JPH0211600A - Production of urine daf - Google Patents
Production of urine dafInfo
- Publication number
- JPH0211600A JPH0211600A JP63163061A JP16306188A JPH0211600A JP H0211600 A JPH0211600 A JP H0211600A JP 63163061 A JP63163061 A JP 63163061A JP 16306188 A JP16306188 A JP 16306188A JP H0211600 A JPH0211600 A JP H0211600A
- Authority
- JP
- Japan
- Prior art keywords
- daf
- urine
- supernatant
- concentration
- adsorbent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002700 urine Anatomy 0.000 title claims abstract description 43
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 239000012528 membrane Substances 0.000 claims abstract description 18
- 238000010438 heat treatment Methods 0.000 claims abstract description 16
- 230000004154 complement system Effects 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 239000003463 adsorbent Substances 0.000 claims abstract description 13
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 13
- 230000004913 activation Effects 0.000 claims abstract description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 9
- 150000001450 anions Chemical class 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- 108010009575 CD55 Antigens Proteins 0.000 claims abstract description 7
- 241000124008 Mammalia Species 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 239000011780 sodium chloride Substances 0.000 claims abstract description 4
- 239000012141 concentrate Substances 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 23
- 239000000126 substance Substances 0.000 claims description 13
- 238000002523 gelfiltration Methods 0.000 claims description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical group CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- 235000011152 sodium sulphate Nutrition 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 abstract description 4
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 3
- 239000003112 inhibitor Substances 0.000 abstract 1
- 229920006395 saturated elastomer Polymers 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 38
- 210000003743 erythrocyte Anatomy 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 230000002485 urinary effect Effects 0.000 description 10
- 210000004379 membrane Anatomy 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 8
- 102000016574 Complement C3-C5 Convertases Human genes 0.000 description 7
- 108010067641 Complement C3-C5 Convertases Proteins 0.000 description 7
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 6
- 235000004252 protein component Nutrition 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 206010018910 Haemolysis Diseases 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 101100008638 Caenorhabditis elegans daf-1 gene Proteins 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 239000012506 Sephacryl® Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000000733 Paroxysmal Hemoglobinuria Diseases 0.000 description 1
- 102100036050 Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 150000002763 monocarboxylic acids Chemical class 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 201000003045 paroxysmal nocturnal hemoglobinuria Diseases 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、ヒトを含む哺乳動物の補体系の活性化の抑制
作用を有する物質を尿から製造する方法に関する０本発
明の方法で製造される物質は、デイケイ・アクセレレー
ティング・ファクター（Ｄｅｃａｙ Ａｃｃｅｌｅｒａ
ｔｉｎｇ Ｆａｃｔｏｒ、Ｄ Ａ Ｆ ）と呼ばれる物
質またはこれと関連を持つ物質であり、ＤＡＦの欠損症
である発作性夜間血色素尿症さらには補体系の活性化に
よっておこる腎炎などの各種自己免疫疾患の診断、治療
、予防薬として有用である。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for producing from urine a substance having an effect of suppressing the activation of the complement system of mammals including humans. Decay Accelerating Factor (Decay Accelera)
It is a substance called ting factor (DAF) or a substance related to it, and is used to diagnose various autoimmune diseases such as paroxysmal nocturnal hemoglobinuria, which is a deficiency of DAF, and nephritis caused by activation of the complement system. It is useful as a therapeutic and preventive drug.
（従来の技術）
ＤＡＦとは、Ｈｏｆｆｍａｎｎにより、ヒト赤血球膜中
に発見された、補体系を活性化する酵素であるＣ３コン
ベルターゼの活性を消失させる作用（ｄｅｃａｙ ａｃ
ｃｅｌｅｒａｔｉｎｇ ａｃｔｉｖｉｔｙ）を示す２種
類の物質（Ｉａｍｕｎｏｃｈｅｍｉｓｔｒｙ、６巻、３
！１１〜４０３頁（１９６９年）およびＩｍｍｕｎｏｃ
ｈｅｍｉｓｔｒｙ、 ６巻、４０５〜４１９頁（１９６
９年））の一方にその後毛付けられた名称である。Ｎ１
ｃｈｏｌ５０，０００ｎ−Ｗｅｌｌｅｒらは、モルモッ
ト赤血球およびヒト赤血球からＤＡＦを精製し、このも
のが分子量が７０ 、０００〜７５，０００の糖蛋白質
であることを報告している（Ｊ、 Ｉｍｍｕｎｏｌ、。(Prior art) DAF is an enzyme that quenches the activity of C3 convertase, an enzyme that activates the complement system, discovered by Hoffmann in human red blood cell membranes.
Two types of substances exhibiting celerating activity (Iamunochemistry, Vol. 6, 3)
! pp. 11-403 (1969) and Immunoc.
hemistry, vol. 6, pp. 405-419 (196
This is the name that was later given to one side of the 9th year). N1
chol50,000n-Weller et al. purified DAF from guinea pig red blood cells and human red blood cells and reported that it is a glycoprotein with a molecular weight of 70,000 to 75,000 (J. Immunol.
１２７巻、 ２０３５〜２０３９頁（１９８１年）およ
びＰｒｏｃ。127, pp. 2035-2039 (1981) and Proc.
Ｎａｔｌ、 Ａｃａｄ、 Ｓｃｉ、 ＬＩＳＡ、 ８０
巻、 ５０６５〜５０７０頁（１９８３年））、さらに
、Ｎ１ｃｈｏｌ５０，０００ｎ−Ｗｅｌｌｅｒらは、Ｄ
ＡＦは最初に発見された赤血球表面のみならず血小板、
好中球、単球、Ｂ細胞およびＴ細胞などの遺産幹細胞に
由来する細胞の表面（Ｂｌｏｏｄ、６５巻、 １２３７
〜１２４４頁（１９８５年）〕さらに、血管内皮細胞の
表面（Ｊ、 Ｉｍｍｕｎｏｌ、 、 １３７巻、 １２
７５〜１２７９頁（１９８６年）〕にも存在すると報告
している。 Ｍｅｄｏｆらは、結膜や角膜、消化管粘膜
外分泌腺、泌尿器系、心外膜、肋膜などの多数の上皮細
胞の表面や涙、唾液、尿などの体液中にも存在すると報
告している（Ｊ、 Ｅｘｐ。Natl, Acad, Sci, LISA, 80
Vol., pp. 5065-5070 (1983)), and furthermore, N1chol50,000n-Weller et al.
