or treatment of inflammatory bowel disease or autoimmune disease.

The composition of the present invention may contain at least one known active ingredient having a cystatin-like protein separated from the tinebrush and having an effect of preventing or treating inflammatory bowel disease or autoimmune disease.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the cystathin-like protein is about 0.05 to 5 mg / kg, preferably about 0.1 to 1 mg / kg, and is preferably administered once or several times a day.

The composition of the present invention may be used alone or in combination with methods for the prevention or treatment of inflammatory bowel disease or autoimmune diseases or using surgery, hormone therapy, drug therapy and biological response modifiers.

The composition of the present invention may be added to a health functional food for the purpose of preventing or improving inflammatory bowel disease or autoimmune disease. When the cystatin-like protein of the present invention is used as a food additive, the cystatin-like protein can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the cystatin-like protein of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as a conventional beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.20 g, preferably about 0.04 to 0.10 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are intended to illustrate the present invention in detail, and the content of the present invention is not limited by the examples and the experimental examples.

In the detailed description of the present invention, the examples and experimental examples are for illustrating the present invention, and the scope of the present invention is not limited to the examples and experimental examples.

Examples. Preparation of cystatin-like protein (rTs-CLP) isolated from recombinant triticum larvae

1. Obtain Ts-CLP

The luteal secretory excretory protein-specific immunoglobulin and the luteal protein-specific immunoglobulin in the progeny mouse serum of the mouse infected with the tritterium were searched and shown in Fig.

As shown in Fig. 1, the luteal secretory excretory protein and the cervical protein were commonly found at about 37.8 kDa.

2. Ts-CLP analysis

The protein thus obtained was analyzed by MALDI-TOF, and its amino acid sequence was analyzed using sinal P 3. The results are shown in FIG. 2 and FIG. 3, respectively.

As shown in FIG. 2, Ts-CLP was composed of 237 amino acids at 714 bp and had about 27.5 kDa and an isoelectric point (pI) of 8.02. And a signal peptide consisting of 24 amino acids at the N terminus and a cleavage site between the 18th and 19th amino acids.

As shown in FIG. 3, polymorphic amino acid sequence analysis revealed that homology with Ts-CLP and the third type of the tannicide cystatin was low but had the closest relationship. Therefore, it was confirmed that the protein was a cystatin-like protein (Ts-CLP) isolated from the tinebrush.

3. rTs-CLP cloning and purification

First, partial sequence was obtained from the EST of the third instar larva, and then the entire sequence of Ts-CLP was obtained by RACE PCR method and the whole gene sequence was amplified based on the general PCR method. As the template strand used in this process, RNA was purified from the third larva using Trizol, and the synthesized cDNA was used. Sense and antisense primers were individually designed (Table 1), and the respective annealing temperatures were set at 55 캜.

Primer sequence Forward primer sense 5'-GTC GAC AAA TGA AGT TGA TGT ACT GCA-3 '
(SEQ ID NO: 3) Reverse primer Antisense 5'-GCG GCC GCT TAG CAG CGA ACT TCA GAG TTT -3 '
(SEQ ID NO: 4)

In order to confirm that the CLP gene was amplified correctly, the amplified CLP gene was inserted into a commercially available T & A cloning vector, and the sequence was confirmed. Then, the amplified CLP gene was inserted into the expression vector pTNT ^TM and pTNT T7 Quick Coupled Proteins were expressed and purified by Transcription / Translation System. Expression conditions were performed using PCR for 30 minutes to 90 minutes. After completion of the reaction, only rTs-CLP was obtained using Affinity Chromatography (Affinity Chromatography).

