L-fucose α1→6 specific lectin

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biochemistry. Disclosed is L-fucose α1→6 specific lectin, which is extracted from a basidiomycete or an ascomycete or an ascomycete, characterised by peak molecular weight of about 4500 m/z, determined via MALDI-TOF mass spectrometry analysis. The novel L-fucose α1→6 specific lectin has high affinity for a L-fucose α1→6 sugar chain, represented by an association constant of 1.0×104 M-1 or higher (at 25°C), and has an association constant of 1.0×103 M-1 or lower (at 25°C) with high-mannose sugar chains and/or glucolipids which do not contain an L-fucose α1→6 sugar chain. In one version, the disclosed L-fucose α1→6 specific lectin is a protein or a peptide which consists of an amino acid sequence selected from SEQ ID NO:2-6. The L-fucose α1→6 specific lectin is used for specific detection of a L-fucose α1→6 sugar chain and effective purification of the L-fucose α1→6 sugar chain or a sugar chain which does not contain L-fucose α1→6.

EFFECT: obtaining L-fucose α1→6 specific lectin.

16 cl, 38 dwg, 8 tbl, 4 ex

 

The technical field

[0001]

The present invention relates to new L-fucose α1→6-specific lectin, method of its production and its use. In particular, the present invention relates to a new lectin derived from a basidiomycete or marsupial of the fungus, the method of its production and method of detection and fractionation of sugar chain with lectin.

The level of technology

[0002]

It is known that the gene α1→6 fucosyltransferase (FUT8), which transfers the L-falosny the remainder to the end of the regenerating N-acetylglucosamine N-glican through α1→6-relationship with fokusirovanie kernel, is shown in connection with the signs of benign hepatocytes. Malignant hepatoma currently detected by electrophoresis on affinity to the lectin using agglutinin from Lens culinaris (LCA), which has affinity to fokusirovannym mono - and Bianchini N-picenum.

[0003]

Antibody-dependent cellular cytotoxicity (hereinafter ADCC activity) is one of the immune functions of man. ADCC activity is the activity through which leukocytes, such as natural cell-killers and monocytes, kill target cells such as cancer cells, using antibodies. ADCC activity is associated with antitumor mechanism of action of medicines on the basis of antibodies, such as Garnati is as gumanitarnogo antibody (therapeutic agent for the treatment of metastatic breast cancer and Rituxan as chimeric antibodies (therapeutic tool for the treatment of non-Hodgkin lymphoma) (off-patent publication 1).

In fact, if these medicines on the basis of antibodies have low ADCC activity, there is a need to use large doses of medicines on the basis of antibodies, which, in turn, creates a number of problems, such as increased cost and side effects (for example, infection due to a weakened immune system).

[0004]

ADCC activity of the antibody to which is transferred α1→6 L-fucose, and antibodies without α1→6 L-fucose varies 50-100 times (off-patent publication 2). In the case of obtaining antibodies to which α1→6 L-fucose is not transferred, it is possible to achieve high ADCC activity.

[0005]

Traditionally, along with the known LCA and other fokusirovannyi lectins, such as, for example, agglutinin from Pisum sativum (PSA), lachip from Aleuria aurantia (AAL), agglutinin from Narcissus pseudonarcissus (NPA), agglutinin from Vicia faba (VFA) and lectin from Aspergillus oryzae (AOL) (Patent publication 1-5).

Off-patent publication 1: Clynes RA et at, Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets/Clans P.A. and other Inhibiting Fc receptors modulate in vivo cytotoxicity against tumor targets NATURE MED 2000 APR; 6(4):443-446

Off-patent publication 2: Toyohide Shinkawa et at., The absence of L-fucose but not the presence of galactose or bisecting N-acetyl glucosamine of human IgGl complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. (Toehider Shinkawa and others, the Absence of L-fucose but not the presence of Gal which of ctazy or division N-acetylglucosamine complex oligosaccharides IgGI person demonstrates the critical role of enhancing antibody-dependent cytotoxicity) J Biol Chem. 2003 Jan 31; 278(5):3466-73. Epub 2002 Nov 8.

Patent publication 1: WO 2002/030954

Patent publication 2: WO 2003/084569

Patent publication 3: Example from the publication of unexamined patent application of Japan No. H02-083337

Patent publication 4: Example 5 from the publication of unexamined patent application of Japan No. 2002-112786

Patent publication 5: Publication of unexamined patent application of Japan No. 2007-161633

Disclosure of inventions

[0006]

Known lectins used for detection of L-fucose α1→6 sugar chain, also have an affinity not only to the L-fucose α1→6 sugar chain, but also to glycolipid sugar chain consisting of L-fucose, attached not due α1→6 sugar chain with a high content of mannose, not containing L-fucose. In particular, the AAL and AOL have, for example, affinity to α1→2 L-fucose and α1→3 L-fucose. Monotonization LCA, PSA and VFA also have affinity to nepokorionnykh mono - and Bianchini N-picenum. Thus, it was not possible to accurately identify the L-fucose α1→6 sugar chain, and also to highlight the L-fucose α1→6 sugar chain. Up to the present time was not known lectin that can bind only the L-fucose α1→6 sugar chain,

[0007]

In light of the above, the present invention is the creation of a new lectin capable of specific binding of L-fucose α1→6 sugar the ETUI. This invention allows you to create a more accurate method of detection of L-fucose α1→6 sugar chain using a new lectin, unlike the conventional case, and a method of fractionation of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sacharides circuit based on the detection of L-fucose α1→6 sugar chain.

[0008]

The authors of the present invention have discovered a new lectin with a very high affinity for a sugar chain, comprising α1→6 linked L-fucose. The authors of the present invention discovered that a new lectin allows the specific detection of L-fucose α1→6 sugar chain, and that this lectin can be used for the purification of L-fucose α1→6 sugar chain or a sugar chain that does not contain L-fucose α1→6 L-fucose α1→2, 1→3,1→4 sugar chain). The term "L-fucose α1→6 sugar chain" means a structure in which L-fucose attached to the end of the regenerating N-acetylglucosamine N-glican communication α1→6. The term "sugar chain not containing L-fucose α1→6" means the sugar chain in the molecule which does not contain L-fucose, attached communication α1→6.

[0009]

In particular, the present invention allows to obtain an L-fucose α1→6 specific lectin that: (1) extracted from the basidiomycete, or marsupial of the fungus, (2) has a molecular weight in accordance with the method of electrophoresis in the high is the bottom of the gel in the presence of sodium dodecyl sulfate (SDS-PAGE), equal 4000-40000, and (3) has an affinity for L-fucose α1→6 sugar chain, defined by the constant Association of 1.0×104M-1or more (at 25 degrees C). Constant Association here means a numeric value as measured by frontal affinity chromatography (FAC) at a temperature of 25 degrees C.

[0010]

L-fucose α1→6 sugar chain may include sialo N-glikana.

[0011]

In addition, it is desirable that the L-fucose α1→6 specific lectin (4) substantially not associated with vysokoporodnymi sugar chain and/or glycolipids, not containing L-fucose α1→6 sugar chain. [0012]

In addition, it is desirable that the L-fucose α1→6 specific lectin (5) had an affinity for α1→6-fokusirovannym mono-, di-, tri-, and tetraantennary N-picenum with the constant Association of 1.0×104M-1or more (at 25 degrees C).

[0013]

Basidiomycete fungus belongs, in particular, to the family Strophariaceae, Tricholomataceae, Amanitaceae or Polyporaceae.

[0014]

Basidiomycete fungi are, for example, Pholiota terrestris, Pholiota squarrosa, Pholiota adiposa, Stropharia rugosoannulata, Naematoloma sublateritium, Lepista sordid and Amanita muscaria.

[0015]

In particular, the amino acid sequence of L-fucose α1→6 specific lectin (6) displays the number 1.

[0016]

The present invention also allows you to get L-fucose α1→6 specific lectin, which represents (a) a protein or Atid, comprising the amino acid sequence described one of the numbers from 2 to 5, or (b) a protein or peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence shown in one of the rooms from 2 to 5, and which is functionally equivalent protein or peptide with the amino acid sequence described one of the numbers from 2 to 5. The term "functionally equivalent" means an affinity for L-fucose α1→6 sugar chain, characterized by the constant Association of 1.0×104M-1or more (at 25 degrees C).

[0017]

Protein or peptide described in paragraph (b)has an amino acid sequence described, for example, number 6.

[0018]

The present invention also allows you to get L-fucose α1→6 specific lectin, which is a protein or a peptide with at least 37% similarity with the amino acid sequence described one of the numbers from 2 to 6, and is functionally equivalent to a protein or peptide with the amino acid sequence described one of the numbers from 2 to 6.

[0019]

The present invention also allows you to create also a method of obtaining L-fucose α1→6 specific lectin, according to which a water extract (water-soluble extract) from and/or marsupial mushroom evaluation of the credits (i) hydrophobic chromatography and reversed-phase chromatography, (ii) affinity chromatography, or (iii) ion-exchange chromatography and gel filtration to obtain lectin, (vi) has a molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and equal 4000-40000, and that (v) is characterized by an affinity characterized by constant Association of the L-fucose α1→6 sugar chain of 1.0×104M-1or more (at 25 degrees C).

[0020]

It is desirable that at least one basidiomycete fungus were selected from families Strophariaceae, Tricholomataceae, Amanitaceae and Polyporaceae.

[0021]

It is desirable that at least one basidiomycete fungus selected from Pholiota terrestris, Pholiota squarrosa, Pholiota adiposa, Stropharia rugosoanmilata, Naematoloma sublateritium, Lepista sordida and Amanita muscaria.

[0022]

Preferably, basidiomycete and/or marsupial fungus used in the preparation of L-fucose α1→6 specific lectin, was a carpophores (fruiting bodies).

[0023]

The present invention also allows you to create a method for the detection of L-fucose α1→6 sugar chain including the process that leads to the effects of sugar chains on L-fucose α1→6 specific lectin.

[0024]

The sugar chain is, in particular, a tumor marker.

[0025]

The present invention also allows you to create a method of fractionation of L-fucose α1→6 sugar chain including the process that leads to the effects of sugar chain n is L-fucose α1→6 specific lectin. In particular, the invention allows to create a method of fractionation of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain by application of immobilized L-fucose α1→6 specific lectin.

[0026]

Sugar chain used for the method of fractionation, associated, for example, with the antibody.

[0027]

The present invention also allows you to create a diagnostic tool and a diagnostic kit for the detection of L-fucose α1→6 sugar chain. The diagnostic tool includes as an active ingredient L-fucose α1→6 specific lectin.

[0028]

A new lectin described in the present invention, has a much higher affinity for sacharides chain, glycopeptides and glycoprotein with α1→6 linked L-vukoti than conventional lectin, and has the constant Association of 1.0×104M-1or more. In particular, it is possible specific recognition only sugar chain with L-fucose α1→6 structure of the chain.

With that level of specificity, L-fucose α1→6 specific lectin can be used in various applications, as shown below.

[0029]

Unlike conventional lectin with affinity for L-fucose α1→6 sugar chain, the present invention allows to detect α1→6 L-fucose sugar chain with greater specificity.

[0030]

Method of fractional is of L-fucose α1→6 sugar chain of the present invention makes it possible to provide on the basis of precise identification of α1→6 L-fucose more rigorous fractionation between the L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain. In the L-fucose α1→6 sugar chain or not containing L-fucose α1→6 sugar chain are cleaned with a high degree of purity. In particular, by using the method described here fractionation to remove the L-fucose α1→6 sugar chain of the drug is based on an antibody comprising a mixture of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain, you can obtain drugs based on antibodies with enhanced ADCC activity. As a result, the drug is based on an antibody can be introduced in a lower dose, thereby successfully be achieved, for example, reduce the cost of the drug and reduce the influence of side effects. In addition, the drug-based antibodies can be set in accordance with a symptom or side effect.

List of figures

[0031]

Fig.1 is a block diagram α1→6 L-fucose oligosaccharide and does not contain α1→6 L-fucose oligosaccharide used in the working example and comparative example of the present invention.

Fig.2 is a block diagram of the L-fucose α1→6 oligosaccharide and not containing L-fucose α1→6 oligosaccharide used in the working example and comparative example of the present invention.

Figure 3 represents the process of cleaning PTL of example 1.

Fig.4 is a graph of the elution in ion-exchange chromatogra the AI PTL of example 1.

Fig.5 is a graph of the elution in affinity chromatography PTL of example 1.

Fig.6 shows the result of electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE) PTL of example 1 (the picture instead of the picture).

Figure 7 shows the process of cleaning SRL of example 2.

Figure 8 shows the scheme of elution with hydrophobic chromatography SRL of example 2.

Figure 9 presents the scheme of elution in reversed-phase chromatography SRL of example 2.

Figure 10 shows the result of electrophoresis SDS-PAGE SRL of example 2 (picture instead of the picture).

Figure 11 shows the result of mass spectroscopy PTL of example 1.

Figure 12 shows the result of mass spectroscopy SRL of example 2.

13 shows the result of Western blotting using PTL of example 1 (the picture instead of the picture).

Figure 14 shows the result of Western blotting using SRL of example 2 (picture instead of the picture).

Figure 15 shows the result of Western blotting using the AAL of comparative example Comparison Example 1 (the picture instead of the picture).

Figure 16 shows the result of Western blotting using AOL from comparative example 2 (the picture instead of the picture).