AF was first discovered not only on the surface of red blood cells, but also on platelets,
Surface of cells derived from heritage stem cells such as neutrophils, monocytes, B cells and T cells (Blood, Vol. 65, 1237
1244 (1985)] Furthermore, the surface of vascular endothelial cells (J, Immunol, vol. 137, 12
75-1279 (1986)]. Medof et al. reported that it is present on the surface of numerous epithelial cells such as the conjunctiva, cornea, gastrointestinal mucosa, exocrine glands, urinary system, epicardium, and pleura, as well as in body fluids such as tears, saliva, and urine (J , Exp.
Ｍｅｄ、、 １６５巻、８４８〜８６４頁（１９８７年
））、そして、その後の研究によればＤＡＦはＣ３コン
ベルターゼのみならず、補体系を活性化する他の酵素Ｃ
５コンベルターゼの活性も阻害することが見出されてい
る（ Ｊ、 Ｅｘｐ、 Ｍｅｄ、 、 １６０巻。Med., vol. 165, pp. 848-864 (1987)), and subsequent studies have shown that DAF is not only a C3 convertase, but also a complex of other enzymes that activate the complement system.
It has also been found to inhibit the activity of 5 convertase (J, Exp, Med, vol. 160.
１５５８〜１５７８頁（１９８４年）〕。pp. 1558-1578 (1984)].
従って、ＤＡＦは、補体系の活性化に重要な役割を果た
しているＣ３および／またはＣ５コンベルターゼを阻害
することにより、補体系か異常に活性化されて生じる局
所または全身的障害を抑制するために有用であると期待
されている。Therefore, DAF is useful for suppressing local or systemic damage caused by abnormal activation of the complement system by inhibiting C3 and/or C5 convertases that play an important role in the activation of the complement system. It is expected that
（発明が解決しようとする課題）
ＤＡＦの精製に関しては、この物質が哺乳動物の生体内
に微量にしか存在しないため、従来、研究室規模での精
製が行なわれているにすぎず（Ｍｅｄｏｆら： Ｊ、
Ｅｘｐ、 Ｍｅｄ、、 １６５巻、８４８〜８６４頁（
１９８７年））、シかもその方法はモノクローナル抗体
をリガンドとするアフィニティクロマトグラフイーな用
いるため、工業的に実用価値のあるものではなかった。(Problems to be Solved by the Invention) Conventionally, purification of DAF has only been carried out on a laboratory scale because this substance exists in only trace amounts in the living body of mammals (Medof et al. : J,
Exp, Med, vol. 165, pp. 848-864 (
(1987)), but the method used affinity chromatography using a monoclonal antibody as a ligand, and was not of industrial practical value.
従って、ＤＡＦを容易な操作で大量に取得する方法の開
発は、上記自己免疫疾患の治療等のためのみならず、そ
の構造決定や生理活性の究明のためにも重要である。Therefore, the development of a method for obtaining large amounts of DAF with easy operations is important not only for the treatment of the above-mentioned autoimmune diseases, but also for determining its structure and investigating its physiological activity.
本発明は、入手が容易な原料である哺乳動物好まし゛く
はヒトの尿から、ＤＡＦを工業的に実施可能な（即ち、
容易且つ大量生産に適する）方法で製造することを目的
とする０本発明によれば、医薬として有用な補体系の活
性化を抑制するＤＡＦを、ヒトの尿から大量に製造する
ことがてきる。The present invention makes it possible to industrially produce DAF from mammalian, preferably human urine, which is an easily available raw material (i.e.,
According to the present invention, it is possible to produce DAF, which is useful as a medicine and suppresses the activation of the complement system, in large quantities from human urine. .
（課題を解決するための手段）
本発明のＤＡＦの製造方法は、尿を濃縮した後、熱処理
に付して沈澱と上清を生ぜしめ、該上清な陰イオン交換
体、好ましくはセルロース系の陰イオン交換体からなる
吸着剤と接触させ１次いで該吸着剤に吸着された成分を
溶出し、ヒトを含む哺乳動物の補体系の活性化を抑制す
る物質を含む溶出画分を回収することを特徴とする。(Means for Solving the Problems) The method for producing DAF of the present invention involves concentrating urine, subjecting it to heat treatment to produce a precipitate and a supernatant, and converting the supernatant into an anion exchanger, preferably a cellulose-based contact with an adsorbent made of an anion exchanger, then elute the components adsorbed to the adsorbent, and collect an eluted fraction containing a substance that suppresses the activation of the complement system of mammals including humans. It is characterized by
なお、本発明でヒト尿中から回収されるＤＡＦは、赤血
球膜表面等に存在するＤＡＦと関連を有し、少なくとも
その活性に関与する構造が同一または類似の物質と考え
られるが、尿中に***される際に、糖成分やアミノ酸組
成に若干の変化か生じている可能性もあるため、本明細
書中では「尿ＤＡＦＪと称する。It should be noted that DAF recovered from human urine in the present invention is related to DAF present on the surface of red blood cell membranes, etc., and is thought to be a substance that is at least the same or similar in structure involved in its activity. In this specification, it is referred to as "urinary DAFJ" because there may be slight changes in the sugar components and amino acid composition during excretion.
本発明で製造される、ヒトを含む哺乳動物の補体系の活
性化を抑制する尿ＤＡＦの０３もしくはＣ５コンベルタ
ーゼ阻害活性の評価は１次の方法により行うことができ
る。Evaluation of the 03 or C5 convertase inhibitory activity of urinary DAF, which is produced according to the present invention and suppresses the activation of the complement system of mammals including humans, can be performed by the following method.