4. Multiple sequence alignment and embryological analysis of rTs-CLP

The amino acid sequence of rTs-CLP obtained in the above 3 is shown in SEQ ID NO: 1, and the nucleotide sequence of rTs-CLP is shown in SEQ ID NO: 2. The amino acid sequence was analyzed using MEGA 5. The sequences used in this analysis were as follows: T. spiralis (gi | 339244349, gi | 111183177, gi | 164521934, gi | 339233392, gi | 339233446, gi | 339234187, gi | 339262984, gi | 1857760 and gi | 325048293), Caenorhabditis elegans (gi | 17562818), Fasciola gigantica (gi | 239937595), Fasciola hepatica (gi | 55978577), Macaca mulatta (gi | 109033376), Sus scrofa (gi | 126022833), Monodelphis domestica (gi | 126325225), Oryctolagus cuniculus 146742108 and gi 194226303), Bos taurus (gi | 154152007), Ovis aries (gi | 148665501), Ornithorhynchus anatinus (gi | 149428804), Equus caballus (gi | 146742108 and gi | 194226303), Mus musculus (gi | 1706279), Taeniopygia guttata (gi | 197128523), Callithrix jacchus (gi | 296189801), Pongo abelii (gi | 297670214), Ailuropoda melanoleuca (gi | 301783717), Meleagris gallopavo (gi | 326912709), Anolis carolinensis | 327278711), Nomascus leucogenys (gi | 332252852), Loxodonta africana (gi | 344294759), Canis lupus (gi | 345796081), Cavia porcellus (gi | 348567133), Taeniopygia guttata (gi | 350538531), Cricet ulus griseus a] (gi | 354506122), Homo sapiens (gi | 4503117 and gi | 4885165), Pan troglodytes (gi | | 57114059) , and Felis catus (57619099 gi). The results of the analysis are shown in FIG. 4 and FIG.

As shown in Fig. 4, rTs-CLP was found to be of the type 3-type cysteine. In addition, Ts-CLP was expressed at all stages of the tymobacteria, and it was confirmed that Ts-CLP was most expressed and secreted, especially in the third stage muscle larvae.

In addition, as shown in FIG. 5, rTs-CLP of the present invention was observed at about 40 kDa in the luteal secretory excretory protein and the ruminal protein even though it was 26 kDa.

EXPERIMENTAL EXAMPLE 1 Measurement of inhibitory activity of rTs-CLP on cysteine protease activity (inhibition of papain activity)

In order to confirm the effect of rTs-CLP of the present invention on cysteine protease inhibitory activity, the following experiment was conducted.

Specifically, Z: N-carbobenzyloxy, which is a peptide recognized by cysteine protease after reacting 20 nM of papain and 1 μg of rTs-CLP at room temperature for 20 minutes; Amino-4-methylcoumarin (Z-Phe-Arg-AMC) was added and its absorbance was measured by a VICTOR3 ^TM machine at intervals of 5 minutes for 30 minutes. The results are shown in FIG.

As shown in FIG. 6, although the rTs-CLP of the present invention lacked domains known to be important for inhibiting cysteine protease, it was confirmed that the activity of papain, a kind of cysteine protease, was inhibited. That is, it was confirmed that the fluorescence absorbance of the sample treated with rTs-CLP of the present invention was decreased, which is thought to be the degradation of the peptide by papain.

Experimental Example 2. Conjugation of innate immune response by rTs-CLP in intestinal epithelial cells

To investigate the innate immune response by rTs-CLP, CT-26 cells were treated with rTs-CLP to observe the mRNA expression patterns and signaling pathways at the protein level. The results are shown in Fig.

As shown in Fig. 7, rTs-CLP increased IL-10 and TGF-β innate immune receptors TLR3 and TLR9, which were regulatory cytokines, and activated ERK, JNK and p38 MAP kinase.

Experimental Example 3. Inhibition of enteritis reaction by rTs-CLP

1. Treatment of rTs-CLP and induction of inflammatory bowel disease

Inflammatory bowel disease was induced by administering Dextran sodium sulfate (DSS) to rTs-CLP-treated and rTs-CLP-treated control mice. At this time, rTs-CLP was dissolved in sterilized water at a concentration of 2 μg per mouse and administered by intraperitoneal injection. The test schedule of the mouse model in which inflammatory bowel disease was induced is shown in Fig. The indicated period refers to the period (days) elapsed since the start of the experiment.

2. Investigation of length and weight changes, fecundity, and health status in mice with inflammatory bowel disease induced by DSS

We observed changes in body weight, fecundity (hardness, presence of blood) and length of the colon in rTs-CLP-treated experimental and control mice. The body weight and fecal status of the mice were scored daily and scored and expressed as the disease activity index (DAI). The DAI values are shown in Table 2 below. The results are shown in Fig.

score Weight reduction rate (%) The state of the feces (hardness) Blood test One 1-3 normal Negative hemoccult 2 4 to 6 Smooth or slightly formed Positive hemoccult 3 7 ~ 9 Very soft Slight bleeding in feces 4 > 9 diarrhea Rectal bleeding

As shown in FIG. 9, diathesis and bloody diarrhea occurred after induction of inflammation in mice with DSS, and the body weight decreased. In addition, the intestinal tract was shrunken due to severe dehydration symptoms and the overall length of the colon was remarkably shortened. However, the rTs-CLP-pretreated mice lost weight.

