JP2723169B2 - Method for producing hemagglutinin - Google Patents
Method for producing hemagglutininInfo
- Publication number
- JP2723169B2 JP2723169B2 JP6085600A JP8560094A JP2723169B2 JP 2723169 B2 JP2723169 B2 JP 2723169B2 JP 6085600 A JP6085600 A JP 6085600A JP 8560094 A JP8560094 A JP 8560094A JP 2723169 B2 JP2723169 B2 JP 2723169B2
- Authority
- JP
- Japan
- Prior art keywords
- activity
- weight
- hemagglutinin
- final concentration
- agglutinating
- 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.)
- Expired - Lifetime
Links
- 101710154606 Hemagglutinin Proteins 0.000 title claims description 16
- 101710093908 Outer capsid protein VP4 Proteins 0.000 title claims description 16
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 title claims description 16
- 101710176177 Protein A56 Proteins 0.000 title claims description 16
- 239000000185 hemagglutinin Substances 0.000 title claims description 16
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 241000206572 Rhodophyta Species 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 9
- 238000005185 salting out Methods 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 241001661641 Verrucosa Species 0.000 claims description 3
- 239000000356 contaminant Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 26
- 230000004523 agglutinating effect Effects 0.000 description 16
- 239000011780 sodium chloride Substances 0.000 description 13
- 210000003743 erythrocyte Anatomy 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 239000008363 phosphate buffer Substances 0.000 description 7
- 101710186708 Agglutinin Proteins 0.000 description 6
- 101710146024 Horcolin Proteins 0.000 description 6
- 101710189395 Lectin Proteins 0.000 description 6
- 101710179758 Mannose-specific lectin Proteins 0.000 description 6
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 6
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 6
- 239000000910 agglutinin Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 230000035931 haemagglutination Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000004220 aggregation Methods 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 238000005194 fractionation Methods 0.000 description 4
- 239000012521 purified sample Substances 0.000 description 4
- 239000012047 saturated solution Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108010062580 Concanavalin A Proteins 0.000 description 3
- 230000004931 aggregating effect Effects 0.000 description 3
- 238000005345 coagulation Methods 0.000 description 3
- 230000015271 coagulation Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000003067 hemagglutinative effect Effects 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010044715 asialofetuin Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108060002885 fetuin Proteins 0.000 description 2
- 102000013361 fetuin Human genes 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- 241000189617 Chorda Species 0.000 description 1
- 241001467331 Gracilaria sp. Species 0.000 description 1
- 241001507103 Hypnea japonica Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 210000003371 toe Anatomy 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、オゴノリ属紅藻類（Ｇ
ｒａｃｉｌａｒｉａ ｓｐ．）を原料として、特異的な
性質をもつ新規な赤血球凝集素を製造する方法に関する
ものである。The present invention relates to a red algae of the genus Ogonori (G
raciliaria sp. ) As a raw material, and a method for producing a novel hemagglutinin having specific properties.
【０００２】[0002]
【従来の技術】赤血球凝集素は、各動物の赤血球に対し
特異的な挙動を示すので、医療、製薬、生化学分野など
における検査用試薬や分離用材料として広く用いられて
いる。この赤血球凝集素は、動物由来のものと植物由来
のものとに大別されるが、大量に入手しうること、処理
しやすいことなどを考慮して、植物由来のものが実用上
注目されている。2. Description of the Related Art Hemagglutinin exhibits a specific behavior with respect to erythrocytes of each animal, and is therefore widely used as a test reagent or a separation material in medical, pharmaceutical, biochemical fields and the like. This hemagglutinin is roughly classified into those derived from animals and those derived from plants. I have.