ａ）Ｃ３コンへルターゼ によ
Ｈｏｎｇらの方法（Ｊ、Ｉｍ＋＊ｕｎｏ１．、１２７巻
、１０９〜１１４頁（１９８１年）〕によってにモノカ
ルボン酸処理したモルモット血清とヒツジ感作赤血球で
調製したＣ３コンベルターゼを用いて、Ｎ１ｃｈｏｌ５
０，０００ｎ−ＷｅｌｌｅｒらのＤＡＦ活性測定方法（
Ｊ、Ｉｍｍｕｎｏｌ、、 １２７巻、 ２０３５〜２０
３９頁（１，９８１年））に従い、赤血球膜上で抗原抗
体反応により補体系を活性化させ、その結果生じる溶血
反応を３０％抑制する量を１単位として表す。a) C3 convertase C3 prepared from monocarboxylic acid-treated guinea pig serum and sheep sensitized red blood cells according to the method of Hong et al. Using convertase, N1chol5
0,000n-Weller et al.'s DAF activity measurement method (
J, Immunol, vol. 127, 2035-20
39 (1,981)), one unit is the amount that activates the complement system through an antigen-antibody reaction on the red blood cell membrane and suppresses the resulting hemolytic reaction by 30%.
ｂ） ＤＡＦ の によＭｅｄｏ ｆ
らのイムノブロッティング法（Ｊ。b) Medo f by DAF
The immunoblotting method of J. et al.
Ｅｘｐ、Ｍｅｄ、 １６５巻、８４８〜８６４頁（１９
８７年）〕に従い、ヒト赤血球膜ＤＡＦに対するウサギ
抗体との結合性を、ヒト赤血球１ｉｊＤＡＦ相当量とし
て表す。Exp, Med, vol. 165, pp. 848-864 (19
[1987], the binding of rabbit antibodies to human red blood cell membrane DAF is expressed as the equivalent amount of human red blood cell 1ijDAF.
ｃ） Ｃ３ａ によ 雪
５ｅｙａらの方法（Ｊ、Ｉｍｍｕｎｏｌ、 １３５巻、
２５６１〜２６６７頁（１９１３５年）〕に従い、ヒト
の補体成分より調製した液相Ｃ３コンベルターゼを用い
、０３分解後産生したＣ３ａ量をラジオイムノアッセイ
法により測定し、Ｃ３ａｆｉｆｉ抑制活性をその蛋白量
で表す。c) C3a method of Yuki et al. (J, Immunol, vol. 135,
2561-2667 (19135)], the amount of C3a produced after 03 degradation was measured by radioimmunoassay using liquid phase C3 convertase prepared from human complement components, and the C3afifi inhibitory activity was expressed as the amount of protein. .
次に１本発明の詳細な説明する。Next, one aspect of the present invention will be explained in detail.
ヒト尿を濃縮するに先だって、必要であれば簡単なろ過
等の前処理を行って差し支え無いが、一般には、前処理
を行う必要はない、濃縮は限外ろ過法又は限外ろ過法と
沈澱法、好ましくは硫安沈澱法を組み合すせて行う、限
外ろ過は、尿中の蛋白質成分の濃度を高めて、次に行う
硫安沈澱を容易にするとともに、尿ＤＡＦよりも分子量
の小さい不純物を取り除くために行う。硫安沈澱は、尿
ＤＡＦを含めて、尿中の主要蛋白質成分を沈澱させて回
収するために行う。Before concentrating human urine, it is possible to perform simple pretreatment such as filtration if necessary, but in general, pretreatment is not necessary.Concentration can be done by ultrafiltration or ultrafiltration and precipitation. Ultrafiltration, preferably carried out in combination with an ammonium sulfate precipitation method, increases the concentration of protein components in the urine, facilitates the subsequent ammonium sulfate precipitation, and removes impurities with a smaller molecular weight than urine DAF. done to remove. Ammonium sulfate precipitation is performed to precipitate and recover major protein components in urine, including urinary DAF.
本発明に用いる限外ろ過膜の具体例として。As a specific example of the ultrafiltration membrane used in the present invention.
ＵＫ−５０（アトパンチツク東洋社、日本）、ウルトラ
フィルター５０，０００ （アトパンチツク東洋社１日
本）、ダイアフローメンブランＰ Ｍ ３０（ブレース
ジャパン社１日本）、ダイアフローメンブランＸＭＳＯ
（グレースジャバン社、日本）およびＵ Ｆ −１００
Ｐ Ｓ （東ソー社１日本）が挙げられる。しかし、こ
れらに限らず分子量約５万以上の成分を通過させない任
意の限外ろ過膜を使用することが可能である。UK-50 (Atpanczuk Toyosha, Japan), Ultra Filter 50,000 (Atpanczuk Toyosha 1 Japan), Diaflow Membrane PM 30 (Brace Japan 1 Japan), Diaflow Membrane XMSO
(Grace Java Co., Ltd., Japan) and UF-100
P S (Tosoh Corporation 1 Japan) is mentioned. However, the present invention is not limited to these, and any ultrafiltration membrane that does not allow components having a molecular weight of about 50,000 or more to pass through can be used.
硫安沈澱は、限外ろ過膜を用いた一次濃縮ののち、尿に
硫酸アンモニウムを５０〜７０％飽和濃度、好ましくは
約６０％飽和濃度で添加して尿ＤＡＦを含む蛋白質成分
を沈澱させることにより行う、限外ろ過および硫安沈澱
により、尿の蛋白質成分は２，０００〜４，０００倍に
濃縮される。Ammonium sulfate precipitation is performed by adding ammonium sulfate to urine at a saturation concentration of 50 to 70%, preferably about 60%, to precipitate protein components including urine DAF, after primary concentration using an ultrafiltration membrane. By , ultrafiltration and ammonium sulfate precipitation, the protein components of urine are concentrated 2,000 to 4,000 times.
