TW201116292A - Agent for prevention or treatment of inflammatory bowel diseases - Google Patents

Abstract

Provided is a prophylaxis or treatment agent for inflammatory bowel diseases, comprising polypeptides as active ingredient; the above polypeptides has at least 70% sequence homology to human paraoxonase, and has an activity selected from the group consisting of lactonase activity, paraoxonase activity, arylesterase activity and reactive oxygen scavenging activity.

Description

201116292 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a protein preparation. More specifically, it relates to a prophylactic or therapeutic agent for an inflamed intestinal disease (inf lammatory bowel di sease) such as ulcerative colitis or Crohn's disease. [Prior Art] The inflammatory bowel disease is a general term for chronic diseases in which the cause of inflammation in the digestive tract is unknown. The diseases represented are ulcerative colitis and Crohn's disease. Ulcerative colitis is a disease in which the mucosal disorder of the large intestine is the main disease, and the cause is still unknown. It is currently believed to involve complex interactions between hereditary factors and environmental factors. Some antigens cause an excessive immune response in the intestinal tract through the cells responsible for immunity in the digestive tract, thus continuing to develop disease and inflammation. The symptoms of ulcerative colitis are weak mucosa of the large intestine, easy bleeding, ulceration or ulceration in the mucosa. Usually, patients may have abdominal pain, sticky blood and other symptoms. The symptoms of ulcerative colitis can be exemplified by bloody stools, sticky stools, diarrhea, or bloody sputum, depending on the extent and severity of the lesion. Although the condition is mild, it is mostly a small amount of blood and there is no sputum, but if it is serious, it is a serious watery diarrhea accompanied by bleeding like ketchup, or a state of blood in the exudate and mucus. Other symptoms include abdominal pain, fever, loss of appetite, weight loss, and anemia. Also often accompanied by arthritis, urethra stones, iritis or conjunctivitis, pancreatitis or hyperamylasemia

S 3 322362 201116292 ‘ Extra-tube complications. When starting a drug therapy for ulcerative colitis, it is important to know the severity of the condition. The drug therapy is basically a combination therapy, and the 5-aminosalicylic acid (5_ASA) preparation and the adrenocortical steroid agent using the basic drug can induce remission (remissi〇n__inducti〇n) and maintain it. However, 'in some cases, it will worsen or recur, so sometimes azathioprine or 6-thiol (6_Mp), enema preparation method, white ball removal therapy, and cyclosporin (cyclin sp〇rin) will be used. . However, there is currently no established medical treatment for ulcerative colitis. In addition, Crohn's disease is mainly found in young people, a granulomatous inflammatory lesion accompanied by ulceration and fibrosis, which may occur anywhere in the digestive tract from the mouth to the door. Clinical signs vary depending on the location and extent of the lesion. It can cause systemic symptoms such as fever, nutritional disorders, anemia, or systemic complications such as arthritis, iritis, and liver dysfunction. In terms of the symptoms of Crohn's disease, the lesions invade the mucosa of the digestive tract to all layers of the serosa. Common symptoms include abdominal pain, diarrhea, weight loss, fever, and anal lesions. When Crohn's disease occurs, there are symptoms of intestinal occlusion, intestinal perforation, and large blood. In addition, there are no abdominal symptoms at the time of onset, but there are symptoms of anal lesions or fever. In the case of extra-gastrointestinal complications, for example, anemia, hypoproteinemia, ankylosing spondylitis, aphthous ulcer, nodular skin disease, urban inflammation, and (four) obstruction. Abdominal At present, there is no established medical treatment for Crohn's disease. Because the purpose of treatment is to control the activity of the disease and maintain the relief state, and improve the patient's Q〇L (quality 0f life, quality of life). Therefore, the current collection

S 322362 201116292 k Combine drug therapy, nutritional therapy, and surgical therapy to maintain nutritional status, suppress symptoms, and prevent recurrence of inflammation. It is a preparation that is expected to be more effective and safer. On the other hand, the P0N1, paraxonase 1 is a representative enzyme called the balasolase (hereinafter sometimes referred to as "PON") enzyme family. The common activity of Ρ0Ν is lactone hydrolysis activity, and pQNl can hydrolyze lactones, esters, such as a wide range of substrates such as paraoxon organic acid salts. There have been many reports on the relationship between P0N1 levels in vivo and disease. For example, in vivo, P0N1 is synthesized by the liver and binds to Ap〇 A-1 in blood to be present on high-density lipoprotein (HDL) particles. It is known that p〇N 1 can inhibit the development of arteriosclerosis by inhibiting oxidation of low-density lipoprotein (LDL) (Non-Patent Document 1). However, there is still no report that Ρ0Ν 1 can be effectively used as a prophylactic or therapeutic agent for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. PRIOR ART DOCUMENT 1: Non-Patent Document 1: Chem. Biol. Interact. 1993, Jun; 87. 161-7b [Summary of the Invention] The object of the present invention is to provide an effective ulcerative colitis. A prophylactic or therapeutic agent for inflammatory bowel diseases such as Crohn's disease. (Means for Solving the Problems) 322362 5 201116292 w In order to solve the above problems, the inventors of the present invention have obtained the following findings after repeated research. (i) Boxes • P0N1 is administered to a model animal that induces ulcerative colitis by sodium dextrin sulfate (DSS), and dysfunction or inflammation of the large intestine can be suppressed. (u) By administering P0N1 to a model animal which induces Crohn's disease by 2,4,6-trinitrobenzenesulfonic acid (TNBS), dysfunction or inflammation of the large intestine can be inhibited.

Ciii) Even if the poNi variant (the C284A variant of Example 16 and the K84E/R306Q, - of Example 17) were administered to the above-mentioned Crohn's disease model animal, the results were the same as in the case of administration of P0N1, and the function of the large intestine was enabled. Barriers and inflammation are inhibited. The P〇N1 variant system is a polypeptide having an internal S-enzyme activity and a balasolase activity similarly to p〇N1. (iv) even if the above-mentioned Crohn's disease model animal is administered with a derivative of P0N1, which is a polypeptide of sequence 8 of evQlutiQn p(10)1 (ePQN1) or a polypeptide of SEQ ID NO: 1 (Example 2〇), the result is In the same manner as in the case of P0N1, the dysfunction and inflammation of the large intestine can be suppressed. H/ The above-mentioned eP〇N1 line has sequence identity with human P〇N1 (h〇m〇1〇gy). In the same manner as ρ〇Ν1, it is a polypeptide having lactonase activity, balasolase's activity, arylesterase activity, and activity of scavenging reactive oxygen species. a) even if the above-mentioned Crohn's disease model animal is administered with Palladium-selective, and active-active milk, and has no lactonase activity and aroma i-protease's tongue-like P〇Nl variant (Example 21) The D269E variant), the results are the same as in the case of £322362 201116292, and POMl, which can inhibit the dysfunction and inflammation of the large intestine. (h) administration of a P0N1 variant having a tongue-like and non-lactonease activity, a balasolase activity, and an aromatase' tongue, even in the above-mentioned Crohn's disease model animals (Example 22) The H115/134Q variant), 7 ', and the slave are the same, which can inhibit the dysfunction and inflammation of the large intestine. In the present specification, a polypeptide consisting of the amino acid sequence of P0N1, wherein one or a plurality of amino acids are deleted, inserted, or substituted, is also referred to as a P0N1 variant. The present invention has been completed based on the above findings and further studies, and provides a preventive or therapeutic agent for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Item 1. A prophylactic or therapeutic agent for an inflammatory bowel disease, comprising a polypeptide as an active ingredient; the polypeptide having a sequence of 70% or more with a human body balasolase, and having a lactonease activity selected from the group consisting of At least one activity in the group is determined by the activity of arylene esterase _ ( ary 1 esterase) and the activity of scavenging reactive oxygen species. The prophylactic or therapeutic agent for an inflammatory bowel disease according to Item 1, wherein the polypeptide is a polypeptide of the following (a), (b) or (c), (a) SEQ ID Nos. 1, 6, and 8 or The amino acid sequence of (b) the amino acid sequence of SEQ ID NO: 1 '6, 8 or 1 构成 is formed by the amino acid sequence of 10 (in which one or more amino acids are deleted, inserted, or taken

322362 S 201116292 and wherein the polypeptide has at least one of the active group (C) sequence numbers 1, 6, 8 or selected from the group consisting of lactonase activity, balasolase activity, and aromatic esterase activity. A polypeptide consisting of an amino acid sequence of 10 (one or a plurality of amino acids are deleted, inserted, or substituted), and the polypeptide has an activity of scavenging active oxygen. The prophylactic or therapeutic agent for an inflammatory bowel disease according to Item 1 or 2, wherein the polypeptide has at least one selected from the group consisting of lactonase activity, balasolase activity, and aryl esterase activity. Activity. Item 4. The prophylactic or therapeutic agent for an inflammatory bowel disease according to Item 1 or 2, wherein the polypeptide has an activity of scavenging active oxygen, a activity of balasolase, and an enzyme activity of aroma. The prophylactic or therapeutic agent for inflammatory bowel disease according to any one of items 1 to 4, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. Item 6. The prophylaxis or chemotherapy of an inflammatory bowel disease according to any one of items i to 5, wherein the prophylactic or therapeutic agent for inflammatory bowel disease is an injection. Item 7. A prophylactic or therapeutic agent for ulcerative colitis which contains a balasolase as an active ingredient. Item 8. A prophylactic or therapeutic agent for Crohn's disease, which comprises a balazonase as an active ingredient. Having a sequence of the same as the human balazonase, the same self-lactamase activity, the balasolase activity, and the activity of the oxygen, at least one of the 9-polypeptides, used to prevent silk The treatment of inflammatory bowel disease, more than 70% of the sex, and has the choice, aromatic esterase activity and clearance of live activity. Item 10. The polypeptide of Item 9, wherein the inflammatory bowel disease is a cancerous 322362 8 201116292 enteritis or Crohn's disease. Further, the present invention provides the following prophylactic or therapeutic agents for ulcerative colitis or Crohn's disease. Item la. A prophylactic or therapeutic agent for ulcerative colitis which contains a balasolase as an active ingredient. Item 2a. The prophylactic or therapeutic agent for ulcerative colitis according to item 1, wherein the balasolase is a polypeptide of the following (a) or (b): (a) the amino acid sequence of SEQ ID NO: 1. Polypeptide (b) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein one or more amino acids are deleted, inserted, or substituted, and the polypeptide has lactonase activity or balasolone Enzyme activity. Item 3a. The prophylactic or therapeutic agent for ulcerative colitis according to item la or 2a, wherein the prophylactic or therapeutic agent for ulcerative colitis is an injection. Item 4a. A prophylactic or therapeutic agent for Crohn's disease, which comprises a balasolase as an active ingredient. Item 5a. The prophylactic or therapeutic agent for Crohn's disease according to Item 4, wherein the balasolase is a polypeptide of the following (a) or (b): (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein one or more amino acids are deleted, inserted, or substituted, and the polypeptide has lactonase activity or balasolase active. Item 6a. The prophylactic or therapeutic agent for Crohn's disease according to Item 4a or 5, wherein the prophylactic or therapeutic agent for Crohn's disease is an injection. (effect of the invention)

S 9 322362 201116292 By the present invention, it is possible to provide a medicine which can be effectively prevented or treated for an inflammatory bowel disease, particularly ulcerative colitis and Crohn's disease. The invention can inhibit inflammation and tissue damage of the large intestinal mucosa caused by ulcerative colitis, and effectively improve symptoms such as diarrhea or bleeding. By the present invention, inflammation and tissue damage of the large intestinal mucosa caused by Crohn's disease can be suppressed, and symptoms such as weight loss, squat or bleeding can be effectively improved. Further, the medicine of the present invention is substantially safe from causing no side effects. [Embodiment] Hereinafter, the present invention will be described in detail. The prophylactic or therapeutic agent for inflammatory bowel disease of the present invention comprises a polypeptide as an active ingredient; the polypeptide has a sequence identity of 70°/ with human body balasolase (human PON). Above, it has at least one activity selected from the group consisting of lactonase activity, balasolase activity, aromatic esterase activity, and activity of scavenging reactive oxygen species. The polypeptide of the active ingredient of the present invention has one or more activities of lactonease activity, balasolase activity, aroma enzyme activity, and activity of scavenging active oxygen. The polypeptide of the active ingredient of the present invention is preferably one having at least the activity of removing active oxygen. The human body PON is preferably a human body PON1. That is, the preferred active ingredient in the present invention has a sequence identity with human PON1 of 70% or more, and has an activity selected from the group consisting of internal acetase activity, balasolase activity, aromatic S enzyme activity, and scavenging active oxygen. At least one active polypeptide in the group. The human PON1 is preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6. 10 322362 201116292 β <Active ingredient><Ρ0Ν> The active ingredient of the prophylactic or therapeutic agent for inflammatory bowel disease of the present invention may, for example, be hydrazine. Just included face, Ρ0Ν2, Ρ(10)3, etc. Further, the source species of ρ〇Ν is not particularly limited, and is preferably ρ(10) derived from humans. Further, it is preferably a human-derived, more preferably: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; (8) a polymorphic structure consisting of the amino acid sequence of the heart number i, one of which Or a plurality of amino acids are deleted, inserted, or substituted, and the polypeptide has at least one activity selected from the group consisting of: (4) enzymatic activity, balaol-activity, and aroma (iv) activity; column number 1 A polypeptide composed of an amino acid sequence in which one or a reduced amino acid is deleted, inserted, or substituted, and the polypeptide has an activity of scavenging active oxygen. The above: the sequence of the amino acid sequence of the peptide can also be the following or a plurality of amino acids are missing, inserting, 2 money 'one of them have a miscellaneous 13⁄4 Bara Ozon County t cut' And the polypeptide is one or more preferably from 1 to 5, more preferably i: from 1 to 10, more preferably one in the present specification. Particularly preferred are 1 to 2 polypeptides composed of the amino acid sequence of the component (4) of the present invention; (a polypeptide consisting of the sequence of the sequence number 6 and having a sequence number of 6 The amino acid sequence is inserted, or substituted, and the polypeptide is: a plurality of amino acid deletions, oxasone activity, and aryl esterase activity selected from the group consisting of lactonase activities, and At least one activity; 322362 201116292 or (C) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, wherein one or more amino acids are deleted, inserted, or substituted, and the polypeptide has scavenging activity The activity of oxygen. The amino acid sequence of SEQ ID NO: 6 is the amino acid sequence of the first position in the amino acid sequence of SEQ ID NO: 1 in the sequence of amino acid sequence SEQ ID NO: 1. Amino acid sequence from position 2 to 355. When P0M1 is produced by in vivo or cultured cell lines, P0N1 usually cleaves the first thiol acid in the cell and then secrets it extracellularly after intracellular synthesis. Specifically, for example, human p〇N1 is taken as an example 'normal sequence number 1 After the intracellular synthesis of the polypeptide consisting of the amino acid sequence, the first thiol acid is cleaved in the cell and then secreted outside the cell to obtain the polypeptide of SEQ ID NO: 6. <P0N1 derivative> The polypeptide as an active ingredient in the prophylactic or therapeutic agent for inflammatory bowel disease of the present invention is preferably an artificial PON 1 derivative produced by molecular evolution engineering. The pon 1 derivative is preferably disclosed in i, A. Etal., Proc. Natl. Acad. Sci. USA 101 (2), 482-487 (2004) and referred to as ev〇lutionPONl (also referred to as ePON1 in this specification). ePONl is obtained as follows. P0N1 derivatives: cDNAs of mice, humans, rats, and rabbits (having more than 80% identity, respectively), using a molecular evolution engineering technique to generate a chimeric cDNA library by random family shuffling Further, a heterologous Escherichia coli expression is selected as a clone. The P0N1 derivative in the present invention preferably has the same balasolase activity, lactonase activity and aromatic esterase as the human P0N1. One or more of the activities In addition, the P0N1 derivative in the present invention preferably has an activity of scavenging active oxygen. Specifically, the P0N1 derivative may be exemplified by G2E6 (SEQ ID NO: 8) and G3C9C SEQ ID NO: 10 ), G3H8 (G3H8 amino acid sequence has been registered in GenBank, Accession No. AAR95985), G1A5 (G1A5 amino acid sequence has been registered in GenBank, Accession No. AAR95981), G1C4 (G1C4 amino acid sequence has been registered) The amino acid sequence of ePONl〇G2E6 (SEQ ID NO: 8) and the amino acid sequence of G3C9 (GenBank, Accession No. AAR95982), G2D6 (the amino acid sequence of G2D6 has been registered in GenBank, Accession No. AAR95983) No. 1) has been revealed in

