WO1995018149A1

WO1995018149A1 - Novel lectin and process for producing the same

Info

Publication number: WO1995018149A1
Application number: PCT/JP1994/002140
Authority: WO
Inventors: Akihiro Kawakubo; Hiroyuki Makino; Masanori Ninomiya; Nozomu Bitou
Original assignee: Marine Greens Laboratory Co., Ltd.
Priority date: 1993-12-24
Filing date: 1994-12-19
Publication date: 1995-07-06

Abstract

A novel lectin characterized in that (a) it has a molecular weight of about 25 thousand as determined by gel filtration, (b) it has a molecular weight of about 29 thousand as determined by SDS electrophoresis, (c) it has an isoelectric point (pI) of from 4.5 to 5.7 as determined by isoelectric electrophoresis, (d) it does not have sugar chains, (e) it has an amino acid sequence of Gly-Arg-Tyr-Thr-Val-X-Asn-Gln-Trp-Gly (wherein X represents Gln or Lys) at the N-terminus, (f) it has a specific agglutination activity against tripsinized rabbit erythrocytes, sheep erythrocytes and tripsinized sheep erythrocytes, and (g) it has a binding specificity for fetuin, asialofetuin, thyroglobulin and yeast mannan. It has physicochemical and biochemical properties different from those of known lectins. It is promising as a test reagent, an immunomodulator, and a specific adsorbent for separating and analyzing sugars because of the unique sugar-chain binding specificity thereof, as a rejection inhibitor for organ transplantation, an antitumor drug, and a preventive or remedy for autoimmune diseases and so forth because of the blastogenic activity thereof against lymphocytes, and as a growth factor for cell culture of lymphocytes and the like. According to the invention process, it can readily be separated from algae of the genus Eucheuma and purified in large amounts.

Description

Specification

Novel lectin and method for producing the same

Technical field

The present invention relates to a novel lectin and a method for producing the same. In particular, the present invention relates to a novel lectin derived from seaweed, in particular, a seaweed belonging to the genus Kirinsai (genus Euchea or Kappaphycus), and a method for producing the same.

Background art

Lectin is a general term for sugar-binding proteins or glycoproteins that are not immunological products and are found in animals, plants, bacteria, etc. In particular, lectins derived from land plants have long been known as hemagglutinins. However, in recent years, several lectins have been found not only in land plants but also in seaweeds, and some of these lectins not only recognize erythrocytes but also recognize complex carbohydrates. It was discovered (Hori et al., Botani ca Marina Vol. 29, pp. 323-328, 1986), and its physiological significance and properties have attracted attention.

Recent research has shown that some species of seaweed have some hemagglutinating activity (Hori et al., Botani ca Marina 31, pp. 133-138, 1988). Has already been purified and its properties as lectins have been confirmed (Shiomi et al., Journal of the Japanese Society of Fisheries Science, Vol. 46, No. 11, pp. 1369-1373, 1980, etc.) _c However, lectins from seaweed at an industrial level When considering production, there are various problems such as yield, purification method, and securing of algal mass on an industrial scale.

Currently, many kinds of lectins derived from animals and plants are sold as research reagents, and development of other practical applications is under consideration, but for seaweed lectins, lectins derived from the genus Mill are sold as two kinds of reagents. It is only.

On the other hand, the seaweed of the genus Kirinsai has only been used as a raw material for carrageenan algae and seaweed salad, and the active ingredients contained therein have not been developed except for the use of polysaccharides. Therefore, from the viewpoint of effective utilization of such seaweed resources, the technology of producing high value-added substances such as lectins from these seaweeds is very useful. Disclosure of the invention

In view of such circumstances, the present inventors have collected a wide variety of seaweeds and conducted intensive research on lectins in seaweeds.As a result, they have found novel lectins in seaweeds of the genus Kirinsai and established a method for their production. Thus, the present invention has been completed.

That is, the present invention

(a) the molecular weight by gel filtration is about 2,500,

(b) sodium dodecyl sulfate (hereinafter referred to as SDS) having a molecular weight of about 29,000 by electrophoresis,

(c) the isoelectric point by isoelectric focusing is in the range of pI 4.5 to pI 5.7,

(d) has no sugar chain,

(e) the amino acid sequence of the N-terminal region is Gly-Arg-Tyr-Thr-Val-X-Asn-Gln-Trp-Gly (X is Gin or Lys),

(f) Trypsin-treated ギ has specific agglutinating activity on heron erythrocytes, sheep erythrocytes and trypsin-treated sheep erythrocytes,

(g) The present invention provides a novel lectin characterized by exhibiting binding specificity to phytin, ash mouth phytin, thyroglobulin, and yeast mannan. Further, the present invention provides a method for producing the above-mentioned novel lectin, that is, using seaweed belonging to the genus Kirinsai as a raw material, extracting it with an extraction solvent selected from a buffer solution and a solvent-water mixture, and extracting the extract with solvent precipitation and gelation. An object of the present invention is to provide a method for producing a novel lectin, which is characterized in that the lectin is separated and purified by filtration, preferably further through one step of ion exchange chromatography.

First, the lectin of the present invention will be described. The novel lectin of the present invention has the above-mentioned characteristics (a) to (g).

Hereinafter, the lectin of the present invention will be specifically described based on these characteristics. (a), (b) Molecular weight

The lectin of the present invention exhibits a molecular weight of about 25,000 by gel filtration, and a molecular weight of about 29,000 by SDS electrophoresis.

The determination of the molecular weight by the gel filtration method in the present invention is performed by using a gel filtration color for molecular weight measurement. High-performance liquid chromatography using HPLC [hereinafter referred to as HPLC, conditions; column: Superdex 75 HR (10x300 mm), eluent: 0.05 M phosphate buffer (pH 7.2) containing 0.1 M NaCl] . More specifically, standard proteins with known molecular weights in advance (aldolase (MW158, 000), transferrin (MW800, 000), and serum albumin (MW68, 000)) , Ovalbumin (MW 43,000), carbonic anhydrase (MW32, OOiOS-lactoglobulin (MW18, 00), aprotinin (MW 6,500), vitamin B12 (MW 1, 355))] can be used to determine the molecular weight of the lectin of the present invention.