17 shows the result of Western blotting using LCA of comparative example 3 (the picture instead of the picture is (a).

Figure 18 shows the result when only whites are stained SVV (Kumasi bright blue) for control (picture instead of the picture).

Figure 19 shows the result of the detection of glycoproteins by the method of enzyme-linked immunosorbent assay ELISA using PTL of example 1.

In Fig.20 shows the result of the detection of glycoproteins using ELISA method using SRL of example 2.

Fig.21 shows the result of the detection of glycoproteins using ELISA method using the AAL of comparative example 1.

In Fig.22 shows the result of the detection of glycoproteins using ELISA method using AOL from comparative example 2.

In Fig.23 shows the result of the detection of glycoproteins using ELISA method using LCA of comparative example 3.

In Fig.24 shows the result of detection of the difference in the sugar chain between the α fetoprotein L1 and L3 using ELISA method using PTL of example 1.

In Fig.25 shows the result of detection of the difference in the sugar chain between the α fetoprotein L1 and L3 using ELISA method using SRL of example 2.

26 shows the result of detection of the difference in the sugar chain between the α fetoprotein L1 and L3 using ELISA method using the AAL of comparative example 1.

On figure 27 shows the result of detection of the difference in the sugar chain between the α fetoprotein L1 and L3 using ELISA method using AOL from the comparator is on example 2.

In figure 28 shows the result of detection of the difference in the sugar chain between the α fetoprotein L1 and L3 using ELISA method using LCA of comparative example 3.

On 29 shows the process of cleaning NSL from example 3.

On RIS chart shows elution with hydrophobic chromatography NSL from example 3.

In Fig.31 shows a graph of the elution in reversed-phase chromatography NSL from example 3.

In figure 32 shows the result of electrophoresis SDS-PAGE NSL from example 3 (picture instead of the picture).

Figure 33 shows the process of cleaning LSL from example 4.

At 34 is a diagram elution with hydrophobic chromatography LSL from example 4.

On picture.35 shows a graph of the elution in reversed-phase chromatography LSL from example 4.

36 shows the result of SDS-PAGE LSL from example 4 (picture instead of the picture).

On 37 shows the result of mass spectroscopy NSL from example 3.

On figure 38 shows the result of mass spectroscopy LSL from example 4.

The best way to use inventions

[0032]

The next section presents an example of an L-fucose α1→6 sugar chain, which is linked L-fucose α1→6 specific lectin.

[0033]

[Chemical formula 1]

[In the formula, Man is mannose, GlcNAc means N-acetylglucosamine, a Fuc means of L-fucose.]

[0034]

In addition, L-fucose α1→6 saharn the e circuit includes, for example, the free oligosaccharide, glycoamino acid, glikopeptid, glycolipid, glycoprotein, proteoglycan and cells. In addition, L-fucose α1→6 sugar chain fluorescently can be painted, for example, using CyDye, 4-ethylaminomethyl (ABEE) and aminopyridine. N-sugar chain includes, for example, Vysokomol chain, the chain complex and hybrid type. In addition, N-sugar chain can also be obtained by partial decomposition of the sugar chain by chemical means using, for example, acid or hydrazine, or by using the simultaneous or successive use of enzymes sialidase, galactosidase, N-acetylglucosaminidase, fucosidase and mannosidase to partial decomposition of the sugar chain. Or N-sugar chain can also be obtained by the addition of sugar, such as glucose, or a functional group such as acetyl group, sulfate group or a phosphate group, a sugar chain.

[0035]

(1) Basidiomycete or marsupial mushroom, which are L-fucose α1→6 specific lectin include, for example, to the family Strophariaceae, Tricholomataceae, Polyporaceae and Amanitaceae. Strophariaceae includes, in particular, Pholiota terrestris, Stropharia rugosoannulata, Naematoloma sublateritium, Pholiota squarrosa, Pholiota adipose and Pholiota adiposa. Tricholomataceae includes, in particular, Lepista sordida. Polyporaceae includes, in particular, Trichaptum elongatum and Microporus affinis. Amanitaceae includes, in particular, Amanita muscaria. Among tabaiwalu or marsupial fungi are particularly preferred in terms of efficiency of extraction of lectin and specificity for binding to sugar, family Strophariaceae, Tricholomataceae and Amanitaceae.

Other preferred basidiomycete and marsupials mushrooms are Pholiota terrestris, Pholiota squarrosa, Pholiota adiposa, Stropharia rugosoannulata, Naematoloma sublateritium, Lepista sordid and Amanita muscaria.

[0036]

L-fucose α1→6 specific lectin has (2) molecular weight determined by electrofresh in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE), 4000-40000, preferably 4000-20000. Molecular mass SDS-PAGE is measured, for example, by the method

Lemmy (Laemmi) (Nature, volume 227, page 680, 1976).

[0037]

L-fucose α1→6 specific lectin has (3) the constant of Association (Ka) with L-fucose α1→6 sugar chain, equal to 1.0×104M-1and more preferably of 1.0×105M-1or more or more preferably of 1.0×106M-1and more. In particular, compared with AAL, AOL, LCA, NPA and PSA, which are known to have affinity to α1→6 linked L-fucose, L-fucose α1→6 specific lectin has an exceptionally high constant Association. This means that the L-fucose α1→6 specific lectin is associated with the L-fucose α1→6 sugar chain with a much higher selectivity than conventional lectin.

[0038]

L-fucose α1→6 sugar chain can have sialic acid on nereguliruem final part of the chain. Normal specific lectin with Fuksas in the kernel (e.g., LCA, NPA and PSA) has a low affinity for L-fucose α1→6 Saha is Noah chains with sialic acid at the non end. On the other hand, L-fucose α1→6 specific lectin superior to conventional from the standpoint of high affinity to sugar chains, as discussed above.

[0039]

The next section describes the method of calculation of Association constants by using frontal affinity chromatography (F). This method is based on the following principle. When the diluent with a certain concentration of fluorescent colored sugar chain (for example, shown in figures 1 and 2) is fed into the column, where the immobilized lectin, a sugar chain in a short time out from the column, if between the lectin and sugar chain no interaction occurs. Then immediately see the front of the elution. If there is an affinity for the lectin, sugar elution circuit occurs with a delay.

[0040]

Preparation of the column with the lectins used in the device, as follows. 1. The purified lectin was dissolved in the 0.1-0.2 M buffer solution of NaHCO3(pH 8,3-8,5). 2. Lectin immobilized primary amino group, a carrier is, for example, an activated NHS-sepharose. 3. Further, the lectin is blocked with a solution of Tris-buffer, including, for example, a primary amine or ethanolamine. 4. Lectin-sepharose suspendered 10 m moles of Tris-buffer containing 0.8% NaCl (pH 7.4, TBS). Next, the resin with immobilized lectin is loaded into a miniature column is (φ2 mm* 10 mm, 31.4 μl). 5. Miniature geyser, which is resin with immobilized lectins is fixed by the holder, then the column with the lectin is connected to the automatic analyzer FAC (FAC-1, SHIMADZU CORPORATION).

[0041]

In balanced column lectin is poured at a flow rate of 0,125 ml/min in the amount of 300 μl pyridinemethanol sugar chain (PA-sugar chain), diluted analytical buffer solution (10 mmol Tris-buffer containing 0.8% NaCl (pH of 7.4, TBS)) to the concentration (2.5 nanomoles) sufficiently lower than the dissociation constant (Kd) lectin. Next, using the detector fluorescite radiation is identified PA-sugar chain that goes from the column (wavelength excitation/wavelength fluorescent radiation: 310 nm/380).

[0042]

Based on the data of the detector, using the front elution (V0) sugar chain (PA-rhamnose), does not interact with the lectin as a model, as an indicator of the intensity of interaction is calculated delay (V-V0) front elution (V) of a sugar chain that interacts with the lectin. Next, on the basis of the following criteria equations FAC, based on V-V0and Btcalculated constant of Association (Kabetween the sugar chain and lectin. If the intensity of the interaction (the value of V-V0and the constant Association is higher, there is a higher affinity between the lectin and L-fucose α1→6 sugar chain

[0043]

[Equation 1]

[In this equation, A denotes a substance used for elution, A0mean initial concentration of substance A, B - immobilized ligand, V - eluting volume, V0- eluting "front" volume for substances that do not interact with the immobilized ligand B, Bt- effective amount of the ligand, Kdthe dissociation constant (the inverse of the constant Association)].

[0044]

The specificity of the lectin-binding sugar is also confirmed by the use of red blood cells, which are specific agglutination by lectins to study the type of sugar, which can inhibit the agglutination of red blood cells and its concentration.

[0045]

In addition, it is desirable that the L-fucose α1→6 specific lectin (4) practically did not correlate with vysokotonnazhnoe sugar chain and/or glycolipid, not including L-fucose α1→6 sugar chain. Thus, the L-fucose α1→6 specific lectin has a higher binding specificity. The expression "virtually associated" here means the constant Association of 1.0×103M-1or less, preferably constants of the Association of 1.0×10 2M-1or less and, in particular, the constant Association of 0.

[0046]

In addition, it is desirable that the L-fucose α1→6 specific lectin (5) had an affinity for α1→6 fokusirovannym, mono-, di-, tri - and tetraantennary N-picenum. This affinity is characterized by constant Association of 1.0×104M-1or more (at 25 degrees C), preferably the constant Association of 1.0×105M-1and more.

[0047]

Examples of structures α1→6 fokusirovannyi mono-, di-, tri - and tetraantennary N-glycans with an affinity to those described here lectin below.

[Chemical formula 2]

[0048]

L-fucose α1→6 specific lectin described in the present invention, is a conventional amino acid sequence, in particular, represented by the number 1. 4th, 5th, 6thand 7thXaas in the sequence of 1 means Asp/Asn/Glu/Thr, Thr/Ser/Ala, Tyr/Phe and Gln/Lys/Glu, respectively, and the diagonal dividing line means "or".

[0049]

A specific example of an L-fucose α1→6 specific lectin described in the present invention, a protein or a peptide represented by the numbers 1-6.

[0050]

Lectin described sequence number 2, represents a new lectin extracted from Pholiota terrestris (hereinafter PTL). 10thand 17is th Xaas rooms 2 can be any amino acid residue, preferably Cys. 20ththe 23rd, 27*, 33rd, 35thand the 39thXaas are a Tyr/Ser, Phe/Tyr, Arg/Lys/Asn, Asp/Gly/Ser, Asn/Ala and Thr/Gln, respectively.

[0051]

Lectin, represented by number 3, is a new lectin extracted from Stropharia rugosoannulata (next SRL). 10thand 17thXaas rooms 3 can be any amino acid residue, preferably Cys. 4th, 7th, 9th, 13th, 20th, 27th, 29th, 33rd, 34thand the 39thXaas are Pro/Gly, Glu/Lys, Val/Asp Asn/Asp/Glu, His/Ser, Lys/His, Val/Ile, Gly/Asn/Ser, Ala/Thr and Arg/Thr, respectively.

[0052]

Lectin, presents ordinal 4, represents a new lectin extracted from Lepista sordida (hereinafter LSL). 10thand 17thXaas rooms 4 can be any amino acid residue, preferably Cys. 1th, 4th, 7th, 8th, 9th, 13th, 16th, 20th, 22th, 25th, 27th, 31thand the 34tXaas are Ala/Gin, Pro/Lys Ala/Ser, Met/Ile/Val, Tyr/Thr, Asp/Asn, Lys/Glu, Ala/Asn, Val/Asp/Asn, Asp/Asn, Arg/His/Asn, Gin/Arg and Thr/Val, respectively.

[0053]

Lectin, presents the ordinal number 5, represents a new lectin extracted from Naematoloma sublateritium (hereinafter NSL). 10thand 17th Xaas 5 can be any amino acid residue, preferably Cys. 13th, 14thand 16tXaas are Asp/Thr, Ser/Ala, and Gln/Lys, respectively.

[0054]

Lectin, presents the ordinal number 6, represents a new lectin extracted from Naematoloma sublateritium (hereinafter NSL). The sequence number 6 describes a variant in which one Asn attached to the peptide with the number 5. Thus, the 10thand 18thXaas rooms 6 can be any amino acid residue, but preferably Cys. 14th, 15thand 17thXaas are Asp/Thr, Ser/Ala, and Gln/Lys, respectively.

[0055]

Since the proteins or peptides having the amino acid sequence of SEQ ID NO: 2-6 are new, the present invention is devoted to the L-fucose α1→6 specific to the lectin, which is (a) a protein or peptide with an amino acid sequence selected from SEQ ID NO: 2-5 or (b) a protein or peptide in which one or several amino acids are deleted, added or substituted in one of the sequences SEQ ID NO: 2-5, which is functionally equivalent protein or peptide with amino acid sequence SEQ ID NO: 2-5. The term "functionally equivalent" here means the affinity constant of the Association of 1.0×104M-1or more to the L-fucose α1→6 sugar chain, preferably 1.0×105M-1or more and more preferably 1.0×106 M-1and more. The variant example shown in paragraph (b), is a protein or peptide having the amino acid sequence of SEQ ID NO: 6.

[0056]

The present invention also devoted gene that defines (a) a protein or peptide with an amino acid sequence selected from SEQ ID NO: 2-5 or (b) a protein or peptide in which one or several amino acids are deleted, added or substituted in one of the sequences SEQ ID nos: 2-5 and which is functionally equivalent protein or peptide with an amino acid sequence selected from SEQ ID nos: 2-5. The expression "functionally equivalent" has the same meaning as above.