Aharoni’A. et al., Proc. Natl. Acad. Sci. U.S.A. 101 (2), 482-487 (2004). The DNA sequence encoding G2E6 is shown in SEQ ID NO: 7, and the DNA of the sequence G3C9 is shown in SEQ ID NO: 9. The particularly preferred P0N1 derivative of the present invention is: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 (hereinafter referred to as ep〇Nl-G2E6) or the amino acid sequence of SEQ ID NO: 10. a polypeptide consisting of the polypeptide (hereinafter referred to as ePON1-G3C9), (b) the amino acid sequence of SEQ ID NO: 8 or 1 in which one or a plurality of amino acids are deleted, inserted, or substituted, And the polypeptide has at least one selected from the group consisting of lactonase activity, balasolase activity and aromatic esterase activity; or more preferably (c) amino acid of SEQ ID NO: 8 or 10. A polypeptide consisting of a sequence in which one or a plurality of amino acids are deleted, inserted, or substituted, and the polypeptide has an activity of scavenging active oxygen. The ep〇Ni-G2E6 and ePONl-G3C9 of the P0N1 derivative are called the human body 13 S* 322362 201116292 P0N1 derivative because they do not lose the identity with the amino acid sequence of the human P0N1. The term "the range in which the identity is not lost" is, for example, one having a sequence identity of about 70% or more, preferably about 75% or more, more preferably about 80% or more, and particularly preferably about 82% or more. The identity of the amino acid sequence can be determined by the NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) by the following conditions (expected 値 = 10; allowable interval; matrix = BLOSUM62; filtering) =OFF) to calculate. For example, if the sequence identity with human P0N1 is sought by this method, eP0Nl-G2E6 is 82% and eP0Nl-G3C9 is 85%. The polypeptide of the present invention having a sequence identity with human P0N of 70% or more, preferably having the sequence identity with human p〇N (preferably the polypeptide of human P0N1 ' is SEQ ID NO: 6). About 75% or more is more preferably about 8% or more, further about 82% or more, further about 85% or more, further about 9% or more, further about 95% or more, and particularly preferably about 98% or more. The best is about 99% or more. The polyvalent form of the active ingredient of the present invention having a sequence identity with human P0N of 7 Å/° or more is preferably, for example, at least one of the following sequences: Amino acid sequence of human P0N1 represented by SEQ ID NO: 1. (A) amino acid sequence of position 36 to =, amino acid sequence of (B) positions 347 to 353, (C) amino acid sequence of positions 219 to 248, (D) 152 to The amino group I sequence at position 165, the amino acid sequence at positions 56 to 69 of (E), and the amino acid sequence at positions 320 to 330 of (F). More preferably, those having the above (B) are more preferably those having the above (A) to (6), and more preferably having the above (A) to (9)

S 14 322362 201116292, especially for those having the above. The above sequence is a common one, and it is preferable to have the above (A) to (8) effect components. Have the above-mentioned common order goods month (4) Serial number:! 6. The M's shape is preferably the following polymorphism: above the polypeptide; (8) the sequence number of the amine, the sequence constitutes the polypeptide 'where the above (4), compared with:: upper =; = (A) to (D ), more preferably, μ, +, γ is the upper 4 (A) S (E), and the sequence other than the common sequence is 7 7 to (F)). A has one or a plurality of amine groups. An acid insertion deletion or substitution, and the polymorph has at least one of a group selected from the group consisting of endoenzyme activity, balasolase activity, and aromatic vinegar activity; and (c) a polypeptide consisting of the amino acid sequence of the sequence number 6, 8 or 1 ' wherein the above (A) and (B) are preferably the above (4) to (6), more preferably the above (A) to (9), and more preferably In the above (A) to (8), it is particularly preferred that the sequence towel other than the common sequence of the above (1) to ((9) has one or more amino acids which are deleted, inserted, or substituted, and the polypeptide has a scavenging activity of active oxygen. For example, the amino acid sequence of SEQ ID NO: 8 or 1 ,, wherein one or more amino acids in the sequence other than the common sequence of (A) to (F) above occur. Loss, insertion, or substitution, and having at least one activity selected from the group consisting of lactonase activity, balasolase activity, and aromatic esterase activity, and a' is preferably G3H8 (GenBank) , Accession No. AAR95985), GlA5 (GenBank, Accession No. AAR95981),

ePON1 such as GlC4 (GenBank' Accession No. AAR95982) and G2D6 (GenBank, Accession No. AAR95983). The polypeptides also have activity to scavenge reactive oxygen species. s 15 322362 201116292 The polypeptide of the active ingredient of the present invention is preferably a polypeptide having at least one activity selected from the group consisting of lactonase activity, balasolase activity and aromatic esterase activity. The polypeptide is preferably, for example, the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, 6, 8, or 10' and (b) an amino group of SEQ ID NO: 1, 6, 8, or 10. a polypeptide consisting of an acid sequence in which one or more amino acids are deleted, inserted, or substituted, and the polypeptide has a selected from the group consisting of lactonase activity, balasolase activity, and aromatic esterase activity. At least one activity in the group. More preferably, the polypeptide has an activity of scavenging active oxygen. The active ingredient in the present invention is also applicable to a polypeptide having activity of scavenging active oxygen and having no lactonase activity, balasolase activity and aromatic esterase activity. Preferably, the polypeptide comprises, for example, the following polypeptide: an amino acid sequence of SEQ ID NO: 6, 6, or 10 in which one or more amino acids are deleted, inserted, or substituted, and the polypeptide has It removes the activity of active oxygen and has no lactonase activity, balasolase activity and aromatic esterase activity. For example, the first position of methionine (Met) in the amino acid sequence of the sequence number 丨 can be eliminated, and the 115th position and the 134 are replaced by other amino acids (for example, glutamine amine (Gin)). A histidine acid (His) is obtained to obtain a polypeptide having activity of scavenging active oxygen and having no lactonase activity, balasolase activity and aromatic esterase activity. The term "lactamase activity" as used in the present invention means having the activity of hydrolyzing 7-thiobutyrolactone. Having lactonase activity, specifically, can be confirmed by the reaction of 16 322362 201116292 4-thiol butyric acid with 5, 5'-dithiobis(2-nitrobenzoic acid) The change in absorbance of sulfur-2-nitrobenzoic acid at 450 nm was measured as an indicator that 7-butyrolactone was hydrolyzed to form 4-thiol butyric acid. More specifically, the measurement method of the cysteine thiolactone activity measuring kit "Alfresa AutoHTLase" (Alfresa Pharma) is usually used, and when the cysteine thiolactone activity is confirmed, it is determined as Has lactone activity. The term "having barathlonase activity" in the present invention means having the activity of hydrolyzing balasolone. The balasolase activity is specifically confirmed by measuring the change in absorbance of p-nitrophenol at 405 nm which is produced by hydrolysis of balasolone as an index. When the balasolase activity was confirmed, it was judged to have a balasolase activity. The term "having an aromatic esterase activity" in the present invention means having an activity of hydrolyzing phenyl acetate. There is an aromatic esterase activity, and specifically, it can be confirmed by detecting the change in absorbance at 270 nm of phenol produced by hydrolysis of phenylacetate as an index. When the aromatic esterase activity was confirmed, it was judged to have an aromatic esterase activity. The term "having an activity of scavenging active oxygen" in the present invention means having an activity of eliminating hydrogen peroxide which is an active oxygen. The activity of scavenging reactive oxygen species can be measured according to the method of Root and Me tea 1 f s 17 322362 201116292. Root and Metcalf methods have been revealed in j Clin

Invest 60, 1266-1279 (1977). Specifically, the sample is reacted with a known amount of hydrogen peroxide and the residual hydrogen peroxide is measured. The residual amount of hydrogen peroxide can be converted into an oxidized type by the action of horseradish catalase (HRP) by reducing the amount of reduced-type halogen (sc〇P〇letin) which emits fluorescence. The change in light intensity (excitation wavelength 366 nm, emission wavelength (10)) was used as an index to detect. When the activity of scavenging active oxygen was confirmed, it was judged to be the activity of sex oxygen. f White matter content (protein content) can usually be determined by absorbance method. Having == When the absorbance of the protein solution (measurement wavelength 280 nm) is 1, = is 1 mg/ml. Therefore, the preparation is suitably treated with distilled water at a concentration of absorbance a at the measurement wavelength. Then & a multiply the dilution factor, thereby obtaining the protein of the preparation containing the purified (4) ___*^ carried out by chemical synthesis or conventionally based on the sociology (4), for example, p〇N can be obtained from various mammalian plasma, this: Intracerebral sputum, etc., is combined with a conventional protein purification method into the kidney: 蔵, the amino acid sequence of SEQ ID NO: 1 is purified. And the peptide of SEQ ID NO: 6 is suspected of ", _ _ 卩 10 10 10 】 】 】 】 】 】 8 8 8 8 8 8 8 8 8 沔 沔 沔 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 322 The P0N1 derivative can be produced, for example, according to the method disclosed in Proc. Natl. Acad. Sci. USA 101 (2), 482-487 (2004). In this document, the Escherichia coli expression system is used, but it can also be described later. The animal cells (CH〇_K 1 cells) described in the examples are produced. The animal cells used in the production of polypeptides such as hydrazine and ρ〇Ν1 derivatives of the active ingredient of the present invention are other than CH0-K1. The NS0, SP2/0 'CH0DG44, HEK293, and PER.C6 are exemplified, but are not limited thereto. Further, for example, a polypeptide consisting of SEQ ID NO: 1, 6, 8, or 1 ,, wherein one or a plurality of amines are present The deletion, insertion, or substitution of the basal acid can be carried out according to the conventional genetic engineering method according to the conventional gene sequence of p〇N or eP〇N1. The obtained polypeptide can also be further improved by the method generally used. The purification treatment may be carried out, for example, 跬api ^ Qiaoru, treatment with a modifier such as urea, a surfactant, etc., ultrasonic treatment, treatment by enzyme digestion, salting out or solvent precipitation , dialysis, centrifugal separation knife, ultrafiltration, colloid filtration, SDS-PAGE, isoelectric point electrophoresis, ion exchange, mostly octagonal analysis, hydrophobic chromatography, affinity chromatography, reverse phase The present invention is not limited thereto. <Pharmaceutical preparation> The preparation may be usually a non-oral preparation, or an oral administration agent. The active ingredient of the present invention is The protein-white matter' is preferably a non-oral administration agent which is easily absorbed. Non-oral and sputum can be exemplified by injections, inhalants, _, _, etc. Among them, injections are preferred. <Injectables> 322362 19 201116292 The injection may be an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, a drip injection, etc. The injection may be usually an aqueous solution, a suspension or the like, or a freeze-dried preparation. It is prepared by appropriately mixing a polypeptide of an active ingredient with a pharmaceutically acceptable additive (a diluent, a pH adjuster, an isotonizing agent, a surfactant, a stabilizer, etc.) by a conventional means such as a preparation. The injection can be prepared by dissolving, suspending or emulsifying a polypeptide such as an active ingredient in a sterile aqueous or oily liquid (injectable carrier) for use in a usual injection. The aqueous solution for injection may, for example, contain physiological saline. Osmotic pressure fluid such as glucose or other auxiliary drugs. A suitable dissolution aid such as an alcohol (for example, ethanol), a polyol (for example, propylene glycol, polyethylene glycol), or a nonionic surfactant (for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated) may be used in combination. Castor oil)) and so on. Examples of the oily liquid include sesame oil, soybean oil, and the like. Phenyl benzoate, benzofuranol or the like may be used in combination with a dissolution aid. The freeze-dried preparation is used by being dissolved in a solution such as distilled water for injection or physiological saline at the time of use. The amount of the active ingredient in the injection may be, for example, from about 0.1% to about 1%, preferably from about 0.1% to about 100% by weight. More preferably, it is about 1 // g/ml to 50 mg/m. <Inhalation> Examples of the inhalant include a spray, a powder for inhalation, and a liquid for inhalation (for example, a solution for inhalation, a suspension for inhalation). Agents, etc.), capsule inhalers, and the like. The liquid preparation for inhalation may also be in the form of being dissolved or suspended in water or used in the appropriate medium of S. The inhalant can be produced according to a conventional method. For example, it can be produced by preparing a polypeptide such as p(10) as an active ingredient into a dry powder or dissolving it in a liquid to form a liquid, and then blending it with an inhalation propellant, a carrier, or both, and refilling it with a suitable suction. Human container... When the polypeptide is pulverized, it may be formed into a fine powder together with lactose, starch, magnesium stearate or the like to form a homogeneous mixture' or granulated to prepare a powder. Further, when the polypeptide is in the form of a liquid, the polypeptide may be dissolved in a liquid carrier such as water, a physiological saline solution or a buffer solution. ^ Inhalation propellant can use conventional propellants, for example, instead of Freon (a 11ernat i ve f reon ), liquefied gas propellant (such as gas and gas carbide, liquefied petroleum, diethyl ether, dioxane, etc.), compressed carcass (e.g., a soluble gas (e.g., carbon dioxide, nitric oxide gas, etc.), an insoluble gas (e.g., nitrogen), etc.). Inhalants may also be formulated with additives as needed. When it is used as a liquid preparation for inhalation, examples of the additive include a preservative (chlorinated chlorinated ammonium chloride, benzoic acid ester, etc.), a coloring agent, a buffering agent (sodium acetate, sodium acetate, etc.), and the like. Wheat osmotic pressure agent (for example, sodium chloride, concentrated glycerin, etc.), tackifier (for example, bismuth polymer, etc.), absorption enhancer, and the like. Further, when it is used as a powder for inhalation, the additive may, for example, be a lubricant (for example, magnesium stearate, light phthalic anhydride, talc, sodium lauryl sulfate, etc.), or a flavoring agent (for example, citric acid, menthol, ammonium glycyrrhizinate) Salt, glycine, orange powder, etc., binding agents (such as starch, dextrin, methylcellulose, hydroxypropylcellulose, polyethylene alpha-pyrrolidone, polyethylene glycol, white sugar, etc.) ), excipients (eg white sugar, lactose, grapes 21 322362 ^ 201116292