この濃縮は硫安沈澱法に限らず、アセトン沈澱法等の一
般に蛋白質の活性に影響の少ない適当な沈澱方法て行う
ことが可能である。このようにして得られた沈澱物は少
量の純粋な水に溶解する。This concentration is not limited to ammonium sulfate precipitation, but can be carried out by any suitable precipitation method that generally has little effect on protein activity, such as acetone precipitation. The precipitate thus obtained is dissolved in a small amount of pure water.
前記工程て得られた尿ＤＡＦを含む水溶液を１次に熱処
理する。これは、約７０℃〜１００℃の温度で約５〜２
０分、好ましくは約８０℃の温度で約１０分開法ＤＡＦ
を含む水溶液を加熱することにより行う、加熱は、７０
°Ｃ以上の温度から効果がみられ、１００°Ｃでは５分
の処理で十分である０本発明者らは、この処理により、
他の工程では除去できない不純蛋白質成分の多くが変性
沈澱して除去され、約４倍の精製が可能であることを見
出した。硫安沈澱による濃縮後に、簡単な熱処理て４倍
程度の精製を行うことは、極めて低濃度でしか尿ＤＡＦ
を含まない尿を原料として、工業的規模で該物質を生産
するために必要不可欠なことであり、この熱処理を行う
ことにより、引き続く吸着剤による精製を極めて効率的
に行うことか可能となった。なお、尿ＤＡＦは上記熱処
理の条件では変性を受けることなく、安定に溶液中に存
在するが、上記の過酷な熱処理によっても、蛋白質であ
る尿ＤＡＦか活性を失わないという事実については過去
に報告がなく、全く新しい知見である。The aqueous solution containing urine DAF obtained in the above step is first heat-treated. This is about 5-2 at a temperature of about 70℃-100℃
0 minutes, preferably about 10 minutes at a temperature of about 80°C.
Heating is performed by heating an aqueous solution containing 70
The effect is seen at temperatures above °C, and at 100 °C, 5 minutes of treatment is sufficient.
It has been found that many of the impure protein components that cannot be removed by other steps are removed by denaturation and precipitation, making it possible to purify the method by about 4 times. After concentration by ammonium sulfate precipitation, purification by a factor of 4 through simple heat treatment means that urine DAF can only be purified at extremely low concentrations.
This is essential for producing this substance on an industrial scale using urine that does not contain it as a raw material, and by performing this heat treatment, it has become possible to perform the subsequent purification using an adsorbent extremely efficiently. . Furthermore, urine DAF does not undergo denaturation under the above heat treatment conditions and remains stable in the solution; however, it has been reported in the past that urine DAF, which is a protein, does not lose its activity even under the above severe heat treatment. This is completely new knowledge.
熱処理で得られた尿ＤＡＦを含有する水溶液を、続いて
、陰イオン交換体からなる吸着剤に吸着させ、塩化ナト
リウム、塩化カリウムまたはリン酸塩等の濃度勾配によ
りイオン強度を増加させて溶出し、尿ＤＡＦを含有する
画分を集める。陰イオン交換体からなる吸着剤の例とし
て、ＤＥＡＥセファセル（Ｓｅｐｈａ−ｅｅｌ） （フ
ァルマシア社、スウェーデン）、ＤＥＡＥセルロース（
Ｃｅｌｌｕｌｏｓｅ） Ｄ Ｅ ５２ （ワットマン社
、米国）、ＤＥＡＥセルロースＤ Ｅ ３２ （ワット
マン社、米国）、セレックス（Ｃｅｌｌｅｘ） Ｄ （
バイオラット社、米国）およびＴＳＫゲル（ｇｅｌ）
トヨバール６５０Ｍ （東ソー社、日本）等のセルロー
スイオン交換体が挙げられるが、蛋白質の分離に一般に
用いられる吸着剤であれば、他のものも使用可俺である
。The aqueous solution containing urine DAF obtained by heat treatment is then adsorbed onto an adsorbent made of an anion exchanger, and eluted by increasing the ionic strength with a concentration gradient of sodium chloride, potassium chloride, or phosphate. , collect the fraction containing urine DAF. Examples of adsorbents made of anion exchangers include DEAE Sepha-eel (Pharmacia, Sweden), DEAE cellulose (
Cellulose) DE 52 (Whatman, USA), DEAE cellulose DE 32 (Whatman, USA), Cellex D (
Biorat Inc., USA) and TSK gel (gel)
Examples include cellulose ion exchangers such as Toyovar 650M (Tosoh Corporation, Japan), but other adsorbents commonly used for protein separation may also be used.
以上の工程での、尿ＤＡＦの収率は、最初の尿中の活性
のほぼ７０％以上である。比活性の上昇は約１０〜２０
倍であり、従って、得られた生成物は１診断、治療、予
防のための薬として使用可能である。The yield of urinary DAF in the above steps is approximately 70% or more of the initial activity in urine. The increase in specific activity is approximately 10-20
Therefore, the obtained product can be used as a drug for diagnosis, treatment and prevention.
本発明によれば、前記工程で得られた尿ＤＡＦは、所望
によりゲルろ過および高速液体クロマトグラフィーによ
り、さらに精製することが可能である。ゲルろ過の前に
、その精製効率を上昇させるためには、前記工程で得ら
れた尿ＤＡＦをゲルろ過に適する量まで濃縮する。According to the present invention, the urine DAF obtained in the above step can be further purified by gel filtration and high performance liquid chromatography, if desired. Before gel filtration, in order to increase its purification efficiency, the urine DAF obtained in the above step is concentrated to an amount suitable for gel filtration.
その際、濃縮は任意の適当な方法で行ってよいが、前記
と同様の限外ろ過膜を用いると都合がよい、ゲルろ過に
用いる担体は、分子１５０，０００、ｏｏ。At that time, the concentration may be carried out by any suitable method, but it is convenient to use the same ultrafiltration membrane as above.The carrier used for gel filtration has a molecular weight of 150,000,000.