The term “SDS electrophoresis” as used herein means polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) performed in the presence of sodium dodecyl sulfate. The determination of the molecular weight by the SDS electrophoresis in the present invention can be carried out according to the SDS-PAGE by Laeimli's method (Nature, 227, 680, 1976). Specifically, the lectin of the present invention was subjected to heat treatment at 100 ° C. for 3 minutes in the presence of SDS (0.5%) and 2-mercaptoethanol (0.5%), followed by SDS (0% In the presence of 1, electrophoresis was performed using a 10-mm 5% to 20% gradient polyacrylamide slab gel, and then the location of the transferred lectin sample was simultaneously analyzed with a standard protein of known molecular weight (e.g. Albumin (MW 66,000), ovalbumin (MW 45,000), glyceraldehyde-13-phosphate dehydrogenase (MW36,000) from cow heron muscle, cow Erythrocyte-derived carbonic anhydrase (MW290,000), bovine kidney trypsinogen (MW24,000), soybean-derived trypsin inhibitor (MW200,100), milk-derived milk —Detection by staining with lactoglobulin (MW 14,200)) And the molecular weight can be determined. From the results of these molecular weight measurements, the lectin of the present invention showed a molecular weight of about 25,000 by the above-mentioned gel filtration method, and a monomer having a molecular weight of about 29,000 by the above-mentioned SDS electrophoresis method. It seems to be the body.

(c) Isoelectric point The lectin of the present invention has a PI of 4.5 to 5.7 when its isoelectric point is measured by polyacrylamide gel isoelectric focusing. Preferably, at least one selected from p1 4.75, pI4.95, pI5.05, pI5.20 and pI5.50 in the polyacrylamide gel isoelectric focusing method is used. Both of them have the same isoelectric point.

Determination of isoelectric point by polyacrylamide gel isoelectric focusing in the present invention is carried out according to a conventional method, using 2% Ampholine (pH 4 to 6.5) as an amphoteric carrier and using a 7% polyacrylamide gel. It can be performed by running at 460 V constant voltage for 20 hours.

(d) Sugar chain presence

The lectin of the present invention is obtained by a phenol-sulfuric acid method using glucose as a measurement standard sample at least CDubois.M., Gilles.KA, Hamilton, JK, Rebers.PA and Smith, F. (1956), Anal.Chem. 28, 357] shows that the sugar content is 0% within the experimental error, and from this it is determined that the sugar chain is not present. The sugar content can be determined by the following equation.

{Sugar content // (protein content + sugar content)} X 100 = sugar content (%)

There is no limitation on the measurement of the amount of protein, and any known method can be used.

(e) Amino acid sequence in N-terminal region

The lectin of the present invention has a Gly-Arg-Tyr-Thr-Val-Gln-Asn-Gln-Trp-Gly or Gly-Arg-Tyr-Thr-Val-Lys-Asn-Gln-Trp-Gly in the N-terminal region. It is a protein having the following amino acid sequence:

The amino acid sequence can be analyzed using an amino acid analyzer such as Applied Biosystems 477A PTH Analyzer.

That is, the lectin included in the present invention has an amino acid sequence consisting of 10 amino acid residues in the N-terminal region, except that the sixth amino acid residue from the N-terminal side is glutamine or lysine. Are characterized by having a common amino acid sequence. Furthermore, in addition to the features of the amino acid sequence of the N-terminal region, the lectins included in the present invention Since the molecular weights obtained by SDS electrophoresis or gel filtration are the same and their isoelectric points differ from pi 4.5 to pI 5.7, some amino acid residues (at least (The sixth amino acid residue from the N-terminal side) are considered to be different from each other.

(f) Agglutinating activity on erythrocytes

The lectin of the present invention has a feature of specifically agglutinating ovine erythrocytes, trypsin-treated ギ egret erythrocytes and trypsin-treated whiskery erythrocytes, and, on the other hand, ego erythrocytes, ゥ red erythrocytes, goose erythrocytes, and trypsin-treated ゥ red blood cells. It has no agglutinating activity on either erythrocytes or trypsin-treated goose erythrocytes. The agglutinating activity on these erythrocytes can be determined by measuring using a serial two-fold dilution method [B0TANICA MAR INA, Vol. 31, pp. 133-138, (1988)].

(g) Sugar bond specificity

The lectin of the present invention does not show specific binding to monosaccharides and oligosaccharides, has binding specificity to asialophetin, phytin, thyroglobulin, and yeast mannan, and has an α1-acid It also has weak binding specificity for glycoproteins. Among them, thyroglobulin and distmannan have particularly strong binding specificities. That is, since thyroglobulin and yeast mannan are rich in high mannose type sugar chains among glycoproteins, the lectin of the present invention recognizes Ν-glycoside type sugar chains in glycoproteins, and In particular, it is understood that the high Mannos type is strongly recognized.

In addition, the sugar-binding specificity of the lectin can prevent the agglutination of the erythrocytes by using a specific erythrocyte which can specifically agglutinate the lectin, for example, a sheep erythrocyte, a trypsin-treated heron erythrocyte or a tribcine-treated shed erythrocyte. It can be confirmed by examining sugars and glycosides.

Furthermore, it has been confirmed that the lectin of the present invention has blastogenic activity on mouse and human lymphocytes (see Test Example 1). The measurement of lymphocyte transformation activity of the lectins since the simple and certain objective results, the lectin of the present invention was added to mouse lymphocytes or human lymphocytes were cultured, into cells ³ .eta. A method of determining thymidine incorporation by radioactivity measurement is preferably used.

It can be said that the lectin of the present invention having such an effect is clinically useful for in vitro detection and evaluation of cell-mediated immunity.

The lectin of the present invention is not particularly limited in its origin, etc., as long as it has the above characteristics, but is preferably a marine algae, especially a marine genus belonging to the genus Kirinsai (genus Eucheuma or Kappaphycus). It is obtained by separation and purification.

The seaweed that can be a source of the lectin of the present invention preferably includes seaweeds of the genus Kirinsai (genus Eucheuma or Kappaphycus) that grow in Japan and all over the world.

By the way, in recent years, seaweed, which had been classified as the red alga Kirinsai genus (scientific name: Eucheuma genus), has recently been separated into Euche genus a and Kappaphycus genus.

(TAXONOMY OF ECONOMIC SEAWEEDS, Vol. 2, pp. 157-219, 1988). In the present invention, the genus Kirinsai refers to a genus of the genus Euche a and the genus Kappaphycus. Further, in the present specification, when each genus name is distinguished and described, the genus name is abbreviated to E. for the genus Eucheun ^ and the genus name is K. for Kappaphycus.