[0057]

The degree of similarity between proteins and peptides, SEQ ID nos: 2-6. is at least 37% (see table 14). Thus, the present invention also allows you to get L-fucose α1→6 specific lectin, which is a protein or peptide with similarity score of at least 37% or more with the amino acid sequence selected from SEQ ID NO: 2-6, and which is functionally equivalent protein or peptide with an amino acid sequence selected from SEQ ID nos: 2-5. The expression "functionally equivalent" has the same meaning as above.

[0058]

L-fucose α1→6 specific lectin can be extracted from the basidiomycete, and/or marsupial fungus, for example, using a suitable combination of f the th method of extraction, method of separation and purification method. In particular, the possible process using an aqueous solvent in order to obtain water-soluble extract from and/or marsupial of the fungus. From this extract get lectin (vi) molecular weight determined by the method of SDS-PAGE and equal 4000-40000, preferably 4000-20000 and (v) the affinity for L-fucose α1→6 sugar chain described by constant Association, equal to 1.0×104M-1and more preferably of 1.0×105M-1and more and more preferably of 1.0×106M-1or more (at 25 degrees C).

[0059]

It is desirable that the basidiomycete fungus was selected from at least one out of the family Strophariaceae, Tricholomataceae, Polyporaceae and Amanitaceae. In particular, Strophariaceae include, for example, Pholiota terrestris (Pholiota terrestris Overholts), Pholiota squarrosa (Pholiota squarrosa (Fr.) Kummer), Pholiota adiposa (Pholiota adiposa (Fr.) Kummer), Stropharia rugosoannulata (Stropharia rugosoannulata Farlow in Murr.), Naematoloma sublateritium (Naematoloma sublateritium (Fr.) Karst or Hypholoma sublateritium (Fr.) Quel), Tricholomataceae include, for example, Lepista sordida (Lepista sordida (Schum.: Fr.) Sing.), to Polyporaceae include, for example, Trichaptum elongatum (Trichaptum elongatum), Microporus affmis (Microporus vemicipes), Amanitaceae include, for example, Amanita muscaria (Amanita muscaria). For all these basidiomycete and/or marsupial mushrooms, preferably using carpophores (fruiting bodies).

[0060]

Method extract basidiomycete has no restrictions, up to day while IU the od provides, for example, the contact of the aqueous solvent with carpophores of a basidiomycete. From the point of view of efficiency of extraction is the preferred method in which the carpophores basidiomycete sprayed in an aqueous medium to obtain a suspension. As a method of sputtering can be used the traditional method using, for example, a mixer or homogenizer.

[0061]

The aqueous solvent may be a buffer solution or a mixture of water or buffer solution and an organic solvent that can be mixed, for example, with water, and preferably represents a buffer solution or a mixture of organic solvent and buffer solution.

[0062]

View of the buffer solution is not limited, it can be a known buffer solutions, among which particularly preferred application of the buffer solution with interval buffering pH 3-10 and even more preferably a buffer solution with an interval of buffering the pH of 6-8. In particular, application of phosphate buffer, nitrate buffer, acetate buffer and Tris buffer, of which from the point of view of efficiency of extraction of the preferred phosphate buffer.

[0063]

The buffer solution is not limited to a specific maximum concentration of salt. From the point of view of efficiency of extraction and interval superiro is of the desired salt concentration of 1-100 mmol or more, the preferred salt concentration 5-20 mmol.

[0064]

In buffer solution, you can add salt. For example, isotonic solution of sodium chloride, buffered phosphoric acid and the resulting addition of salt to the phosphate buffer, it is desirable to use as the aqueous solvent for the purposes of the present invention.

[0065]

The organic solvent may be any organic solvent that is miscible with water without any restrictions, in particular, preferably the use of acetone, methanol, ethanol, 2-propanol and acetonitrile. It is preferable to mix an organic solvent with water or buffer solution at a concentration of 10-40% (mass).

[0066]

It is desirable that the extraction process is additionally included, in particular, the removal process from a mixture of an aqueous solvent and carpophores basidiomycete substances, insoluble in water. Method for removing insoluble substances may represent, for example, filtration or centrifugation, and from the point of view of efficiency of removal preferably centrifugation.

[0067]

It is desirable that the extraction process was provided by sputtering of carpophores basidiomycete, for example, phosphate buffered salt for removal of insoluble materials by centrifugation order floor the treatment of the extract in aqueous solvent.

[0068]

Method for L-fucose α1→6 specific lectin may provide for more effective cleaning with the use of any of the following cleaning methods.

[0069]

(Method of purification 1)

The extract in aqueous solvent, the resulting implementation of this process, is subjected to the method of ammonium sulfate precipitation, with the aim of obtaining latinamerica faction. Next, the resulting latinamerica fraction is purified using hydrophobic chromatography and reversed-phase chromatography.

[0070]

(Method of purification 2)

The extract in aqueous solvent, the resulting implementation of this process, is subjected to affinity chromatography using a carrier representing thyroglobulin, immobilized on agarose.

[0071]

(Method of purification 3)

The extract in aqueous solvent obtained by this process is handled by precipitation with ammonium sulfate to obtain latinamerica fraction, which is subjected to dialysis in contrast to the methods of distillation and freeze drying. Next, the coarse fraction lectin is dissolved in a solution of Tris-buffer and then subjected to ion-exchange chromatography. Next, the remaining active fraction is concentrated and further separated by gel filtration.

[0072]

Meth is on the receipt, described in the present invention, may include a processing stage dialysis fractions, including lectin obtained by purification, and the stage of lyophilization of a solution of lectin obtained from dialysis treatment. Eventually allows simple selection of the lectin. The processing stage dialysis and lyophilization can be performed using known traditionally used methods.

[0073]

L-fucose α1→6 specific lectin, which represents (a) a protein or peptide with an amino acid sequence selected from SEQ ID NO: 2-5 or (b) a protein or peptide in which one or several amino acids are deleted, added or substituted in one of the sequences selected from SEQ ID nos: 2-5 and which is functionally equivalent protein or peptide with an amino acid sequence selected from SEQ ID nos: 2-5, can be obtained not only by extraction from natural plants, but also by artificial expression in non-natural media or chemical synthesis. The above substance is also within the technical scope of the present invention. Expression in the media and chemical synthesis can be performed using a conventionally used known methods.

[0074] the Present invention also allows you to create a method for the detection of L-fucose α1→6 sugar chain is using the L-fucose α1→6 specific lectin. L-fucose α1→6 specific lectin recognizes L-fucose α1→6 sugar chain with greater specificity than in the normal case, and can communicate with it. Thus, the L-fucose α1→6 specific lectin is desirable to use for the specific detection of compounds with a sugar chain, including L-fucose α1→6 sugar chain, for example, polysaccharides, glycolipids or glycoproteins.

[0075] In the L-fucose α1→6-specific lectin used for detection, it is preferable to use labeled lectin. Labeled lectin described in the present invention, includes, as a minimum, the L-fucose α1→6 specific lectin and a means of tagging and marked so that the labeled lectin to be detected.

[0076] the Set of tagging is not limited, they can refer to any known method of tagging, including, for example, labeling with a radioisotope or, for example, the binding connections of the label.

[0077] the Set of compounds of the labels is not particularly limited and may include the connection traditionally used for this purpose, including direct and indirect connection-label, an enzyme or a fluorescent compound. Among specific examples of compounds-tag Biotin, digoxigenin, peroxidase from horseradish, fluoresceinisothiocyanate and CyDye. These compounds tags associated with lectin using the traditional method.

[0078] Preferably, L-fucose α→6 specific lectin was a lectin from a basidiomycete, preferably PTL, SRL, SRL, LSL and AML, and more preferably PTL and SRL. As shown in the example, PTL and SRL different from the usual L-fucose-specific lectins that PTL and SRL is not associated with L-vukoti other than α1→6 linked L-fucose, and vysokotonnazhnoe sugar chain not containing L-fucose. Thus, PTL and SRL - the best option when using L-fucose α1→6 specific lectin method described in the present invention.

[0079] Detection of L-fucose α1→6 sugar chain may be performed, for example, using lectinology chromatography using immobilized L-fucose α1→6 specific lectin. Lachinova chromatography is a affinity chromatography, which uses the property of the lectin specifically bind to a sugar chain. In the case of a combination lectinology chromatography with HPLC (HPLAC) can be expected analysis with high throughput.

[0080]

The media on which immobilized L-fucose α1→6 specific lectin is a gel-forming material, such as agarose, dextran, cellulose, starch or polyacrylamide. These gel-forming material may be a commercial material without any particular limitation, including, for example, sepharose 4B and sepharose 6B (GE Healthcare Bioscience).

[0081]

The column used in lachinova the chromatography represents a column in which lectin immobilized, for example, on the microplate or nanomembrane.

[0082]

Immobilized L-fucose α1→6 specific lectin has a concentration typically in the range from 0.001 to 100 mg/ml and preferably from 0.01 to 20 mg/ml When the carrier is agarose gel, the carrier is activated, for example, CNBr and subsequently binds to the lectin. Lectin can also be mobilitat gel with activated spacer. Or lectin can also be mobilitat on the gel using a formyl group with subsequent restoration NaCNBH3. Or you can also use commercial activated gel, for example, NHS-sepharose (GE Healthcare Bioscience).

[0083]

Sample L-fucose α1→6 sugar chain is poured into a column through which then transmit the buffered cleaning solution and equilibrium. One type of buffer solution has a concentration of from 5 to 500 mmol, preferably from 10 to 500 mmol, and pH from 4.0 to 10.0, preferably from 6.0 to 9.0, and contains NaCl from 0 to 0.5 mol, preferably from 0.1 to 0.2 mol, and CaCl2, MgCl2or MnCl2from 0 to 10 mmol, preferably from 0 to 5 mmol.

[0084]

After washing the unbound materials buffer L-fucose α1→6 sugar chain eluted neutral sedentarism buffer solution, which contributes effectively is at the elution sugar chain desorbers agent, such as sodium chloride or hapten-sugar. This buffer solution may be a buffer solution specified above. Deformirujuschij agent has a concentration of preferably from 1 to 500 mmol, and more preferably from 10 to 200 mmol.

[0085]

Along with the above method, the sugar chain is detected also by means of chromatography, pectinophora chip, enzyme-linked immunosorbent assay (ELISA), aggregated, method, surface plasma resonance, for example using the Biacore system®, or electrophoresis, for example, a method well known to specialists in this field of technology.

[0086]

On a sample, comprising the sugar chain is not subject to any specific restrictions. Sample sugar chain may include, for example, blood, blood plasma, serum, tears, body fluids, fluid in the chest, urine, air-conditioned environment in cell culture and secretion of genetically modified animals.

[0087]

A concrete example of an L-fucose α1→6 sugar chain as the object of detection by this method is the sugar chain is synthesized using α1→6 fucosyltransferase (FUT8). L-fucose α1→6 sugar chain may be, for example, α-fetoprotein, α5β1-integrin, TGFβ-receptor or EGF-receptor. Preferably, the sugar chain used in the method of detection, not only is La a tumor marker.

[0088]

Accurate detection of α1→6 pulselearning α-fetoprotein useful for early diagnosis of cancer of the liver cells, which clinically complicates cirrhosis, histological observations of cancer of the liver cells, the precise determination of therapeutic effect, early detection of embryonic tumors and determine the rate of recovery of the liver in fulminant hepatitis. α5β1-integrin, which is transferred α1→6 linked L-fucose, is also considered as an indicator in the diagnosis of liver cancer.

[0089]

The object detection method described in the present invention are including, in addition to cancer of the liver cells, diagnosis of tumors (e.g. prostate cancer, breast cancer, stomach cancer, cancer of the small intestine, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, uterine cancer, ovarian cancer, soft tissue sarcoma, bone cancer, melanoma, glioblastoma, astrocytoma, medulloblastoma, acute lymphoma, malignant lymphoma, the disease Hodgkins, non-Hodgkin's lymphoma, acute myelogenous leukemia, chronic lymphocytic leukemia), allergies, autoimmune disorders, and cardiovascular diseases such as emphysema.

[0090]

L-fucose α1→6 specific lectin is a new lectin, which differs from the known to the traditional lectin their physico-chemical and biochemical properties, such as, for example, specific binding to a sugar chain. In particular, since the L-fucose α1→6 specific lectin has the ability specific recognition of L-fucose α1→6 communication, L-fucose α1→6 specific lectin can be used as a diagnostic tool, the test reagent adsorbing agent for analysis with separation of carbohydrates and immunoregulatory funds. Thus, the present invention allows to obtain a diagnostic tool that includes an L-fucose α1-6-specific lectin as an active ingredient and is used to detect L-fucose α1→6 sugar chain is synthesized using L-fucose α1→6 transferase, and a diagnostic kit comprising the diagnostic tool. Diagnostic tool or a diagnostic kit used, for example, for the diagnosis of cancer of the liver cells.

[0091]

The present invention also allows you to create a method of fractionation of L-fucose α1→6 sugar chain, comprising the step of applying the L-fucose α1→6 specific lectin as a binding medium for L-fucose α1→6 sugar chain with the purpose of fractionation of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain.

[0092]

Preferably as L-fucose α1→6 specific lectin used in the method of fractionation of L-fucos the α1→6 sugar chain, used lectin from a basidiomycete, more preferably PTL, SRL, SRL, LSL and AML, and more preferably PTL and SRL.