Sugar, mannitol, mountain MA storage (such as stupid 2 alcohol, maltose, cellulose, etc.), coloring agent, chlorpyrifos, etc.), = sodium hydrogen sulfite, p-benzoic acid vinegar, pair A blanket-receiving agent (a gallbladder _ Γ 化 化 ( ( ( ( ( ( ( ( ( ( ( ( ( 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The content of the component of the active ingredient 3 is, as a liquid preparation for inhalation, a dry weight of, for example, from about 0.01/g/ml to about 50 tons; ο.1-1111 More preferably, it is about 5% by weight of the active ingredient, and when it is used as a powder for inhalation, it can be changed to 20% by weight, and the dry weight of the household A can be, for example, about 〇·〇15 wt% in the preparation. . t is from about 0.1 to U% by weight, more preferably about 〇. 5 to <adhesive agent> is listed on the A holding body with the active ingredient in the multi-month too = agent can be = 'adhesive paste _asteI ° 甲 Μ Μ + . 海 海, gelatin, corn house powder, tragacanth, methyl cellulose, ethyl fiber 飨 +, grade m P Na, 隹素瑷 methyl cellulose, Sanxian gum, Antler rubber, polymannose, Yangying wall, fine xylose extract, methyl starch, polyvinyl alcohol, sodium polyacrylate, methoxybate-shun. , Polyethylene women's scales, Polyethylene thinner, several ethylene polymers, propyl cellulose, propyl methyl cellulose, polytriglucose and other polymers; white Vaseline, yellow Vaseline, paraffin, ground wax, Microcrystalline wax and other interesting substances; colloidal tobacco (such as the trade name Plastibase, Bristol Myers 匕. and other higher fatty acids; cetyl alcohol, octyl 12 t bb A ^ / polyethylene glycol (such as MACROGOL 4000 and other hard alcohol Equivalent alcohols, decanediol, glycerol (ie 322362 22 201116292 = oil), t dipropylene glycol, 1,3-butanediol, concentrated glycerol and other polyols; Fatty acid esters such as oleic acid esters and glyceryl stearate; phosphate buffers, etc. In addition, it is also possible to add a locusing aid, an inorganic filler, a pH adjuster, a moisturizer, a preservative, a stick, an antioxidant, and a cool An additive such as a chemical agent, the content of the polypeptide of the active ingredient is converted into a dry weight of the polypeptide, and is usually about 20% by weight, preferably about 0.1 to 10% by weight, and more preferably about 0. 5 to 5% by weight. <Suppository> The agent is prepared by combining the polypeptide of the active ingredient with a commonly used suppository base. The content of the polypeptide of the active ingredient of the plug is changed into a different amount. The dry weight of the polypeptide may, for example, be generally from about 0.01 to 2 〇 heavy denier % 'preferably about 〇1 s q JH main 10 weight %, more preferably about 0.5 to 5% by weight. Agents> Only, Μ °" lists powders, granules, lozenges, capsules, emulsions, liquids, syrups, enteric solvents, etc. Among them, it is an enteric solvent. U is attributed to the above active ingredients + Formulated with pharmaceutically; j carrier or additive. For example, it can be combined with: white sugar, lactose, and glucosinolate , Dian powder, mannose _ shaped agent; gum arabic, gelatin, sputum = hydroxypropyl cellulose, methyl cellulose adhesive; slow methyl i, vegetarian, temple powder disintegrant; citric acid _, sodium, glycerin, etc.. It can also be coated with (4) glue, or coated in the gum arabic, palm Μ ^ u ^ plexus. In addition, the liquid preparation is dissolved by the active ingredient of the female Or prepared by dispersing in water, ethanol, glycerin, monosaccharide, or a mixture thereof, etc. 322362 201116292 r The content of the polypeptide of the active ingredient in the oral administration agent is converted into the dry weight of the polypeptide, for example, usually It is about 1 to 2% by weight, preferably about 0. 1 to 10% by weight, and more preferably about 5 to 5% by weight. <Usage method>#The amount of the prophylactic or therapeutic agent for inflammatory bowel disease of the present invention varies depending on the disease or the subject's symptoms, age, etc., but the polypeptide of the active ingredient is changed to dry on the 1st day. The weight is usually preferably from about Q· to 5〇〇mg/kg, more preferably from about 1Ag/kgi1〇〇mg/kg, and even more preferably from about g/kg to 50 mg/kg. /kg to i 〇mg/kg. This amount is preferably divided into 1 to 3 injections per day or continuous administration by drip. <Target> The subject to which the prophylactic or therapeutic agent for inflammatory bowel disease of the present invention is administered is an animal in which an inflammatory bowel disease has occurred or an animal in which an inflammatory bowel disease may occur. Preferably, the animal has developed an inflammatory bowel disease. The animal can be exemplified by (9) 'm rat, bovine, pig, horse and other mammals. It is preferably human. In the present invention, "prevention" includes avoiding the onset, suppressing the incidence, and suppressing the development of symptoms. In addition, "treatment" includes the treatment of sores, mitigation or improvement of symptoms. Examples of the inflammatory bowel disease in the present invention include ulcerative colitis, cloning, and the like. A preferred aspect of the therapeutic or therapeutic agent of the present invention is a prophylactic or therapeutic agent for ulcerative colitis, or a prophylactic or therapeutic agent for Crohn's disease. Containing more than 7〇% of the sequence identity with the human balasolase, and 322362 24 201116292 has an activity selected from the group consisting of enzyme activity, balasolase activity, aromatic esterase activity and scavenging reactive oxygen species. A prophylactic or therapeutic agent for ulcerative colitis which is an active ingredient of at least one active polypeptide in the group is one of preferred embodiments of the present invention. And having a sequence identity with human body balasolase of 70% or more, and having an activity selected from the group consisting of an enzyme activity, a balasolase activity, an aromatic esterase activity, and an activity of scavenging reactive oxygen species. One or more preferred embodiments of the present invention are the prophylactic or therapeutic agents for Crohn's disease in which at least one active polypeptide is used as an active ingredient. The polypeptide of the active ingredient is preferably the above-mentioned PON, PON1 derivative or the like. <Prophylactic or therapeutic agent for ulcerative colitis> The amount of the prophylactic or therapeutic agent for ulcerative colitis of the present invention varies depending on the symptoms or age of the subject, but the peptide of the active ingredient is administered. The amount converted to dry weight is usually preferably from about 0.1/ig/kg to 500 mg/kg, more preferably from about 1/zg/kg to 100 mg/kg, still more preferably from about 10 // g/kg to 50 mg/ Kg, particularly preferably from about 10 // g/kg to 10 mg/kg. This amount is preferably divided into 1 to 3 administrations or continuous administration by drip. The prophylactic or therapeutic agent for ulcerative colitis is preferably a patient suffering from ulcerative colitis. It is also suitable for intestinal occlusion, intestinal perforation, toxic colonic disease, colorectal cancer, etc., which prevent complications of ulcerative colitis. In other words, the present invention also encompasses a method for preventing or treating ulcerative colitis in which a polypeptide such as PNN is administered to a mammal (especially a human). <Prophylactic or therapeutic agent for Crohn's disease> The amount of the prophylactic or therapeutic agent for Crohn's disease of the present invention is

I 25 322362 201116292 The symptom or age of the subject varies, but the daily dose of the polypeptide of the active ingredient is preferably converted to a dry weight in an amount of about 0.1%/kg/kg to 500 mg/kg, more preferably about 1 / g / kg to 100 mg / kg, and further about 10 / g / kg to 50 mg / kg, particularly preferably about 10 # g / kg to 10 mg / kg. This amount is preferably administered in 1 to 3 divided doses or continuously administered by drip. The prophylactic or therapeutic agent for Crohn's disease is preferably administered to a patient with Crohn's disease. It is also suitable for the prevention of complication of Crohn's disease, anal fistula and anal fissure, or arthritis, iritis, gangrenous pyoderma or nodular erythema. In other words, the present invention also encompasses a method for preventing or treating Crohn's disease by administering a polypeptide such as the above PON to a mammal (especially a human). The invention also comprises the method for preventing or treating inflammatory bowel disease, having more than 70% sequence identity with the human balasolase, and having a selected from the group consisting of lactonase activity, balasolase activity, and aromatic vinegar enzyme activity. And at least one active polypeptide in the group in which the activity of the active oxygen is removed. The inflammatory bowel disease can be exemplified by ulcerative colitis or Crohn's disease. The above polypeptide, a preferred embodiment thereof, a method of using the above polypeptide, and the like are the same as those described above for the prophylactic or therapeutic agent for inflammatory bowel disease. (Examples) Hereinafter, the present invention will be more specifically described by way of Examples, but the present invention is not limited thereto. <Example 1> (Purification of human balasolone 1 from plasma) Humans who have substantially removed hepatitis virus and other pathogenic microorganisms £ 26 322362 201116292 Gold paste as a raw material 'Using human plasma to utilize Cohen's (c 〇hn) A low temperature ethanol separation method to obtain a distinction (fracti〇n) IV-1. The differential IV-1 was dissolved in a low temperature range of 2 to 8 ° C in a 20 mM phosphate buffer containing i5 mM mM sodium chloride. Then, centrifugation was carried out under the conditions of 1200 g, 4 Torr, and 10 minutes to recover the protein component of the supernatant. The supernatant was then recovered at 3 〇〇〇〇g, 4. Centrifugal separation was carried out under conditions of 50 minutes, and the solution layer of the intermediate layer (upper layer, lipid layer, lower layer: shoji) was recovered. Then, the recovered solution was added dropwise to Superdex 200 (manufactured by GE Healthcare) which had been equilibrated with 2 mM phosphate buffer (ρΗ6·8 to 7.4). Then, the solution was eluted with the same solution as the equilibrium, and the second peak measured by UV280mn was regarded as a polymer region distinction and selected. Then, the solution was added dropwise to a solution of 2% lean acid buffer (manufactured by pH 6.8), washed with the same solution as the equilibrium, and then neutralized with 2 mM mM phosphoric acid containing 0.4 M sodium chloride. The solution (pH 6 8 to 7.4) was subjected to elution. The solution was replaced with a 2 mM phosphate buffer (pH 6 8 to 7) using a centrifugal ultrafiltration membrane (manufactured by MiUp〇re Co., Ltd.) having a molecular weight cut off (M〇lecular_weight cut ff). 4), and get the concentrate of the ship. The obtained concentrated droplets were added to the unadsorbed fraction of poros 50 HS (Applied Biosystems) which had been equilibrated with 20 mM phosphate buffer (ρΗ6.8). The unadsorbed fraction was added to Gushui acid buffer (10).8 to 7.4. Balanced CHT_Greystone Taosui Co., Ltd.) in the same solution as the equilibrium / net 彳, and then containing 0.5M sodium hydride 20 mM phosphate buffer ( pH6 8 to 74)

27 S - 322362 201116292 • Wash with 0.1 M phosphate buffer (pH 6.8 to 7.4), and then elute with 0.4 μL phosphate buffer (ρΗ6·8 to 7.4). Next, the guanidine solution was replaced with 20 mM phosphate buffer (pH 6.8 to 7.4) using a centrifugal ultrafiltration membrane (Mi 1 lpore potential) having a molecular weight cut off of 10,000, and lml was selected.

Concentrated solution. Then make HiTrap NHS-activated HP

Manufactured with a human P0N1 antibody (manufactured by Cosmo Bi〇 Co., Ltd.) at a concentration of 200 /zg to prepare a P0N1 antibody column, and the concentrated solution contained therein was added dropwise to a 20 mM phosphate buffer (pH 6.8 to 7 4). The P0N1 antibody column was then washed with the same solution as the equilibrium, and then eluted with 2M glycine acid (pH 3.0) to prepare a solution containing p〇N1. The solution is then rapidly neutralized to pH 6.8 to 7.4 by using 5 M Tris citrate buffer (pH 9.5 to 10.5), and then centrifuged ultrafiltration and membrane with a molecular weight cut off of 10,000 (Millp〇re) A 20 mM phosphate buffer containing 150 mM sodium hydride was injected and the solution was replaced by ultrafiltration. Then, a sterile filtration membrane (manufactured by Milipore Co., Ltd.) having a pore size of 0.22 #m was used for sterile filtration to obtain a preparation of balasolone 1 (pH: 7.3, 〇D (wavelength: 280 nm): 0.3, liquid amount: 200 // 1). <Example 2> (Expression vector for constructing recombinant human body balasolone 1) The human balazonase 1 gene was obtained by the method comprising: a human balasolase 1 gene mutant The pCj^-TOPO vector (described in OPEN BIOSYSTEMS (Clone ID 30915169)) was transformed into the normal human balasolone 1 gene by mutation-induced PCR. Then, by using the base sequence of the ttgctagcccccgaccatggcgaagctg (sequence s 28 322362 201116292 number 2), the base sequence consisting of the front primer (f 〇i*ward pr i mer) and ttgcggccgctcattagtggtggtggtggtggtgcttttcgaactgcggg tggctccagagctcacagtaaagagc (sequence number 3) The PCR method of the reverse primer was obtained by adding a Nhe I restriction enzyme site to the 3' end of the human balazonase 1 gene, and a 6xHis Tag sequence to the 5' end side, Not I. Restriction of the cleavage point, and a fragment of the termination code. The fragment was cleaved with the restriction enzyme Nhe I and the restriction enzyme Not I, and then ligated to the expression vector pcDNA3.1-(+) by the T4 DNA ligase, which was also cleaved by the restriction enzyme Nhe I and the restriction enzyme Not I. -dhfr, and constructed a gene expression vector for the recombinant human balazonase 1. The gene expression vector pcDNA3·1-(+)-dhfr was inserted into the pcDNA3.Bu (+) vector (manufactured by Invitrogen Co., Ltd.) into a expression vector of mouse dhfr expression casette. The mouse dhfr gene expression IE was selected by PCR using pSV2-dhfr (ATCC 37146) as a template. At this time, the front primer was cccacgtgtcgcgacaattagtcagcaaccatagtcc (SEQ ID NO: 4), and the inverted primer was cccacgtgtcgcgaggacaaaccacaactagaatgc (SEQ ID NO: 5). The obtained PCR fragment was ligated into the pCR-2. 1 vector (manufactured by Invitrogen) by TA colony to prepare a vector pCR-2·1-dhfr to which the dhfr gene to which Nru I was added was produced. Then, the obtained vector pcDNA3.1-(+) and the vector pcr-2. dhfr were cleaved with restriction enzyme Nru I and the individual fragments were ligated to prepare an expression vector s 29 322362 201116292 pcDNA3.l-dhfr 〇 The present expression vector has an anti-neomycin gene expression as a selective drug marker (mar ker) and a dhfr gene expression with an augmented gene. For the selection of anti-new gene genes} The use of dhfr gene is used in the preparation of biopharmaceuticals, such as 舀6-like 1: 丨 (^11), 〇418, neomycin sulfate, etc. The agent for increasing the gene is generally used with metorexate ° <Example 3> (Expression using 293F cells) The recombinant human body balasolone 1 produced by Example 2 is used for the expression vector utilization. The FreeStyle 293 Expression System (Invitrogen) was temporarily performed according to the manual attached to the same kit to prepare a culture supernatant containing the recombinant human body balasolone 1. In addition, the culture supernatant was The manual attached to the FreeStyle 293 Expression System was prepared by mixing and expressing the expression strain with the culture medium. <Example 4> (Bio-recombinant human balasolone 1 expression strain (293F cells) Culture supernatant preparation Preparation of human balasolone 1) The 293F cell culture supernatant which expresses and secretes human balasolase 1 is equivalent to 20 mM phosphate buffer (pH 6.6 to 7.0) containing 1 M sodium chloride. Mix and stir The precipitate was removed by a 0.45/m filter (manufactured by Millpore Co., Ltd.), and the filtrate was applied to a His trap HP (Presidented with 20 mM phosphate buffer (ρΗ6·8 to 7.4) containing 0.5 M gasified sodium. GE Healthcare s 322362 30 201116292, manufactured by the company, followed by washing with the same solution as the equilibration, followed by 20 mM phosphate buffer (pH 7. 〇 to 7.5) containing 〇·5M sodium chloride and 25 mM imidazole. Washed. Finally, the solution was separated by His trap HP adsorption using a buffer containing 2 〇·5M sodium chloride and 5 〇〇龅 imidazole (pH 7. 0 to 7.5). Next, the His trap HP The adsorption-separated eluent was concentrated using a centrifugal excess molecular weight cut-off of 10,000 (manufactured by Mi iip〇re), and then added to Superdex 200 16/60 which had been equilibrated with 20 mM acid-filled buffer containing 150 mM sodium chloride. (manufactured by GE Healthcare), and then eluted with the same solution as the equilibrium to prepare a 6 mg/ml solution of the balasolase 1. Then, a sterile filtration membrane having a pore size of 0.22 (manufactured by Millpore Co., Ltd.) was used. Sterile filtration is carried out to obtain a balasolone 1 preparation. The analysis results of the balasolase 1 preparation prepared in Example 4 are shown in Table 1. Among them, the protein content of the balasolase 1 was determined by the absorbance method. The aromatic enzyme activity was performed using ARYLESTERASE/PARAOXONASE ASSAY. KIT (manufactured by ZeptoMetrix Co., Ltd.) was measured. The pH system was measured in accordance with the Pharmacopoeia of the Pharmacopoeia, and the endotoxin content was measured using Endospecy (manufactured by Biochemical Industries, Ltd.). The morphology was confirmed visually.