Examples of seaweeds belonging to the genus Kirinsai used in the present invention include, for example, togekirinsai (E. serra), euphorma kottonii cottoni i), katamenkirin rhinocerat gelat inae), amasakirinsai amakusaensis), ^

Eucheum genus such as dent iculatunu E. muri catum, E. cupresoideum, E. spinosunu E. arnoldi i- E. alvarezi i, E. striatum, and procrusteanum,

Seaweed belonging to the genus Kappaphycus, such as K. cottoni i, K. striatum ^ K. alvarezi i, and K. arnoldi i. Preferable ones are P. serra), E. plumeria cottoni i), E. gelat inae and A. makusaensis.

The original algae for carrageenan production, which was conventionally called ^ cottoni i, has recently been separated from Euche genus and proposed to be called Kappaphycus procrusteanum (SEAWEED CULTAVATION AND MARINE RANCHING, 75-88). P. 1993), but in the present invention, it will be referred to as E. cottonii. The lectin of the present invention can be isolated and obtained from these seaweeds by appropriately combining known extraction methods, separation methods, purification methods, and the like. Representative production methods are described in detail below. I do.

The method for producing a lectin of the present invention comprises extracting seaweed, particularly seaweed belonging to the genus Kirinsai, with an extraction solvent selected from a buffer solution and a solvent-water mixture, and subjecting the extracted solution to solvent precipitation and gel filtration. This method is characterized in that it is separated and purified, and if necessary, ion exchange chromatography may be performed.

The seaweeds used in the method preferably include seaweeds belonging to the genus Kirinsai (genus Eucheuma or Kappaphycus). E. gelatinae, E. amakusaensis, E. dent iculatum ^ E. rauricatum, E.

seaweed belonging to the genus Kappaphycus such as cupresoideunu E. spinosum, E. arnoldi is E. alvarezi i, E. striatum, etc. Is mentioned. Judging from the content of lectin and the availability of lectin, etc., it is preferable to use serra).. ユユ cotton cotton cotton, カタカタ E E E cotton, E. denticulat 圆, E. spinosunu E. alvarezii, K. procrusteanum and cottoni i ゝ alvarezii, and particularly preferred are E. serra, E. serra, C. cottonii and (E. gelat inae) and E. amakusaensis.

In the production of lectin, it is preferable to first collect these seaweeds, wash them well with seawater, freeze-dry them, and then pulverize them into seaweed powder.

Next, the obtained seaweed powder is extracted using a suitable extraction solvent.

As the extraction solvent, any of various buffers, various organic solvents that can be mixed with water, or a mixed solution of an organic solvent and water can be used. Preferably, a mixed solution of a buffer or an organic solvent and water is used. Examples of the buffer include, but are not particularly limited to, various known buffers, and a buffer having a buffering ability in the pH range of 3 to 10 is preferable.

As the organic solvent, acetone, methanol, ethanol or isopropanol is preferable. Among them, it is particularly preferable to use a mixed solution of ethanol and water of 10% to 40%.

Specifically, for example, a 25% ethanol aqueous solution is added to seaweed powder, and extraction is performed while maintaining the temperature as low as necessary, to obtain a seaweed extract. Next, the seaweed extract is separated into a supernatant and an extraction residue by filtration or centrifugation, and the supernatant is recovered as an extract containing the lectin of the present invention (primary extract) ). Alternatively, a 25% aqueous solution of ethanol may be added again to the extraction residue to perform a second extraction and collect as an extract containing the lectin of the present invention (secondary extract).

Further, the extract containing the lectin of the present invention is subjected to a solvent precipitation operation, for example, salting out with ammonium sulfate (hereinafter referred to as ammonium sulfate salting out) or precipitation with an organic solvent. Can be recovered.

Ammonium sulfate salting out can be carried out by a conventional method, and is usually carried out by collecting a fraction precipitated with 0 to 80% saturated ammonium sulfate, preferably 20 to 80% saturated ammonium sulfate. Precipitation with an organic solvent can be carried out by an ordinary method using any water-soluble organic solvent, regardless of the type of extraction solvent used in the previous extraction operation. Preferred organic solvents include acetone, methanol, ethanol, isopropanol and the like, with ethanol being particularly preferred. For example, when extracting with an ethanol-water mixture, if the final concentration of ethanol is adjusted to 70% or more by adding an appropriate amount of cold ethanol, the protein containing the lectin of the present invention precipitates as a precipitate. .

The precipitate fraction obtained by these methods is collected by a conventional method, suspended in a buffer solution (PH 6 to 8), dialyzed against the buffer solution, and the solvent is removed to obtain a crude solution of the lectin of the present invention. Can be obtained.

Next, the lectin of the present invention can be easily isolated and purified by subjecting the crude lectin solution from which the solvent has been removed to gel filtration. As the filler used in the gel filtration, it is preferable to use various products having an exclusion limit molecular weight of 100,000 or less. Preferred is gel filtration column chromatography using a column filled with such a packing material. The eluent is not particularly limited as long as it does not affect the physical and biochemical properties of the lectin.For example, the eluent contains a salt of about 0 to 1 M. Phosphate buffer (pH 6 to 9).

Specifically, a crude lectin solution is applied to a column for gel filtration, and then the eluate is supplied while the eluent is supplied. It can be carried out by monitoring the agglutinating activity of each fraction on erythrocytes using erythrocytes showing activity. The agglutinating activity on erythrocytes can be determined by the serial two-fold dilution method CBOTANICA MARINA, Vol. 31, pp. 133-138, (1988)].

As a result, it is possible to obtain, as the lectin fraction of the present invention, a fraction in which the maximum peak of ultraviolet absorption (280 nm) coincides with the peak of agglutination activity for erythrocytes. The lectin fraction can be obtained from both the primary extract and the secondary extract described above.

By the above operation, a lectin purified to 90% or more purity, which can be confirmed as a single band in SDS electrophoresis, can be obtained. In addition, depending on the type of seaweed used as a raw material, a lectin purified to a purity of 99% or more can be produced and obtained by such an operation.

The lectin produced by the above method has the following characteristics irrespective of the type of seaweed used as a starting material.

(a) the molecular weight by gel filtration is about 25,000,

(b) the molecular weight by SDS electrophoresis is about 29,000,

(c) by isoelectric focusing, showing one or more isoelectric points in the range of pI4.5 to pI5.7,

(d) has no sugar chain,

(E) the amino acid sequence of the N-terminal region is Gly-Arg-Tyr-Thr-Val-X-Asn-Gln-Trp-Gly (X is Gin or Lys)

(g) Shows binding specificity for phytin, ash mouth phytin, thyroglobulin, and yeast mannan.