【０００３】これまで、この植物由来の赤血球凝集素と
しては、陸上植物由来のものとしてタチナタマメからの
コンカナバリンＡ（Ｃｏｎ Ａ）や小麦からの小麦胚芽
レクチン（ＷＧＡ）などや［「ジャーナル・オブ・バク
テリオロジー（Ｊ．Ｂａｃｔｅｒｉｏｌ．）」第３２
巻、第２２７〜２３７ページ（１９３６年）］、海洋植
物由来のものとしてオゴノリ（Ｇｒａｃｉｌａｒｉａ
ｖｅｒｒｕｃｏｓａ）からのＧＶＡＩ、カギイバラノリ
（Ｈｙｐｎｅａ ｊａｐｏｎｉｃａ）からのＨｙｐｎｉ
ｎ Ａ、Ｂ、Ｃ及びＤ［「ブレタン・オブ・ザ・ジャパ
ニーズ・ソサエティ・オブ・サイエンティフィック・フ
ィッシェリイズ（Ｂｕｌ．Ｊａｐ．Ｓｏｃ．Ｓｃｉ．Ｆ
ｉｓｈｅ．）」第４７巻、第１０７９〜１０８４ページ
（１９８１年）］などが知られている。Hitherto, hemagglutinin derived from plants has been known as a terrestrial plant, such as concanavalin A (Con A) from jack bean, wheat germ lectin (WGA) from wheat, and [Journal of Bacterio J. Bacteriol. "No. 32
Vol., Pp. 227 to 237 (1936)], as a plant derived from marine plants (Graciliaria).
verrucosa) and Hypni from Hypnea japonica.
n A, B, C and D ["Bultan of the Japanese Society of Scientific Fisheries (Bul.Jap.Soc.Sci.F.)
ishe. 47), pp. 1079-1084 (1981)].
【０００４】しかしながら、陸上植物由来のものは、凝
集活性の高い標品は比較的容易に得ることができる
が、、単糖類や二糖類のような単純な糖によっても赤血
球凝集活性が阻害されるため、認識糖鎖選択性が低いと
いう欠点があるし、また海洋植物由来のものは、単糖類
や二糖類によって赤血球凝集活性が阻害されず、フェツ
イン、アシアロフェツインのような糖タンパク質によっ
て阻害されるため、認識糖鎖選択性が高いと考えられる
が、凝集活性の高い標品を得ることが困難であるという
欠点を有する上に、両者ともイオン強度の変化により凝
集活性の制御を行うことができないという欠点をもって
いる。[0004] However, terrestrial plant-derived preparations having a high agglutinating activity can be obtained relatively easily, but the hemagglutinating activity is also inhibited by simple sugars such as monosaccharides and disaccharides. Therefore, there is a disadvantage that recognition sugar chain selectivity is low, and those derived from marine plants are not inhibited by hemagglutination activity by monosaccharides and disaccharides, but are inhibited by glycoproteins such as fetuin and asialofetuin. Therefore, it is thought that the recognition sugar chain selectivity is high, but it has a disadvantage that it is difficult to obtain a sample having high aggregating activity, and both of them can control the aggregating activity by changing the ionic strength. It has the disadvantage of not being able to.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、このような
事情のもとで、イオン強度により凝集活性が制御でき、
認識糖鎖選択性に優れ、かつ比活性が高い新規な赤血球
凝集素を提供することを目的としてなされたものであ
る。SUMMARY OF THE INVENTION Under such circumstances, the present invention can control the aggregating activity by ionic strength.
The purpose of the present invention is to provide a novel hemagglutinin having excellent recognition sugar chain selectivity and high specific activity.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、植物由
来、特に海洋植物由来の赤血球凝集素について、種々研
究を重ねた結果、オゴノリ属紅藻類（Ｇｒａｃｉｌａｒ
ｉａ ｓｐ．）から、特定な条件下で抽出された赤血球
凝集素が、凝集活性が高く認識糖類選択性が高い上に、
イオン強度により凝集活性を制御しうることを見出し、
この知見に基づいて本発明をなすに至った。The present inventors have conducted various studies on haemagglutinin derived from plants, particularly marine plants, and as a result, have found that the red alga of the genus Ogonori (Gracilar).
ia sp. ), Hemagglutinin extracted under specific conditions has high agglutinating activity and high recognition saccharide selectivity,
Finding that coagulation activity can be controlled by ionic strength,
The present invention has been accomplished based on this finding.