〜９０，００Ｇの物質の分離に適する任意のものが使用
可能である０例えば、バイオゲル（ＢｉｏＧｅｌ）Ａ−
０，５ｍ （バイオラット社、米国）、セファクリル（
Ｓｅｐｈａｃｒｙｌ） Ｓ ：１ＯＯＨＲ（ファルマ
シア社、スウェーデン）、セルロファインＧＳＬ−１０
００ｓｆ （生化学工業社１日本）、ＴＳＫゲルトヨバ
ールＨＷ５５（東ソー社、日本）などが挙げられる。溶
出液としては、例えばリン酸化緩衝生理食塩水などが使
用できる。Any suitable for the separation of substances of ~90,00G can be used, e.g. BioGel A-
0.5m (Biorat, USA), Sephacryl (
Sephacryl) S: 1OOHR (Pharmacia, Sweden), Cellulofine GSL-10
Examples include 00sf (Seikagaku Kogyo Co., Ltd. 1 Japan), TSK Gertjobar HW55 (Tosoh Co., Ltd., Japan), and the like. As the eluent, for example, phosphorylated buffered saline can be used.
ゲルろ過により、尿ＤＡＦの活性が、先に溶出される分
子量約１２万〜３０万の両分および後から溶出される分
子量約７万の画分の２つに分かれるが、いづれもＤＡＦ
活性を有するので、Ｎｕｓｓｅｎｚｗｅｉｇらが推定し
た膜固着型ＤＡＦおよび分泌型Ｄ Ａ Ｆ （Ｎａｔｕ
ｒｅ、３２５巻５４５〜５４９頁（１９８７年）〕に相
当すると考えられる。以下、最初の両分をＤＡＦｌ、後
の両分をＤＡＦｎと呼ぶ、このゲルろ過の工程後の尿Ｄ
ＡＦは、濃縮工程後の尿中のものに比して約１００倍精
製されており、ヒツジ感作赤血球を用いる溶血反応にお
ける測定（前記ａ）の０３コンペルターゼ活性抑制によ
る評価〕で、約３００ｎｇが１単位の活性を有している
。By gel filtration, the activity of urine DAF is divided into two fractions: a fraction with a molecular weight of approximately 120,000 to 300,000 eluted first, and a fraction with a molecular weight of approximately 70,000 eluted later, but both of them are DAF.
activity, the membrane-anchored DAF and secreted DAF (Natu
re, Vol. 325, pp. 545-549 (1987)]. Hereinafter, the first two fractions will be referred to as DAFl, and the latter two fractions will be referred to as DAFn.Urine D after this gel filtration process.
AF is approximately 100 times more purified than that in urine after the concentration process, and approximately 300 ng was measured in a hemolytic reaction using sheep-sensitized red blood cells (evaluation by inhibition of 03 compertase activity in a) above). It has 1 unit of activity.
このようにして得られた、尿ＤＡＦは、所望によりさら
に高速液体クロマトグラフィで精製することが可能であ
る。その際のクロマトグラフィー担体としては、蛋白質
分離用の任意の担体が使用可能である９例えば、ＴＳＫ
ゲル（ｇｅｌ）・フェニル（Ｐｈｅｎｙｌ） −５Ｐ
Ｗ （東ソー社１日本）、プロティン・パック（Ｐｒｏ
−ｔｅｉｎＰａｋ）ｇ−フェニＪしくＰｈｅｎｙｌ）
（ウォーターズ社、米国）などが挙げられる。クロマ
トグラフィーの溶出は、硫酸アンモニウムや硫酸ナトリ
ウムの濃度勾配で行うことができる。ＤＡＦＵの活性は
溶出剤の濃度が約ＬＭのところて溶出される。The urine DAF thus obtained can be further purified by high performance liquid chromatography if desired. As the chromatography carrier in this case, any carrier for protein separation can be used.9For example, TSK
Gel/Phenyl-5P
W (Tosoh Corporation 1 Japan), Protein Pack (Pro
-teinPak)g-Phenyl)
(Waters Inc., USA). Elution in chromatography can be performed using a concentration gradient of ammonium sulfate or sodium sulfate. The activity of DAFU is eluted at an eluent concentration of approximately LM.
高速液体クロマトグラフィーから得られるＤＡＦＵの精
製溶液は、熱処理工程後のものに比べて、比活性の点で
約４０〜６０Ｄ倍精製されており、ヒツジ感作赤血球を
用いる溶血反応による測定で、約１１００ｎが１単位の
活性を有している。熱処理後の全活性に対する高速液体
クロマトグラフィーから得られるＤＡＦＩＩ活性の回収
率は約１０〜２０％である。The purified solution of DAFU obtained from high-performance liquid chromatography is about 40 to 60 times more purified in terms of specific activity than that after the heat treatment process, and as measured by a hemolytic reaction using sheep sensitized red blood cells, it is about 1100n has 1 unit of activity. The recovery rate of DAFII activity obtained from high performance liquid chromatography relative to the total activity after heat treatment is about 10-20%.
得られた、２つの尿ＤＡＦの精製溶液は、そのまま使用
することもでき、あるいは凍結乾燥して保存してもよい
。The two obtained purified solutions of urinary DAF can be used as they are, or can be lyophilized and stored.
（実施例）
以下、実施例により本発明をより具体的に説明するが、
本発明はこれらの実施例に限定されるものではない。(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.
実施例１
ａ） の およ ゛
正常人尿１８３皇（ｐＨ５，１）をＵ Ｋ −５０膜に
よる限外ろ過で１見に濃縮し、これに硫酸アンモニウム
を３９０ｇ（６０％飽和）加え、４℃にて一晩静置した
。生じた沈澱物を遠心分離により集め、約７０ｓ１の蒸
留水に溶解し、これをイオン々換水に対して４°Ｃで２
４時間以上透析した。透析中に析出した沈澱物は、遠心
除去した。ヒツジ感作赤血球を用いる溶血反応で測定し
たところ、尿ＤＡＦの収量は３０５，８４０単位であり
、比活性は６５単位／ｒｓｇ蛋白質てあつた。透析後の
遠心分離により得られた上清を８０℃で１０分間熱処理
し、水中で冷却し、熱変性した蛋白質の沈澱を遠心分離
して、尿ＤＡＦを上清に回収した。この熱処理における
活性の収率は、はぼ１００％であり、比活性は約４倍上
昇した。Example 1 a) Normal human urine 183 ml (pH 5.1) was concentrated to one level by ultrafiltration using a UK-50 membrane, 390 g of ammonium sulfate (60% saturation) was added thereto, and the mixture was heated at 4°C. It was left undisturbed overnight. The resulting precipitate was collected by centrifugation, dissolved in about 70 s of distilled water, and mixed against deionized water at 4°C for 2 hours.