In addition, the lectin fraction obtained by the above gel filtration method can be further subjected to ion exchange chromatography, preferably anion exchange chromatography, if necessary. Specifically, the lectin fraction after gel filtration is subjected to anion-exchange chromatography using a carrier having a weak anion-exchange group such as getylaminoethyl (DEAE) group. Fractionate by the gentle elution method. By such a method, the lectin fraction obtained by the gel filtration method can be further fractionated into a plurality of fractions having only slightly different isoelectric points according to the lectin contained in the fraction. That is, when the lectin fraction obtained by the gel filtration method contains a lectin having a plurality of isoelectric points, it should be isolated and purified into individual lectins having respective isoelectric points by the ion exchange chromatography. Can be.

The lectin that can be produced by such a method is characterized in terms of molecular weight, presence or absence of sugar chains, amino acid sequence, hemagglutinating activity and sugar-binding specificity, by various properties of the lectin obtained by gel filtration ((a), (b) and (d) to (g)), and has the characteristic that the isoelectric point measured by the isoelectric point electrophoresis method indicates one point in the range of PI 4.5 to pI 5.7. .

The number of lectins contained in the lectin fraction obtained by the gel filtration method varies depending on the type of giraffe used as a raw material, and the isoelectric point of each lectin contained also varies depending on the type of giraffe. There is a slight difference between 5 and pi 5.7. For example, in the case of using Papaver rhinoceros as a raw material, a lectin exhibiting an isoelectric point of pi 4.75 or pi 4.95 by isoelectric focusing If the lectin with pI 5.05 or pi 5.20 is derived from the Japanese rhinoceros or linden, it will have pi 4.95, pi 5.20 or pi 5.50 Lectin Is obtained.

Lectins obtained by gel filtration, lectins obtained by anion-exchange chromatography, and mixtures of these lectins were confirmed to have blastogenic activity on mouse and human lymphocytes, respectively. It has been.

The lectin of the present invention is a novel lectin having different physicochemical properties from conventional lectins, and also different biochemical properties such as specificity of sugar chain binding. In addition, like the well-known lectins PHA (phytohemagglutinin), concanapalin A, etc., they utilize their unique sugar chain binding specificity to test reagents, immunomodulators, and specific adsorbents for separation and analysis of carbohydrates. In addition to its use, it is expected to be applied to various other fields using its affinity for sugar chains.

It is also expected to be used as a growth factor when mass-culturing cells derived from lymphocytes and other cells that produce useful substances.

Furthermore, since the lectin of the present invention has a blastogenic activity on lymphocytes, it can be used in mammals such as humans, mice, dogs, dogs, and mice, for example, when transplanting organs or bone marrow. Used as an inhibitor of rejection, an antitumor agent capable of exerting an antitumor effect through lymphocyte activation, or as a preventive or therapeutic agent for autoimmune diseases such as rheumatoid arthritis, immunodeficiency, etc., or as a reagent in medicine or pharmaceuticals be able to.

The lectin of the present invention may be used alone, or as a mixture of a plurality of substances having different isoelectric points, mixed with a pharmaceutically acceptable carrier, excipient, diluent, etc., to give powders, capsules, tablets, ointments, etc. And administered to patients. Alternatively, a freeze-dried preparation may be prepared by a means known per se. Furthermore, it can also be used as cosmetics. For this purpose, the lectin of the present invention is used in an amount of 0.1 to 100 mg / day, which is appropriately selected in consideration of a patient's disease, symptoms, weight, sex, age, etc., usually per adult. Is administered once to several times a day.

Furthermore, according to the production method of the present invention, such a useful lectin can be separated and purified from seaweeds, particularly seaweeds belonging to the genus Kirinsai, by a simpler method to obtain a large amount thereof, resulting in industrial production of lectins. be able to. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a chromatogram obtained by subjecting a crude lectin solution derived from Rhododendron rhizome to gel filtration chromatography. FIG. 2 shows a chromatogram obtained by subjecting an active fraction (lectin ES 0) of a crude lectin solution derived from Pterodactylus to a DEAE ion exchange chromatography after gel filtration chromatography. FIG. 3 shows a chromatogram obtained by subjecting the active fraction (lectin EC0) of the crude lectin solution derived from Yukiyama Cotney after gel filtration chromatography to DEAE ion exchange chromatography. FIG. 4 shows a chromatogram of an active fraction (lectin EG0) obtained by gel filtration chromatography of a crude lectin solution derived from Rhinoceros oryzae, which was applied to DEAE ion exchange chromatography. FIG. 5 shows a chromatogram of an active fraction (lectin EA0) after gel filtration chromatography of a crude lectin solution derived from Acacia officinalis subjected to DE AE ion exchange chromatography. In each of the figures, “—” means absorbance (280 nm), and …… means the hemagglutination activity titer.

Hereinafter, examples and test examples will be described in order to explain the present invention in more detail, but the present invention is not limited by these.

Examples 1-4

Separation and purification of lectins derived from P. serra), lectins derived from Yukiyuma cotney cottonii), lectins derived from Eucheuma gelatinae or lectins derived from Amakusaensis, and their physicochemical and biochemical tests. Will be described.

(1) Preparation of algal bodies

(1-1) Spruce ginseng growing on the Pacific coast of Shikoku was collected, washed well with seawater, freeze-dried, and crushed with a crusher to obtain seaweed powder.

(1-2) Eucoma cotonii, which is cultured in large quantities as carrageenan algae in tropical seas, is collected, washed well with seawater, freeze-dried, and pulverized with a pulverizer to obtain seaweed powder. I got

(1-3) Harvesting rhinoceros ginseng growing on the Pacific coast of Kyushu was collected, washed well with seawater, freeze-dried, and crushed with a crusher to obtain seaweed powder. (1-4) Amaranthus rhinoceros growing in Shikoku and Kyushu was collected, washed well with seawater, freeze-dried, and crushed with a crusher to obtain seaweed powder.

(2) Extraction of lectin from algae

(2-1) 100 g of a 20% aqueous ethanol solution was added to 100 g of the powdered seaweed rhinoceros algae of the above (1-1), and the mixture was extracted with stirring overnight in a refrigerator to obtain a seaweed extract. Was. The supernatant and the extraction residue were separated by centrifugation, and the supernatant was collected. The same amount of a 20% aqueous ethanol solution was again added to the extraction residue to perform secondary extraction, and the supernatant was recovered in the same manner. As a result, a primary extract of 840 ml and a secondary extract of 900 ml were obtained.

(2-2) To 100 g of the Eucalyptus cotonii seaweed powder of (1-2) above was added 100% of a 23% aqueous ethanol solution, and the mixture was extracted while stirring overnight in a refrigerator. An extract was obtained. The supernatant and the extraction residue were separated by centrifugation, and the supernatant was collected. The same amount of a 23% aqueous ethanol solution was again added to the extraction residue to perform secondary extraction, and the supernatant was recovered in the same manner. As a result, a primary extract of 83 Om1 and a secondary extract of 8995 m1 were obtained.