[0093]

Method of fractionation described in the present invention is, for example, lectinology chromatography using immobilized L-fucose α1→6 specific lectin. Method details match the details described for the method of detection. If L-fucose α1→6 sugar chain cleaned, L-fucose α1→6 specific lectin is associated with the media in the column representing the agarose or cellulose, with a functional group, and then poured the sample with a sugar chain. After passing the sample through the column is collected adsorbed L-fucose α1→6 sugar chain. If not cleaned containing L-fucose α1→6 sugar chain, going this sample sugar chain, which is not adsorbed during the transmission of the sample sugar chain through the column.

[0094]

The object purified by fractionation method described in the present invention, can be of two types - L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain. A concrete example is the sugar chain of the antibody and preferably a sugar chain of human IgG.

[0095]

The purity of the sugar chain, fractionated by the method of fractionation (i.e., the ratio in the case L α1→6 sugar chain, L-fucose α1→6 sugar chain and the total number of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain and the ratio, in the case of not containing L-fucose α1→6 sugar chain that does not contain L-fucose α1→6 sugar chain and the total number of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain), is generally from 90 to 100%, preferably from 95 to 100% and particularly preferably from 99 to 100%.

[0096]

The present invention also allows you to get a drug on the basis of antibodies, comprising as an active ingredient L-fucose α1→6 sugar chain or not containing L-fucose α1→6 sugar chain with purity, generally from 90 to 100%, preferably from 95 to 100% and particularly preferably from 99 to 100%. In particular, the drug-based antibodies, including antibody is removed during the transfer to him α1→6 L-fucose is expected to provide enhanced ADCC activity. Among the options: Rituxan (chimeric antibody, non-Hodgkin's lymphoma), Herceptin (humanitariannet antibody, breast cancer), Erbitux (chimeric antibody, colon cancer, head and neck cancer), Zevalin (mouse antibody, non-Hodgkin's lymphoma), Campath (humanitariannet antibody, B-cell chronic lymphocytic leukemia), Bexar (mouse antibody, non-Hodgkin's lymphoma) and Avastin (humanitariannet antibody, for metastatic colon cancer).

0097]

Medicine on the basis of the antibodies obtained by the fractionation method described in the present invention may be used as provided by the traditional method (as regards, for example, pharmacologically approved carrier, excipient, the method of drug administration or dosage form), except for the fact that medicine is based on an antibody obtained by the fractionation method described in the present invention can be used in low doses and low dose, since this tool has a higher specific activity than the traditional means.

[0098]

The present invention also allows you to get L-fucose α1→6 sugar chain or not containing L-fucose α1→6 sugar chain with a purity of from 90 to 100%, fractionated by the proposed method, as well as a medical tool, comprising as an active ingredient L-fucose α1→6 sugar chain or not containing L-fucose α1→6 sugar chain with a purity of from 90 to 100%. Purity means that in the case of L-fucose α1→6 sugar chain, the ratio of L-fucose α1→6 sugar chain and the total number of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain and, in the case of not containing L-fucose α1→6 sugar chain, the ratio does not contain L-fucose α1→6 sugar the second circuit and the total number of L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain. Preferably, the medical facility was a drug on the basis of antibodies.

[0099]

The present invention also allows you to create a method of selection of the L-fucose α1→6 sugar chain. This method includes the stage of interaction of the liquid containing the sugar chain, with L-fucose α1→6 specific lectin and collecting the L-fucose α1→6 sugar chain adsorbed L-fucose α1→6 specific lectin. This method of selection is useful to search for a new marker with L-fucose α1→6 sugar chain. You can also use the L-fucose α1→6 specific lectin, which can be used for detection method described in the present invention, for simple selection marker comprising an L-fucose α1→6 sugar chain.

[0100]

Preferably, the L-fucose α1→6 specific lectin used in the method of selection was a lectin from a basidiomycete, particularly preferably one of the family Strophariaceae, Tricholomataceae, Polyporaceae and Amanitaceae and more preferably PTL, SRL, SRL, LSL, and/or AML, among which the most preferred are the PTL and SRL.

[0101]

The present invention also allows you to create a method of selection of the L-fucose α1→6 sugar chain-specific lectin. This method is used, in particular, immobilized L-fucose α1→6 specific lectin. Provides exposure to liquids containing several saharn the x circuits on immobilized lectin. Next, the test profile sugar chains adsorbed on immobilized lectins (e.g., gel electrofret) is compared with a reference profile of a sugar chain adsorbed by the lectin, in the case where the fluid acts on immobilized PTL or SRL. Thus, the extracted L-fucose α1→6 specific lectin with a profile that matches the reference. As to identify a sugar chain adsorbed on each sample lectins is not required, the target lectin can be selected by a very simple procedure. Extracted the L-fucose α1→6 specific lectin can be used as a reference for the next sampling. Maybe use another method to use cDNA with the purpose of full or partial encoding amino acid sequence SEQ ID NO: 1. as a primer for cDNA capture of L-fucose α1→6 specific lectin from the sample to the lectins.

[0102]

Sugar chain includes at least one of the L-fucose α1→6 sugar chain, as shown in Fig.1 and Fig.2, and preferably includes at least one of not containing L-fucose α1→6 sugar chain to which is transferred L-fucose, non-attachable communication α1→6, and at least one sugar chain not containing L-fucose (for example, vysokomernoa sugar chain)Putem add it to confirm the absence of affinity for a sugar chain, non-L-fucose α1→6 sugar chain.

[0103]

Profile of adsorbed sugar chain can be measured using a method well known to experts in the art, for example, by using various types of chromatography, mass analysis, gel electrophoresis, pectinophora chip, immunofermentnogo assay (ELISA), surface plasma resonance, such as Biacore®, or electrophoresis.

Example

[0104]

In the next section, the present invention is described in more detail by examples and comparative examples. However, it is not limited to the examples presented.

[0105]

[Examples 1 and 2] (the Receipt, measurement characteristics and description of PTL and SRL) (1)

Getting PTL (example 1)

On the basis of the purification process, shown in figure 3, a lectin from Pholiota terrestris (PTL) was purified from the fungus Pholiota terrestris.

[0106]

(Extraction)

All procedures were performed at 4°C. Dried by sublimation powders Pholiota terrestris was subjected to extraction with 50 ml of 10 mm Tris buffer (pH of 7.2) at 4 degrees C for 2 hours. The resulting liquid was centrifuged (15000 rpm./min, 20 min, 4 ° C). Next, the supernatant was filtered through cheesecloth to obtain the first extract. The residue was subjected to repeated extraction with 50 ml of 10 mm Tris buffer (pH of 7.2) at 4 degrees C during the night. The liquid is then centrifuged (15000 rpm./min, 20 min, 4 gra the USA C) and the supernatant was filtered through cheesecloth to obtain a second extract. Then the extracts were filtered together through filter paper to obtain an extract of Pholiota terrestris.

[0107]

(Ion exchange chromatography)

Extract (87 ml) was poured into the column with DEAE-separate (GE Healthcare Bioscience) and set the balance using 10 mm Tris buffer (pH of 7.2). After washing the column with buffer, the bound fraction was desirerable 0.1 M NaCl in the buffer. Next faction with hemagglutinin activity (shown ←→ figure 4) was subjected to dialysis against distilled water and lyophilization.

[0108]

(Affinity chromatography)

Dried dialysate was again dissolved in 10 mm sodium phosphate buffer (pH of 7.4, hereinafter simply PBS). Then the extract solution was poured into a column with thyroglobulin, immobilized on agarose, and the balance was achieved by using the same buffer. After washing the column PBS associated fraction was determinables using 0.2 M ammonia. Then the fraction with hemagglutinin activity (shown ←→ figure 5) were collected, subjected to ultrafiltration and lyophilization to obtain 1.07 mg PTL.

[0109]

(Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE)

Procedure SDS-PAGE was carried out using the device for electrophoresis Phastsystem (GE Healthcare Bioscience) and gel with a gradient 8-25 (GE Healthcare Bioscience). The test solution and the molecular weight marker used in the plantations in the amount of 1 μl.

Electrophoresis was carried out on the basis of the Protocol of the product using the traditional method. Figure 6 shows the results of SDS-PAGE for PTL. Fig.6 band M, lanes 1 and 2 indicate the following. Lane M: molecular weight marker (APRO), band 1: PTL, 2-mercaptoethanol (-), lane 2: PTL, 2-mercaptoethanol (+), gel: Gradient 8-25 Sample: 1 µl/lane, dye: Kumasi brilliant blue (CBB)

[0110]

When carrying out SDS-PAGE with 8-25% gel confirmed that the main component is PTL.

[0111]

(2) Receiving SRL (example 2)

On the basis of the purification process, shown in Fig.7, the lectin from Stropharia rugosoannulata (SRL) was purified from the fungus Stropharia rugosoannulata.

[0112]

(Extraction)

All procedures were performed at 4°C. Dried by sublimation powders Stropharia rugosoannulata (400 g) was subjected to extraction with 1.6 l of PBS at 4 degrees C for 2 hours. The resulting liquid was centrifuged (15000 rpm./min, 20 min, 4 ° C). Next, the supernatant was filtered through cheesecloth to obtain the first extract. The residue was subjected to repeated extraction with 0.8 l of PBS at 4 degrees C during the night. The liquid is then centrifuged (10000 rpm./min, 20 min, 4 ° C) and the supernatant was filtered through cheesecloth to obtain a second extract. Then the extracts were mixed to form a liquid extract Stropharia rugosoannulata.

[0113]

(Deposition of ammonium sulfate)

Solid (NH 4)2SO4(1.3 kg) was added to the obtained supernatant (2.4 l) and 80% saturation. After leaving overnight at 4 degrees C the precipitate was collected by centrifugation (10000 rpm./min, 20 min, 4 ° C) and dialyzed against distilled water and lyophilization to obtain the fraction of Stropharia rugosoannulata, obtained by fractionation of 80% ammonium sulfate.

[0114]

(Hydrophobic chromatography)

Stropharia rugosoannulata, obtained by fractionation of 80% ammonium sulfate, was poured in a Butyl-TOYOPEARL 650M (TOSOH CORPORATION), the balance of which was set with 2M ammonium sulphate, PBS, for purification by hydrophobic chromatography. This type of chromatography involves the collection, ultrafiltration and lyophilization of the fractions, buervenich distilled water, to obtain the coarse fraction of lectin from Stropharia rugosoannulata (shown ←→ figure 8).

[0115]

(Reverse-phase chromatography)

Coarse fraction lectin from Stropharia rugosoarmulata was poured into the column C8 (Wako Pure Chemical Industries, Ltd.), the balance which was achieved with the help of 0.05% triperoxonane acid (TFA)/acetonitrile (100/0). When using this type of chromatography collected fraction, elyuirovaniya of 0.05% TFA/acetonitrile (70/30) (shown ←→ figure 9). Next, the solvent was removed by evaporation at room temperature, and the resulting dry powder was collected by receiving 7.5 mg SRL.

[0116]

SDS-PAGE (PhastGel gradient 8-25) was performed using system Phastsystem (GE Healthcare Bio-Sciences). The test solution and the molecular weight marker was taken in quantities of 1 µl. Electrophoresis was carried out on the basis of the Protocol of the product using the traditional method. Figure 10 shows the results of SDS-PAGE for SRL. 10, lane M, lanes 1 and 2 indicate the following. Lane M: molecular weight marker (APRO), band 1: SRL, 2-mercaptoethanol (+), lane 2: SRL, 2-mercaptoethanol (-), gel: gradient 8-25 Sample: 1 μl per strip, color: silver.

[0117]

When carrying out SDS-PAGE with 8-25% gel confirmed that the main component is SRL.

[0118]

(3) Properties PTL and SRL

(Mass-spectrometric analysis of MALDI-TOF)

PTL of example 1 and SRL of example 2 in the amount of 10 μg respectively separately dissolved in TA (a mixture with a volume ratio of 0.1%of TFA and acetonitrile 2:1). Next, a saturated sample, dissolved in TA - and lachinova TA-solutions were mixed in a volume ratio of 4:1 and the resulting mixture was added dropwise in an amount of 1.0 µl of the substrate with the sample. To measure the molecular weight of the PTL and SRL in the LP mode used the device Autoflex (Broker Daltonics K.K.). As shown by the result, the molecular weight is about 4500 m/z (Fig.11 and Fig.12).

[0119]

(The analysis of amino acid sequence)

For PTL of example 1 and SRL of example 2 were analyzed by linakis is now a sequence with apparatus Protein Peptide Sequencer PPSQ-21 (SHIMADZU CORPORATION). The results observed numbers of sequences 2 and 3, respectively. All sequences were new.

[0120]

PTL and SRL were tested for agglutinating activity of erythrocytes rabbit, horse, pig, sheep, human (A, B, O) and rabbit erythrocytes treated with actinozoa E. the Result in the form hemagglutinine activity are shown in table 1.

[0121]

[Table 1]
PTLSRL
Unit: titer (1.0 mg/ml)Unit: minimum hemagglutinins concentration (µg/ml)Unit: titer (1.0 mg/ml)Unit: minimum hemagglutinins concentration (µg/ml)
Rabbit645.21645.21
HorseNTNT1282.61
Pig1 20.83210.4
Sheep<1>333<1>333
PeopleA<1>333<1>333
B<1>333<1>333
O<1>333<1>333
Rabbit processing actinase E25600.135120.651

NT: Tests were not carried out

[0122]

As can be seen from the above results of analysis of amino acid composition and test hemagglutinin activity, PTL of example 1 and SRL of example 2 had a new lectin.