【０００７】すなわち、本発明は、オゴノリ属紅藻類
（Ｇｒａｃｉｌａｒｉａ ｓｐ．）からのリン酸塩緩衝
液抽出液に、先ず最終濃度３０〜４０重量％になるまで
硫酸アンモニウムを加えて第１段目の塩析を行い、沈殿
した夾雑物を除去したのち、さらにその抽出液に最終濃
度７０重量％程度になるまで硫酸アンモニウムを加えて
第２段目の塩析を行い、沈殿として得られる粗活性画分
を分取し、次いでこれからクロマトグラフィーにより分
子量１００，０００以上の画分を分離、回収することを
特徴とする高活性赤血球凝集素の製造方法を提供するも
のである。[0007] That is, the present invention relates to a method for preparing a first-stage salt by adding ammonium sulfate to a phosphate buffer extract from a red alga of the genus Gracilaria sp. To a final concentration of 30 to 40% by weight. After removing precipitated contaminants, ammonium sulfate was further added to the extract to a final concentration of about 70% by weight to perform a second-stage salting-out, and a crude active fraction obtained as a precipitate was obtained. It is intended to provide a method for producing highly active hemagglutinin, comprising separating and collecting a fraction having a molecular weight of 100,000 or more by chromatography and then fractionating the fraction by chromatography.
【０００８】本発明方法における原料としては、オゴノ
リ属紅藻類が用いられるが、特にオゴノリ（Ｇｒａｃｉ
ｌａｒｉａ ｖｅｒｒｕｃｏｓａ）、ツルシラモ（Ｇｒ
ａｃｉｌａｒｉａ ｃｈｏｒｄａ）、それらの亜種が好
ましい。これらの紅藻類は、寒海にも存在するが特に暖
海に多く、わが国ではほとんどすべての海岸地帯に分布
しており、寒天の増量物や刺身のつまなどに用いられて
いる。[0008] As a raw material in the method of the present invention, red alga of the genus Ogonori is used.
Laria verrucosa), Tsurushiramo (Gr.
aciaria chorda) and their subspecies are preferred. These red algae are also present in the cold sea but particularly in the warm sea, and are distributed in almost all coastal areas in Japan, and are used as agar augmentation and sashimi toes.
【０００９】本発明方法に従えば、上記の紅藻類原料に
（イ）水溶性画分の抽出工程、（ロ）粗活性画分の分取
工程、及び（ハ）凝集素の精製工程を順次施すことによ
り、所望の赤血球凝集素を得ることができる。According to the method of the present invention, (a) a step of extracting a water-soluble fraction, (b) a step of fractionation of a crude active fraction, and (c) a step of purifying agglutinin are sequentially performed on the red algae raw material. By performing the application, a desired hemagglutinin can be obtained.