In the disease activity index (DAI), the rTs-CLP treated group showed lower disease levels than the control group. The rTs-CLP pretreated mice were significantly lower in DAI than the control mice, which were treated with DSS alone and induced enteritis.

In the experimental group, the degree of contraction of the intestinal tract due to frequent dehydration symptoms was inhibited by pretreatment with rTs-CLP. In the rTs-CLP-treated group, the length of the intestine was significantly longer than that of the control group, and it gradually returned to normal level with the lapse of time.

In addition, histological changes in the colon of the mice infected with inflammatory bowel disease were observed, and the results are shown in Fig. Scoring criteria for intestinal inflammation are shown in Table 3 below.

One 2 3 4 Inflammation number 0 1-2 3-4 5 < Inflammation range <100 μm 100 탆 <500 탆 500 μm <1 mm 1 mm < Tissue destruction 0 1-2 3-4 5 < Immune cell invasion 1-3 4-7 8-11 11 <

As shown in Fig. 10, the intestinal epithelial cells and subcutaneous layer were destroyed and thickened by DSS, and infiltration of inflammatory cells was observed. On the other hand, it was observed that the number of mice treated with rTs-CLP pretreatment was reduced.

Experimental Example 4: Control of cytokines by rTs-CLP in a mouse model in which inflammatory bowel disease was induced

1. Cytokine regulation in the spleen and mesenteric lymph nodes

(IL-6, INF-γ, IL-1β, TNF-α, IL-10 and TGF-β) were isolated and cultured in spleen and mesenteric lymph nodes of mice with inflammatory bowel disease. And the immune response was observed. The results are shown in Fig.

As shown in FIG. 11, IL-6, IFN-y, TNF-alpha and IL-1 beta, which are inflammatory cytokines, were significantly increased in mice treated with DSS alone compared with mice not treated with any of them.

Treatment of DSS with rTs-CLP in a mouse model of inflammatory bowel disease-induced inhibition of inflammatory cytokine expression. IL-6, IFN-γ and IL-1β, which are inflammatory cytokines, were suppressed in the mouse spleen and IL-10, which is a regulatory cytokine, was inhibited by rTs- TGF-b was increased. In the mesenchymal lymph nodes of mice treated with rTs-CLP, inflammatory bowel disease-induced mouse models showed IFN-γ, TNF-α, IL-1β suppression and TGF-β increase.

2. Expression patterns of cytokines in the colon

The expression pattern of intracellular cytokines in rTs-CLP-treated mice was confirmed in a mouse model in which inflammatory bowel disease was induced by the same method as described above. The results are shown in Fig.

As shown in Fig. 12, it was confirmed that IL-2 and IL-1? Increased by DSS treatment were inhibited by rTs-CLP treatment.