[0123]

(4) Evaluation Sagarmatha specificity PTL and SRL

Various monosaccharides, oligosaccharides and polisaharidy, presented in table 2, and glycoproteins, are presented in table 3, were tested for inhibition of haemagglutination assays to assess Sagarmatha specificity PTL of example 1 and SRL of example 2.

[0124]

In 96-well titration the microplate with a U-shaped bottom has prepared a series of double dilution of 10 μl of solutions of monosaccharide, oligosaccharide, polysaccharide and glycoprotein. Then a solution of the lectin with a pre-installed title 4 was added in an amount of 10 μl into the appropriate wells. Then the tablet was kept for sensitization at room temperature for one hour. Then 10 μl of a 4% suspension of erythrocytes was added to appropriate wells and kept the plate at room temperature for one hour. Next was evaluated visually dilution factor of the investigated solution, which completely suppressed the hemagglutination. The minimum concentration at which the observed suppression, called the minimum overwhelming concentration. The lower the minimum inhibitory concentration, the higher the specificity of the lectin. The results are presented in tables 2 and 3.

[0125]

For comparison, the estimated Sagarmatha specificity using the following commercially available lectins: comparative example 1: AAL (SEIKAGAKU BIOBUSINESS CORPORATION - J-OIL MILLS, Inc.), comparative example 2: AOL (TOYO CHEMICAL INDUSTRY CO., LTD. - Gekkeikan Sake Company, Ltd.), comparative example 3: LCA (SEIKAGAKU BIOBUSINESS CORPORATION - J-OIL MILLS, Inc.) and comparative example 4: PSA (SEIKAGAKU BIOBUSINESS CORPORATION - J-OIL MILLS, Inc.). The results are presented in tables 2 and 3.

[0126]

[Table 2]
Example 1 PTL (mmol)Example 2 SRL (mmol)Comparative example 1 AAL (mmol)Comparative example 2 AOL (mmol)Comparative example 3 LCA (mmol)Comparative example 4 PSA (mmol)
Glucose>100>100>100>100>100>100
Galactose>100>100>100>100>100>100
Mannose>100>100501005050
L-fucose>100>1000.3910.391>100>100
Xylose>100>100>100>100>100>100
The rhamnose>100>100>100100>100>100
N-acetylglucosamine>100>100>100>100>100>100
N-atsetilgalaktozamin>100>100>100>100>100>100
Methyl α-mannoside>100>100>1005050
Maltose>100>100>100>100100>100
Fructose>100>1002525>100>100
Sucrose>100>100>100>100>100>100
Melibiose>100>100>100>100>100>100
Raffinose>100>100>100>100>100>100
N-acetylneuraminic acid>100>100>100>100>100
N-glycolylneuraminic acid>100>100>100>100>100>100
Lactose>6>6>6>6>6>6
Serial lactose>6>6>6>6>6>6

[0127]

tr>
[Table 3]
Example 1 PTL (MCP/ml)Example 2 SRL (ál/ml)Comparative example 1 AAL (ál/ml)Comparative example 2 AOL (ál/ml)Comparative example 3 LCA (ál/ml)Comparative example 4 PSA (ál/ml)
The serum albumin bovine blood>250>250>250>250>250>250
The mucin (porcine)>250>25015.662.5>250>250
Sialomucins (pork)>250>25031.362.5>250>250
Fetuin>250>250>250>250>250>250
Avaliation>250>250>250>250250>250
α1-acid glycoprotein>250>250&t; 250>250>250>250
Transferrin>250>250>250>250>250>250
Thyroglobulin250250125>25062.562.5
The mucin (bovine)>250>250>250>250>250>250
Casein>250>250>250>250>250>250
Lactoferrin>250>250>250>250>250>250
Ribonuclease&t; 250>250>250>250>250>250
Immune globulin human G>250>250>250>250>250>250
Immune globulin human A>250>250>250>250>250>250

[0128]

As can be seen from tables 2 and 3, PTL of example 1 and SRL of example 2 was associated only with the thyroglobulin with α1→6 linked L-Fuksas. On the other hand, the AAL of comparative example 1 was associated not only with thyroglobulin, but also with sugars, such as L-fucose, fructose, as well as glycoprotein, for example, mucin with L-Fuksas in O-linked sugar chain. AOL of comparative example 2 was associated with sugars, such as L-fucose and fructose, and glycoprotein, for example, mucin with L-Fuksas in O-linked sugar chain. LCA of comparative example 3 and PSA of comparative example 4 was associated not only with thyroglobulin, but also with sugars, such as the mannose and methyl α-mannoside. We can say that the PTL of example 1 and SRL of example 2 are L-fucose α1→6 specific lectin that is not associated with L-Fuksas and mannose, and communicates only with α1→6 linked L-Fuksas.

[0129]

(5) Measurement of Association constants PTL and SRL with L-fucose α1→6 sugar chain. Constant Association PTL of example 1 and SRL of example 2 with L-fucose α1→6 sugar chain was measured using the following procedure.

[0130]

(Preparation of oligosaccharide)

Pyridylmethylamine (PA) sugar chain shown in figure 1 and 2, were used for analysis of frontal affinity chromatography (FAC). PA-sugar were purchased from companies TAKARA BIO INC., SEIKAGAKU BIOBUSINESS CORPORATION and Masuda ChemicalIndustries co., LTD. PA-caxap received by pyridylmethylamine using GlyeoTAG®(TAKARA BIO INC.) unlabelled sugar chain or a sugar chain obtained, for example, enzymatic cleavage.

[0131]

(Preparation of lectin column)

Lectin was dissolved in 0.2 M buffer solution of NaHCO3(pH 8,3), containing 0.5 M NaCl, and was associated with NHS-activated separate (GE Healthcare Bioscience) according to the manufacturer's instructions. Then separate with immobilized lectins suspended in 10 mm Tris buffer (pH of 7.4, TBS), containing 0.8%of NaCl, and the resulting substance was filled with miniature column (φ2 mm×10 mm of 31.4 μl).

[0132]

(Frontal affinity chromatography)

Frontal affinity chromatography was performed using an automatic instrument FAC (FAC-1, SHIMADZU CORPORATION). In particular, prepared as described above column with lectins were installed in the holder stainless steel, and a holder attached to the unit FAC-1. The flow rate and the column temperature was maintained at 0,125 ml/min and 25 degrees C, respectively. After the establishment of equilibrium in the column with TBS excessive volume of 0.5 ml to 4 ml) of PA-sugar chain (3,75 nm or 7.5 nm) was continuously applied to the column using an automatic device for sampling.

[0133]

The intensity of fluorescence of the eluate PA-sugar (wavelength of 310 nm excitation and wavelength fluorescent radiation 380 nm) were recorded to measure the intensity of the interaction between [the difference between the front edge of the eluate and the standard polysaccharide (PA-rhamnose): V-V0]. On the basis of intensity of interaction and the effective amount of the ligand was calculated constant Association Ka. The results are presented in tables 4-9.

[0134]

For comparison, using procedures similar to the above, was also calculated Association constants for AAL (comparative example 1), AOL (comparative example 2), LCA (comparative example 3) and PSA (comparative example 4), which are considered lectins specific to cow fucose. The results are shown in tables 4-7.

[0135]

[Table 4]
no sugar chainExample PTL 1 (M-1)Example 2 SRL (M-1)Comparative example 1 AAL (M-1)Comparative example 2 AOL (M-1)Comparative example 3 LCA (M-1)Comparative example 4 PSA (M-1)
Sugar chain containing α1→6 L-fucose0155.0×1054.9×1049.1×1041.3×1055.6×1041.2×105
2014.6×1056.4×1045.1×1041.2×1055.0×1051.0×105
2024.0×1056.1×1045.4×104of 1.2×1051.1×1054.4×104
2033.3×1055.2×1046.6×1041.3×1057.3×1042.5×104
4013.5×1055.7×1045.5×1041.4×1054.2×1049.2×104
4022.0×1054.4×1041.1×1041.6×1055.9×1044.8×104
403of 3.4×1055.6×1045.2×1041.3×1059.9×1043.8×104
4043.9×1055.8×1046.5×1041.5×1055.8×104 4.5×104
4053.2×1055.4×1045.6×1041.3×1054.7×1043.6×104
4062.2×1053.8×1044.7×1041.1×1051.8×1041.3×104
4072.8×1051.0×1044.5×1041.1×1051.0×103<1.0×103
4102.2×1053.9×1043.8×1041.0×105,<1.0×103<1.0×103
4132.8×1051.0×1043.2×1047.9×104<1.0 is 10 3<1.0×103
4182.2×1054.3×103<1.0×1046.3×104<1.0×103<1.0×103
6012.4×1051.0×1046.1×1041.4×1053.0×1043.1×104
6021.2×1053.2×1045.2×1041.4×1052.4×1042.9×104

[0136]

[Table 5]
no sugar chainExample PTL 1 (M-1)Example 2 SRL (M-1)Comparative example 1 AAL (M-1)Comparative example 2 AOL (M-1)Comparative p is emer 3 LCA (M -1)Comparative example 4 PSA (M-1)
Sugar chain containing another L-fucose, other than α1→6 L-fucose419<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
420<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
718<1.0×103<1.0×1036.5×1041.7×103<1.0×103<1.0×103
719<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
720<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
721<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
722<1.0×103<1.0×103<1.0×1035.6×104<1.0×103<1.0×103
723<1.0×103<1.0×1033.5×1047.6×104<1.0×103<1.0×103
726<1.0×103<1.0×103<1.0×1031.2×104<1.0 is 10 3<1.0×103
727<1.0×103<1.0×1031.3×1037.6×104<1.0×103<1.0×103
728<1.0×103<1.0×103<1.0×1031.2×104<1.0×103<1.0×103
729<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
730<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
731<1.0×103<1.0×103<1.0×0 3<1.0×103<1.0×103<1.0×103
739<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
909<1.0×103<1.0×1033.1×1054.4×104<1.0×103<1.0×103
910<1.0×103<1.0×1039.0×1045.6×104<1.0×103<1.0×103
931<1.0×103<1.0×103<1.0×1039.9×104<1.0×103<1.0×103
932<1.0×10 <1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
933<1.0×103<1.0×1032.1×1053.1×104<1.0×103<1.0×103

[0137]

<1.0×103
[Table 6]
no sugar chainExample PTL 1 (M-1)Example 2 SRL (M-1)Comparative example 1 AAL (M-1)Comparative example 2 AOL (M-1)Comparative example 3 LCA (M-1)Comparative example 4 PSA (M-1)
Sugar chain without L-fucose001<1.0×103<1.0×103<1.0×103<1.0×103<1.0×10sup> 3<1.0×103
002<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
003<1.0×103<1.0×103<1.0×103<1.0×1037.2X1037.8×103
004<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
005<1.0×103<1.0×103<1.0×1037.3×103<1.0×1037.4×103
006<1.0×103<1.0×103<1.0×10 3<1.0×1031.4×1047.1×103
007<1.0×103<1.0×103<1.0×103<1.0×103<1.0×1037.8×103
008<1.0×103<1.0×103<1.0×1036.9×1041.8×1041.0×104
009<1.0×103<1.0×103<1.0×103<1.0×1031.8×1047.9×103
010<1.0×103<1.0×103<1.0×103<1.0×1031.3×104<1.0×103
011<1.0×103<1.0×103<1.0×1031.4×104<1.0×103
012<1.0×103<1.0×103<1.0×103<1.0×1032.5×1041.7×104
013<1.0×103<1.0×103<1.0×103<1.0×1031.7×104<1.0×103
014<1.0×103<1.0×103<1.0×103<1.0×1031.7×1048.6×103
101<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
104<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
105<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
107<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
108<1.0×103<1.0×103<1.0×103<1.0×103 <1.0×103<1.0×103
301<1.0×103<1.0×103<1.0×1031.8×104<1.0×103<1.0×103
304<1.0×103<1.0×103<1.0×1032.0×104<1.0×103<1.0×103
305<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
307<1.0×103<1.0×103<1.0×103<1.0×103<1.0×1032.4×103
308<1.0×103<1.0×103 <1.0×103of 2.0×104<1.0×103<1.0×103
313<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
314<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
323<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
501<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
502 <1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
503<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
504<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103

[0138]

903
[Table 7]
no sugar chainExample I PTL (M-1)Example 2 SRL (M-1)Comparative example 1 AAL (M-1)Comparative example 2 AOL (M-1)Comparative example 3 LCA (M-1)Comparative example 4 PSA (M-1 )
Sugar chain without L-fucose701<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
702<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
703<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
704<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
705<1.0×103<1.0×103 <1.0×103<1.0×103<1.0×103<1.0×103
706<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
707<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
708<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
709<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
710 <1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
711<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
712<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
713<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
715<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103 <1.0×103
716<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
717<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
724<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
725<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
728<1.0×103<1.0×103<1.0×103 <1.0×103<1.0×103<1.0×103
732<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
733<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
734<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
735<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
736<1.0×103 <1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
737<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
738<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
901<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
902<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
905<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
906<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103
907<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103<1.0×103

[0139]

As can be seen from tables 4-7, AAL of comparative example 1 and AOL from comparative example 2, the contact does not contain α1→6 glycolipids L-fucose sugar chains (sugar chain 718, 722, 723, 727, 909, 910 and 933), and t is the train with L-fucose α1→6 sugar chain (sugar chain 15, 201-203 and 401-418). LCA of comparative example 3 and PSA of comparative example 4 are associated with a large number of sugar chains do not contain L-fucose α1→6 sugar chains are sugar chains 003, 005-014). On the other hand, PTL of example 1 and SRL of example 2 bind strongly with L-fucose α1→6 sugar chain and are not associated with not containing L-fucose α1→6 sugar chain and a sugar chain, not containing L-fucose. In addition, PTL of example 1 has a higher constant Association than usual lectin (constant Association Ka=1.0×105M-1or more). Moreover, PTL of example 1 and SRL of example 2 also bind strongly to the crust fokusirovannyi trentenne N-glycanase (sugar chain 407-413) and tetraantennary N-Picanol (sugar chain 418). Even in the case of adding sialic acid (sugar circuits 601 and 602) shows that the constant Association of the L-fucose α1→6 sugar chain is not reduced.