【００１０】前記各工程の好適な実施態様について説明
すると、まず（イ）工程においては、原料の紅藻類に緩
衝液、例えば塩化ナトリウム含有リン酸緩衝液を加えて
ホモゲナイズしたのち、遠心分離処理し、上澄である粗
抽出液を得る。次に（ロ）工程においては、前記（イ）
工程で得られた抽出液に、まず最終濃度が３０〜４０重
量％程度の飽和溶液になるように硫酸アンモニウムを加
えて１段目の塩析を行い、生成した沈殿を遠心分離処理
により除去する。この操作で色素などの夾雑物が沈殿画
分として除去される。次いで、遠心分離処理で得た上澄
に最終濃度７０重量％程度の飽和溶液になるように硫酸
アンモニウムを加えて２段目の塩析を行い、生成した沈
殿を遠心分離処理により分別したのち、この沈殿画分を
塩化ナトリウム含有リン酸緩衝液などの緩衝液で再溶解
して粗活性画分を得る。A preferred embodiment of each of the above steps will be described. First, in step (a), a buffer solution, for example, a phosphate buffer solution containing sodium chloride is added to the starting red algae, homogenized, and then centrifuged. To obtain a crude extract which is the supernatant. Next, in the (b) step, the (a)
First, ammonium sulfate is added to the extract obtained in the step so as to obtain a saturated solution having a final concentration of about 30 to 40% by weight, and the first step of salting out is performed, and the generated precipitate is removed by centrifugation. By this operation, impurities such as dyes are removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by the centrifugal separation treatment so as to obtain a saturated solution having a final concentration of about 70% by weight, salting out was performed in the second stage, and the formed precipitate was separated by centrifugal separation. The precipitated fraction is redissolved in a buffer such as a phosphate buffer containing sodium chloride to obtain a crude active fraction.
【００１１】最後に、（ハ）工程においては、前記
（ロ）工程で得られた粗活性画分を、塩化ナトリウム含
有リン酸緩衝液などの緩衝液に対して透析後、クロマト
グラフィーにより分離し、精製赤血球凝集素を得る。こ
の際、クロマトグラフィーとしては、イオン交換クロマ
トグラフィー又はゲルろ過クロマトグラフィーあるいは
それらの組合せが好ましく用いられる。Finally, in the step (c), the crude active fraction obtained in the step (b) is dialyzed against a buffer such as a phosphate buffer containing sodium chloride and then separated by chromatography. To obtain purified hemagglutinin. At this time, as the chromatography, ion exchange chromatography or gel filtration chromatography or a combination thereof is preferably used.
【００１２】本発明方法で得られる赤血球凝集素は、新
規物質で（ａ）プロナーゼ処理したヒツジ赤血球を凝集
させる性質を有し、かつこの凝集活性が単純な単糖類又
は二糖類では阻害されないが、フェツインやアシアロフ
ェツインのような糖タンパク質で阻害され、（ｂ）ウサ
ギ赤血球に対する凝集活性がイオン強度により変化し、
かつ（ｃ）球状タンパク質を標準分子量物質として使用
したときのゲルろ過クロマトグラフィーにおいて、分子
量１００，０００以上に相当する画分に溶出するという
特徴を有している。The hemagglutinin obtained by the method of the present invention has the property of agglutinating (a) sheep erythrocytes treated with a novel substance with pronase, and this agglutinating activity is not inhibited by simple monosaccharides or disaccharides. Inhibited by glycoproteins such as fetuin and asialofetuin, (b) agglutinating activity on rabbit erythrocytes is changed by ionic strength,
And (c) it is characterized in that it elutes in a fraction corresponding to a molecular weight of 100,000 or more in gel filtration chromatography using a globular protein as a standard molecular weight substance.
【００１３】[0013]
【発明の効果】本発明方法により得られる赤血球凝集素
は、紅藻類由来の新規なものであって、イオン強度によ
り凝集活性が制御でき、認識糖鎖選択性に優れ、かつ比
活性が高いなどの特性を有している。この特性を利用す
れば、イオン強度により、凝集素の凝集活性を制御する
ことができるため、臨床分野、医療分野、生化学工業分
野などにおいて、例えば検査用試薬や分離材料などとし
て使用することができる。The hemagglutinin obtained by the method of the present invention is a novel hemagglutinin derived from red algae, whose agglutinating activity can be controlled by ionic strength, its recognition sugar chain selectivity is excellent, and its specific activity is high. It has the following characteristics. If this property is used, the agglutinating activity of agglutinin can be controlled by ionic strength, so that it can be used as a test reagent or a separation material in the clinical field, medical field, biochemical industry field, etc. it can.