Dialysis was performed for over 4 hours. The precipitate deposited during dialysis was removed by centrifugation. As measured by a hemolytic reaction using sheep sensitized red blood cells, the yield of urinary DAF was 305,840 units, and the specific activity was 65 units/rsg protein. The supernatant obtained by centrifugation after dialysis was heat-treated at 80° C. for 10 minutes, cooled in water, and the heat-denatured protein precipitate was centrifuged to collect urine DAF in the supernatant. The yield of activity in this heat treatment was nearly 100%, and the specific activity increased about 4 times.
ｂ）゛ によ
上記ａ）で得られた熱処理上清に上清の１０分の１量の
０．２Ｍリン酸ナトリウム緩衝液（ｐＨ７，５）を加え
、全体を１４３１として、同緩衝液でｐＨ７，５に平衡
化したＤＥＡＥ−セファセルフ０Ｇ＋＊ｌを充填したカ
ラム（５，５ｘ ３量ｃｍ）に通し、同緩衝液中のθ〜
３００ｍＭ生理食塩水の一次濃度勾配により、流速５０
１／時間で１６１ずつ分画しながら溶出した。溶出の様
子を第１図に示す。b) To the heat-treated supernatant obtained in a) above, add 1/10 of the supernatant volume of 0.2M sodium phosphate buffer (pH 7.5) to make the total 1431, and add the same buffer. Pass through a column (5.5 x 3 volume cm) packed with DEAE-Sephaself 0G+*l equilibrated to pH 7.5, and
A first-order concentration gradient of 300 mM saline resulted in a flow rate of 50
It was eluted in fractions of 161 at a time of 1 hour. The elution process is shown in Figure 1.
得られた尿ＤＡＦの活性は、約３１６，０００単位（比
活性５４２単位／−ｇ）で、前記濃縮工程後の活性量に
対する収率は、約ｇθ％であった。The activity of the obtained urine DAF was about 316,000 units (specific activity 542 units/-g), and the yield based on the amount of activity after the concentration step was about gθ%.
実施例２ ル およ クロマラフ −によ
ル
実施例１で得た尿ＤＡＦをさらに精製するため、ＵＫ−
５０膜で限外ろ過し、 ６．２＋ｓｌまで濃縮し、その
５．２１をリン酸塩緩衝生理食塩水で平衡化したバイオ
ゲルＡ−０゜５■を１４０１充填したカラム（２，Ｓｘ
９３ｃｍ）に注入し、リン酸化緩衝生理食塩水で流速
２０１７時間で３．５＋ｓｌずつ分画しながら溶出した
。活性測定、５ＤＳ−ポリアクリルアミドゲル電気泳動
および前記Ｍｅｄｏｆらのイムノブロクティングにより
、尿ＤＡＦの両分を追跡して回収した。このゲルろ過で
、尿ＤＡＦは第２図に示すとおり２つの両分に分かれた
ので、フラクション５０〜８０番までの活性部分をＤＡ
ＦＩ、フラクション８１〜１００番までの活性部分なり
ＡＦｎとしてそれぞれ別個に集め、それぞれをＵ Ｋ
−５０膜を用いた限外ろ過により濃縮した後、凍結乾燥
して、精製された尿ＤＡＦを得た。ＤＡＦＩおよびＤＡ
ＦＩＩの収量は、それぞれ４０膜ｇおよび２０膜ｇであ
り、各々比活性が２．８９６単位／ｍｇ蛋白質および
：ｌ、８０６単位／ｌａｇであった、濃縮工程で回収さ
れた活性を基にした総括性回収率は６０％（うち、ＤＡ
ＦＩが６０％。Example 2 In order to further purify the urinary DAF obtained in Example 1 by chromaph and chromaph,
A column (2,Sx
93 cm) and eluted with phosphorylated buffered saline at a flow rate of 2017 hours while fractionating 3.5+sl. Both fractions of urinary DAF were tracked and collected by activity measurements, 5DS-polyacrylamide gel electrophoresis, and immunoblotting as described by Medof et al. Through this gel filtration, the urine DAF was divided into two parts as shown in Figure 2, so the active part of fractions 50 to 80 was filtered into DA
FI and fractions 81 to 100 were collected separately as the active part or AFn, and each was collected in the UK.
After concentration by ultrafiltration using a -50 membrane, the product was lyophilized to obtain purified urine DAF. DAFI and DA
The yields of FII were 40 g and 20 g, respectively, with specific activities of 2.896 units/mg protein and
:l, 806 units/lag, the overall recovery rate based on the activity recovered in the concentration step was 60% (of which DA
FI is 60%.
ＤＡＦ■が４０％）で、精製度は１２〜１５倍であった
。DAF■ was 40%), and the degree of purification was 12 to 15 times higher.