[0140]

(6) Detection of glycoproteins using PTL and SRL

(i) Preparation of glycoprotein

Were prepared glycoproteins (1)-(9) with the following main structures of sugar chains and (10) bovine serum albumin that do not contain sugar.

[0141]

(1) as a glycoprotein containing L-fucose α1→6 N-linked glikana, thyroglobulin (pork), having the following formula:

[Chemical formula 3]

[0142]

(2) to the operation of the glycoprotein, containing L-fucose α1→6 N-linked glikana, lactoferrin (bovine), having the following formula:

[Chemical formula 4]

[0143]

(3) as a glycoprotein containing L-fucose α1→6 N-linked glikana, immune globulin G (human), having the following formula:

[Chemical formula 5]

[0144]

(4) as glycoprotein that does not contain L-fucose α1→6 N-linked glycans, transferrin (human), having the following formula:

[Chemical formula 6]

[0145]

(5) as glycoprotein that does not contain L-fucose α1→6 N-linked glycans, α1-acid glycoprotein (human), having the following formula:

[Chemical formula 7]

[0146]

(6) as a glycoprotein containing vysokonadezhnye sugar chain, invertase (yeast), having the following formula:

[Chemical formula 8]

[0147]

(7) as a glycoprotein containing L-fucose O-linked sugar chain, mucin (porcine), having the following formula:

[Chemical formula 9]

[0148]

(8) as a glycoprotein containing L-fucose O-linked Saha the main chain, the mucin (bovine), having the following formula:

[Chemical formula 10]

[0149]

(ii) Detection of glycoproteins using Western blotting

PTL of example 1, SRL of example 2, AAL of comparative example 1 AOL from comparative example 2 and LCA of comparative example 3 was subjected to bitenova tagging.

[0150]

(Biotinylation lectin)

Lectin was measured and dissolved in 0.1 M sodium bicarbonate solution (5 mg/ml). Then the reagent for biotinidase was dissolved in dimethyl sulfoxide, and the resulting solution was added to a solution of the lectin for the reaction between them. Further reactant person to distil and liofilizirovanny to obtain labeled with Biotin lectin.

[0151]

(SDS-PAGE and blotting glycoprotein)

The solution obtained by dissolving the sample peptide in 10 mm sodium phosphate buffer (pH of 7.4, PBS) at 2 mg/ml, is supplied to microtrace 18 ál. 6 μl of liquid sodium dodecyl sulfate (SDS) (investigational buffer solution (MA-); Wako Pure Chemical Industries, Ltd.) and 1.25 μl of 2-mercaptoethanol (Bio-Rad Laboratories, Inc.) was added to each fluid and the resulting mixture was boiled for 5 minutes.

After electrophoresis in polyacrylamide gel substances were transferred to PVDF membrane (Immobilon IPVH 304 F0, Millipore K.K.).

[0152]

(Staining labeled with Biotin lectin)

The film was immersed in a 10 mm solution of Tris-buffer, with the holding of 0.8% NaCl with 1% BSA (pH 7,4, 1% BSA + TBS), and were shaken at room temperature for one hour. Then, the foil was cleaned three times with 10 mm solution of Tris-buffer (pH of 7.4, TBS)containing 0.8% NaCl. Then the film was immersed in a solution labeled with Biotin lectin (2 μg/ml) and was shaken at room temperature for one hour. Then the film three times purified using the TBS. After that, the film was immersed in a solution labeled with HRP (horseradish peroxidase streptavidin at 1 µg/ml (Vector Laboratories) and were shaken at room temperature for 30 minutes. After the film was purged three times with TBS, to run samples with staining used the set for painting with the use of immune-tag POD (Wako Pure Chemical Industries, Ltd.). Sample staining is designed for detection of glycoprotein using Western blotting using labeled with Biotin lectin. 13-17 presents pictures, which depict the following glycoproteins painted with PTL (Fig.13), SRL (Fig.14), AAL (Fig.15), AOL (Fig.16) or LCA (Fig.17).

[0153]

13-17 bands 0-6 mean the following. Lane 1: thyroglobulin, lane 2: lactoferrin, band 3: immune globulin, band 4: transferrin, band 5: α1-acid glycoprotein, band 6: invertase, stripe 0: bovine serum albumin (Standart).

[0154]

To control also performed staining protein of STS. Fot the graphy staining is presented in Fig.18. 18 strips 1-6 M and mean the following. Lane M: molecular weight marker, lane 1: thyroglobulin, lane 2: lactoferrin, band 3: immunoglobulin G, lane 4: transferrin, band 5: α1-acid glycoprotein, band 6: invertase, stripe 0: bovine serum albumin

[0155]

[Table 8]
GlycoproteinFeaturesExample 1 PTLExample 2 SRLComparative example 1 AALComparative example 2 AOLComparative example 3 LCA
(1) Thyroglobulin (pork)N-linked of glycan containing α1→6 L-fucose
(2) Lactoferrin (bovine)N-linked of glycan containing α1→6 L-fucose
(3) Immune globulin G (man) N-linked of glycan containing α1→6 L-fucose
(4) Transferrin (human)N-linked of glycan, not containing α1→6 L-fucosexxxxx
(5) α1-acid glycoprotein (human)N-linked of glycan containing α1→3 L-fucosexxxxx
(6) Invertase (yeast)Vysokomernoa sugar chainxxxxx
(0) Albumin serum (bovine)No sugar chainxxxxx
○ Discovered
x Not found

[0156]

As can be seen from the results shown in table 8 and in figures 13-18, in the case of LCA comparative example

were found not only glycoprotein containing L-fucose α1→6 sugar chain and glycoprotein containing vysokotonnazhnoe chain (invertase). On the contrary, in the case of PTL of example 1 and SRL of example 2 was detected only glycoprotein containing sL-fucose α1→6 sugar chain, and does not contain L-fucose α1→6 sugar chain and a sugar chain that does not contain L-fucose, were not found at all.

[0157]

(iii) Detection of glycoproteins using enzyme-linked immunosorbent assay (ELISA)

PTL of example 1, SRL of example 2, AAL of comparative example 1 AOL from comparative example 2 and LCA of comparative example 3, labeled with Biotin, was used for detection of glycoprotein enzyme-linked immunosorbent assay (ELISA).

[0158]

Glycoprotein and albumin as a protein that does not contain sugar chain and the role of the standard, was dissolved in 0.1 M buffer solution of carbonic acid (pH 9,5) at 1 mg/ml of the resulting solution was added to titration microplate (Nunc 439454) and incubated at 4 degrees C during the night. Then the solution was purged three times with 0.05% Tween/PBS. After this was added to the wells with 1% BSA/PBS and incubated solution at 37 degrees C for one hour. Then after about three times isdi solution of 0.05% Tween/PBS was added to wells labeled with Biotin lectin, diluted appropriately with 1% BSA/0.05% Tween/PBS and incubated solution at 37 degrees C for one hour. After three cleaning solution of 0.05% Tween/PBS was added to wells, and the solution labeled with HRP (horseradish peroxidase streptavidin (diluted 1% BSA/0.05% Tween/PBS and incubated solution at 37 degrees C for 30 minutes. After three cleaning solution of 0.05% Tween/PBS was added to the substrate TMB-Peroxidase (KPL) and the resulting solution was incubated at room temperature and without access to light for 10 minutes.

[0159]

The reaction was stopped with 1 M phosphoric acid. Then using the reader for microplates (MPR-A4i, TOSOH CORPORATION) was measured by the absorption at 450 nm. Based on this value was calculated rate for reaction ([absorbance at 450 nm for the wells, which were solid-phase glycoprotein involved in the reaction] - [absorbance at 450 nm for wells in which no solid phase glycoprotein involved in the reaction]. Next, for each lectin expected, the interaction intensity (relative value) with each glycoprotein based on the assumption that the value for glycoprotein (thyroglobulin) is 100%. The results of the calculations are presented in table 9 and figure 19-23.

[0160]

[0161]

As can be seen from tables 9 and Fig.19-3, in the case of AAL from comparative example 1 and AOL from comparative example 2 were found not only glycoprotein containing L-fucose α1→6 sugar chain, but that is undesirable, O-linked sugar chain (mucin).

On the contrary, in the case of PTL of example 1 and SRL of example 2 was detected only glycoprotein containing L-fucose α1→6 sugar chain and not containing L-fucose α1→6 sugar chain and a sugar chain that does not contain L-fucose, were not found.

In addition, lectins from comparative examples 1-3 demonstrate the intensity of the interaction with lactoferrin and immunoglobulin G (IgG), lower than that observed for thyroglobulin. On the contrary, the lectins from examples 1 and 2 showed the same intensity of interaction with lactoferrin, as in the case of thyroglobulin.

[0162]

(iv) the Discovery of a sugar chain marker using ELISA method

α-fetoprotein ("AFP") is a glycoprotein contained in the serum N-linked sugar chain. AFP is virtually absent in the serum of healthy human adult. On the other hand, the serum of patients with benign liver disease is characterized by a high content of α-fetoprotein L1-sugar chain(AFP-L1).

Next, α-fetoprotein L3-sugar chain (AFP-L3) is detected in a patient suffering from Raco the liver. Usually the difference in the sugar chains is measured using LCA, and the measurements are used to diagnose liver diseases.

[0163]

[Chemical formula 12]

[0164]

Labeled with Biotin PTL of example 1, SRL of example 2, AAL example 1, AOL from comparative example 2 and LCA of comparative example 3 were used to assess the binding properties of α-fetoprotein ELISA method.

[0165]

0.1 M buffer solution of carbonic acid (pH 9.5) is used for cultivation of α-fetoprotein (obtained from serum, cord blood, mainly L1-sugar chain) and α-fetoprotein-L3 (prepared from air-conditioned environment of cancer cells human liver) to 0.01 µg/ml of the resulting solution was added to microtiter tablet (Nunc) and incubated at 4 degrees C for one night. After three cleaning solution of 0.05% Tween/PBS was added to the wells with 1% BSA/PBS and incubated solution at 37 degrees C for one hour. After triple rinsing solution of 0.05% Tween/PBS in the wells was added to the solution labeled with Biotin lectin, suitably diluted with 1% BSA/0.05% Tween/PBS, and incubated solution at 37 degrees C for one hour. After three cleaning solution 0.05% Tween/PBS was added a solution of HRP labeled streptavidin diluted 1% BSA/0.05% Tween/PBS, and incubated solution is ri 37 degrees C for 30 minutes. After three cleaning solution of 0.05% Tween/PBS, was added to the substrate TMB-Peroxidase (KPL) and the resulting solution was incubated at room temperature and without access to light for 10 minutes. The reaction was stopped by 1 M phosphoric acid. Next, using the reader for microplates (MPR-A4i, TOSOH CORPORATION) was measured by the absorption at 450 nm.

[0166]

Measured the absorbance at 450 nm for a tablet, on which was the reaction of α-fetoprotein and alpha-fetoprotein-L3 with lectin. Based on this value, expected value for the reaction ([absorbance at 450 nm for the wells, which were solid-phase glycoprotein involved in the reaction] - [absorbance at 450 nm for wells in which no solid phase glycoprotein involved in the reaction]. Next, for each lectin was calculated intensity of the interaction. The results of the calculations are presented in table 10 and figure 24-28.

[0167]

[Table 10]
Example 1 PTLExample 2 SRLComparative example 1 AALComparative example 2 AOLComparative example 3 LCA
α-Fetoprotein0,039 0,031-0,0490,0110,010
α-Fetoprotein-L31,5481,6250,7950,8980,455

[0168]

As can be seen from table 10, PTL of example 1 and SRL of example 2 can detect the change in the sugar chain marker (α-fetoprotein-L1 - sugar chain and L3-sugar chain) with a level of accuracy similar to or higher than the detection LCA of comparative example 3. Thus, PTL of example 1 and SRL of example 2 can be used as a diagnostic agent or as part of a diagnostic kit for the detection of changes in the sugar chain marker. In addition, lectin described in the present invention, has high specificity and can detect compounds with L-fucose α1→6 sugar chain among a group of compounds containing sugar chains, non-L-fucose α1→6 sugar chain, with higher accuracy than other lectins.

[0169]

[Examples 3 and 4] (the Receipt and measurement properties of the NSL and LSL)

(1) Obtaining NSL (example 3)

Using the cleanup process, presented on 29, lectin Naematoloma sublateritium (NSL) was purified from Naematoloma sblateritium.