S 31 322362 201116292 Table 1 Analytical results of the balasolone 1 preparation derived from 293F cells Bararaosin 1 protein content (mg/ml) 0.6 Aromatic acetase activity (U/L) 43800 PH 7. 2 Toxin content (EU/ml) 2. 2 Form almost colorless and transparent The preparation prepared above can be used directly as an injection. &lt;Example 5&gt; (Construction of a stable expression strain using CH0/DG44 cells) The recombinant human Balazonase obtained by the method of Example 2 was electroporated (Gene Pulsern; manufactured by Bi〇-Rad). 1 Expression vector was introduced into CH0/DG44 cells. One day after the gene transfer, culture was carried out in a selection medium containing G418 at a concentration of 1 mg/ml to obtain a recombinant human Balazonase 1 stable expression strain into which the expression vector was introduced. Then, the medium was gradually cultured by using the medium in which the concentration of the cancer inhibitor was gradually increased, and the genetically amplified strain DG44/P0M-His CL45 was obtained. Specifically, the concentration is increased in the order of the medium containing 5 〇 nm of the cancer-killing medium, the medium containing the 250 nm cancer-killing medium, and the medium containing 1, 〇〇〇nm cancer-killing spirit, and finally selected by the limiting dilution method. Colonization. The culture supernatant containing the recombinant human body balasolone was prepared by mixing and mixing j) G44/P0N1-His CL45 and EX-CELL302 medium (manufactured by SAFC) in a 2 L culture tank or a 15 L culture tank. <Example 6 &gt; £ 32 322362 201116292 (The culture supernatant of human body balasolase 1 expression strain (CH〇/DG44 cells) prepared by genetic recombination prepares a preparation containing human balasolase 1) The CH〇/DG44 cell culture supernatant secreting human body balasolase 1 was subjected to removal of the precipitate by a 0.45/m filter (manufactured by Millpore Co., Ltd.). The filtrate was added to Ni Sepharose HP (manufactured by GE Healthcare), which was previously equilibrated with 2 M mM phosphate buffer (pH 6.8 to 7.4) containing 5 M sodium chloride. Then, the solution was washed with the same solution as the equilibration, and further washed with a 20 mM phosphate buffer (pH 7.0 to 7.5) containing 0.5 M sodium chloride and 35 mM imidazole. Finally, it was eluted with 2 mM sulphate buffer (pH 7.0 to 7.5) containing 5 M sodium sulfide and 500 mM imidazole. The lysate which adsorbed Ni Sepharose HP adsorption was replaced with 2 mM phosphate buffer (pH 6.8 to 7.4) using a centrifugal ultrafiltration membrane (manufactured by Milip® Re) having a molecular weight cut off of 10,000, and then added dropwise CHN hydroxyapatite ceramic Typen 4〇em (manufactured by Bio-Rad Co., Ltd.) equilibrated with 2 mM phosphate buffer. Then, using a centrifugal ultrafiltration membrane (manufactured by Millpore Co., Ltd.) having a molecular weight cut off of 10,000, the unadsorbed fraction and the elution of 2 mM mM phosphate buffering 117. 〇 to 7.5 containing 1 M of sodium chloride were replaced by 2 〇 111. The acid buffer (0116.8 to 7.4) was then added dropwise to Poros 50 HS (manufactured by Applied Biosystems) which had been equilibrated with 2% phosphate buffer (pH 6.8 to 7.4). Then, the non-adsorbed separation was concentrated using a centrifugal ultrafiltration membrane (manufactured by Millpore Co., Ltd.) having a molecular weight cut off of 10,000, and then added dropwise to Superdex 200 16/60 (GE) which had been equilibrated with 20 mM acid buffer containing 150 mM of fluorided steel. In the company of Healthcare Co., Ltd.). Then, it was eluted with the same solution as the equilibration, and the solution was concentrated to a concentration of 3.9 mg/ml using a centrifugal type 33 322362 201116292 ultrafiltration membrane (manufactured by Millpore Co., Ltd.) having a molecular weight cut off of 10,000. Subsequently, sterile filtration was carried out using a sterile filtration membrane (manufactured by Mi 1 lpore Co., Ltd.) having a pore size of 0.22 / zm to obtain a preparation of balasolase 1. The analysis results of the balasolone 1 preparation prepared in Example 6 are shown in Table 2. Among them, the protein content of the balasolase 1 was determined by an absorbance method. The enzyme activity of the cysteine thiosinol was measured using A1 f resa Auto HTLase (manufactured by Alfresa Pharma Co., Ltd.), and the acetaminophenase activity was measured using ARYLESTERASE/PARAOXONASE ASSAY KIT (manufactured by ZeptoMetrix Co., Ltd.). The pH system was measured in accordance with the Japanese Pharmacopoeia, and the endotoxin content was measured using Endospecy (manufactured by Biochemical Industries, Ltd.). The morphology was confirmed visually. Analytical results of the balasolone 1 preparation derived from CH0/DG44 cells. Barrasonase 1 protein content (mg/ml) 4. 0 Aromatic S enzyme activity (U/L) 915000 liters of cysteine sulfur Endoesterase activity (IU/L) 4375 pH 7. 3 Endotoxin content (EU/ml) 0. 32 Form almost colorless and transparent The preparation prepared above can be used directly as an injection. <Example 7&gt; (Construction of human balazonase 1 expressing cells) (Constructing human P0N1 expressing cells)

S 34 322362 201116292 The cDNA of human P0N1 uses the sequence disclosed by NCBI GenBank Accession Number BC074719. The pDM of the human p〇N1 was inserted into the expression vector pΕΕ12·4 of the animal cell regulated by the hCMV-MIE promoter, and the pEE12. 4-hPON1 of the human PON1 (hPONl) expression vector was constructed. The ρΕΕ12·4-hP〇N1 was introduced into CHO-Π cells by electroporation, and selectively cultured with sulfonimide (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "human p〇N1 expressing cell") was used in the following examples. <Example 8> (Purification of human balasolase 1) Human P0N1 expressing cells were cultured in CD-CH0 medium (manufactured by I nv i trogen) as an initial medium to produce human balasolone 1 . The culture conditions were pH 7.1, 37 Ϊ, and CHO CD EfficientFeed B (Invitrogen) 40 ml/L/day was added between the 5th and the 9th day of culture. The culture supernatant was recovered by culturing the first 〇曰, and it was filtered with a filter of 〇45/m, and then used for the following purification. The solution which has been filtered by the filter of 0.54/zm is added dropwise to the strong anion exchange column (10) Q Sphere Q; BiG-Rad UbQra steals the company's product. The sodium and lmM calcium chloride 25 mM THs hydrochloric acid solution (pH 7.5) was washed, and then eluted with a 25 mM Tris hydrochloric acid solution (pH 7.5) containing M. 25 M sodium chloride and hydrazine. The solution was added dropwise to a hydroxyapatite column (CHT hydroxyapatite ceramic type Type j; manufactured by Bi〇_Rad Lab〇ratories), and washed with 25 mM THs hydrochloric acid solution (pH 7 5), and then 0. 1Μ phosphate buffer (pH 7.5) containing human p〇N1

S 322362 35 201116292 'The distinction is made for dissolution. Subsequently, the solution was ultrafiltered using a 3 〇 ultrafiltration membrane, and the solvent was replaced with a 20 mM Tris hydrochloric acid solution (pH 7.2) containing lmM chloride. This solution was added dropwise to a strong cation exchange column (UN〇 Sphere S; manufactured by Bio-Rad Laboratories Co., Ltd.) to recover the unabsorbed fraction. The solution was subjected to a 30 K ultrafiltration membrane to remove the sputum, and the solvent was replaced with a solution containing 0.15 M sodium chloride and 1 mM chlorinated 25 mM Tris hydrochloric acid (pH 7.5). The solution was then subjected to sterile filtration treatment on a 0.22/zm filter to obtain a balasolone 1 preparation. The analysis results of the balasolone 1 preparation prepared in Example 8 are shown in Table 3. Among them, the content of the balasolase 1 protein was determined by the absorbance method. The cysteine thiolactone activity was measured using A1 fresa Auto HTLase (Alfresa Pharma), and the balasolase activity was measured in accordance with Journal of Lipid Research Vol. 41, 2000 P1358-1363. The pH system was measured in accordance with the Japanese Pharmacopoeia, and the endotoxin content was measured using Endospecy (manufactured by Seikagaku Kogyo Co., Ltd.). The form (appearance) is visually confirmed. Table 3 Analytical results of the balasolase 1 preparation Barrasonase 1 protein content (mg/ml) 10 liters of cysteine thiolactone activity (IU/L) 29220 Balain-free enzyme activity ( U/L) 5200000 PH 7. 5 Endotoxin content (EU/ml) 0. 29 Form almost colorless and transparent

S 36 322362 201116292 can be used directly as an injection core preparation (purification result), Examples 1, 4. Non-reducing or suturing each of the 18 enzymes for electrophoresis, and 2 rsDS_polyacrylamide 2 to 2 _ gel (5) 〇 Λ 仃 仃 silver staining or CBB staining. The results are shown in the first Α:=D map. The figure is a graph showing the results of silver staining of the prepared balasolone: a coating agent (non-reducing sample). Fig. 1 is a view showing the results of CBB dyeing of the balasolase 1 preparation (reduced sample) of Example 4 in the table. Fig. 1 is a diagram showing the results of (10) staining of the balasolase (formula) prepared in Example 6. Fig. 1D is a diagram showing the actual consumption of ΓΓ拉奥松酶1 preparation (non-reduced sample): fruit. The arrow in Fig. 1 indicates the Bainson's enzyme. In addition, in the first to D pictures, the band on the left is the molecular weight marker. In addition, each of the balasolase i obtained in the examples i, 4, 6 and 8 was subjected to SDS_PAGE, and the protein on the colloid was used as a film, and then a small oxygen resistance was used. The human body balasolone 1 antibody (P0N-5D-1, manufactured by BML) was subjected to a reaction. The HRP-labeled anti-mouse immunoglobulin antibody (10) I system was used to carry out the second reaction, and the color was developed by the method. The results are the same as follows: The figure is a diagram of the Western blot method of the Baci Oxonase 1 preparation (non-original sample) prepared in Example 1. Figure 2B" (4) 4 _ = The diagram of the western ink dot method of the Barra Oslozyme 1 preparation (reduced sample) is the Bala (4) 1 彳 (还麟样) West 322362 37 201116292 The map of the dot method. Fig. 2D is a diagram of the Western blot method of the balasolase 1 preparation (non-reduced sample) prepared in Example 8. As shown in Fig. 1 and Fig. 2, it is presumed that the band of Baraxonase 1 was purified' and the band was stained by Western blotting using the above mouse anti-human balasolase 1 antibody. .

V Baraoxasin 1 which is an active ingredient of the balasolase 1 preparation prepared in Examples 1, 4, 6 and 8 is the thiol acid of the first position in the amino acid sequence of SEQ ID NO: 1. The eliminated sequence is the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. <Example 10 &gt; (Confirmation of the effect of prevention or treatment of ulcerative colitis 1) Eighteen 8-week-old male C57BL/6J mice (manufactured by Nippon SLC Co., Ltd.) were prepared for the experiment. Of these, 15 were administered a 2% dextrin sulfate (DSS) solution in drinking water from DayO to 7 in order to establish an ulcerative colitis mode. The recombinant human body balasolone 1 ' prepared by the method of Example 8 was administered intravenously in a single dose of 1 mg/kg (P0N1 lmg/kg administration group; n = 5) at DayO to 7. For other groups, the main drug of the commercially available ulcerative colitis drug, 5-aminosalic acid (5-ASA; SIGMA), was used at a dose of 100 mg/kg (5-ASA 100 mg/kg; n=5). From DayO to 7 for a single oral injection. Only 5 mice administered with 2% DSS solution were used as a control group. Further, 5 mice which were not administered with 2% DSS solution and P0N1 preparation (or 5-ASA preparation) were used as the normal group. In other words, the normal group was a non-administered mouse population of 2% DSS solution, P0N1 preparation, and 5-ASA preparation. 38 322362 201116292 The disease activity index score (DAI score; Disease activity index score) was measured on DayO and Days 3 to 8 once a day. The DAI score consists of the following three items: weight loss (0 points: less than 1%; 1 point: less than 1 to 5%; 2 points: less than 5 to 10%; 3 points: less than 10% to 20% % ; 4 points: 20% or more), fecal morphology (0 points: no abnormalities; 2 points: soft stool; 4 points: lower jaw), fecal bleeding (0 points: no abnormalities; 2 points: occult blood; 4 points: bleeding), Evaluation by 12 points. After the start of the animal experiment, Day 8 was sacrificed by inhaling the excess oxidative anesthesia, and the large intestine tissue was taken. Then, the large intestine tissue was cut longitudinally, and the contents were washed in physiological saline solution, and the mucosa was observed under a microscope, and the Macroscopic score was measured (0 points: no abnormality; 1 point: hyperemia, mucosal hypertrophy; 2 points: hyperemia) Mucosal hypertrophy, hemorrhage; 3 points: 1 ulcer or inflammation; 4 points: 2 ulcers or inflammation; 5 points: 2cm or more ulcers or inflammation). Each score system calculates the mean ± standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, the Dunnett test was performed on the effect of the P0N1 administration group or the 5-ASA administration group (SAS Preclinical Package Version 5.00.010720, Windows (registered trademark)). Edition SAS System Release 8.02TS level 02M0 (SAS Institute Japan)). The risk rate is less than 5% (*) and the hazard rate is lower than the judgment. The results are shown in Figure 3 and Table 4. In Fig. 3, the white point is the normal group; the black point is the control group; the triangle is the P0N1 lmg/kg administration group; and the square is the 5-ASA 100 mg/kg administration group.

S 39 322362 201116292 Table 4

Macroscopic score

Macroscopic score Normal group 0. 0+0. 0 Control group: ulcerative colitis group 2. 5+0. 9 P0N1 lmg/kg Administration group 0.4±0.2* 5-ASA 100mg/kg Administration group 1. 6+ 0. 6 As shown in Figure 3 and Table 4, it was confirmed that the DAI score and the Macroscopic score were increased in the control group compared with the normal group. However, inflammation or ulceration of the large intestine confirmed in the control group, or symptoms such as weight loss, squat or bleeding were significantly inhibited in the P0N1 administration group. From this experiment, the prevention and treatment effects of ulcerative colitis caused by the P0N1 preparation were confirmed. In addition, no side effects generally thought to be caused by administration of the PON 1 preparation were found in the P0N1 formulation administration group. <Example 11 &gt; (Confirmation of the effect of prevention or treatment of ulcerative colitis 2) 24 male C57BL/6J mice (manufactured by SLC, Japan) of 8 weeks old were prepared for the experiment. Of these, 18 were administered a 2% dextrin sulfate (DSS) solution in drinking water from Day 0 to 5 in order to establish an ulcerative colitis mode. Using the genetically modified human balazonase 1 prepared in Example 8,

The dose was 0.01 mg/kg (PONl 0·01 mg/kg administration group; n=6) for intravenous administration in a single dose from DayO to 5. For other groups, the commercially available ulcerative colitis drug, 5-aminosalic acid (5-ASA; SIGMA), was administered at a dose of 100 mg/kg (5-ASA 100 mg/kg; n=6). A single oral investment in DayO to 5 for consecutive days. The mice that were only administered with 2% DSS solution 6 40 s 201116292 were only used as a control group. Further, 6 mice which were not administered with 2% DSS solution and P0N1 preparation (or 5._ASA preparation) were used as the normal group. In other words, the normal group was a non-administered mouse population of 2% DSS solution, P0N1 preparation, and 5-ASA preparation. The disease activity index score (DAI score; Disease activity index score) was measured on DayO, Dayyl, Day2, Day4, and Day5 once a day. The evaluation items and benchmarks of the DAI score are the same as in the tenth embodiment. Day 5 after the start of the animal experiment was sacrificed by inhaling the animal with an excess of oxidative anesthesia. Each score system calculates the average soil standard deviation. Regarding the statistical analysis of the DAI score, the Dunnett assay was performed in the same manner as in Example 1 相对 in terms of the effect of the P0N1 administration group or the 5-ASA administration group with respect to the control group. A risk rate of less than 5% (*) was judged to be significantly different. The results are shown in Figure 4. In Fig. 4, the white point is the normal group; the black point is the control group; the triangle is the P0N1 〇·〇lrog/kg administration group; and the square is the 5-ASA 100 mg/kg administration group. As shown in Fig. 4, it was confirmed that the DAI score of the control group was increased as compared with the normal group. However, the inflammation or ulceration of the large intestine confirmed in the control group, or the symptoms such as weight loss, squat or bleeding were significantly inhibited in the P0N1 administration group. From this experiment, the prevention and treatment effects of Crohn's disease caused by the P0N1 preparation were confirmed. Further, no side effects due to administration of the P0N1 preparation were found in the 'PON1 preparation administration group.

S 41 322362 201116292 <Example 12 &gt; (Confirmation of the effect of prevention or treatment of Crohn's disease 1) Eighteen eight-week male Balb/c sputum (manufactured by SLC, Japan) were prepared for the experiment. 21 of them were used for the establishment of the Crohn's disease model, such as the use of 2'4'6-dinitrobenzoic acid (Dingxian 5) solution and intrarectal administration in the amount of 2. 〇 / mouse. The recombinant human body balasolone enzyme prepared in Example 8 was administered intravenously at a dose of 1 mg/kg (P〇Nl img/kg administration group; n=7) at j)ay〇 to 2 Sub-investment. Use other commercially available clones for other groups.