(2-3) 100 ml of 25% aqueous ethanol solution was added to 100 g of the powdered seaweed rhinoceros algae of (1-3), and the mixture was extracted while stirring overnight in a refrigerator. I got The supernatant and the extraction residue were separated by centrifugation, and the supernatant was collected. The same amount of a 25% aqueous ethanol solution was again added to the extraction residue to perform secondary extraction, and the supernatant was recovered in the same manner. As a result, a primary extract of 84 Om1 and a secondary extract of 890 ml were obtained.

(2-4) 100 ml of a 25% ethanol aqueous solution was added to 100 g of the above-mentioned seaweed powder, and extracted with stirring in a refrigerator overnight to obtain a seaweed extract. The supernatant and the extraction residue were separated by centrifugation, and the supernatant was collected. The same amount of a 25% aqueous ethanol solution was again added to the extraction residue to perform secondary extraction, and the supernatant was recovered in the same manner. As a result, 845 ml of the primary extract and 890 ml of the secondary extract were obtained.

(3) Recovery of lectin from extract

(3-1) In order to recover lectin from the extract derived from Rhododendron rhinoceros described in (2-1) above, an appropriate amount of cold ethanol was added to the extract to a final concentration of 83%, and lectin was added. The protein containing the protein was precipitated as a precipitate. The precipitate was collected by centrifugation, suspended in 5 OmM phosphate buffer (pH 7.2), dialyzed against the buffer, and ethanol was removed to obtain a crude lectin solution.

(3-2) In order to recover lectin from the extract derived from C. elegans in (2-2) above, an appropriate amount of cold ethanol was added to the extract to a final concentration of 80%. Was precipitated as a precipitate. The precipitate was recovered by centrifugation, suspended in 5 OmM phosphate buffer (pH 7.2), dialyzed against the same buffer to remove ethanol, and crude lectin solution was recovered. I got

(3-3) In order to recover lectin from the extract derived from P. chinensis in (2-3) above, an appropriate amount of cold ethanol is added to the extract to a final concentration of 81%, and the lectin is contained. Evening protein was precipitated as a precipitate. The precipitate was collected by centrifugation, suspended in 5 OmM phosphate buffer (pH 7.2), and the buffer was subjected to precipitation to remove ethanol to obtain a crude lectin solution. .

(3-4) To recover lectin from the extract derived from Acacia catechu (2-4) above, an appropriate amount of cold ethanol was added to a final concentration of 81%, and the protein containing lectin was precipitated. Was deposited. The precipitate was collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.2), dialyzed against the same buffer to remove the ethanol, and a crude lectin solution was obtained. Was.

(4) Purification of lectin by gel filtration

(4-1) The crude liquid of lectin derived from Rhododendron persica obtained in (3-1) was subjected to gel filtration mouth chromatography. A column packed with 75 pg Superdex manufactured by Pharmacia Co. (inner diameter 26 mm, charged length 6 O cm) was used. Specifically, this column, buffered with 5 · mM phosphate buffer (pH 7.2) containing 0.1 IV [NaCl], was added to the crude lectin solution extracted from the rhododendron (3-1). The protein solution is measured at 280 nm using a UV monitor and the protein content is measured at 1-minute intervals. Each fraction was monitored for agglutination activity. As a result, the largest peak near the retention time of about 60 minutes was coincident with the peak of the aggregation activity. As a result of the gel filtration chromatography, about 80 Omg of lectin derived from Rhododendron was obtained from the primary extract and about 16 Omg from the secondary extract. This lectin was converted to a single band by SDS electrophoresis (electrophoresis in the presence of 0.1% SDS using a 1.0 mm thick 5 to 20% gradient polyacrylamide slab gel at a constant current of 20 mA for 70 minutes). The purity was 95% and 99.9%, respectively, as measured by DEAE ion exchange chromatography and analytical gel filtration chromatography described in (5) and (6). The recovery rate from the extract was 95%, calculated from the agglutinating activity on erythrocytes. In the end, 96 Omg of lectin derived from Rhododendron was obtained from 100 g of the freeze-dried powder. The lectin derived from P. terrestris purified by gel filtration chromatography was designated ES0 and used in the following experiments. Fig. 1 shows the above results.

(4-2) Under the same conditions as above, the crude lectin solution derived from Yukiyama Cotney obtained in (3-2) was subjected to gel filtration chromatography, and the agglutination activity of each fraction was determined using hidge erythrocytes. Was monitored.

As a result, a lectin showing a single band was obtained by SDS electrophoresis (about the same conditions as above) of about 174 mg from the primary extract and about 2 Omg from the secondary extract. ) And (6) were 95% and 97% by DEAE ion exchange chromatography and analytical gel filtration chromatography, respectively. The recovery from the extract was 90%, calculated from the agglutinating activity on erythrocytes. Less than 20 mg of lectin was recovered from 100 g of freeze-dried Yuki Yuma cotney powder. This lectin purified by gel filtration chromatography was designated as EC0 and used in the following experiments.

(4-3) Under the same conditions as described above, the crude lectin solution derived from the red beetle obtained in (3-3) was subjected to gel filtration chromatography, and the agglutinating activity was monitored using hidge erythrocytes.

As a result, from the primary extract, a lectin derived from Kamenokirin rhizome showing a single band was obtained by SDS electrophoresis of about 21 Omg (same conditions as above), and the purity was as described in (5) and (6). DEAE ion exchange chromatography and analytical gels By filtration chromatography, they were 92% and 93%, respectively. The recovery from the extract was 90% in terms of the agglutinating activity on erythrocytes. Approximately 200 mg of lectin was recovered from 100 g of the lyophilized powder. This lectin purified by gel filtration chromatography was used as EG0 in the following experiments.

(4-4) Under the same conditions as described above, the crude lectin solution derived from Amaxilla was obtained in (3-4) was subjected to gel filtration chromatography, and the agglutinating activity was monitored using hidge erythrocytes.

As a result, a lectin from Acacia chinensis showing a single band was obtained from the primary extract by SDS electrophoresis of about 21 Omg (same conditions as above).

6

Was 91% and 93% by DEAE ion exchange chromatography and gel filtration chromatography for analysis as described in (5) and (6), respectively. The recovery from the extract was 90% in terms of the agglutinating activity on erythrocytes. Approximately 200 mg of lectin was recovered from 100 g of the lyophilized powder. This lectin purified by gel filtration chromatography was designated as EA0 and used in the following experiments.