<110> Pusan National University Industry-University Cooperation Foundation <120> Composition comprising cystatin-like protein isolated from          Trichinella spiralis for or treating inflammatory          bowel disease or autoimmune desease <130> 1.127P <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 237 <212> PRT <213> rTs-CLP, recombination Trichinella spiralis-Cystatin like protein <400> 1 Met Lys Leu Met Tyr Cys Met Leu Val Val Leu Phe Leu Ala Phe Asn   1 5 10 15 Glu Ala Gln Gln Ile Asp Tyr Ile Lys Arg Ala Gln Asp Ala Leu Phe              20 25 30 Leu Trp Asn Trp Asn Arg Pro Asp Pro Tyr Lys Lys Ile Ile Thr Val          35 40 45 Arg Asn Arg Arg Ile Glu Gly Thr Arg Ile Ile Phe Gly Val Asp Leu      50 55 60 Gln Asp Thr Val Cys Ile Ala Ser Gln Val Arg Val Tyr Ser Tyr Asp  65 70 75 80 Asp Met Val Arg Val Cys Pro Pro Arg Gln Gly Ser Arg Ile Lys Tyr                  85 90 95 Cys Ala Ile Tyr Tyr Glu Met Gly Asp Ile Arg Thr Thr Gln Val Tyr             100 105 110 Cys Glu Ser Asp Arg Glu Leu Val Leu Val Ala Glu Gly Gly Arg Ser         115 120 125 Asp Pro Glu Ile Gly Gly Thr Gly Gly Thr Ile Lys Tyr Asp Val Ser     130 135 140 His Gln Asp Phe Gln Asp Ile Val Arg Arg Gly Val Phe Glu Trp Asp 145 150 155 160 Lys Lys Arg Asn Asp Gly Lys Tyr His Leu Val Lys His Val Ile Glu                 165 170 175 Gly Ser Arg Ser Gly Ile Leu Ser Ser Phe Gln Ile Val Leu Asp Asp             180 185 190 Ala Ser Cys Pro Val Arg Thr Ser Val Phe Asp Asn Tyr Gln Asp Val         195 200 205 Tyr Arg Arg Cys Ala Gly Gln Gly Leu Ser Arg Gln Cys Val Leu Glu     210 215 220 Phe Arg Tyr Leu Asp Glu Lys Asn Ser Glu Val Arg Cys 225 230 235 <210> 2 <211> 1171 <212> DNA <213> rTs-CLP, recombination Trichinella spiralis-Cystatin like protein <400> 2 ttaggtgaca ctatagaata caagctactt gttctttttg cactcgagaa ttcacgcgtg 60 gtacctctag agtcgacaaa tgaagttgat gtactgcatg ttagtggtcc tttttcttgc 120 atttaatgaa gctcagcaaa ttgattacat caaaagagca caagatgcat tatttttgtg 180 gaattggaat cgaccagatc catataaaaa gataatcact gtcagaaatc gtcgtatcga 240 gggaacaaga attatttttg gcgttgattt gcaagacact gtttgcatcg cttcacaagt 300 aagagtttat tcatatgacg atatggtgag ggtgtgtcca cctagacaag gatcccgaat 360 caaatattgt gcaatctact atgaaatggg agacattcga acaacgcaag tgtattgtga 420 atcagacaga gaacttgtgc tggttgcgga aggagggcgt tctgatccag aaatcggagg 480 gacaggagga accataaagt atgatgtttc tcatcaagac tttcaagata tagttcgaag 540 aggcgtgttt gaatgggata aaaagaggaa tgatggaaag tatcatcttg tgaaacatgt 600 cattgaaggt tcgcgtagtg gaatactgag cagttttcaa attgttttgg atgatgcatc 660 ttgccccgtc agaacaagtg tgtttgataa ctaccaagat gtctacagac gttgcgctgg 720 acagggactt tccaggcagt gtgtacttga attcagatat ctggatgaga aaaactctga 780 agttcgctgc taagcggccg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa actagcataa 840 ccccttgggg cctctaaacg ggtcttgagg ggttttttgg atccgggctg gcgtaatagc 900 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg 960 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 1020 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 1080 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 1140 ctttacggca cctcgacccc aaaaaacttg a 1171 <210> 3 <211> 27 <212> RNA <213> sense primer <400> 3 gtcgacaaat gaagttgatg tactgca 27 <210> 4 <211> 30 <212> RNA <213> anti-sense primer <400> 4 gcggccgctt agcagcgaac ttcagagttt 30

Claims (6)

1. A pharmaceutical composition for preventing or treating an inflammatory bowel disease or autoimmune disease, which comprises a cystatin-like protein separated from a cruel trichomes and consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the cystatin-like protein is isolated from the third stage larva of the tilapia. 2. A pharmaceutical composition for preventing or treating inflammatory bowel disease or autoimmune disease.
The pharmaceutical composition for preventing or treating inflammatory bowel disease or autoimmune disease according to claim 1, wherein the cystatin-like protein is isolated from the secretion of the tritter and the excreted protein.
2. A pharmaceutical composition for preventing or treating inflammatory bowel disease or autoimmune disease, which comprises a cystatin-like protein separated from a tymy cymbidium and encoded by a nucleotide sequence represented by SEQ ID NO: 2 as an active ingredient. 1. A food composition for preventing or ameliorating an inflammatory bowel disease or autoimmune disease, which comprises a cystatin-like protein separated from a tymy fish and consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 2. A food composition for preventing or ameliorating an inflammatory bowel disease or autoimmune disease, which comprises a cystatin-like protein separated from a tymy cigarette and encoded by a nucleotide sequence represented by SEQ ID NO: 2 as an active ingredient.

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