[0170]

(Extraction)

Dried by sublimation powders Naematoloma sublateritium (40 g) was subjected to extraction with 0.8 l of PBS at 4 degrees C for 2 hours. The resulting liquid was centrifuged (10000 rpm./min, 20 min, 4 ° C). Next, the supernatant was filtered through cheesecloth to obtain the first extract. The residue was subjected to repeated extraction with 0.4 l of PBS at 4 degrees C during the night. The liquid is then centrifuged (10000 rpm./min, 20 min, 4 ° C) and the supernatant was filtered through cheesecloth to obtain a second extract. Then the extracts were mixed to form a liquid extract Naematoloma sublateritium.

[0171]

(Deposition of ammonium sulfate)

Solid (NH4)2SO4(0.8 kg) was added to the obtained supernatant (1.5 l) to 80% saturation. After leaving overnight at 4 degrees C the precipitate was collected by centrifugation (10000 rpm./min, 20 min, 4 ° C) and subjected to dialysis against distilled water and lyophilization to obtain fractions Naematoloma sublateritium, obtained by fractionation of 80% ammonium sulfate.

[0172]

(Hydrophobic chromatography)

Naematoloma sublateritium, obtained by fractionation of 80% ammonium sulfate, was poured in a Butyl-TOYOPEARL 650M (TOSOH CORPORATION), balanced with 2M ammonium sulphate, PBS, for purification by hydrophobic chromatography. This type of chromatography involves the collection, the ultra-filter is tion and lyophilization of the fractions, buervenich distilled water, to obtain the coarse fraction of lectin from Naematoloma sublateritium (shown ←→ RIS).

[0173]

(Reverse-phase chromatography)

Coarse fraction lectin from Naematoloma sublateritium was loaded into the column C8 (Wako Pure Chemical Industries, Ltd.), the balance which was achieved with the help of 0.05% triperoxonane acid (TFA)/acetonitrile (100/0). When using this type of chromatography collected fraction, elyuirovaniya of 0.05% TFA/acetonitrile (70/30) (shown ←→ Fig.31). Next, the solvent was removed by evaporation at room temperature, and the resulting dried powders were collected with getting NSL.

[0174]

SDS-PAGE (PhastGel, Gradient 10-15) was performed using system Phastsystem (GE Healthcare Bio-Sciences). The test solution and the molecular weight marker was taken in quantities of 1 µl. Electrophoresis was carried out on the basis of the Protocol of the product using the traditional method. In figure 32 shows the results of SDS-PAGE for NSL, where lanes 1 and 2 indicate the following. Lane M: molecular weight marker, lane 1: NSL, 2-mercaptoethanol (+), lane 2: NSL, 2-mercaptoethanol (-), gel: gradient 10-15 (GE Healthcare Bioscience), Sample: 1 μl per strip, color: silver.

[0175]

When carrying out SDS-PAGE with 10-15% gel confirmed that the main component is the NSL.

[0176]

(2) Obtaining LSL (example 4)

On the basis of the purification process shown figure 33, lectin from Lepista sordida (LSL) would is purified from the fungus Lepista sordid.

[0177]

(Extraction)

Dried by sublimation powders Lepista sordida (40 g), obtained by freeze-drying to about 400 g Lepista sordida, was subjected to extraction with 0.8 l of PBS at 4 degrees C for 2 hours. The resulting liquid was centrifuged (10000 rpm./min, 20 min, 4 ° C). Next, the supernatant was filtered through cheesecloth to obtain the first extract. The residue was subjected to repeated extraction with 0.4 l of PBS at 4 degrees C during the night. The liquid is then centrifuged (10000 rpm./min, 20 min, 4 ° C) and the supernatant was filtered through cheesecloth to obtain a second extract. Then the extracts were combined to form a liquid extract Lepista sordida.

[0178]

(Deposition of ammonium sulfate)

Solid (NH4)2SO4(0.8 kg) was added to the obtained supernatant (0.5 l) to 80% saturation. After leaving overnight at 4 degrees C the precipitate was collected by centrifugation (10000 rpm./min, 20 min, 4 ° C) and dialyzed against distilled water and lyophilization to obtain fractions Lepista sordid obtained by the fractionation of 80% ammonium sulfate.

To 0.5 l of fluid was added about 0.8 kg of ammonium sulfate to achieve a concentration of 80% saturation of ammonium sulfate and the resulting liquid was stirred. Then it was confirmed by complete dissolution. After that, the liquid left over night at 7 degrees C. This is t the solution was centrifuged (10000 rpm./min, 20 min, 4 ° C) and to the precipitate was added a small amount of distilled water. Next, the resulting solution suspended with the purpose of collecting the fraction Lepista sordid obtained by the fractionation of 80% ammonium sulfate. The collected fraction Lepista sordid obtained by the fractionation of 80% ammonium sulfate, was subjected to dialysis against pure water using a dialysis membrane (fraction 6000-8000).

[0179]

(Hydrophobic chromatography)

Fraction Lepista sordid obtained by precipitation with 80% ammonium sulfate were loaded into Butyl-TOYOPEARL 650M (TOSOH CORPORATION), balanced 2M ammonium sulfate-PBS for purification by hydrophobic chromatography. This type of chromatography involves the collection, ultrafiltration and lyophilization of the fractions, buervenich distilled water, to obtain the coarse fraction of lectin from Lepista sordida (shown ←→ 34).

[0180]

(Reverse-phase chromatography)

Coarse fraction lectin from Lepista sordida was loaded into the column C8 (Wako Pure Chemical Industries, Ltd.), the balance which was achieved with the help of 0.05% triperoxonane acid (TFA)/acetonitrile (100/0). When using this type of chromatography collected fraction, elyuirovaniya of 0.05% TFA/acetonitrile (70/30) (shown ←→ picture.35). Next, the solvent was removed by evaporation at room temperature, and the resulting dried powders were collected with getting LSL.

[0181]

SDS-PAGE (PhastGel, G is adient 10-15) was performed using system Phastsystem (GE Healthcare Bio-Sciences). The test solution and the molecular weight marker was taken in quantities of 1 µl. Electrophoresis was carried out on the basis of the Protocol of the product using the traditional method. 36 shows the results of SDS-PAGE for LSL. 36 band M band 1 mean the following. Lane M: molecular weight marker, lane 1: LSL (hydrophobic chromatography, reversed-phase): 2-mercaptoethanol (+), gel: Gradient 10-15 (GE Healthcare Bioscience), sample: 1 μl per strip, color: silver.

[0182]

When carrying out SDS-PAGE with 10-15% gel confirmed that the main component is LSL.

[0183]

(3) properties of NSL and LSL

(Mass-spectrometric analysis of MALDI-TOF)

NSL from example 3 and LSL of example 4 in an amount of 10 μg, respectively, were separately dissolved in TA (a mixture with a volume ratio of 0.1% TFA and acetonitrile 2:1) using a procedure analogous to the procedure in example 1. Next, the mixture obtained by dissolving the saturated sample, TA - and lachinova TA-solution at a volume ratio of 4:1 was added dropwise in an amount of 1.0 µl of the substrate with the sample. To measure the molecular weight of the NSL and LSL in the LP mode used the device Autoflex (Bruker Daltonics K.K.).

As shown by the result, the molecular weight is about 4500 m/z (37 and 38).

[0184]

(The analysis of amino acid sequence)

For NSL in example 3 were analyzed by amino acid sequence with what omashu appliance Protein Peptide Sequencer PPSQ-21 (SHIMADZU CORPORATION). NSL from example 3 was a mixture of two proteins that have the amino acid sequence of SEQ ID NO: 5-6. These sequences were new.

[0185]

Similarly for LSL from example 4 were analyzed by amino acid sequence with apparatus Protein Peptide Sequencer PPSQ-21 (SHIMADZU CORPORATION). The result was an amino acid sequence of SEQ ID NO: 4. This sequence was also new.

[0186]

(4) Analysis Sagarmatha specificity NSL and LSL

For NSL in example 3 and LSL of example 4 was evaluated Sagarmatha specificity in relation to the L-fucose α1→6 sugar chain by the same method as in example 1. In particular, the calculated Association constants (Ka) for NSL and LSL. The results are presented in tables 11-13.

[0187]

[Table 11]
no sugar chainExample 3 NSL (M-1)Example 4 LSL (M-1)
Sugar chain containing α1→6 L-fucose0153.6×1041.9×105
2013.8×1042.3×105
2023.9×1042.3×105
2033.0×1042.0×105
4015.1×1042.2×105

4023.6×1041.5×105
4035.2×1042.2×105
4044.3×1042.6×105
4055.0×1042.2×105
4062.3×1041.6×105
407NTNT
4103.1×1041.6×105
413NTNT
4183.5×1041.6×l05
601NTNT
6021.9×1041.3×l05

NT: Tests were not carried out

[0188]

[Table 12]
no sugar chainExample 3 NSL (M-1)Example 4 LSL (M-1)
Sugar chain containing another L-fucose, other than α1→6 L-fucose419<1.0×103<1.0×103
420<1.0×103<1.0×103
718<1.0×103<1.0×103
719<1.0×103<1.0×103
720<1.0×103<1.0×103
721<1.0×103<1.0×103
722<1.0×103<1.0×103
723<1.0×103<1.0×103
726<1.0×103<1.0×103
727<1.0×103<1.0×103
728<1.0×103<1.0×103
729<1.0×103<1.0×103
730<1.0×103<1.0×103
731<1.0×103<1.0×103
739<1.0×103<1.0×103
909<1.0×103<1.0×103
910 <1.0×103<1.0×103
931<1.0×103<1.0×103
932<1.0×103<1.0×103
933<1.0×103<1.0×103

[Table 13]
Sugar chain not containing L-fucoseno sugar chainExample 3 NSL (M-1)Example 4 LSL (M-1)no sugar chainExample 3 NSL (M-1)Example 4 LSL (M-1)
001<1.0×103<1.0×103701<1.0×103<1.0×103
002<1.0×103<1.0×103702<1.0×103<1.0×10 3
003<1.0×103<1.0×103703<1.0×103<1.0×103
004<1.0×103<1.0×103704<1.0×103<1.0×103
005<1.0×103<1.0×103705<1.0×103<1.0×103
006<1.0×103<1.0×103706<1.0×103<1.0×103
007<1.0×103<1.0×103707<1.0×103<1.0×103
008<1.0×103<1.0×103708 <1.0×103<1.0×103
009<1.0×103<1.0×103709<1.0×103<1.0×103
010<1.0×103<1.0×103710<1.0×103<1.0×103
011<1.0×103<1.0×103711<1.0×103<1.0×103
012<1.0×103<1.0×103712<1.0×103<1.0×103
013<1.0×103<1.0×103713<1.0×103<1.0×103
014<1.0×103<1.0×103 715<1.0×103<1.0×103
101<1.0×103<1.0×103716<1.0×103<1.0×103
103<1.0×103<1.0×103717<1.0×103<1.0×103
104<1.0×103<1.0×103724<1.0×103<1.0×103
105<1.0×103<1.0×103725<1.0×103<1.0×103
107<1.0×103<1.0×103728<1.0×103<1.0×103
108<.0×10 3<1.0×103732<1.0×103<1.0×103
301<1.0×103<1.0×103733<1.0×103<1.0×103
304<1.0×103<1.0×103734<1.0×103<1.0×103
305<1.0×103<1.0×103735<1.0×103<1.0×103
307<1.0×103<1.0×103736<1.0×103<1.0×103
308<1.0×103<1.0×103737<1.0×103<1.0×103
313<1.0×103<1.0×103738<1.0×103<1.0×103
314<1.0×103<1.0×103901<1.0×103<1.0×103
323<1.0×103<1.0×103902<1.0×103<1.0×103
501<1.0×103<1.0×103903<1.0×103<1.0×103
502<1.0×103<1.0×103905<1.0×103<1.0×103
503<1.0×103<1.0×103907<1.0×103 <1.0×103
504<1.0×103<1.0×103

[0190]

In the case of NSL from example 3 and LSL of example 4 was found only L-fucose α1→6 sugar chain, and does not contain L-fucose α1→6 sugar chain and a sugar chain that does not contain L-fucose, were not found. In addition, NSL from example 3 and LSL of example 4 also was closely associated with three or tetraantennary L-fucose α1→6 sugar chain. Moreover, even when adding sialic acid constant Association with L-fucose α1→6 sugar chain is not decreased.

[0191]

And finally, table 14 shows the results of calculation of the similarity between proteins or peptides for rooms sequences 2-6. As can be seen from the results, the L-fucose α1→6 specific lectin can be obtained by achieving at least 37% similarity with the amino acid sequence selected from SEQ ID nos: 2-6.

[0192]

Sequence 6
[Table 14]
Sequence 2Sequence 3Sequence 4Posledovatelnosti
Sequence 2100%61-74%52-73%69-79%65-70%
Sequence 3-100%46-64%58-72%43-64%
Sequence 4--100%46-64%37-55%
Sequence 5---100%87-92%
Sequence 6----100%

1. L-fucose α1→6 specific lectin that: (1) extracted from the basidiomycete, or marsupial of the fungus, (2) has a peak value of molecular weight of about 4500 m/z as determined by mass spectrometric analysis of MALDI-TOF, and (3) has an affinity for L-fucose α1→6 sugar chain, which began to rise by the constant Association of 1.0×10 4M-1or more (at 25 º C), and (4) has the constant Association of 1.0×103M-1or less (at 25 º C) with vysokoporodnymi sugar chain and/or glycolipids, not containing L-fucose α1→6 sugar chain.