(5) Fractionation by DEAE ion exchange chromatography

(5-1) The lectin ES 0 derived from Rhododendron persica obtained in (4-1) was subjected to DEAE ion exchange chromatography.

Specifically, ES 0 obtained by gel filtration chromatography was first desalted and buffered by dialyzing it against a 20 mM sodium carbonate buffer (pH 9.5). Eluent A (20 mM sodium carbonate buffer, pH 9.5) and eluent were used as the eluent, using TSK-ge 1 DEAE-5 PW (manufactured by Tosoichi Co., Ltd., size: 7.5 x 7.5 cm). Solution B (20 mM sodium carbonate buffer, pH 9.5 + 0.5 M sodium chloride) was used. The buffered ES 0 is applied to the column equilibrated with eluent A. 0-5 minutes: eluent A 100%, 5-40 minutes: eluent B 0-50%, flow rate 1. Elution was performed with a linear gradient (linear gradient) under the condition of Om 1 in. The protein content of the eluate was determined by detecting the absorbance at 280 nm, At the same time, the agglutinating activity of the eluted fraction on hedging erythrocytes was measured. The results are shown in FIG. _2c. As a result, two active fractions having different elution times from ES0 were obtained. Each fraction was fractionated as ES 1 and ES 2 and subjected to the following tests.

(5-2) The lectin EC0 derived from Yukiyuma cotoni obtained in (4-2) was subjected to DEAE ion exchange chromatography under the same conditions as above. As a result, two active fractions having different elution times were obtained. The results are shown in Figure 3. Each fraction was collected as EC 1 and EC 2 and subjected to the following tests.

(5-3) The lectin EG0 derived from the red rhinoceros ginseng obtained in (4-3) was subjected to DEAE ion exchange chromatography under the same conditions as described above. As a result, three active fractions having different elution times were obtained. Fig. 4 shows the results. Each fraction was collected as EG1, EG2, and EG3 and subjected to the following tests.

(5-4) The lectin EA0 derived from Acacia catechu obtained in (4-3) was subjected to DAE ion exchange chromatography under the same conditions as described above. As a result, as shown in FIG. 5, three active fractions having different elution times from EA0 were obtained. Each fraction was collected as EA1, EA2 and EA3 and subjected to the following tests.

(6) Molecular weight measurement by gel filtration chromatography

(6-1) The active fractions ES 1 and ES 2 obtained in ES 0 and (5-1) were subjected to gel filtration HPLC equipped with a Superdex 75 HR column (10x300 nmi), and contained in each fraction. The molecular weight of the lectin was measured.

Specifically, using the above column buffered with a 0.05 M phosphate buffer (pH 7.2) containing 0.1 M NaCl, aldolase (MW158, 000), transferrin ( MW 80,000), pepsin albumin (MW 68,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 32,000), / The molecular weight was measured using 5-lactoglobulin (MW18, 000), abrotinin (MW6, 50,000), and vitamin B12 (MW1, 355). Results: All of ES 0, ES 1 and ES 2 had a molecular weight of about 25,000.

(6-2) In the same manner as (6-1), EC 0 and the active fraction EC 1, obtained in (5-2), Molecular weight was determined by gel filtration HP LC for each of the ECs. As a result, all of EC0, EC1, and EC2 had a molecular weight of about 25,000.

(6-3) In the same manner as in (6-1), the molecular weight of each of the active fractions EG1, EG2 and EG3 obtained in EGO and (5-3) was measured by gel filtration HPLC. did. As a result, all of EG0, EG1, EG2 and EG3 had a molecular weight of about 25,000.

(6-4) In the same manner as in (6-1), the molecular weight of each of the active fractions EA1, EA2 and EA3 obtained in EA0 and (5-4) was measured by gel filtration HPLC. Was. As a result, EA 0, EA, EA 2 and EA 3 all had a molecular weight of about 25,000.

(7) Molecular weight measurement by SDS-PAGE

(7-1) SDS-PAGE was performed according to the method of Laemmli (Nature, 227, 680, 1976). Specifically, ES 0, ES 1 and ES 2 obtained in (5-1) were subjected to heat treatment at 100 ° C. for 3 minutes in the presence of 0.5% SDS and 0.5% 2-mercaptoethanol. Thereafter, electrophoresis was carried out using a 1.0-20 mm thick 5-20 concentration gradient polyacrylamide slab gel in the presence of 0.1% SDS. For the measurement of molecular weight, serum albumin (MW 66,000), ovalbumin

(MW 45,000), glyceraldehyde-3-phosphate dehydrogenase derived from rabbit heron muscle (MW 36,000), and carbonic anhydride derived from bovine erythrocytes (MW 29,0 0), trypsinogen derived from bovine spleen (MW 24,000), trypsin inhibitor derived from soybean (MW 20, 100), and heractoglobulin derived from milk (MW 14,200) A molecular weight marker was used. The protein is stained for 2 hours in a mixture of 7.5% acetic acid and 50% methanol containing 0.25% Coomassie Brilliant Blue R-250, and decolorization is performed using a solution containing 7.5% acetic acid and 5% methanol. I went.

As a result, all of ES 0, ES 1 and ES 2 had a molecular weight of about 29,000.

(7-2) EC obtained in (5-2) by SDS-PAGE as in (7-1) The molecular weights of 0, EC 1 and EC 2 were measured. As a result, each of EC 0, EC 1 and EC 2 had a molecular weight of about 29,000.

(7-3) The molecular weights of EG0, EG1, EG2 and EG3 obtained in (5-3) were measured by SDS-PAGE in the same manner as in (7-1). As a result, all of EG0, EG1, EG2 and EG3 had a molecular weight of about 29,000.

(7-4) In the same manner as in (7-1), the molecular weights of EA0, EA, and EA2 and EA3 obtained in (5-4) were measured by SDS-PAGE. As a result, all of EA0, EA1, EA2 and EA3 had a molecular weight of about 29,000.

(8) Isoelectric point measurement by isoelectric focusing method

(8-1) Isoelectric points of ES 0, ES 1 and ES 2 were measured by polyacrylamide gel isoelectric focusing. Specifically, 2% Ampholine, pH 4.0 to 6.5 was used as an amphoteric carrier, and electrophoresis was performed in a 7% polyacrylamide gel at a constant voltage of 460 V for 20 hours. As a running buffer, a 0.01 M phosphoric acid solution was used on the anode side, and a 0.01 M sodium hydroxide solution was used on the cathode side. After electrophoresis, the gel was fixed and stained to detect proteins.