The main drug of the drug, 5-ASA (5-aminosalic acid; SIGMA), was administered in a daily dose of DayO to 2 in an amount of i〇〇mg/kg (5-ASA 100 mg/kg administration group; n=7). Oral administration. Only 7 mice to which TNBS solution was administered were used as a control group. Further, 7 mice which were not administered with the TNBS solution and the P0N1 preparation (or the 5-ASA preparation) were used as the normal group. In other words, the normal group was not administered to the TNBS solution, the P0N1 preparation, and the 5-ASA preparation. The disease activity index score (DAI score; Disease activity index score) was measured on Day 0 to 3 once a day. The DAI score evaluation item and the reference system were the same as those in Example 1. After the start of the animal experiment, on Day 3, the animals were sacrificed by inhaling an excess of carbon dioxide anesthesia, and the large intestine tissue was taken. The Macroscopic score was then determined in the same manner and in the same manner as in Example 10. Each score system calculates the average soil standard deviation. The statistical analysis of the DAI score and the Macroscopic score was performed in the same manner as in Example 10 with respect to the effect of the control group 'PON1 administration group or 5-ASA administration group s 42 322362 201116292. The risk rate is below 5% (*) and the hazard rate is below the judgement as a significant difference. The results are shown in Figure 5 and Table 5. In Fig. 5, the white point is the normal group; the black point is the control group; the triangle is the P0N1 lmg/kg administration group; and the square is the 5-ASA 100 mg/kg administration group. table 5

Macroscopic score

Macroscopic score Normal group 0. 0±0. 0 Control group: Crohn's disease group 3·3±1·2 P0N1 lmg/kg Administration group 2. 0+0. 4 5-ASA 100mg/kg Administration group 2. 3±0. 7 As shown in Fig. 5 and Table 5, it was confirmed that the DAI score and the Macroscopic score of the control group were increased compared with the normal group. However, the inflammation or ulcer of the large intestine confirmed in the control group. Symptoms such as weight loss, lower pain, or bleeding were significantly inhibited in the PON 1 administration group. From this experiment, the prevention and treatment effects of Crohn's disease caused by the PON 1 preparation were confirmed. Further, no side effects due to administration of the P0N1 preparation were found in the 'PON1 preparation administration group. <Example 13 &gt; (Confirmation of the effect of prevention or treatment of Crohn's disease 2) 42 male C57BL/6J mice (manufactured by Nippon SLC Co., Ltd.) of 8 weeks old were prepared for the experiment. Of these, 36 were used for the establishment of the Crohn's disease model, and TNBS solution was used as in 7 并 and administered intrarectally in an amount of 2·Oing/mouse. 43 s 322362 201116292 The recombinant human body balasolone i prepared by the method of Example 8 was administered at 0.01 mg/kg (PONl 0-01 mg/kg administration group; n=6), 0.1 mg/kg (P0N1). 0. lmg/kg administration group; n=6), lmg/kg (P0N1 lmg/kg administration group; n=6), 10 mg/kg (P〇Nl lOmg/kg administration group; n=6) Various doses were administered intravenously in DayO to 3 for a single administration. For other groups, the main drug 5-ASA (5-aminosalic acid; SIGMA), which is a commercially available Crohn's disease drug, was used at a dose of 100 mg/kg (5-ASA 100 mg/kg administration group; n=6). DayO to 3 for a single oral investment. Six mice that were only administered TNBS solution were used as a control group. Further, 6 mice which were not administered with the TNBS solution and the P0N1 preparation (or the 5-ASA preparation) were used as the normal group. In other words, the normal group was not administered to the TNBS solution, the P0N1 preparation, and the 5-ASA preparation. Determination of disease activity indicators from DayO to 4 on a daily basis

DAI score 'Disease activity index score. The evaluation items and benchmarks of the DAI score are the same as in the tenth embodiment. After the start of the animal experiment, Day 4 was sacrificed by anesthetizing the animal by inhaling excess carbon dioxide, and the large intestine tissue was taken. Then, the Macroscopic score was measured in the same manner and in the same manner as in Example 1 . Each score system calculates the average soil standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, the Dunnett assay was performed in the same manner as in Example 1 with respect to the control group, the effect of the P0N1 administration group or the 5-ASA administration group. The risk rate is below 5% (*) and the hazard rate is below the judgement as a significant difference. The results are shown in Figure 6 and Table 6. In Figure 6, the white point is the normal group;

S 322362 201116292 Black spot is the control group; black square (♦) is P0N1 0. 01mg/kg administration group; white square (◊) is P0N1 0. lmg/kg administration group; black triangle is P0N.1 lmg/kg The administration group; the white triangle was the P0N1 10 mg/kg administration group; the tetragonal (_) was the 5-ASA 100 mg/kg administration group. Table 6

Macroscopic score Normal group 0. 0+0. 0 Control group: Crohn's disease group 3. 8±0. 3 PUN 1 〇. 〇1 mg/kg Administration group 2. 8±0. 3 lmg/kg Administration group 1. 8±0. 3** P0M1 lmg/kg Administration group 0. 7±0. 2** P0N1 lOmg/kg is administered as group 0. 5±0. 2** 5-ASA l〇〇mg/kg The administration group 1. 2±0· 4** As shown in Fig. 6 and Table 6, it was confirmed that the DAI score and the Macroscopic score were increased in the control group compared with the normal group. However, the inflammation or ulcer of the large intestine confirmed in the control group, or the symptoms such as weight loss, lower pain, or bleeding were significantly inhibited in the respective p〇N1 administration groups with the amount. From this experiment, the prevention and treatment effects of Crohn's disease caused by the p〇N1 preparation were confirmed. Further, in the p〇N1 preparation administration group, no side effects which are generally considered to be caused by the administration of the cation preparation were not found. <Example 14> (Confirmation 1 of the preventive effect against Crohn's disease) 25 male C57BL/6 mice (manufactured by Nippon SLC Co., Ltd.) of 8 weeks old were prepared for the experiment. 】Recombinant human balasolone 45 322362 201116292 1' with the gene prepared by Example 8 at 1 mg/kg (P0N1 lmg/kg administration group; n=5), or 1 〇mg/kg (p〇 The N1 lOmg/kg administration group; n=5) was administered intravenously in a single dose of 5 rats per group. For other groups, the main drug of the commercially available Crohn's disease drug is 5-85-(5-aminosalic acid; 51〇1^), to 1〇〇1112/1^(5_八^1〇〇1112) /1^ Investing in the group; n=5) The amount used in Day 〇 to 3 for a single oral injection. The P0N1 lmg/kg administration group, the p〇N1 1〇mg/kg administration group, and the 5-ASA 100 mg/kg administration group were administered with TNBS solution and administered with p〇N1 or 5_ASA for 3 minutes. A single dose in the rectum was administered in an amount of 〇〇mg/kg. Only 5 mice administered TNBS solution were used as a control group. Further, 5 mice which were not administered with the TNBS solution, the PON1 preparation, and the 5_ASA preparation were used as the normal group. Determination of disease activity indicators from DayO to 4 on a daily basis

DAI score 'Disease activity index score 〇 The evaluation item and benchmark of the DAI score are the same as those in the first embodiment. After the start of the animal experiment, Day 4, after the animal was sacrificed by inhaling excess manganese dioxide for anti-anesthesia, the large intestine tissue was taken. Then, the Macroscopic score was measured in the same manner and in the same manner as in Example 1 . Each score system calculates the average soil standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, the Dunnett assay was performed in the same manner as in Example 1 in terms of the effect of the P0N1 administration group or the 5-ASA administration group with respect to the control group. A risk ratio of less than 5% (*) was judged to be significantly different. The results are shown in Fig. 7 and Fig. 8 (Fig. 7 and Fig. 8, *: 322362 46 201116292 ρ &lt; 0· 05). In Fig. 7, 'white point is normal group; black point is control group; triangle is 5-ASA l〇〇mg/kg administration group; quadrilateral (_) is Ρ0Ν1 lmg/kg combination group; square shape (♦) is PON 1 1 〇mg/kg administration group. The number of deaths in each group is as follows. • Day 2 ··1 control group, 5 ASA group 1 • Day 4 : 5-ASA group 1 As shown in Fig. 7 and Fig. 8, it can be confirmed that the control group has DAI compared with the normal group. Score and Macroscopic score have increased. However, symptoms such as inflammation, ulceration, body weight loss, diarrhea, and hemorrhage of the large intestine confirmed in the control group were significantly inhibited in the P〇N丨l〇mg/kg administration group. From this experiment, the preventive effect of Crohn's disease caused by the p0N1 preparation was confirmed. In addition, the side effects of the PON 1 preparation were generally not found in the PON 1 formulation administration group. <Example 15 &gt; (Confirmation of the therapeutic effect of Crohn's disease) 1 Eighteen male C57BL/6 mice (manufactured by SLC Co., Ltd.) of 8 weeks old were prepared for the experiment. Of these, 15 were used in DayO to establish a Crohn's disease model and a single intrarectal administration was performed in a dose of 10 mg/kg in TNBS. After 4 hours of administration of TNBS solution (slight symptoms), the recombinant human body balasolone prepared in Example 8 was used at a dose of 10 mg/kg (P〇Ni 4 hours after a single administration; n= 5) The amount is administered in a single intravenous dose. After 24 hours of administration of the TNBS solution to other groups (severe symptoms), the recombinant human body balasolase 1 prepared in Example 8 was administered at 10 mg/kg (P〇Nl was administered to the group 24 hours later; n =5) The amount was administered intravenously on Dayyl 47 322362 201116292 to 3 for several days. Only 5 mice administered TNBS solution were used as a control group. Further, 5 mice which were not administered with the TNBS solution and the P0N1 preparation were used as the normal group. Determination of disease activity indicators from DayO to 4 on a daily basis

DAI score; Disease activity index score. The evaluation items and benchmarks of the DAI score are the same as those in the first embodiment. After the start of the animal experiment, Day 4, after sacrificed by inhaling an excess of carbon dioxide anesthesia, took the large intestine tissue. The Macroscopic score was then determined in the same manner and on the basis of Example 1A. Each score system calculates the average soil standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, the effect of the single-dose group after 4 hours of P0N1 and the daily dose of the group after the poNl 24 hours was compared in the same manner as in Example 1 with respect to the control group. Dunnett check. A risk rate of less than 5% (*) was judged to be significantly different. The results are shown in Fig. 9 and Fig. 1 (Fig. 9 and Fig. 1 and *: P &lt; 0.05). In Fig. 9, the white point is the normal group; the black point is the control group; the triangle shape is P0N1, and the single administration group is 4 hours later; the square shape is p〇N1 24 hours after the sputum administration group. Among them, the data of Dayl of the group that was administered to the group after 24 hours of P0N1 was about to be submitted before the administration of P0N1. The number of death individuals in each group is as follows. • Day 2: 2 in the control group • Day 3 : P0N1 After 24 hours, the group was only administered for 48 322362 201116292 • Day 4 : 1 in the control group, P0N1, 4 in a single dose after 4 hours, P0N1, 24 hours later Lianyu injection group 1 only showed that the DAI score and Macroscopic score increased compared with the normal group as shown in Fig. 9 and Fig. 10; However, when the symptoms such as inflammation, ulceration, body weight loss, diarrhea, and hemorrhage of the large intestine are mild compared with the control group (after 4 hours of administration of Tnbs solution), the above symptoms can be administered by p0N1 alone. Significant inhibition was achieved (P0N1 was administered to the group 4 hours later). Further, even if the above symptoms were severe at 1 (after 24 hours of administration of the TNBS solution), it was confirmed that the symptoms were gradually improved by the daily administration of P0N1 (the day after the ρ〇Νι 24 hours). From this experiment, the therapeutic effect of Crohn's disease caused by the P0N1 preparation was confirmed. Further, no side effects due to the administration of the P0N1 preparation were found in the 'PON1 preparation administration group. <Example 16> An amino acid sequence of human balazonase 1 was produced (in the sequence number ,, the first position of methionine (Met) was eliminated, and the 284th cysteine (Cys) A polypeptide consisting of an amino acid sequence substituted with alanine (Ala) (C284A variant). (Constructing human body balasolase 1CC284A) variant expressing cells) (Constructing human P0NKC284A) expressing cells) Human P0N1 (C284A) The cDNA was used as a template using the sequence disclosed by NCBI GenBank Accession Number BC07471S, and point mutation was induced using Genetailor Site-Directed Mutagenesis Systems 49 322362 s 201116292 (Invitrogen), and the pair of cysteine encoding the 284th cysteine was used. Substituted as a base pair encoding alanine. Then, the CDNA of human P0N1CC284A) was inserted into the expression vector pEE12. 4 of the animal cell regulated by the hCMV-MIE promoter, and pEE12.4-hP0N1 (C284A) of the human P0N1 (C284A) (hPON1) expression vector was constructed. pEE12.4-hP〇Nl (C284A) was introduced into CH0-K1 cells by electroporation, and selectively cultured with sulfonimide (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "human P〇Nl (C284A) expression cell") was used for the following examples. (Purification of C284A variant of human recombinant Baraoxasone 1) In addition to the human P0N1 (C284A) expressing cells described above, the human P0N1 expressing cells were replaced, and the recombinant human body was purified in the same manner as in Example 8. A C284A variant of Laorson's enzyme 1 was obtained, and a C284A variant preparation of the recombinant human balasolone 1 was obtained. The C284A variant of the human recombinant Barasolsone 1 obtained by the recombinant gene obtained was analyzed in the same manner as in Example 8. Specifically, the protein content of the C284A variant of the recombinant human balasolase 1 was determined by an absorbance method. The cysteine thiolactone activity was measured using Alfresa Auto HTLase (Alfresa Pharma). The balasolone enzyme activity was measured in accordance with the method described in Journal of Lipid Reaserch Vol. 41, 2000 pl358_1363. The arylesterase activity was measured using ARYLESTERASE/PARAOXONASE ASSAY KIT (manufactured by ZeptoMetrix Co., Ltd.). The pH is determined according to the Japanese Pharmacopoeia 50 322362 201116292. The form (appearance) was confirmed visually. The results are shown in Table 7. Table 7 Analysis results of C284A variant preparations C284A variant protein 4. 1 liter of cysteine thiol 1 enzyme activity (IU/l) 7954 Barathon _ enzyme activity (u/lT~~- 1921000 aroma Ester ΙΪ activity (U/L) ~ ....... 3819000 pH 7. 5 Form (Appearance) - Almost colorless and transparent The preparation prepared above can be used directly as an injection. (Purification results) A recombinant sample was prepared from the C284A variant of the recombinant human balasolone 1 produced by the gene of 16, and subjected to electrophoresis using a SDS-polyacrylamide 5 to 2% gradient gel in the same manner as in Example 9. CBB staining was performed and the results are shown in Figure UA. The band on the left is the molecular weight marker. This is the C284A s variant of the above-mentioned recombinant human Balasonase, which is based on SDS_pAGE in this sample. The protein on the colloid was transferred to P. (manufactured by ATT0 Co., Ltd.), and the reaction was carried out by using an anti-human balazonase 1 antibody ^-4CM, manufactured by Brewing Co., Ltd.). Subsequently, the anti-mouse immunoglobulin antibody (manufactured by KPL Co., Ltd.), which has been deleted, was used for secondary reaction, and the color was developed by the φ TMB method. The results of the Western blot method of the C284A variant (reduced sample) are shown in Fig. 11B. As shown in Fig. 11A and Fig. 11B, it is presumed that the band of the C284A variant of human body Balason 322362 51 201116292 1 was purified, and the band was subjected to the above-mentioned anti-human balazonase 1 antibody. Western blotting method. Thus, it was confirmed that the C284A variant of human balasolone 1 was obtained. <Example 17 &gt; In the preparation of the amino acid sequence of human balazonase 1 (SEQ ID NO: 1), the first position of thiol acid (Met) was eliminated, and the 84th position of the amino acid (Lys) a polypeptide consisting of the amino acid sequence of the 306th arginine (Arg) substituted with glutamic acid (Glu) (K84E/R306Q variant). (Constructing human body balasolase 1CK84E/R306Q) variant expression cells) (constructing human PON1CK84E/R306Q) expressing cells)

The human body PON1CK84E/R306Q) cDNA is used in NCBI

The sequence disclosed by GenBank Accession Number BC074719 was used as a template to induce point mutation using Genetailor Site-Directed Mutagenesis Systems (Invitrogen), and the base pair encoding the 84th amino acid group was substituted with a base pair encoding glutamic acid. The base pair encoding arginine at position 306 is substituted with a base pair encoding glutamine (Gin). Then, the cDNA of human PON1 (K84E/R306Q) was inserted into pEE12. 4 of the expression vector for animal cells regulated by the hCMV-MIE promoter, and the peE12 of human PON1CK84E/R306Q) expression vector was constructed. 4-hPONl (K84E/ R306Q). pEE12 4_hP〇N1 (K84E/R306Q) was introduced into CH0-K1 cells by electroporation, and selectively transformed with guanidine imidate (MSX) to obtain a transformant. Use the obtained transformant (hereinafter referred to as "human PON1CK84E/R306Q" to express cells") for the following

Example experiment of S 52 322362 201116292. (Purification of the K84E/R306Q variant of the recombinant human balazonase 1) In addition to the human PON1CK84E/R306Q) expressing cells to replace the human P0N1 expressing cells, the rest was purified in the same manner as in Example 8. The K84E/R306Q variant of the human balasolase 1 was obtained, and the K84E/R306Q variant preparation of the recombinant human balasolone 1 was obtained. The K84E/R306Q variant preparation of the human recombinant balasolone 1 obtained by the recombinant gene obtained was analyzed in the same manner as in Example 16. The results are shown in Table 8. Table 8 Analysis results of K84E/R306Q variant preparations K84E/R306Q variant protein content (mg/ml) 5.4 liters of cysteine thiolactone activity (IU/L) 3618 Barrason enzyme activity (U/ L) 1174000 pH 7. 5 Form (Appearance) Almost colorless and transparent The preparation prepared above can be directly used as an injection. (Purification result) A reduced sample was prepared from the K84E/R306Q variant of the recombinant human balasolone 1 obtained in Example 17, and SDS-polypropylene amine 5 was used in the same manner as in Example 9. A 20% gradient gel was electrophoresed and subjected to CBB staining. The results are shown in Fig. 12A. The band on the left is a molecular weight marker. s, 53 322362 201116292 Further, the Western blotting method was carried out in the same manner as in Example 16 except that the K84E/R306Q variant of the above-described genetically recombined human balasolase i was used. The results of the Western blot method of the K84E/R306Q variant (reduced sample) are shown in Fig. 12B. As shown in Fig. 12A and Fig. 12B, it is presumed that the K84E/R306Q and the anamorphic band of the human balasolase 1 were purified, and the band was subjected to the above-mentioned anti-human balazonase 1 antibody. Western blotting method. From this, it was confirmed that the K84E/R306Q variant of human balasolone 1 was obtained. <Example 18 &gt; (Green recognition 3 for the prophylactic or therapeutic effect of Crohn's disease) 25 rats of 8-week-old male C57BL/6 mice (Japan SLC Co., Ltd.) were prepared for the experiment. Twenty of them used TNBS solution in Day to establish a Crohn's disease model and administered a single intrarectal dose in an amount of 10 mg/kg. The C284A variant preparation of the recombinant human balasolone 1 or the recombinant K84E/R306Q variant of the recombinant human balazonase 1 prepared by the recombinants prepared in Examples 16 and 17 respectively, i〇mg/kg (C284A 10 mg/kg administration group; n=5, K84E/R306Q 10 mg/kg administration group; n=5) A single intravenous administration was carried out in a manner of 5 per group. Similarly, the recombinant human body balasolone 1 prepared in Example 8 was used and a single intravenous injection was administered to the other 5 at a dose of 10 mg/kg (PON 10 mg/kg administration group; n=5). versus. Only 5 mice administered TNBS solution were used as a control group. Further, 5 mice which were not administered with the TNBS solution, the P0N1 preparation, and the P0N1 variant preparation were used as the normal group.