【００１４】[0014]
【実施例】次に、実施例により本発明をさらに詳細に説
明するが、本発明はこれらの例によってなんら限定され
るものではない。Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.
【００１５】実施例１ （イ）水溶性画分の抽出工程 ツルシラモ（徳島県吉野川河口域産）を０．１５Ｍ塩化
ナトリウム水溶液で洗浄後、天日乾燥して乾燥物を得
た。この乾燥物１００ｇに０．１５Ｍ塩化ナトリウム含
有１００ｍＭリン酸緩衝液（ｐＨ６．９）７００ｍｌを
加えてホモゲナイズしたのち、このホモゲナイズした液
を４℃で６時間放置後、遠心分離して上澄である粗抽出
液を得た。Example 1 (a) Extraction step of water-soluble fraction Tsurusilamo (produced from the estuary of Yoshino River, Tokushima Prefecture) was washed with a 0.15 M aqueous sodium chloride solution and then dried in the sun to obtain a dried product. To 100 g of the dried product, 700 ml of 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride was added and homogenized, and the homogenized solution was left at 4 ° C. for 6 hours, centrifuged, and the supernatant was obtained. A crude extract was obtained.
【００１６】（ロ）粗活性画分の分別工程 次いで、この粗抽出液に、最終濃度３５重量％飽和溶液
になるように硫酸アンモニウムを加えて１段目の塩析を
行った。硫酸アンモニウムを添加終了後、４℃で１時間
放置したのち、生成した沈殿を遠心分離して除去した。
この操作で色素などの夾雑物が沈殿画分として除去され
た。次に、遠心分離で得た上澄に、最終濃度７０重量％
飽和溶液になるように硫酸アンモニウムを加えて２段目
の塩析を行った。硫酸アンモニウムを添加終了後、４℃
で一晩放置したのち、生成した沈殿を遠心分離して分別
した。分別した沈殿画分を、０．１５Ｍ塩化ナトリウム
含有１００ｍＭリン酸緩衝液（ｐＨ６．９）で再溶解
し、粗活性画分を得た。得られた粗活性画分のウサギ赤
血球に対する赤血球凝集活性は２５６単位であり、比活
性は３３７２．９単位／ｍｇプロテイン、活性回収率は
６２．４％であった。ここで、凝集活性の単位は、凝集
活性が検出できる試料の最大希釈率の逆数と定義した。
これらの結果を表１に示す。(B) Separation Step of Crude Active Fraction Next, ammonium sulfate was added to this crude extract so as to obtain a 35% by weight saturated solution at the final concentration, and the first stage of salting out was carried out. After the addition of ammonium sulfate, the mixture was allowed to stand at 4 ° C. for 1 hour, and the formed precipitate was removed by centrifugation.
By this operation, impurities such as dyes were removed as a precipitate fraction. Next, the supernatant obtained by centrifugation was added to a final concentration of 70% by weight.
Ammonium sulfate was added so as to obtain a saturated solution, and the second stage of salting out was performed. After addition of ammonium sulfate, 4 ° C
, And the resulting precipitate was separated by centrifugation. The fractionated precipitate fraction was redissolved in 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride to obtain a crude active fraction. The obtained crude active fraction had a hemagglutinating activity on rabbit erythrocytes of 256 units, a specific activity of 3372.9 units / mg protein, and an activity recovery of 62.4%. Here, the unit of the agglutinating activity was defined as the reciprocal of the maximum dilution ratio of the sample from which the agglutinating activity could be detected.
Table 1 shows the results.