As a result, the isoelectric points of ES 0 were p I 4.75 and p i 4.95, £ 1 was 14.75, and E S2 was p I 4.95.

(8-2) The isoelectric point was measured in the same manner as in (8-1). As a result, EC (Hip 1.55 and pI 5.20, EC1 had p5.05 and EC2 had pI 5.20).

(8-3) The isoelectric point was measured in the same manner as in (8-1). As a result, EGO was pI 4.95, pI 5.20 and pI 5.50, EG1 was p4.95, EG2 was pI5.20, and EG3 was pI5.50. .

(8-4) The isoelectric point was measured in the same manner as in (8-1). As a result, EA0 was pI 4.95, PI 5.05 and pI 5.20, EA1 was p 4.95, EA2 was pI 5.20, and EA3 was pI 5.50.

(9) Measurement of sugar content

(9-1) Phenol sulfate method CDubois.M., Gilles.K.A., Hamilton, J.K.,

Rebers.PA and Smith, F. (1956), Anal.Cem. 28, 357], ES 0, E The amount of sugar contained in S1 and ES2 was measured. Glucose was used as a standard sample for measuring the amount of sugar. The evening mass was measured using a Bio-Rad DC Protein Atskit. The sugar content was determined by the following equation.

{Sugar content (protein content + sugar content)} X 100 = sugar content (%)

As a result, ES1 was 0.01% and ES2 was 0.02%, and it is considered that the lectin derived from Togekirinsai has no sugar chain. For ES0, reproducible data was not obtained. This is presumably because saccharides unrelated to the lectin may remain slightly mixed during the purification process.

(9-2) The sugar content of EC 0, EC 1 and EC 2 was determined in the same manner as in (9-1) .The sugar content was 0.5% for EC 0, 0.03% for EC 1 and EC 2 Based on the fact that the power was 0.02% _c, it is considered that the lectin derived from Equine cotonia has no sugar chain.

(9-3) The sugar content of EG1, EG2 and EG3 was determined in the same manner as in (9-1) .The sugar content was 0.15% for EG1, 0.01% for EG2 and 3 was 0.01%. Based on this, it is considered that the lectin derived from Rhinoceros ginseng has no sugar chain.o

(9-4) The sugar content of EA1, EA2, and EA3 was determined in the same manner as in (9-1) .The sugar content was 0.15% for EA1, 0.01 for EA2, and EA3. Was 0.01%. From this, it is considered that the lectin derived from Amacakirinsai has no sugar chain.

(10) Amino acid sequence analysis of N-terminal region

Lectins derived from Papaver rhinoceros rhinoceros (ES 0, ES 1 and ES 2), lectins derived from Yuki-Yuma Cotney (EC 0, EC 1 and EC 2), and lectins derived from Papaver rhinoceros rhinoceros (R. EG 3) -derived lectin and Amaxilla lectin-derived lectins (EA 0, EA 1, EA 2 and EA 3) were applied to an Applied Biosystems 477A PTH analyzer, and amino acids from the N-terminus to 1q residue were analyzed. The sequence was analyzed.

The specific results are shown in the table below. The amino acid sequence of 10 residues is Gly-Arg-Tyr-Thr-Val-X-Asn-Gln-Trp-Gly, and the sixth amino acid from the N-terminal indicated by X is glumin or lysine. Were identical except for

ES 1 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

ES 2 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

EC 1 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

EC 2 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

EG 1 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

EG 2 Gly-Arg- Tyr-Thr-Val- Lys-Asn- Gln-Trp-Gly

EG 3 Gly-Arg- Tyr-Thr-Val- ys-Asn-Gln-Trp-Gly

E A 1 Gly-Arg- Tyr-Thr-Val- Gln-Asn- Gln-Trp-Gly

EA 2 Gly-Arg- Tyr-Thr-Val- Lys-Asn- Gln-Trp-Gly

EA 3 Gly-Arg- Tyr-Thr-Val- Lys-Asn- Gln-Trp-Gly

(11) Agglutinating activity on various erythrocytes

Lectins derived from Papaver rhinoceros rhinoceros (ES 0, ES I and ES 2), lectins derived from Yucca cotonii (ECO, EC 1 and EC 2), and lectins derived from Papaver rhinoceros rhinoceros rhinoceros (Rice chinensis) (EG0, EG1, EG2 and EG3) The agglutinating activity on various erythrocytes was measured for each of the lectin and the lectins (EA0, EA1, EA2 and EA3) derived from Rhododendron. Erythrocytes, sheep erythrocytes, ゥ erythrocytes, goose erythrocytes, trypsin-treated ギ heron erythrocytes, trypsin-treated hedge erythrocytes, trypsin-treated ゥ erythrocytes, and trypsin-treated geese erythrocytes were used as erythrocytes. The measurement was carried out by the continuous two-fold dilution method CBOTANICA MARINA, Vol. 31, pp. 133-138, (1988)]. Table 1 shows the results. It was confirmed that the lectins derived from each of the above-mentioned Kirinsai genera specifically agglutinate hedged erythrocytes, trypsin-treated heron erythrocytes, and trypsin-treated shed erythrocytes, regardless of the type of seaweed. Table 1 Agglutinating activity of lectins from the genus Giraffe on various erythrocytes

+: Hemagglutination activity-: No hemagglutination activity

(12) Sugar bond specificity

Lectins derived from Rhododendron ginseng (ES 0 ES 1 and ES 2), Lectins derived from Echinoma crispina (EC 0 EC 1 and EC 2), and Lectins derived from Lactobacillus reticulata (EG0 EG 1 EG2 and EG3) And the lectins (EA0 EA1 EA2 and EA3) derived from Acacia chinensis were examined for their sugar-binding specificity. Specifically, first prepare various concentration solutions of various monosaccharides, oligosaccharides and glycosides listed in Table 2, add each lectin adjusted to the minimum aggregation concentration to each of these solutions, and add to room temperature. For 2 hours. Next, hedged erythrocytes were added to them, the agglutination of the hedged erythrocytes was examined, and the sugar concentration that inhibits the agglutinating activity of each lectin on erythrocytes was determined to determine the sugar-binding specificity of each lectin.

The concentrations (gZml) required for various sugars to inhibit the agglutination reaction by 50% are shown for the fractions of lectins derived from Rhododendron rhinoceros, Yukiyuma cotney, Rhododendron rhinoceros rhinoceros and Akumasakirin rhinoceros. Shown along with 2.