S 54 322362 201116292 The disease activity index score (DAI score; Disease activity index score) was measured once every day from DayO to 2. The evaluation items and baselines of the MI score were the same as those in Example 1. Day 3 ' after the animal experiment was sacrificed by inhaling the animal with an excess of carbon dioxide anesthesia, the large intestine tissue was taken. Then, the Macroscopic score was measured in the same manner and in the same manner as in Example 10. Each score system calculates the mean ± standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, Dunnett's assay was performed in the same manner as in Example 10 in terms of the effect of the P0N1 administration group or the variant administration group with respect to the control group. The risk rate is below 5% (*) and the hazard rate is below the judgement as a significant difference. The results are shown in Fig. 13 and Fig. 14 (Fig. 13, wood: P &lt; 〇. 05, **P &lt; 0. 01). In Fig. 13, the white point is the normal group; the black point is the control group; the triangle is the P0N1 10 mg/kg administration group; the square (_) is the C284A l〇mg/kg administration group; the square shape (order) is K84E/R3 〇6Q10mg/kg administration group 0 The number of death individuals in each group is as follows. • Day 3: Control group 1 As shown in Fig. 13 and Fig. 14, it was confirmed that the DAI score and the Macroscopic score increased in the control group compared with the normal group. However, symptoms such as inflammation, ulceration, tissue loss, squatting, and hemorrhage of the large intestine confirmed in the control group were significantly inhibited in the P0N1 variant administration group. The degree of inhibition is approximately the same as that of the p〇N1 administration group. Therefore, it can be seen that the prevention of Crohn's disease caused by the PON 1 preparation or the therapeutic effect of 322362 55 201116292 can also be confirmed in the p〇N1 variant preparation. . Further, in the P0N1 preparation administration group and the p〇N1 variant preparation, side effects which are generally considered to be caused by administration of the P0N1 preparation or the P0N1 variant preparation were not found. <Example 19> An ep〇Nl-G2E6 (SEQ ID NO: 8) of a human P0M1 derivative was produced. (Construction of human P0N1 derivative (ep〇Nl-G2E6) expressing cells) The cDNA of eP0Nl-G2E6 was constructed using the amino acid sequence (SEQ ID NO: 8) of the NCBI GenBank Accession Number AAR95984 (invitrogen) , SEQ ID NO: 7), wherein the performance of CH0 cells has been optimized. Then, the cDNA of eP0N1-G2E6 was inserted into the expression vector pEE12.4 of the animal cell regulated by the hCMV-MIE promoter, and the pEE12 4-AY499191 of the ePON-G2E6 expression vector was constructed. pEE12.4-AY499191 was introduced into CH0-K1 cells by electroporation, and selectively cultured with sulfinamide sulfonimide (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "eP0N1-G2E6 expressing cell") was used for the following examples. (Purification of eP0N1-G2E6) The ePON1-G2E6 expressing cells were cultured in CD-CH0 medium as an initial medium to produce a human balasolone 1 derivative eP0N1-G2E6. The culture conditions were pH 7.1, 37 ° C, and CHOCDEfficientFeedB was added at 40 ml/L/day between the 5th day and the 9th day of culture. The culture supernatant was recovered on the 10th day of the culture, and it was subjected to filtration treatment with a 遽.45# m ruthenium membrane, and then used for the following purification. 56 322362 201116292 The solution after filtration through the membrane of 0·45 // m is added dropwise to a strong anion exchange column (UNO Sphere Q) t, which contains 〇. 15M gasification nano and lmM chlorine After washing with a 25 mM Tris hydrochloric acid solution (pH 7.5) of #5, it was further eluted with a 25 mM Tris hydrochloric acid solution (pH 7.5) containing 0. 25 M sodium chloride and 1 mM calcium chloride. The solution was then ultrafiltered using a 3 〇 ultrafiltration membrane, and the solvent was replaced with a 2 mM Tris hydrochloric acid solution (pH 7.2) containing 1 mM calcium chloride. This solution was added dropwise to a strong cation exchange column (UN〇 Sphere S) to recover the unadsorbed fraction. The solution was subjected to ultrafiltration using a 3 〇 ultrafiltration membrane, and the solvent was replaced with a solution of 0.15 M sodium chloride and lmM gasification mother 25 mM Tris salt solution (pH 7.5). Then, the solution was subjected to sterile filtration treatment with a filter of 〇. 22 / / m to obtain an ePON1-G2E6 preparation. The eP〇Nl-G2E6 preparation obtained in Example 19 was analyzed in the same manner as in Example 16. The results are shown in Table 9. Table 9 Analysis results of ePONl-G2E6 preparation eP0Nl-G2E6 Protein content (mg/mL) 7. 3 liters of cysteine thiolactone activity (IU/L) 20148 Barathlon enzyme activity (U/L_) 35894000 ^•Acetylesterase activity (U/L) 8819000 pH 7. 5 Form C Appearance) Almost colorless and transparent The preparation prepared above can be directly used as an injection. (Purification result) 57 322362 t 201116292 The ePONl-G2E6 coating of the human P0N1 derivative obtained in Example 19 was used as a reconstituted sample. In the same manner as in Example 9, 5 to 20% of SDS-polyglycolamide was used. The gradient gel was electrophoresed and subjected to CBB staining. The result is shown in Figure 15A. The band on the left is the molecular weight marker. Further, the above ePON1-G2E6 was subjected to the Western dot method in the same manner as in the embodiment 46. The results of the Western blot method of the eP0Nl-G2E6 (reduced sample) are shown in Fig. 15B. As shown in Fig. 15A and Fig. 15B, the band of ePON G2E6 was presumed to be purified, and the band was stained by Western blotting using the above anti-human balazonase 1 antibody. From this, it was confirmed that eP0Nl-G2E6 was obtained. <Example 20 &gt; eP0N1-G3C9 (SEQ ID NO: 10) of a human P0N1 derivative was produced. (Construction of human P0N1 derivative (ePONl-G3C9) expressing cells) The cDNA of ePONl-G3C9 is an artificial base sequence (SEQ ID NO: 10) encoding the amino acid sequence (SEQ ID NO: 10) disclosed by NCBI GenBank Accession Number AAR95986 (Invitrogen, sequence No. 9), which has been optimized for the performance of CH0 cells. Then, the cDNA of eP0N1-G3C9 was inserted into the expression vector PEE12.4 of the animal cell regulated by the hCMV-MIE promoter, and the pP12-4-YY499193 of the eP0N1-G3C9 expression vector was constructed. The pΕΕ12.4-ΑΥ499193 was introduced into CH0-K1 cells by electroporation, and selectively cultured with methionine (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "ePONl-G3C9 expressing cells") was used for the following examples of s 58 322362 201116292. (Purification eP0N1-G3C9) The ePON1-G3C9 expressing cells were cultured and purified in the same manner as in Example 19 to prepare an ePON-G3C9 preparation. The eP0N1-G3C9 preparation obtained in Example 20 was analyzed in the same manner as in Example 16. The results are shown in Table 10. Table 10 Analysis results of eP0Nl-G3C9 preparation eP0Nl-G3C9 Protein content (mg/ml) 7. 9 liters of cysteine thiolactone activity (IU/L) 25912 Balasols enzyme activity (U/L) 37530000 Aromatic esterase activity (U/L) 13117000 pH 7. 5 Form (appearance) Almost colorless and transparent The preparation prepared above can be directly used as an injection. (Purification result) A reduced sample was prepared from eP0N1-G3C9 of the human P0N1 derivative obtained in Example 20, and subjected to electrophoresis using a SDS_polyacrylamide 5 to 20% gradient gel in the same manner as in Example 9. And CBB staining was performed. The result is shown in Figure 16A. The band on the left is the molecular weight marker. Further, the above ePON Bu G3C9 was subjected to the Western dot method in the same manner as in the sixteenth embodiment. The results of the Western blot method of the eP0N1-G3C9 (reduced sample) are shown in Fig. 16B. As shown in Fig. 16A and Fig. 16B, the band 59 322362 201116292, which is presumed to be eP0N1-G3C9, was purified, and the band was stained by the Western blot method using the above anti-barasolase 1 antibody. From this, it was confirmed that eP0N1_G3C9 was obtained. <Example 21 &gt; In the preparation of the amino acid sequence of human balasolase 1 (SEQ ID NO: 1), the first position of methionine (Met) was eliminated, and the aspartic acid at position 269 was A polypeptide consisting of an amino acid sequence substituted with glutamic acid (D269E variant). (Construction of human balasolone 1 (D269E) variant expressing cells) (Constructing human P0N1 (D269E) expressing cells) Human cDNA of PONKD269E) using the sequence disclosed by NCBI GenBank Accession Number BC074719 as a template, using Genetailor Site -Directed Mutagenesis Systems (manufactured by Invitrogen) induces point mutation and replaces the base pair encoding the 687th aspartic acid with a base pair encoding glutamic acid. Then, the human P0N1 (D269E) cMA was inserted into the animal cell expression vector pEE12. 4 regulated by the hCMV-MIE promoter, and the human P0N1 (D269E) (hPONl) expression vector pEE12.4-hP0Nl (D269E) was constructed. . pEE12, 4-hP0N1 (D269E) was introduced into CH0-K1 cells by electroporation, and selectively cultured with sulfonimide (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "human P0NKD269E) expression cell" was used for the following examples. (P269 recombination of the human recombinant P283N269E) was used to replace the human P0N1 expression cells, and the recombinant human body Balason was purified in the same manner as in Example 8. The D269E variant of enzyme 1 was obtained, and the D269E variant preparation of human balasolone 1 of genetic recombination s 60 322362 201116292 was obtained. The D269E variant heterologous preparation of the recombinant human balasolone 1 obtained was analyzed in the same manner as in Example 8. Specifically, the protein content of the D269E variant of the recombinant human balasolase 1 was determined by the absorbance method. The u-cysteine thiolactonease activity was carried out using Alfresa Auto HTLase (Alfresa Pharma). Determination. The balasolone enzyme activity was measured in accordance with the method described in Journal of Lipid Reaserch Vol. 41, 2000 pi358-1363. The arylesterase activity was measured using ARYLESTERASE/PARAOXONASE ASSAY KIT (manufactured by ZeptoMetrix Co., Ltd.). The PH system was measured in accordance with the Japanese Pharmacopoeia. The form (appearance) was confirmed visually. The results are shown in Table 11. Table 11 Analysis results of D269E variant preparation D269E variant protein content (mg/mi) 6. 1 liter of cysteine thiolactone activity (IU/L) below detection limit (less than 50) Pine enzyme activity (U/L) 147000 Aromatic esterase activity (U/L) 0 pH 7.5 Form (appearance) Almost colorless and transparent <Example 22 &gt; Preparation of amino acid sequence of human balasolase 1 (sequence number In 1), the first position of thiol acid (Met) is eliminated, and the 115th and 134th histidines (His) are respectively composed of amino acid substituted by glutamine (Gin). Polypeptide (H115/134Q variant).

S 61 322362 201116292 (Construction of human balasolone 1CH115/134Q) variant expression cells) (Construction of human P0N1 (H115/134Q) expression cells) Human P0N1 (H115/134Q) cDNA was used with NCBI GenBank Accession Number BC074719 Using the disclosed sequence as a template, a point mutation was induced using Genetailor Site-Directed Mutagenesis Systems (Invitrogen), and the base pairs encoding the ii5th and 134th histidines were respectively substituted for the test for encoding glutamine. Base pair. Then, the cDNA of human P0N1CH115/134Q) was inserted into the expression vector pEEi2.4 of animal cells regulated by hCMV-MIE promoter, and pEE12.4-hP〇 of human P0N1(H115/134Q_)(hP0Nl) expression vector was constructed. Nl (H115/134Q). PEE12.4-hPONl (HI 15/134Q) was introduced into CH0-K1 cells by electroporation, and selectively cultured with methionine hydrazide (MSX) to obtain a transformant. The obtained transformant (hereinafter referred to as "human PONUH115/134Q" expression cells" was used for the following examples. (purification of recombinant h17/134q variant of human balasolase 1) &lt; In addition to using human P0N1OH15/134Q) expressing cells to replace human P0N1 expressing cells, the rest was purified in the same manner as in Example 8. The H115/134Q variant of human balazonone 1 is obtained, and the H115/134Q variant preparation of human balazonone 1 is obtained. The 15/134 卩 variant preparation of the human recombinant balasolone 1 obtained by the recombinant gene obtained was analyzed in the same manner as in Example 21. The results are shown in Table 12. 322362 62 201116292 Table 12 Analysis results of H115/134Q variant preparations H115/134Q variant protein content (mg/ml) 6.2 liters of cysteine thiolactone activity (IU/L) below detection limit (lower than 50) Barrason's enzyme activity (U/L) 0 Aromatic vinegar enzyme activity (U/L) 0 pH 7.5 Form (appearance) Almost colorless and transparent (purification result) The recombinant human body obtained by the method of Example 21 A non-reducing sample was prepared by using the D269E variant of Laoxasin 1 and the H115/134Q variant of the recombinant human balasolone 1 obtained in Example 22, and using SDS- in the same manner as in Example 9. The polyacrylamide 5 to 20% gradient gel was electrophoresed and subjected to CBB staining. The results of CBB staining of the D269E variant are shown in Figure 17A. The results of CBB staining of the H115/134Q variant are shown in Figure 17B. In the 17A and 17B, the left band is a molecular weight marker. <Example 23> (Confirmation of the effect of prevention or treatment of Crohn's disease 4) For the experiment, 45 male C57BL/6 mice (manufactured by SLC, Japan) of 8 weeks old were prepared. Forty of them used TNBS solution in DayO to establish a Crohn's disease model and administered a single intrarectal dose at a dose of 100 mg/kg. Using the ePONl-G2E6 preparation and the ePONl-G3C9 preparation prepared in Examples 19 and 20, intravenous administration of Day 0 to 2 was carried out at a dose of 1 mg/kg or 10 mg/kg, respectively, in groups of 5 (G2E6 £ 63 322362 201116292 lmg/kg administration group; n=5, G2E6 10mg/kg administration group; n=5, G3C9 lmg/kg administration group; n=5, G3C9 10mg/kg administration group; n =5). Similarly, the H115/134Q variant preparation of the recombinant human balazonase 1 prepared by using the genetic modification prepared in Example 21 and Example 22, or the recombinant human body balasolase 1 and D269E variant preparation, Intravenous administration of Day 0 to 2 in a dose of 1 〇mg/kg, 5 rats per group (H115/134Q 10 mg/kg administration group; n=5, D269E 10 mg/kg administration group; n =5). In addition, the recombinant human body balasolone 1 prepared in Example 8 was administered in a dose of 10 mg/kg to the other 5 days of Day 0 to 2 for intravenous administration (P0N1 10 mg/kg administration). Group; n=5). Further, 5 mice which were not administered with the TNBS solution, the P0N1 preparation, and the P0N1 variant preparation were used as the normal group. The disease activity index score (DAI score; Disease activity index score) was measured on DayO to 3 once a day. The evaluation items and benchmarks of the DAI score are the same as those of the example i〇. After the start of the animal experiment, Day3 was sacrificed by anesthetizing the animal by inhaling excess carbon dioxide, and the large intestine tissue was taken. Then, the Macroscopic score was measured in the same manner and in the same manner as in Example 1 . Each score system calculates the average soil standard deviation. Regarding the statistical analysis of the DAI score and the Macroscopic score, Dunnett's assay was performed in the same manner as in Example 10 in terms of the effect of the P0N1 administration group or the variant administration group with respect to the control group. A risk ratio of less than 5% (*) and a risk ratio of less than 1% (material) were judged to be significantly different. The results are shown in Tables 13 and 14, and Figs. 18 and 19. Table £ 322362 201116292 13 shows the results of the Dunnett test for the DAI scores of each group (mean ± standard deviation). The results shown in Table 13 are plotted to become the map shown in Fig. In Fig. 18, (〇) is the normal group; it is the control group; (▲) is the P0N1 l〇mg/kg administration group; (_) is the eP〇N1_G2E6 lmg/kg