【００１７】（ハ）凝集素の精製工程 次に、このようにして得られた粗活性画分を、０．１５
Ｍ塩化ナトリウム含有１００ｍＭリン酸緩衝液（ｐＨ
６．９）に対して透析後、ＴＳＫｇｅｌＤＥＡＥ‐５Ｐ
Ｗを用いたイオン交換クロマトグラフィーにより分離
し、精製標品を得た。得られた精製標品のウサギ赤血球
に対する赤血球凝集活性を示す最小タンパク質濃度は
０．８７６３μｇ／ｍｌであった。以上の結果から、本
発明方法によると、紅藻類由来の赤血球凝集素が、その
活性を保持したまま効果的に得られることが分かる。(C) Step of Purifying Agglutinin Next, the crude active fraction thus obtained was
M sodium chloride containing 100 mM phosphate buffer (pH
After dialysis against 6.9), TSKgelDEAE-5P
Separation was performed by ion exchange chromatography using W to obtain a purified sample. The minimum protein concentration showing hemagglutination activity on rabbit erythrocytes of the obtained purified sample was 0.8763 μg / ml. From the above results, it can be seen that according to the method of the present invention, red algae-derived hemagglutinin can be effectively obtained while maintaining its activity.
【００１８】精製標品について、ウサギ赤血球に対する
凝集活性のイオン強度依存性を検討したところ、０．１
５Ｍ塩化ナトリウム濃度での凝集活性は２０４８単位で
あり、一方０．４Ｍ塩化ナトリウム濃度での凝集活性は
８単位であった。これらの結果を表２に示す。The ionic strength dependence of the agglutination activity on the rabbit erythrocytes of the purified sample was examined.
Aggregation activity at 5M sodium chloride concentration was 2048 units, while at 0.4M sodium chloride concentration was 8 units. Table 2 shows the results.
【００１９】比較例１ 実施例１‐（ロ）の粗活性画分の分別工程において、硫
酸アンモニウム添加による２段階の塩析による分別処理
の代わりに、５０重量％エタノールによる分別処理
［「フィトケミストリー（Ｐｈｙｔｏｃｈｅｍｉｓｔｒ
ｙ）」第２７巻、第２０６３〜２０６７ページ（１９８
８年）参照］を行った以外は、実施例１と同様にして粗
活性画分を得た。この粗活性画分のウサギ赤血球に対す
る赤血球凝集活性は４単位、比活性は５３．４単位／ｍ
ｇプロテイン、活性回収率は５．０％であった。これら
の結果を表１に示す。Comparative Example 1 In the fractionation step of the crude active fraction of Example 1- (b), instead of the fractionation treatment by two-stage salting out by adding ammonium sulfate, a fractionation treatment with 50% by weight ethanol [“Phytochemistry ( Phytochemistr
y) "Volume 27, Pages 2063-2067 (198
8)), but a crude active fraction was obtained in the same manner as in Example 1. Hemagglutination activity of this crude active fraction on rabbit erythrocytes is 4 units, and specific activity is 53.4 units / m.
g protein, activity recovery was 5.0%. Table 1 shows the results.
【００２０】比較例２ 「Ｃｏｍｐ．Ｂｉｏｃｈｅｍ．Ｐｈｙｓｉｏｌ．」第１
０２Ｂ巻、第４４５〜４４９ページ（１９９２年）に記
載されている方法に従って、紅藻類由来の赤血球凝集素
を得た。得られた粗活性画分の赤血球凝集活性は１６単
位、比活性は１４９．５単位／ｍｇプロテイン、活性回
収率は１９．５％であった。これらの結果を表１に示
す。また、精製標品のウサギ赤血球に対する赤血球凝集
活性を示す最小タンパク質濃度は３２．６μｇ／ｍｌで
あり、実施例１の約１／４０の比活性に相当した。Comparative Example 2 "Comp. Biochem. Physiol."
Hemagglutinin derived from red algae was obtained according to the method described in Volume 02B, 445-449 (1992). The obtained crude active fraction had a hemagglutination activity of 16 units, a specific activity of 149.5 units / mg protein, and an activity recovery of 19.5%. Table 1 shows the results. Further, the minimum protein concentration of the purified sample showing the hemagglutinating activity on rabbit erythrocytes was 32.6 μg / ml, which corresponded to the specific activity of about 1/40 of Example 1.