ロマ −
ゲルろ過で得られたＤＡＦｎ画分をＴＳＫゲル・フェニ
ル−５ＰＷ（内径７．５ｅｍ：長さ７．５ｅｍ）の疎水
クロマトグラフィーにより精製した。ＤＡＦｎ画分を１
Ｍ硫酸アンモニウムを含む０．１Ｍリン酸ナトリウム緩
衝液中でｐＨ７，０に調製し、同一緩衝液で平衡化した
カラムにのせ、１Ｍ〜ＯＭ硫酸アンモニウムのグラジェ
ント（４０分、流速０．５ｍｌ／分）で溶出した。第３
図に示すとおり、ＤＡＦｎ活性は単一ピークとして溶出
され、その比活性は９ 、３２５単位／ｓｇに上昇した
。The DAFn fraction obtained by Roma-gel filtration was purified by hydrophobic chromatography on TSK gel phenyl-5PW (inner diameter 7.5 em: length 7.5 em). DAFn fraction 1
A gradient of 1M to OM ammonium sulfate (40 min, flow rate 0.5 ml/min) was prepared on a column equilibrated with the same buffer and adjusted to pH 7.0 in 0.1 M sodium phosphate buffer containing M ammonium sulfate. It was eluted. Third
As shown in the figure, DAFn activity was eluted as a single peak, and its specific activity increased to 9,325 units/sg.
以上のようにして得たＤＡＦ ＩおよびＤＡＦ■のＮ末
アミノ酸配列を気相プロテインシークエンサー（アップ
ライトバイオシステム社製４７０Ａ型ンで、ＤＡＦＩＩ
のアミノ酸組成な自動アミノ酸分析機（日立 ８３５型
）でそれでれ分析して第１表および第２表に示した。The N-terminal amino acid sequences of DAF I and DAF■ obtained as described above were analyzed using a gas phase protein sequencer (Model 470A manufactured by Upright Biosystems) with
The amino acid compositions were analyzed using an automatic amino acid analyzer (Hitachi Model 835) and are shown in Tables 1 and 2.
Ｎ末アミノ酸配列はＤＡＦ−１，ＤＡＦ−ｎとも赤血球
膜ＤＡＦのＮ末アミノ酸配列と一致してい−た。The N-terminal amino acid sequences of both DAF-1 and DAF-n matched the N-terminal amino acid sequence of red blood cell membrane DAF.
さらに、前記Ｈｏｎｇらの方法および５ｅｙａらの方法
によりＤＡＦ−１，ＤＡＦ−■の活性および性質を調べ
たところ、赤血球１１ｊＤＡＦと同様の活性および免疫
学的性質を有することを確認した。Furthermore, when the activities and properties of DAF-1 and DAF-■ were investigated using the methods of Hong et al. and 5eya et al., it was confirmed that they had the same activity and immunological properties as red blood cell 11j DAF.
第１表
（アミノ酸の表記は一文字表記法によった。）第２表
アミノ酸
ＤＡＦ ■（ａ）
（ｍｏｌ ％）
以上の実施例１および実施例２を通しての活性および蛋
白質の回収量ならびに比活性の上昇をまとめて第３表に
示す。Table 1 (Amino acids are expressed using the one-letter notation system.) Table 2 Amino acid DAF ■(a) (mol %) Activity and protein recovery amount and specific activity in Examples 1 and 2 above. The increase is summarized in Table 3.
ｓｐ ｈｒ ｅｒ １ｕ Ｐｒ。sp hr er 1u Pr.
ｌｙ
ｌａ
ａｌ
ｙｓ
ｅｔ
１ｅ
ｅｕ
ｙｒ
ｈｅ
ｙｓ
ｉｓ
ｒｇ
ｒｐ
第３表
全単位
比活性（単位／―ｇ）
尿
硫酸アンモニウム
６０％沈澱
熱処理
ＤＥＡＥ−セファセル
バイオゲル＾−〇、５■
ＤＡＦＩ
ＤＡＦ■
＋１ＰＬｃ （フェニル−５ＰＷ）
ＡＦｎ
１５．０１１０
４．６７６
１．３９９
２．３
４４４、５６３
３０５、１１４０
３４６．１フ０
３１６．０１＞７
１２０．３１０
７２．５８５
２１．１９１
２、８９１ｉ
３、１１０６
９．３２５
（ａ） 数値は２４時間加水分解のロスを補正してい
ない。Table 3 Total unit specific activity (unit/-g) Urine ammonium sulfate 60% precipitation heat-treated DEAE-Sephacel biogel^-〇, 5■ DAFI DAF■ +1PLc (Phenyl -5PW) AFn 15.0110 4.676 1.399 2.3 444, 563 305, 1140 346.1fu0 316.01>7 120.310 72.585 21.191 2, 891i 3, 1106 9.325 (a) Values are not corrected for 24 hour hydrolysis losses.
（ｂ） 未同定
（発明の効果〕
本発明によれば、尿ＤＡＦを工業的生産に適温３図は本
発明におけるゲルろ過で得られたＤＡＦｎのＨＰＬＣパ
ターンを示す図である。(b) Unidentified (Effect of the Invention) According to the present invention, temperature suitable for industrial production of urine DAF. Figure 3 is a diagram showing the HPLC pattern of DAFn obtained by gel filtration in the present invention.
する方法で生産することが可能である１本発明は、比較
的安価で大量入手の容易な尿を原料とし、この原料から
高い回収率で、比活性が約３．０００単位／ｍｇ以上の
尿ＤＡＦを大量に製造することが可能である。このよう
に高い比活性のＤＡＦを工業的生産規模で製造する方法
は従来報告されていない、従って、本発明はＤＡＦを診
断および治療の目的て応用する可能性を初めて提供した
ものである。1 The present invention uses urine, which is relatively inexpensive and easily available in large quantities, as a raw material, and produces urine with a specific activity of about 3,000 units/mg or more with a high recovery rate from this raw material. It is possible to manufacture DAF in large quantities. A method for producing DAF with such high specific activity on an industrial production scale has not been previously reported, and therefore, the present invention provides for the first time the possibility of applying DAF for diagnostic and therapeutic purposes.
さらに、本発明は尿ＤＡＦの大量生産を可能としたため
、ＤＡＦの生理活性や化学構造の研究促進にも役立つこ
とが期待される。Furthermore, since the present invention enables mass production of urinary DAF, it is expected to be useful in promoting research into the physiological activity and chemical structure of DAF.