In the administration group, (□) was the eP0Nl-G2E6 10 mg/kg administration group; (♦) was eP (M-G3C9 lmg/kg was administered to the 'group; (◊) was ep0N Bu G3C9 10 mg/kg administration group. x) is H115/134Q 10 mg/kg administration group; (*) is D269E 10 mg/kg 4 and group. Table 14 shows the results of Dunnett test for each group's Macroscopic score (mean ± standard deviation) ^ Table 14 The results shown are plotted as 'Fig. 19'. In Table 13, Table 14, Figure 18, and Figure 19, *: P&lt;〇. 〇5,: p&lt;〇. The number of death individuals is as follows: • Day 3: Control group 2 Table 13 DAI score

Day 1 Day 2 Day 3 Normal group 0. 0±0. 0 0. 0±0. 0 0.0+0. 0 Control group 8. 2±0.8 9. 2±0. 7 9. 7+0. 3 P0N1 10mg /kg The group is 5. 0±0. 3 3. 8±0. 7*木3. 4+0. 6* ePONl-G2E6 lmg/kg Administration group 6. 2±1.0 5. 2+0. 8 5. 2+0. 7 ePON 1-G2E6 1 Omg/kg Administration group 4. 8+0. 9 3. 8±0. 7 wood 2. 8±0. 5« ePONl -G3C9 1 mg/kg Group 4. 2+0. 7* 4. 0±0. 〇3. 2+0. 5* ePONl-G3C9 lOmg/kg Administration group 3. 6±0. 7* wood 3. 4+1. 3.4+ 1.4±0. 8 5. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 +0. 6 4. 4±0. 4 322362 65 201116292 Table 14

Macroscopic score

Macroscopic score Normal group 0. 0±0. 0 Control group 4. 7+0. 3 PON 1 1 Omg/kg Administration group 1.8+0.4 eP0Nl-G2E6 lmg/kg Administration group 2. 0+0. 3 eP0Nl- G2E6 1 Omg/kg Administration group 2. 0+0. 3 eP0Nl-G3C9 lmg/kg Administration group 1.4±0.2* ePONl-G3C9 lOmg/kg Administration group 1·2±0.7** D269E lOmg/kg Group 2. 0+0. 3 H115/134Q lOmg/kg Administration group 2. 0+0. 4 As shown in Table 13, Table 14, Figure 18 and Figure 19, the control group and the normal group can be confirmed. In comparison, there is an increase in DAI score and Macroscopic score. However, the symptoms of tissue damage, weight loss, diarrhea, and hemorrhage of the large intestine confirmed in the control group were in the P0N1 administration group, the eP0N1-G2E6 administration group of the P0N1 derivative, and the eP0N1-G3C9 administration group. The H115/134Q administration group and the D269E administration group of the P0N1 variant were significantly inhibited. From this experiment, it was found that the prophylactic and therapeutic effects of Crohn's disease caused by the P0N1 preparation were also confirmed in the P0N1 derivative preparation and the p〇N丨 variant system. Further, the P0N1 preparation administration group, the P0N1 derivative preparation administration group, and the P0N1 variant preparation administration group did not find any side effects which were generally considered to be caused by administration of the preparations. <Example 24 &gt; (Measurement result of activity of scavenging active oxygen) 66 322362 201116292 The recombinant human body balasolone 1 preparation prepared in Example 8, and the recombinant human body parallel prepared in Example 16 The C284A variant preparation of oxasone 1 , the ep〇Nl-G2E6 preparation obtained in Example 、9, the ep〇m-G3C9 preparation obtained in Example 20, and the recombinant human body obtained in Example 21 The D269E variant preparation of balasolase 1 and the H115/134Q variant preparation of the recombinant human balasolone 1 prepared in Example 22 were assayed for activity of scavenging reactive oxygen species. The activity of scavenging reactive oxygen species was determined according to the method of Root and Metcalf (J Clin Invest 60, 1266-1279 (1977)). Specifically, the hydrogen peroxide (Wako Pure Chemical Industries, Ltd.) prepared in a 4/z Μ aqueous solution was prepared from a sputum and a protein concentration of 4. lmg/ml of the sample 50α 丨 at 37 ° for 10 minutes. Then, the horseradish peroxidized rabbit (was pure chemical industry, biochemical) 50//1 was prepared and adjusted to 8#Μ=菪素(Wako Pure Chemical Industries) again at 37° After C reaction for 5 minutes, the work light = degree (excitation wavelength 366 nm, emission wavelength 460 nm) was measured.

The fluorescence intensity was measured using Tecan's mex) plate reader and Infinity M200. The preparation of the sample is 2 mM

Tris-ΗΠ, 150 mM NaCl, 2 mM CaCl 2 , pH 7. 4 &gt; _ ^, ± 1 &lt; buffer, the preparation of the reagent was 50 mM PBS 'pH 7.4. The results are shown in Table 15 322362 67 201116292 Table 15 Formulation name removal active oxygen activity (%) Example 8 Human body PON1 47. 6 Example 16 C284A variant 47. 2 Example 19 ePONl-G2E6 33. 4 Example 20 ePONl - G3C9 39. 4 Example 21 D269E variant 70. 7 Example 22 H115/134Q variant 67. 0 Examples of the preparation of the present invention include the following preparations. However, the present invention is not limited to these preparation examples. 。 The pH of the human albumin was adjusted to 7.5. The human albumin was added as a stabilizer to be added to the recombinant human body of the balainsonase 1 prepared in Example 8. After sterilization and filtration, it is filled in a vial (vial) and lyophilized to prepare an injection. Further, in the same manner as described above, the preparation containing the polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 obtained in Example 19 or the SEQ ID NO: 10 obtained in Example 20 can be used. A preparation of a polypeptide consisting of the amino acid sequence is formulated as an injection. (Industrial Applicability) The polypeptide of the active ingredient of the present invention such as PON is preferably used as a prophylactic or therapeutic agent for ulcerative colitis as an inflammatory bowel disease, and an active ingredient of a prophylactic or therapeutic agent for Crohn's disease. . BRIEF DESCRIPTION OF THE DRAWINGS The 1A, IB, 1C, and 1D drawings are views showing the SDS-PAGE colloids of PON1 obtained in Examples 1, 4, 6, and 8, respectively. 68 322362 201116292 Figures 2A, 2B, 2C, and 2D are graphs showing the results of the Western blot method of P0N1 obtained in Examples 1, 4, 6, and 8, respectively. Fig. 3 is a graph showing the results of measuring the DAI score and performing Dunnett's assay in order to investigate the preventive or therapeutic effect of P0N1 on ulcerative colitis. Fig. 4 is a view showing the results of measuring the DAI score and performing Dunnett's test for the prevention or treatment effect of P0N1 on ulcerative colitis. Fig. 5 is a view showing the purpose of investigating the preventive or therapeutic effect of P0N1 on Crohn's disease. DA I score and a graph of the results of the Dunne 11 assay. Fig. 6 is a view showing the results of measuring the DA I score and performing Dunn 11: assay for the prevention or treatment effect of Crohn's disease. Fig. 7 shows the purpose of investigating the preventive effect of P0N1 on Crohn's disease. A plot of the DAI score and the results of the Dunnett assay. Fig. 8 is a view showing the measurement of the Macroscopic score and the Dunnett test for investigating the prevention of Crohn's disease by P0N1. Fig. 9 is a graph showing the results of measuring the MI score and performing Dunnett's assay in order to investigate the treatment of P0N1 for Crohn's disease. Fig. 10 is a view showing a celebrity in which the Macroscopic score was measured for the treatment of Crohn's disease and the Dunnett test was performed. 322362 69 201116292 Fig. 11A is a diagram showing the results of SDS-PAGE of the C284A variant of PON1 obtained in Example 16, and Fig. 11B is a graph showing the results of the Western blot method of the C284A variant of the PON1. Fig. 12A is a view showing the results of SDS-PAGE of the K84E/R306Q variant of P0N1 obtained in Example 17, and Fig. 12B is a graph showing the results of the Western blot method of the K84E/R306Q variant of the P0N1. Fig. 13 is a graph showing the results of measuring the DA I score and performing Dunnett assay in order to investigate the effect of the P0N1 and P0N1 variants on the prevention or treatment of Crohn's disease. Fig. 14 is a graph showing the results of measuring the Macroscopic score and performing Dunnett's assay for the purpose of capturing the preventive or therapeutic effects of the P0N1 and P0N1 variants on Crohn's disease. Fig. 15A is a view showing the results of SDS-PAGE of eP0N:l-G2E6 obtained in Example 19; and Fig. 15B is a view showing the result of the Western blotting method of the ePON1-G2E6. Fig. 16A is a view showing the results of SDS-PAGE of eP0N1-G3C9 obtained in Example 20; and Fig. 16B is a view showing the result of the Western blot method of the ePON G3C9. Fig. 17A is a graph showing the results of SDS-PAGE of the D269E variant of P0N1 obtained in Example 21; and Fig. 17B is a graph showing the results of SDS-PAGE of the H115/134Q variant of P0N1 obtained in Example 22. Fig. 18 is a graph showing the results of measuring the DA I score and performing Dunnett assay in order to investigate the effect of the ep〇N1 and PON1 variants on the prevention or treatment of Crohn's disease.

S 70 322362 201116292 Fig. 19 is a graph showing the results of measuring the Macroscop i c score and performing Dunnett's assay in order to investigate the effect of the prevention and treatment of ePON1 and PON1 variants on Crohn's disease. [Main component symbol description] None

S 71 322362 201116292 Sequence Listing &lt;110>Japan Pharmaceutical Co., Ltd. <120> Prophylactic or therapeutic agent for inflammatory bowel disease

&lt;130> N48F3513(TW)P &lt;150&gt; JP2009-217629 &lt;151> 2009-09-18 &lt;160&gt; 10 &lt;170&gt; Patentln version 3.1 &lt;210&gt; 1 &lt;211&gt; 355 &lt;212 〉 PRT <213> Human &lt;400〉 1

Met Ala Lys Leu lie Ala Leu Thr Leu Leu Gly Met Gly Leu Ala Leu 15 10 15

Phe Arg Asn His Gin Ser Ser Tyr Gin Thr Arg Leu Asn Ala Leu Arg 20 25 30

Glu Val Gin Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly lie 35 40 45

Glu Thr Gly Ser Glu Asp Met Glu lie Leu Pro Asn Gly Leu Ala Phe 50 55 60 lie Ser Ser Gly Leu Lys Tyr Pro Gly lie Lys Ser Phe Asn Pro Asn 65 70 75 80

Ser Pro Gly Lys lie Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Thr 85 90 95

Val Leu Glu Leu Gly lie Thr Gly Ser Lys Phe Asp Val Ser Ser Phe 100 105 110

Asn Pro His Gly lie Ser Thr Phe Thr Asp Glu Asp Asn Ala Met Tyr 115 120 125

Leu Leu Val Val Asn His Pro Asp Ala Lys Ser Thr Val Glu Leu Phe 130 135 140 201116292

Lys Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr lie Arg 145 150 155 160

His Lys Leu Leu Pro Asn Leu Asn Asp lie Val Ala Val Gly Pro Glu 165 170 175

His Phe Tyr Gly Thr Asn Asp His Tyr Phe Leu Asp Pro Tyr Leu Gin 180 185 190

Ser Trp Glu Met Tyr Leu Gly Leu Ala Trp Ser Tyr Val Val Tyr Tyr 195 200 205

Ser Pro Ser Glu Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 210 215 220

Gly lie Asn lie Ser Pro Asp Gly Lys Tyr Val Tyr lie Ala Glu Leu 225 230 235 240

Leu Ala His Lys lie His Val Tyr Glu Lys His Ala Asn Trp Thr Leu 245 250 255

Thr Pro Leu Lys Ser Leu Asp Phe Asn Thr Leu Val Asp Asn lie Ser 260 265 270

Val Asp Pro Glu Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 275 280 285

Met Lys lie Phe Phe Tyr Asp Ser Glu Asn Pro Pro Ala Ser Glu Val 290 295 300

Leu Arg lie Gin Asn lie Leu Thr Glu Glu Pro Lys Val Thr Gin Val 305 310 315 320

Tyr Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr Val Ala Ser Val 325 330 335

Tyr Lys Gly Lys Leu Leu lie Gly Thr Val Phe His Lys Ala Leu Tyr 340 345 350

Cys Glu Leu 355 &lt;210> 2 &lt;211&gt; 28 201116292 &lt;2l2&gt; DNA &lt;2l3&gt; Artificial sequence &lt;220&gt;&lt;223> PCR primer &lt;400&gt; 2 ttgctagccc ccgaccatgg cgaagctg 28 &lt;2l0&gt; 3 &lt;2ll&gt; 76 &lt;2l2&gt; DNA &lt;2l3&gt; Artificial sequence &lt;220> &lt;223> PCR primer &lt;400> 3 ttgcggccgc tcattagtgg tggtggtggt ggtgcttttc gaactgcggg tggctccaga 60 gctcacagta aagagc 76 &lt;2l0&gt; 4 &lt;2ll&gt; 37 &lt;2l2&gt; DNA &lt;2l3&gt; artificial sequence &lt;220&gt;&lt;223&gt; PCR primer &lt;400&gt; 4 cccacgtgtc gcgacaatta gtcagcaacc atagtcc 37 0 12 3 11 ΊΧ 1x li 2 2 2 2 &lt;&lt;&lt;&lt; 5 36

DNA artificial sequence &lt;220> &lt;223>PCR primer &lt;400> 5 cccacgtgtc gcgaggacaa accacaacta gaatgc 36 &gt;&gt;&gt;&gt; 0 12 3 1X IX 1x TA 2 2 2 2 &lt;&lt;&lt;&lt; Negative 4 T 0 5 R 3 p / &lt;400&gt; 6

Ala Lys Leu lie Ala Leu Thr Leu Leu Gly Met Gly Leu Ala Leu Phe 15 10 15 3 322362 s 201116292

Arg Asn His Gin Ser Ser Tyr Gin Thr Arg Leu Asn Ala Leu Arg Glu 20 25 30

Val Gin Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly He Glu . 35 40 45

Thr Gly Ser Glu Asp Met Glu lie Leu Pro Asn Gly Leu Ala Phe lie 50 55 60

Ser Ser Gly Leu Lys Tyr Pro Gly He Lys Ser Phe Asn Pro Asn Ser 65 70 75 80

Pro Gly Lys He Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Thr Val 85 90 95

Leu Glu Leu Gly lie Thr Gly Ser Lys Phe Asp Val Ser Ser Phe Asn 100 105 110

Pro His Gly lie Ser Thr Phe Thr Asp Glu Asp Asn Ala Met Tyr Leu 115 120 125

Leu Val Val Asn His Pro Asp Ala Lys Ser Thr Val Glu Leu Phe Lys 130 135 140

Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr He Arg His 145 150 155 160

Lys Leu Leu Pro Asn Leu Asn Asp lie Val Ala Val Gly Pro Glu His 165 170 175

Phe Tyr Gly Thr Asn Asp His Tyr Phe Leu Asp Pro Tyr Leu Gin Ser 180 185 190

Trp Glu Met Tyr Leu Gly Leu Ala Trp Ser Tyr Val Val Tyr Tyr Ser 195 200 205

Pro Ser Glu Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn Gly 210 215 220 lie Asn lie Ser Pro Asp Gly Lys Tyr Val Tyr lie Ala Glu Leu Leu 225 230 235 240 4 322362 £ 201116292

Ala His Lys He His Val Tyr Glu Lys His Ala Asn Trp Thr Leu Thr 245 250 255

Pro Leu Lys Ser Leu Asp Phe Asn Thr Leu Val Asp Asn He Ser Val 260 265 270

Asp Pro Glu Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly Met 275 280 285