【００２１】比較例３ 紅藻類から「Ｃｏｍｐ．Ｂｉｏｃｈｅｍ．Ｐｈｙｓｉｏ
ｌ．」第１０２Ｂ巻、第４４５〜４４９ページ（１９９
２年）記載の方法に従って精製した分子量約５０，００
０の凝集素について、ウサギ赤血球に対する凝集活性の
イオン濃度依存性を検討した。０．１５Ｍ塩化ナトリウ
ム濃度及び０．４Ｍ塩化ナトリウム濃度での凝集活性は
ともに１０２４単位であり、凝集活性のイオン強度依存
性は見られなかった。これらの結果を表２に示す。Comparative Example 3 From red algae, "Comp. Biochem. Physio"
l. 102B, 445-449 (199
2 years) purified according to the method described in US Pat.
Regarding agglutinin of 0, the ion concentration dependence of the agglutinating activity on rabbit erythrocytes was examined. The aggregation activity at the concentrations of 0.15 M sodium chloride and 0.4 M sodium chloride was 1024 units, and the ionic strength dependence of the aggregation activity was not observed. Table 2 shows the results.
【００２２】比較例４ Ｃｏｎ Ａ［和光純薬（株）製］２５ｍｇをリン酸緩衝
液１００ｍｌに溶解し、ウサギ赤血球に対する赤血球凝
集活性のイオン強度依存性を検討した。０．１５Ｍ塩化
ナトリウム濃度及び０．４Ｍ塩化ナトリウム濃度での凝
集活性はともに６４単位であり、凝集活性のイオン強度
依存性は見られなかった。これらの結果を表２に示す。Comparative Example 4 25 mg of Con A (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 100 ml of phosphate buffer, and the ionic strength dependence of the hemagglutination activity on rabbit erythrocytes was examined. The coagulation activity at both the 0.15 M sodium chloride concentration and the 0.4 M sodium chloride concentration was 64 units, and the ionic strength dependence of the coagulation activity was not observed. Table 2 shows the results.
【００２３】[0023]
【表１】 注１）凝集活性は粗活性画分を連続希釈し、凝集活性を
示す最大希釈率から算出した。 ２）活性回収率は粗抽出液全量の凝集活性を１００％と
して算出した。[Table 1] Note 1) Agglutinating activity was calculated from the maximum dilution rate showing the agglutinating activity by serially diluting the crude active fraction. 2) The activity recovery rate was calculated assuming that the aggregation activity of the whole crude extract was 100%.
【００２４】[0024]
【表２】 注１）凝集活性は精製凝集素を連続希釈し、凝集活性を
示す最大希釈率から算出した。[Table 2] Note 1) Agglutinating activity was calculated from the maximum dilution showing the agglutinating activity by serially diluting purified agglutinin.