第１図は本発明におけＤＥＡＥ−セファセルでのイオン
交換クロマトグラフィーの画分と生理活性との関係を示
す図、
第２図は本発明におけるゲルろ過で、ＤＡＦ工とＤＡＦ
ｎが分離する様子を示す図。
持田製薬株式会社
（ほか２名）
第
図
（３，５ｍｊ／試験１）Figure 1 shows the relationship between the fractions of ion exchange chromatography using DEAE-Sephacel and physiological activity in the present invention. Figure 2 shows gel filtration in the present invention.
A diagram showing how n is separated. Mochida Pharmaceutical Co., Ltd. (and 2 others) Figure (3,5mj/Test 1)
Claims (10)
ぜしめ、該上清を陰イオン交換体からなる吸着剤と接触
させ、次いで該吸着剤に吸着された成分を溶出して、ヒ
トを含む哺乳動物の補体系の活性化を抑制する物質を含
む溶出画分を回収することを特徴とする、ディケイ・ア
クセレレーティング・ファクター （Ｄｅｃａｙ Ａｃｃｅｌｅｒａｔｉｎｇ Ｆａｃｔｏ
ｒ，ＤＡＦ）の製造方法。(1) After concentrating the urine, it is subjected to heat treatment to produce a precipitate and a supernatant, the supernatant is brought into contact with an adsorbent made of an anion exchanger, and the components adsorbed on the adsorbent are then eluted. Decay Accelerating Factor (Decay Accelerating Factor), which is characterized by recovering an elution fraction containing a substance that suppresses the activation of the complement system of mammals including humans.
r, DAF) manufacturing method.
ぜしめ、該上清を陰イオン交換体からなる吸着剤と接触
させ、次いで該吸着剤に吸着された成分を溶出して、ヒ
トを含む哺乳動物の補体系の活性化を抑制する物質を含
む溶出画分を回収し、該画分を分子量５０，０００〜９
０，０００の物質の分離に適する担体を用いたゲルろ過
に付し、所望によりさらに高速液体クロマトグラフィー
に付して精製することを特徴とする、ディケイ・アクセ
レレーティン グ・ファクター（ＤＡＦ）の製造方法。(2) After concentrating the urine, it is subjected to heat treatment to produce a precipitate and a supernatant, and the supernatant is brought into contact with an adsorbent made of an anion exchanger, and then the components adsorbed on the adsorbent are eluted. The elution fraction containing a substance that suppresses the activation of the complement system of mammals including humans is collected, and the fraction is divided into a fraction with a molecular weight of 50,000 to 9.
Production of Decay Accelerating Factor (DAF), characterized in that it is subjected to gel filtration using a carrier suitable for the separation of 0,000 substances, and optionally further purified by high performance liquid chromatography. Method.
過させない限外ろ過膜によるろ過濃縮およ び、該ろ過濃縮物に対して５０〜７０％飽和濃度の硫酸
アンモニウムを用いる沈澱濃縮の組み合わせで行なわれ
る、請求項１または２記載の方法。(3) Urine concentration is performed by a combination of filtration concentration using an ultrafiltration membrane that does not allow components with a molecular weight of 50,000 or more to pass through, and precipitation concentration using ammonium sulfate at a saturation concentration of 50 to 70% of the filtration concentrate. 3. The method according to claim 1 or 2.
５０，０００、ダイアフローメンブランＰＭ３０、ダイ
アフローメンブランＸＭ５０およびＵＦ−１００ＰＳか
らなる群から選択される請求項３記載の方法。4. The method of claim 3, wherein the ultrafiltration membrane is selected from the group consisting of UK-50, Ultrafilter 50,000, Diaflow Membrane PM30, Diaflow Membrane XM50, and UF-100PS.
加熱により行なわれる、請求項１または２記載の方法。(5) The method according to claim 1 or 2, wherein the heat treatment is performed by heating at 70°C to 100°C for 5 to 20 minutes.
ｈａｃｅｌ）、ＤＥＡＥセルロース（Ｃｅｌｌｕｌｏｓ
ｅ）ＤＥ５２、ＤＥＡＥセルロースＤＥ３２、セレック
ス（Ｃｅｌｌｅｘ）ＤおよびＴＳＫゲル（ｇｅｌ）トヨ
バール６５０Ｍからなる群から選択されるセルロースイ
オン交換体である、請求項１または２記載の方法。(6) The anion exchanger is DEAE Sephacel (Sep
hacel), DEAE cellulose (Cellulos)
3. The method of claim 1 or 2, wherein e) a cellulose ion exchanger selected from the group consisting of DE52, DEAE cellulose DE32, Cellex D and TSK gel Toyovar 650M.
われる、請求項１または２記載の方 法。(7) The method according to claim 1 or 2, wherein elution from the adsorbent is performed by changing ionic strength.
カリウムの濃度勾配により行なわれ る、請求項７記載の方法。(8) The method according to claim 7, wherein the ionic strength is changed by a concentration gradient of sodium chloride or potassium chloride.
ある、請求項２記載の方法。(9) The method according to claim 2, wherein the eluent of gel filtration is phosphate buffered saline.
ンモニウムまたは硫酸ナトリウムの濃度勾配により行な
われる、請求項２記載の方 法。(10) The method according to claim 2, wherein the elution in high performance liquid chromatography is performed using a concentration gradient of ammonium sulfate or sodium sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63163061A JPH0211600A (en) | 1988-06-30 | 1988-06-30 | Production of urine daf |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63163061A JPH0211600A (en) | 1988-06-30 | 1988-06-30 | Production of urine daf |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0211600A true JPH0211600A (en) | 1990-01-16 |
Family
ID=15766435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63163061A Pending JPH0211600A (en) | 1988-06-30 | 1988-06-30 | Production of urine daf |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0211600A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5353021A (en) * | 1991-09-04 | 1994-10-04 | Matsushita Electric Industrial Co., Ltd. | Apparatus for measuring moving state of vehicle in tunnel |
-
1988
- 1988-06-30 JP JP63163061A patent/JPH0211600A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5353021A (en) * | 1991-09-04 | 1994-10-04 | Matsushita Electric Industrial Co., Ltd. | Apparatus for measuring moving state of vehicle in tunnel |
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