Lys lie Phe Phe Tyr Asp Ser Glu Asn Pro Pro Ala Ser Glu Val Leu 290 295 300

Arg lie Gin Asn lie Leu Thr Glu Glu Pro Lys Val Thr Gin Val Tyr 305 310 315 320

Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr Val Ala Ser Val Tyr 325 330 335

Lys Gly Lys Leu Leu He Gly Thr Val Phe His Lys Ala Leu Tyr Cys 340 345 350

Glu Leu &lt;210> 7 &lt;211&gt; 1092 &lt;212&gt; DNA &lt; 213 &gt; 213 &gt; artificial sequence &lt;220 &lt; 223 &gt; 223 &gt; Barathonase-1 from human, mouse, rat and rabbit Gene family gene recombination of the balasolase-1 variant G2E6 &lt;220〉

&lt;221&gt; CDS &lt;222> (16)..(1080) &lt;223&gt;&lt;400&gt; 7 aagcttgccg ccacc atg gca aag ctg aca get ctg act ctg ctg gga atg 51

Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Met ggg ctg get etc ttc gac ege cag aaa tcc age ttt cag acc agg ttt 99

Gly Leu Ala Leu Phe Asp Arg Gin Lys Ser Ser Phe Gin Thr Arg Phe 15 20 25 aat gtg cac aga gaa gtg aca cca gtt gag ett ccc aat tgc aat ctg 147

Asn Val His Arg Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu 30 35 40 5 322362 195 201116292 gtc aaa ggc att gat aac gga tct gag gac etc gag ate ett cct aac

Val Lys Gly lie Asp Asn Gly Ser Glu Asp Leu Glu He Leu Pro Asn 45 50 55 60 ggt ctg gca ttt att tee tct ggc etc aag tac cca ggt att atg age

Gly Leu Ala Phe lie Ser Ser Gly Leu Lys Tyr Pro Gly lie Met Ser 65 70 75 ttc gat ccc gac aag agt ggc aag att etc ctg .atg gat ctg aac gag

Phe Asp Pro Asp Lys Ser Gly Lys He Leu Leu Met Asp Leu Asn Glu 80 85 90 aaa gaa cca gca gtg age gag ctg gag att ate gga aat aca ctg gat

Lys Glu Pro Ala Val Ser Glu Leu Glu He lie Gly Asn Thr Leu Asp 95 100 105 att agt age ttt aac ccc cat ggc ate tct act ttc ata gac gat gac

He Ser Ser Phe Asn Pro His Gly lie Ser Thr Phe lie Asp Asp Asp 110 115 120 aat acc gtc tac ctg ett gtc gtg aat cat cca gga tea tcc tct aca

Asn Thr Val Tyr Leu Leu Val Val Asn His Pro Gly Ser Ser Ser Thr 125 130 135 140 gtg gag gtt ttt aag ttc caa gag gag gag aaa tcc ctg etc cac ctg

Val Glu Val Phe Lys Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu 145 150 155 aag acc att ega cat aag etc ctg cca agt gtt aac gac ate gtg gcc

Lys Thr lie Arg His Lys Leu Leu Pro Ser Val Asn Asp lie Val Ala 160 165 170 gtt gga cct gaa cat ttc tat gcc acc aac gac cac tat ttt att gat

Val Gly Pro Glu His Phe Tyr Ala Thr Asn Asp His Tyr Phe lie Asp 175 180 185 ccc tat ett aag agt tgg gag atg cac etc ggc ett gca tgg tct ttc

Pro Tyr Leu Lys Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe 190 195 200 gtg act tat tac age ccc aat gac gtg ege gtt gtg gcc gaa ggc ttt

Val Thr Tyr Tyr Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe 205 210 215 220 gac ttc gcc aat ggg att aat ate agt ccc gat ggc aag tac gtg tac

Asp Phe Ala Asn Gly lie Asn lie Ser Pro Asp Gly Lys Tyr Val Tyr 225 230 235 att get gag etc ett gca cac aag ate cac gta tac gag aag cac gcc lie Ala Glu Leu Leu Ala His Lys lie His Val Tyr Glu Lys His Ala 240 245 250 aat tgg acc etc aca cct ett ege gtc ctg age ttc gac aca ctg gtc

Asn Trp Thr Leu Thr Pro Leu Arg Val Leu Ser Phe Asp Thr Leu Val 255 260 265 gac aac ata tea gtg gac ccc gtc acc ggg gac etc tgg gtt ggc tgt 6 243 . 291 339 387 435 483 531 579 627 675 723 771 819 867 322362 201116292

Asp Asn lie Ser Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys 270 275 280 cat cca aat ggt atg cga att ttc ttc tac gat get gag aac cct cc.a 915

His Pro Asn Gly Met Arg lie Phe Phe Tyr Asp Ala Glu Asn Pro Pro 285 290 295 300 gga tet gaa gtc ett ege ate cag gac ate ett age gag gag cct aaa 963

Gly Ser Glu Val Leu Arg lie Gin Asp lie Leu Ser Glu Glu Pro Lys 305 310 315 gtg aca gtg gtg tat get gag aat gga acc gta ctg cag ggt tet aca 1011

Val Thr Val Val Tyr Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr 320 325 330 gtg gee gee gtg tat aag ggc aag ctg ctg ate gga acc gtg ttc cac 1059

Val Ala Ala Val Tyr Lys Gly Lys Leu Leu He Gly Thr Val Phe His 335 340 345 . aag get etc tac tgc gac etc taatgagaat tc 1092

Lys Ala Leu Tyr Cys Asp Leu 350 355 &lt;210〉 8 &lt;211> 354 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt; 223 &gt; 223 &gt; 223 &gt; 223 &gt; Recombination of the balazonase-1 gene, the balasonase-1 variant G2E6 &lt;400> 8

Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Met Gly Leu Ala Leu Phe 15 10 15

Asp Arg Gin Lys Ser Ser Phe Gin Thr Arg Phe Asn Val His Arg Glu 20 25 30

Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly lie Asp 35 40 45

Asn Gly Ser Glu Asp Leu Glu lie Leu Pro Asn Gly Leu Ala Phe lie 50 55 60

Ser Ser Gly Leu Lys Tyr Pro Gly lie Met Ser Phe Asp Pro Asp Lys 65 70 75 80

Ser Gly Lys lie Leu Leu Met Asp Leu Asn Glu Lys Glu Pro Ala Val 85 90 95 7 322362 £. 201116292

Ser Glu Leu Glu lie He Gly Asn Thr Leu Asp lie Ser Ser Phe Asn 100 105 110

Pro His Gly He Ser Thr Phe He Asp Asp Asp Asn Thr Val Tyr Leu 115 120 125

Leu Val Val Asn His Pro Gly Ser Ser Ser Thr Val Glu Val Phe Lys 130 135 140

Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr lie Arg His 145 150 155 160

Lys Leu Leu Pro Ser Val Asn Asp He Val Ala Val Gly Pro Glu His 165 170 175

Phe Tyr Ala Thr Asn Asp His Tyr Phe lie Asp Pro Tyr Leu Lys Ser 180 185 190

Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr Ser 195 200 205

Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn Gly 210 215 220 lie Asn lie Ser Pro Asp Gly Lys Tyr Val Tyr lie Ala Glu Leu Leu 225 230 235 240

Ala His Lys He His Val Tyr Glu Lys His Ala Asn Trp Thr Leu Thr 245 250 255

Pro Leu Arg Val Leu Ser Phe Asp Thr Leu Val Asp Asn He Ser Val 260 265 270

Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly Met 275 280 285

Arg He Phe Phe Tyr Asp Ala Glu Asn Pro Pro Gly Ser Glu Val Leu 290 295 300

Arg lie Gin Asp lie Leu Ser Glu Glu Pro Lys Val Thr Val Val Tyr 305 310 315 320 201116292

Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr Val Ala Ala Val Tyr 325 330 335

Lys Gly Lys Leu Leu lie Gly Thr Val Phe His Lys Ala Leu Tyr Cys 340 345 350

Asp Leu &lt;210> 9 &lt;211&gt; 1092 <212> DNA &lt;213>Artificial Sequence &lt;220&gt;&lt;223&gt;Baraosone-1 gene obtained from human, mouse, rat and rabbit The family gene recombination of the balasolase-1 variant G3C9 &lt;220> &lt;221> CDS &lt;222> (16).. (1080) &lt;223> &lt;400> 9 aagcttgccg ccacc atg get aag Ctg aca get ctg acc ett ctg gga ett 51

Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu 1 5 10 ggc ctg gca ctg ttt gac ggc cag aag tet tcc ttt cag act aga ttc 99

Gly Leu Ala Leu Phe Asp Gly Gin Lys Ser Ser Phe Gin Thr Arg Phe 15 20 25 aat gtg cat ege gaa gta aca cca gtt gaa ttg cca aac tgc aac etc 147

Asn Val His Arg Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu 30 35 40 gtc aag ggc gtc gac aac ggg tea gaa gat ctg gag att ttg cct aac 195

Val Lys Gly Val Asp Asn Gly Ser Glu Asp Leu Glu He Leu Pro Asn 45 50 55 60 ggt ttg get ttt ate tcc tcc gga ctg aag tac cct ggg ata atg agt 243

Gly Leu Ala Phe He Ser Ser Gly Leu Lys Tyr Pro Gly lie Met Ser 65 70 75 ttc gac cct gac aag tet ggc aag ate etc etc atg gac ctg aac gaa 291

Phe Asp Pro Asp Lys Ser Gly Lys lie Leu Leu Met Asp Leu Asn Glu 80 85 90 gaa gac cct gtc gtg etc gag etc ggc ata act ggg aat act ctg gac 339

Glu Asp Pro Val Val Leu Glu Leu Gly lie Thr Gly Asn Thr Leu Asp 95 100 105 att tet tcc ttc aat cct cac ggt att tcc acc ttc acc gac gag gac 387

He Ser Ser Phe Asn Pro His Gly lie Ser Thr Phe Thr Asp Glu Asp 110 115 120 9 322362

I 201116292 aat act gtc tac ctg ctt gtg gtg aac cat ccc gac tct tcc agt acc 435

Asn Thr Val Tyr Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr 125 130 135 140 gtt gaa gtg ttc aag ttc caa gaa gag gag aaa age etc ctt cat ctt 483

Val Glu Val Phe Lys Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu 145 150 155 aaa act att agg cat aaa ctg ttg ccc tea gtt aac gac ate gtc gca 531

Lys Thr lie Arg His Lys Leu Leu Pro Ser Val Asn Asp lie Val Ala 160 165 170 gtg ggc ccc gaa cat ttc tac gcc acc aac gat cat tat ttt gcc gat 579

Val Gly Pro Glu His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp 175 180 185 ccc tac ctg aag tct tgg gaa atg cat ctg ggt etc gca tgg tcc ttt 627

Pro Tyr Leu Lys Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe 190 195 200 gtg act tac tac age ccc aat gac gta ege gtc gtg gcc gag ggc ttc 675

Val Thr Tyr Tyr Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe 205 210 215 220 gat ttc gca aac ggc att aat ate agt ccc gac ggg aag tat gta tat 723

Asp Phe Ala Asn Gly lie Asn lie Ser Pro Asp Gly Lys Tyr Val Tyr 225 230 235 att gca gag ctg ctg gca cac aag ate cac gtg tat gag aaa cac gca 771 lie Ala Glu Leu Leu Ala His Lys lie His Val Tyr Glu Lys His Ala 240 245 250 aac tgg acc ctg act cct etc aag tct etc gac ttt gat acc ctg gtc 819

Asn Trp Thr Leu Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val 255 260 265 gac aac ata age gtg gac cca gtg act ggt gat ttg tgg gtc ggt tgc 867

Asp Asn He Ser Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys 270 275 280 cac cca aac gga atg aga ate ttc tat tat gac ccc aag aac cca cca 915

His Pro Asn Gly Met Arg lie Phe Tyr Tyr Asp Pro Lys Asn Pro Pro 285 290 295 300 ggg tea gaa gtc ttg agg att caa gat ate ctt tcc gaa gag cca aag 963

Gly Ser Glu Val Leu Arg lie Gin Asp lie Leu Ser Glu Glu Pro Lys 305 310 315 gtc aca gtg gtt tat gca gaa aac ggc acc gta ctg cag ggg tcc acc 1011

Val Thr Val Val Tyr Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr 320 325 330 gta get get gtg tat aag ggt aag ctg ctg ate ggc aca gtg ttc cat 1059

Val Ala Ala Val Tyr Lys Gly Lys Leu Leu He Gly Thr Val Phe His 335 340 345 aag get ctt tat tgt gag etc taatgagaat tc 1092 10 322362 201116292

Lys Ala Leu Tyr Cys Glu Leu 350 355 &lt;210> 10 &lt; 211 &gt; 354 &lt; 212 &gt; PRT &lt; 213 &gt; 213 > Artificial Sequence &lt; 220 &gt;&lt; 223 &gt; 223 > #羞会 I occupies rats, rats and rabbits Bara Ozonase of the Family Gene Recombination of the Barathonase-1 Gene &lt;400&gt; 10

Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu Phe 1 5 10 15

Asp Gly Gin Lys Ser Ser Phe Gin Thr Arg Phe Asn Val His Arg Glu 20 25 3〇

Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val Asp 35 40 45

Asn Gly Ser Glu Asp Leu Glu He Leu Pro Asn Gly Leu Ala Phe He 50 55 60

Ser Ser Gly Leu Lys Tyr Pro Gly lie Met Ser Phe Asp Pro Asp Lys 65 70 75 80

Ser Gly Lys lie Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val Val 85 90 95

Leu Glu Leu Gly lie Thr Gly Asn Thr Leu Asp lie Ser Ser Phe Asn 100 105 110

Pro His Gly lie Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr Leu 115 120 125

Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe Lys 130 135 140

Phe Gin Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr He Arg His 145 150 155 160

Lys Leu Leu Pro Ser Val Asn Asp lie Val Ala Val Gly Pro Glu His 165 170 175 11 322362 201116292

Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys Ser 180 185 190

Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr Ser 195 200 205

Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn Gly 210 215 220 lie Asn He Ser Pro Asp Gly Lys Tyr Val Tyr lie Ala Glu Leu Leu 225 230 235 240

Ala His Lys lie His Val Tyr Glu Lys His Ala Asn Trp Thr Leu Thr 245 250 255 .

Pro Leu-Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn lie Ser Val 260 265 270

Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly Met 275 280 285

Arg lie Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val Leu 290 295 300

Arg lie Gin Asp He Leu Ser Glu Glu Pro Lys Val Thr Val Val Tyr 305 310 315 320

Ala Glu Asn Gly Thr Val Leu Gin Gly Ser Thr Val Ala Ala Val Tyr 325 330 335

Lys Gly Lys Leu Leu He Gly Thr Val Phe His Lys Ala Leu Tyr Cys 340 345 350

Glu Leu 12 322362 £

Claims (1)

201116292 VII. Scope of application for patents · · · A prophylactic or therapeutic agent for a disease of enteric disease contains a polypeptide as an active ingredient; the polypeptide has a sequence similarity to the body of the balasolase 70/0 or more' and At least one activity in the group selected from the group consisting of lactonase activity, balasolase activity, aromatic esterase activity, and activity of scavenging reactive oxygen species. 2. A prophylactic or therapeutic agent for an inflammatory bowel disease according to the scope of the application of the invention, wherein the polypeptide is a polypeptide of the following (a), (8) or (4); (a) SEQ ID NO: 1, 6, 8 or a compound consisting of the amino acid sequence of the amino acid sequence (b), the amino acid sequence of the sequence number 1, 6, 8 or 1 ,, wherein one or more amino acids are deleted or inserted Or a substituted and the peptide system has at least one of a group selected from the group consisting of internal acetase activity, balasolase activity, and aroma g enzyme activity. (c) Sequence number number 6, 8 or A polypeptide consisting of a 1 amino acid sequence in which one or a plurality of amino acids are deleted, inserted, or substituted, and the polypeptide has an activity of scavenging active oxygen. 3.=Application of the special case (4) 丨 or 2 cases of prevention of inflammatory bowel disease] The polypeptide has a selected from the group consisting of vinegar enzyme activity, balasolone enzyme and aroma_activity At least a kind of activity. , • ^ Application for the scope of the special case! Or the prophylactic or therapeutic agent for inflammatory bowel diseases according to the above, wherein the polypeptide has an activity of scavenging active oxygen, and has no (tetra), balason, and aromatic activity. The inflammatory bowel disease 322362 201116292 is a prophylactic or therapeutic agent for a disease according to any one of the items 1 to 4, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. 6. The prophylactic or therapeutic agent for inflammatory bowel disease according to any one of claims 1 to 5, which is an injection. 7. A prophylactic or therapeutic agent for ulcerative colitis comprising a balasolase as an active ingredient. 8. A prophylactic or therapeutic agent for Crohn's disease, comprising a balasolase as an active ingredient. 2 322362