Table 2 Sugar-binding specificity of lectins derived from the genus Kirinsai seaweed Sugar-related substances Toke'Kirinsai t. Cottonn

ES0 ES1 ES2 ECO EC1 EC2 EGO EG1 EG2 EG3 EA0 EA1 EA2 EA3

L-arahi

Doxyk Rucos

D-Fructose

Force 'Lactos

N-acetylka 'lactose

D-force 'racfuronic acid

N-Acetyl Rucos

• D-Curcuronic acid

D-Courcouse

D-Culcose 6-phosphate

Lactose

Maltose

Mannitol

D-Mannose

D-Merich ';

D-raffinos

L-rhamnoth

D-Riho '

Pheasant D-s

a one

Ash 7 gar glyco D tin 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 100 フ 1000 fuetin 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 125 62.5 62.5 62.5 125 ash 0 fuetin 31.3 31.3 31.3 31.3 31.3 31.3 31.3 31.3 31.3 125 31.3 31.3 31.3 125 Chiroc 'Lof' phosphorus 3.9 3.9 3.9 3.9 3.9 3.9 3.9 0 3.9 7.8 3.9 1.0 3.9 7.8 Diestmannan 3.9 3.9 3.9 3.9 3.9 3.9 3.9 3.9 3.9 15.6 31.3 3.9 3.9 15.6 31.3 Early: ag / m 1 Aggregation No inhibition Neither lectin shows specific binding to monosaccharides and oligosaccharides, has binding specificity for asialophetin, phytin, thyroglobulin and yeast mannan, and binds to 1-acid glycoprotein. It also showed weak binding specificity. Among them, thyroglobulin and yeast mannan exhibited particularly strong binding characteristics.

From these facts, it was found that the lectins derived from each of the above-mentioned Kirinsai genera recognize N-glycoside type sugar chains of glycoproteins, and particularly strongly recognize high mannose type among them.

Test Example 1 Measurement test of the blastogenic activity of mouse and human lymphocytes of lectins derived from Rhododendron spp.

(a) Measurement of blastogenic activity on mouse lymphocytes

Mouse (BALBZC, female, 17-week-old) A lymphocyte fraction was obtained from the spleen according to a conventional method, and washed three times with RPMI 1640 medium containing 10% fetal calf serum.

1 0 ⁶ lymphocyte cell suspension Zm 1 concentration was prepared. The suspension 1 00 RPM 1 1 640 medium 100〃1 added containing lectin from each Eucheuma prepared to each concentration in 1, were cultured in 5% C0 ₂ under 37 ° C in the same medium. After culturing for 50 hours, ³ H- thymine

(0.5 CiZl 0/1) was added, and the cells were further cultured for 6 hours. The cultured cells of the culture medium was collected on a glass-like fiber filter (Whatman) using a static base Seruha, dried, placed in a counting vial, ³ H incorporated into lymphocytes in liquid scintillation one Chillon counter one -The amount of thymidine was measured. Examination of the steady-state index from the amount of radiation uptake revealed that the lectin from Togekirin 'rhinoceros

(ES 0, ES 1 and ES 2), lectin from E. cucumber (EC 0, EC 1 and EC 2), lectin from katamenokirin (EG0, EG1, EG2 and EG3) and from Akumasakirinsai All lectins (EA0, EAl, EA2 and EA3) were found to have mouse lymphocyte blastogenesis activity.

(b) Measurement of blastogenic activity on human lymphocytes A lymphocyte suspension derived from human peripheral blood at a concentration of 5 × 10 ⁶ cells Zm 1 was prepared from human blood by density gradient centrifugation. The suspension 1 0 0 1 to the lectin from each Eucheuma prepared to each concentration RPM 1 1 64 0 Medium 1 0 0〃1 added, cultured at 5% C 0 ₂ under 37 ° C in the same medium did. After 50 hours of culture, ³ H-thymidine (0.5 i / 10 u \) was added, and the cells were further cultured for 6 hours. The cultured cells in this culture solution were collected on a glass fiber filter (Whatman) using a cell harvester, dried, placed in a counting vial, and incorporated into lymphocytes using a liquid scintillation counter. The amount of thymidine was measured. . Examination of the stimulation index based on the amount of radiation uptake revealed that the lectins derived from the rhododendron (ES0, ES1, and ES2), the lectins (Ec0, EC1, and EC2) from the eastern fox, the catkin giraffe, Lectins derived from EG0, EG and EG2 and EG3 and lectins derived from Acacia chinensis (EA0, EA1, EA2 and EA3) may have blastogenic activity on human lymphocytes. Admitted.

Claims

The scope of the claims

1. Lectin with the following features:

(a) the molecular weight by gel filtration is about 25,000,

(b) the molecular weight by SDS electrophoresis is about 29,000,

(c) the isoelectric point by isoelectric focusing is in the range of pI4.5 to pI5.7,

(d) has no sugar chain,

(g) Shows binding specificity for phytin, asia-mouth phytin, thyroxine, roblin and yeast mannan.

2. The isoelectric point by isoelectric focusing is at least one selected from the group consisting of p1 4.75, pI4.95, pI5.05 pi5.20 and pi5.50 The lectin according to claim 1.

3. The lectin according to claim 1, wherein the lectin is derived from seaweed.

4. The lectin according to claim 3, wherein the seaweed belongs to a genus Kirinsai (genus Eucheuma or Kappaphycus).

5. The seaweed belonging to the genus Giraffe is selected from the group consisting of Eucheuma serra, EuGheuma cottonii, Eucheuma gelatinae and Eucheuma ^ nakusaensis. 5. The lectin according to claim 4, wherein the lectin is at least one.

6. Extract seaweeds belonging to the genus Kirinsai (genus Eucheuma or Kappaphycus) with an extraction solvent selected from a buffer and a solvent-water mixture, and subject the extract to solvent precipitation and gel filtration. 3. The method for producing a lectin according to claim 1, wherein the lectin is separated and purified.

7. The method for producing a lectin according to claim 6, further comprising subjecting the gel filtration to ion exchange chromatography.

8. at least one isoelectric point whose lectin is selected from the group consisting of p1 4.75, pI4.95, pI5.0, pI5.2 and pI5.5 8. The method for producing a lectin according to claim 6 or 7, wherein the method comprises:

9. The method according to any one of claims 6 to 8, wherein any one of the solvents used in the extraction and the solvent precipitation is any one selected from the group consisting of acetone, methanol, ethanol, and isopropanol. Production method of lectin.

10. The method for producing a lectin according to any one of claims 6 to 8, wherein one of the solvents used in the extraction and the solvent precipitation is ethanol.