2. L-fucose α1→6 specific lectin described in claim 1, in which the L-fucose α1→6 sugar chain has nereguliruem the end of sialic acid.

3. L-fucose α1→6 specific lectin described in claim 1, in which the L-fucose α1→6 specific lectin (5) has an affinity for L-fucose α1→6 mono-, di-, tri-, tetraantennary N-picenum, defined by the constant Association of 1.0×104M-1or more (at 25 º C).

4. L-fucose α1→6 specific lectin described in claim 1, wherein basidiomycete fungus belongs to the family Strophariaceae, Tricholomataceae, Amanitaceae or Polyporaceae.

5. L-fucose α1→6 specific lectin described in claim 1, wherein basidiomycete fungus is a Pholiota terrestris, Pholiota squarrosa, Pholiota adiposa, Stropharia rugosoannulata, Naematoloma sublateritium, Lepista sordida or Amanita muscaria.

6. L-fucose α1→6 specific lectin, characterized in claim 3, in which the L-fucose α1→6 specific lectin (6) has the amino acid sequence described by SEQ ID NO:1.

7. L-fucose α1→6 specific lectin, which represents (a) a protein or peptide consisting of the amino acid sequence selected from SEQ ID NO:2-6, (1) is extracted from uidialog mushroom or marsupial mushroom, (2) has a peak value of molecular weight of about 4500 m/z as determined by mass spectrometric analysis of MALDI-TOF, and (3) has an affinity for L-fucose α1→6 sugar chain, defined by the constant Association of 1.0×104M-1or more (at 25 º C), and (4) has the constant Association of 1.0×103M-1or less (at 25 º C) with vysokoporodnymi sugar chain and/or glycolipids, not containing L-fucose α1→6 sugar chain.

8. The method of obtaining L-fucose α1→6 specific lectin, according to which a water-soluble extract from and/or marsupial of the fungus is exposed to (i) hydrophobic chromatography and reversed-phase chromatography, (ii) affinity chromatography, or (iii) ion-exchange chromatography and gel filtration to obtain latinamerica fraction (vi) has a peak value of molecular weight of about 4500 m/z as determined by mass spectrometric analysis of MALDI-TOF, and which (v) is characterized by affinity for L-fucose α1→6 sugar chain, defined by the constant Association of 1.0×104M-1or more (at 25 º C), and (vi) has the constant Association of 1.0×103M-1or less (at 25 º C) with vysokoporodnymi sugar chain and/or glycolipids, not containing L-fucose α1→6 sugar chain.

9. The method of obtaining L-fucose α1→6 specific lectin described in claim 8, in which at least dimbaseline the fungus is selected from the collections Strophariaceae, Tricholomataceae, Amanitaceae and Polyporaceae.

10. The method of obtaining L-fucose α1→6 specific lectin described in claim 8, in which at least one basidiomycete fungus is selected from Pholiota terrestris, Pholiota squarrosa, Pholiota adiposa, Stropharia rugosoannulata, Naematoloma sublateritium, Lepista sordida and Amanita muscaria.

11. The method of obtaining L-fucose α1→6 specific lectin described in item 8, which uses the carpophores (fruiting bodies) basidiomycete and/or marsupial of the fungus.

12. Method detection L-fucose α1→6 sugar chain including the process that leads to the effects of sugar chains on L-fucose α1→6 specific lectin, characterized in claim 1.

13. Method detection L-fucose α1→6 sugar chain described in item 12, in which the sugar chain is a tumor marker.

14. Method of fractionation of L-fucose α1→6 sugar chain including the process that leads to the effects of sugar chains on L-fucose α1→6 specific lectin, characterized in claim 1.

15. Method of fractionation of L-fucose α1→6 sugar chain described in 14, in which a sugar chain is bound to the antibody.

16. Diagnostic tool for the detection of L-fucose α1→6 sugar chain, comprising as an active ingredient L-fucose α1→6 specific lectin, characterized in claim 1.



 

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18 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: present invention refers to prognostic analysis, as well as to a method for applying it for determining a probability of producing a therapeutic response in the affected cells or tissues on treating a disease having an aethiology related to excessive cell proliferation with using cardiac glycoside. The method for prediction consists in determining the relation of Na, K-adenosine triphosphatase α-subunit isoforms in the affected cells or tissues. The above method can be used for the prediction of individual's cancer or tumour sensitivity to the therapeutic treatment with cardiac glycoside.

EFFECT: predicted method can be applied in a method of treating a disease or a disorder having an aethiology related to excess cell proliferation with using the composition containing cardiac glycoside.

59 cl, 9 tbl, 27 ex, 13 dwg

FIELD: medicine.

SUBSTANCE: kit of reagents contains a solution for providing an antigen bioavailability, a blocking solution of horseradish peroxidase, a protein blocking solution, a washing solution, a solution of primary HER2 receptor antibodies, a solution of primary oestrogen receptor antibodies, a solution of primary progesterone receptor antibodies, a solution of anti-species secondary antibodies to primary antibodies, a solution of streptavidin - horseradish peroxidase, diamine benzidine, a substrate solution, a negative reference sample, three positive reference samples of breast cancer for detecting HER2, oestrogen receptors, progesterone receptors. The kit of reagents according to the invention possesses higher sensitivity and specificity. The invention may be used in medical laboratories and research establishments.

EFFECT: possibility to detect oestrogen and progesterone receptors, as well as HER2 receptors either simultaneously, or separately.

3 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: treating respiratory distress syndrome accompanying neonatal ventilation is ensured by measuring glutathione peroxidase (GSH-Px-3) with underlying drug therapy, and the measured value being less than 2.41 mcmole/l requires including intravenous bolus injections of the preparation Selenase in a therapeutic complex at 10 mcg/kg/day, once a day for 10 days.

EFFECT: method provides increasing the survival rate in reducing a number of complications and a length of treatment by ensuring a lower severity of oxidative stress and a higher antioxidant protection of the body.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: wings of ilium are punctured in an anterior and posterior one-third of the wings with two trocars being inserted into each wing. The bone marrow (BM) is collected by simple aspiration, aspiration irrigation or a combination thereof at an underpressure of 0.6 Atm with using a device. The bone marrow preparation device comprises a disposable multi-channel closed system, an aspiration collection unit and a perfusion unit. The group of inventions also refers to a method for assessing the prepared bone marrow. The effect is ensured by automatic control of myeloaspiration by preparing a biological material with using a special designed device for the bone marrow collection.

EFFECT: using the given method for preparing the bone marrow provides preparing the sterile bone marrow rich in viable multipotent mesenchymal stromal and hemopoietic progenitor cells.

7 cl, 1 ex, 1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: for evaluation of disorders of cellular immunity when exposed to phenol the blood sampling and its analysis is carried out using experimental and control samples with preliminary introduction to the test sample of toxicant - phenol in a concentration corresponding to its regional background level. As a criterion factors in the method the index of specific sensitivity of cells is used for cytokine tumour necrosis factor TNF-α and the index of specific sensitivity of cells to cytokine interleukin-10 IL-10, which are defined as follows: the experimental and control blood samples are placed into the vial with a sterile nutrient medium DMEM with heparin and gentamicin in a volume ratio of 1:4, respectively (in a preferred embodiment, the contents in the sterile nutrient medium DMEM of heparin is 2.5 U/ml and gentamicin - 100 mcg|ml), incubation of these samples is carried out at 37°C during 24 hours, followed by centrifugation for 3 minutes at 1000 rev/min, and in the selected supernatant of the test sample the level of IPCIL-10 phenol-induced production of cytokine IL-10 is determined and the level of IPCTNF-α phenol-induced production of cytokine - tumour necrosis factor TNF-α, and in the selected supernatant of the control sample the level of spontaneous cytokine production is determined, namely the SPCIL-10 level of spontaneous production of cytokine IL-10 and SPCTNF-α level of spontaneous production of the cytokine TNF-α, then ISSTNF-α index of specific sensitivity of cells for cytokine TNF-α is calculated, and the ISSIL-10 index of specific sensitivity of cells for cytokine IL-10 is calculated using the following formulas: ISSTNF-α=(IPCTNF-α- SPCTNF-α): SPCTNF-α, ISSIL-10 =(IPCIL-10- SPCIL-10): SPCIL-10, where IPCIL-10 is the level of phenol-induced production of cytokine IL-10; IPCTNF-α is the level of phenol-induced production of cytokine TNF-α; SPCIL-10 is the level of spontaneous production of cytokine IL-10; SPCTNF-α is the level of spontaneous production of the cytokine TNF-α; and when meeting the simultaneous requirement of ISSTNF-α of over 10 and ISSIL-10 of over 2 the disorder of cellular immunity of the organism is determined under the influence of phenol.

EFFECT: increase in reliability of making assessment of impact of phenol on disorder of cellular immunity of the human body.

2 cl, 8 tbl

FIELD: medicine.

SUBSTANCE: diagnostic technique according to the present invention consists in measuring Anti-MCV in the oral fluid (mixed saliva), using mathematical expression F=0.17×Anti-MCV-0.2537 for processing derived values Anti-MCV in the oral fluid, diagnosing rheumatoid arthritis if observing F more than zero, and stating a doubtful diagnosis of rheumatoid arthritis if deriving negative F values.

EFFECT: minimally invasive and reliable diagnostic technique for rheumatoid arthritis.

FIELD: medicine.

SUBSTANCE: in vitro fertilisation (IVF) programme is preceded by luteal-phase endometrial biopsy to determine a relative CD95+ macrophage count in an endometrial leukocytic infiltrate. If the relative CD95+ macrophage count is 48.8% or more, the onset of pregnancy is predicted, while the value being less than 48.8% shows the absence of the onset of pregnancy. The declared method extends the range of prognostic aids, increases the accuracy, sensitivity and specificity of the prediction procedure for the onset of pregnancy in the females suffering from tubal-peritoneal infertility prior to IVF programme.

EFFECT: using the method enables specifying the further approach to managing a patient, taking timely additional therapeutic measures.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method is based on a contact of a membrane test strip with an analysed liquid sample and initiated by the said contact movement on the test strip membranes of reagents, contained in the sample or applied on the membrane and forming immune complexes in membrane pores or its surface in the course of interactions. Intensity of coloration of a mark, which binds in an analytic zone, is detected visually. The claimed system is intended for immunochromatographic determination of antibodies or antigens in liquid samples. A characteristic feature of the claimed method of carrying out an immunochromatographic analysis consists in the following: a multimembrane composite of the test strip contains an additional membrane with a substance applied on it substance, which dissolves in water slowly, for instance a bovine serum albumin (BSA), which is attached to the test strip immediately after the membrane with the applied colloid conjugate. The front of the liquid sample, passing the membrane with colloidal gold, conjugated with a reagent for analite binding, stops, after getting on the membrane with BSA, until BSA is dissolved. During this time the sample is incubated with the colloidal conjugate, which approaches a reaction of analite binding to an equilibrium position.

EFFECT: reduction of detection limit of the analysis.

2 dwg, 1 ex

FIELD: biotechnology.

SUBSTANCE: single-domain mini-antibody is proposed, specifically binding the protein-receptor of epidermal growth factor of human HER2/ERBB2/neu obtained by immunisation of two-humped camel (Camelus bactrianus) by the preparation of tumour cells SKBR3, and characterised by the amino acid sequence. Also the method of detecting protein HER2/ERBB2/neu and its expressing cells is considered. The antibody according to the present invention is able to penetrate in the target cell on which surface HER2/ERBB2/neu is exposed, and may find further application in diagnostics and therapy of diseases associated with overexpression of HER2/ERBB2/neu.

EFFECT: improving efficiency of use of the compound.

2 cl, 6 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method describes determining circulating tumour cells sensitive to adjuvant hormone therapy in blood of patients suffering from breast cancer, and involves recovering and enriching the circulating tumour cells with using magnetically marked antibodies, producing lysates of the circulating tumour cells, recovering iRNA from the lysates of the circulating tumour cells, producing cDNA, producing amplified DNA, determining expression of marker genes; if observing one of the markers GA733-2, MUC-1, HER2, the presence of the circulating tumour cells is stated; and if the markers ESR1, PGR are present, the circulating tumour cells sensitive to hormone therapy is concluded.

EFFECT: method enables providing a considerably higher probability of detecting the circulating tumour cells sensitive to adjuvant hormone therapy, increasing the measurement accuracy and the number of analysed characteristics of the tumour cells, ensuring higher sensitivity of the analysis techniques and more effective diagnosis in managing patients with breast cancer for assessing the prediction and therapeutic efficacy, improving the clinical effectiveness and survival rate of oncologic patients.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: cervical biopsy material tissues taken by colposcopic biopsy are examined for typing a human papillomavirus (HPV), apoptosis values (sFAS, TRAIL), genetic characteristics of expression of the genes regulating apoptosis processes, as well as radical formation processes (pyeloperoxidase activity, nitrate-nitrite levels, total antioxidant tissue capacity by the Fenton-type reaction). A summing risk factor of developing cervical cancer formation is calculated by formula. If the summing risk factor K is less than 3, then a low risk of cancer formation is predicted; if K is 3 or more, the risk of cancer formation is considered to be high.

EFFECT: applicability of the given method before the pathological processes are formed in tissues that let doctors prescribe a preventive course and prevent developing cervical cancer if having a predisposition observed or the risk factors detected, applicability of the given method with a diagnosed cervical pathology for the purpose of predicting the disease and monitoring of the efficacy of the conducted therapy.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

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