【００２５】表１から明らかなように、実施例の粗活性
画分は、比較例１及び２のものに比べて、凝集活性、比
活性、活性回収率がともに高く、活性回収率は比較例１
の約１２倍、比較例２の約３倍、比活性は比較例１の約
６３倍、比較例２の約２３倍である。また、表２から実
施例の精製凝集素は、比較例３及び４のものと異なり、
ウサギ赤血球に対する凝集活性がイオン強度により制御
されることが分かる。As is evident from Table 1, the crude active fraction of the example has higher agglutinating activity, specific activity and activity recovery rate than those of the comparative examples 1 and 2, and the activity recovery rate is comparative example. 1
About 12 times that of Comparative Example 2, about 63 times that of Comparative Example 1, and about 23 times that of Comparative Example 2. Further, from Table 2, the purified agglutinin of Example is different from those of Comparative Examples 3 and 4,
It can be seen that the agglutinating activity on rabbit erythrocytes is controlled by the ionic strength.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 李 忠富 香川県高松市花ノ宮町二丁目３番３号 工業技術院四国工業技術研究所内 (72)発明者 上嶋 洋 香川県高松市花ノ宮町二丁目３番３号 工業技術院四国工業技術研究所内 (56)参考文献 特開 平３−279396（ＪＰ，Ａ) ＢＵＬＬ．ＪＡＰＡＮ．ＳＯＣ．ＳＣ Ｉ．ＦＩＳＨ．，47（８），Ｐ．1079− 1084（1981) ＢＵＬＬ．ＪＡＰＡＮ．ＳＯＣ．ＳＣ Ｉ．ＦＩＳＨ．，47（６），Ｐ．793− 798（1981) ──────────────────────────────────────────────────続 き Continuing on the front page (72) Tadatomi Lee 2-3-3 Hananomiyacho, Takamatsu City, Kagawa Prefecture Inside the Shikoku Institute of Technology (72) Inventor Hiroshi Uejima 2-chome Hananomiyacho, Takamatsu City, Kagawa Prefecture No. 3-3 Inside Shikoku Institute of Industrial Technology, Institute of Industrial Science and Technology (56) References JP-A-3-279396 (JP, A) BULL. JAPAN. SOC. SCI. FISH. , 47 (8), p. 1079-1084 (1981) BULL. JAPAN. SOC. SCI. FISH. , 47 (6), p. 793-798 (1981)
Claims (2)
ａ ｓｐ．）からのリン酸塩緩衝液抽出液に、先ず最終
濃度３０〜４０重量％になるまで硫酸アンモニウムを加
えて第１段目の塩析を行い、沈殿した夾雑物を除去した
のち、さらにその抽出液に最終濃度７０重量％程度にな
るまで硫酸アンモニウムを加えて第２段目の塩析を行
い、沈殿として得られる粗活性画分を分取し、次いでこ
れからクロマトグラフィーにより分子量１００，０００
以上の画分を分離、回収することを特徴とする高活性赤
血球凝集素の製造方法。1. A red alga of the genus Ogonori (Gracilari)
a sp. )), Ammonium sulfate was first added to a final concentration of 30 to 40% by weight to carry out a first-stage salting-out to remove precipitated contaminants. To the final concentration of about 70% by weight, and the second stage of salting-out was carried out until a final concentration of about 70% by weight. A crude active fraction obtained as a precipitate was collected and then subjected to chromatography to obtain a molecular weight of 100,000.
A method for producing highly active hemagglutinin, comprising separating and collecting the above fractions.
ｉｌａｒｉａ ｖｅｒｒｕｃｏｓａ）又はツルシラモ
（Ｇｒａｃｉｌａｒｉａ ｃｈｏｒｄａ）あるいはそれ
らの亜種である請求項１記載の製造方法。2. The red algae belonging to the genus Ogonori (Grac).
2. The production method according to claim 1, wherein the production method is iliaria verrucosa) or tursilamo (Graciliaria choda) or a subspecies thereof.
Priority Applications (1)
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|---|---|---|---|
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6085600A JP2723169B2 (en) | 1994-03-31 | 1994-03-31 | Method for producing hemagglutinin |
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| Publication Number | Publication Date |
|---|---|
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| JP2723169B2 true JP2723169B2 (en) | 1998-03-09 |
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| JP6085600A Expired - Lifetime JP2723169B2 (en) | 1994-03-31 | 1994-03-31 | Method for producing hemagglutinin |
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1994
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Non-Patent Citations (2)
| Title |
|---|
| ＢＵＬＬ．ＪＡＰＡＮ．ＳＯＣ．ＳＣＩ．ＦＩＳＨ．，４７（６），Ｐ．７９３−７９８（１９８１） |
| ＢＵＬＬ．ＪＡＰＡＮ．ＳＯＣ．ＳＣＩ．ＦＩＳＨ．，４７（８），Ｐ．１０７９−１０８４（１９８１） |
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|---|---|
| JPH07278004A (en) | 1995-10-24 |
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