JP5698588B2 - Lacanka lectin, its production method and use - Google Patents

Description

  The present invention relates to a novel lectin that can be obtained from Rahan fruit plant, a method for producing the same, and use thereof.

  Sialic acid is an acidic sugar that is attached to the end of a sugar chain, and is present in vivo at the end of an oligosaccharide on the surface of a cell membrane as a constituent component of a complex carbohydrate in addition to a simple substance. It is related to recognition, immunity, etc. in the cell surface layer.

  As a lectin for detecting sialic acid, conventionally Japanese chick collectin (SSA), western chick collectin (SNA), dog endurectin (MAM), willow matsutake lectin (ACG), kikarasu lectin (TJA-I) and the like are known. Yes. There are very few types of sialic acid specific lectins compared to lectins having other specificities. Despite the fact that lectins are very good probes for conducting sugar chain research, they do not have sufficient types of lectins.

  If a sialic acid-specific lectin is newly found, it can be used for identification of diseases related to sialic acid by utilizing its characteristics. For example, JP 2005-145837 (diagnosis method of Alzheimer's disease, Patent Document 1) includes detecting or measuring a secreted sialyltransferase cleaved by β-secretase contained in a human-derived sample. A method for diagnosing Alzheimer's disease is described. JP 2006-317210 (Diagnosis method of Alzheimer's disease using serum glycoprotein as a biomarker, Patent Document 2) discloses a diagnostic agent for Alzheimer's disease containing a lectin for detecting an α2,6-sialic acid-containing glycoprotein. Have been described.

  It has been reported that sialic acid is also involved in the infection of various viruses. For example, hemagglutinin (glycoprotein) present on the surface of influenza virus has the property of recognizing sialic acid α2-6Gal, a glycoprotein present on the surface of infected cells (eg, cells of human respiratory tract mucosa). It is known that viruses infect cells. When the influenza virus moves from an infected cell to an uninfected cell, it is necessary to cut the sialic acid α2-6 bond, and thus the virus has the enzyme neuraminidase on the cell surface.

  Increased levels of sialic acid have recently been observed for cardiovascular diseases, alcoholism, diabetes and the like, and thus sialic acid is a marker for diagnosing and detecting such lethal diseases Research is progressing.

JP-A-2005-145837 JP 2006-317210 A

  An object of the present invention is to provide a novel sialic acid α2-6 specific lectin and its use.

As a result of intensive research aimed at solving the above-mentioned problems, the present inventor succeeded in isolating a lectin having a binding activity with a sialic acid α2-6 sugar chain from an extract of Luhan fruit plant. Completed. That is, the present invention can be extracted from a Mordicae grosvenori plant and has the following physicochemical properties:
(1) The subunit molecular weight by SDS-polyacrylamide electrophoresis is 20,000 to 70,000.
(2) shows strong agglutination activity against erythrocytes of rabbits and pigs,
(3) having affinity for sialic acid α2-6 sugar chain,
A lacanka lectin is provided.

  In particular, the above-mentioned rakankalectin has a minimum inhibitory concentration of 5 μg / ml or less for 6′-sialyl lactose or 6′-sialyl lactosamine. Here, the minimum inhibitory concentration means the minimum concentration required for the sugar to prevent the aggregation reaction. That is, the smaller the minimum inhibitory concentration, the higher the affinity for lectin.

  The rakankalectin has an affinity for immunoglobulin M and prostate specific antigen.

  The present invention also provides the method for producing the above-mentioned Lacanka lectin, characterized in that it is obtained from an aqueous medium extract of Luohan fruit plant.

  The method for producing a Lacanka lectin of the present invention preferably includes subjecting the Lacanca aqueous medium extract to a protein precipitation method such as an ammonium sulfate precipitation method and affinity chromatography.

  The present invention also provides a method for detecting a sialic acid α2-6 sugar chain, which comprises reacting a sugar chain with a rakankalectin.

  The present invention also provides a method for fractionating a sialic acid α2-6 sugar chain, characterized by allowing lakankalectin to act.

  The present invention also provides a research reagent or a research kit for detecting a sialic acid α2-6 sugar chain, which contains rakankalectin as an active ingredient.

  The present invention also provides a diagnostic agent or diagnostic kit for detecting a sialic acid α2-6 sugar chain, which contains rakankalectin as an active ingredient.

  The present invention also provides a specific binding agent for sialic acid α2-6 sugar chain, comprising lacanka lectin as an active ingredient.

  The present invention also provides a method for fractionating a sialic acid α2-6 sugar chain, characterized by allowing lakankalectin to act.

  The present invention also provides a pharmaceutical composition comprising lacanka lectin.

  The present invention also provides a sanitary article carrying a lacan lectin.

  The sanitary article is suitable for applications that shield pathogens having binding properties to sialic acid α2-6 sugar chains.

  The sanitary product is, for example, any one of a mask and a filter for an air cleaner.

  The present invention also provides foods to which purified lacanka lectin is added.

  The said food is suitable for the use which has the antagonistic property of the pathogen which has binding property to the sialic acid alpha2-6 sugar chain. The food is in the form of any one of beverages such as troches, strawberries, drops, gums, teas, coffees and juices.

  The “rahan fruit plant” that is the raw material of the lectin of the present invention has a food experience with foods (beverages and sweeteners) and is widely distributed in the market. The present invention capable of producing a sialic acid α2-6 sugar chain-specific lectin from a naturally-derived safe and easily available raw material has high utility value. The provision of a novel sialic acid α2-6 sugar chain-specific lectin is very significant for future sugar chain research. In addition, since research on sialic acid is involved in various biological reactions, such research is being carried out all over the world. Since the lectin of the present invention can specifically find the “sialic acid α2-6 sugar chain” structure, it can be expected to be used in the research of many researchers.

  The sialic acid α2-6 sugar chain-specific lectin can be used for detection of sialic acid α2-6 sugar chains and purification of glycoproteins having sialic acid α2-6 sugar chains. It can also be used in a method for diagnosing cancer or disease involving sialic acid α2-6 sugar chain. In addition, the present invention can be applied to search for new tumor markers, etc., and can lead to the discovery of tumor markers.

  Furthermore, the sialic acid α2-6 sugar chain-specific lectin can be applied to infection prevention methods and sanitary goods for pathogens such as influenza virus.

1 is a diagram showing a purification process of lacanka lectin (MGL) in Example 1. FIG. 2 is an elution diagram of affinity chromatography of MGL of Example 1. FIG. It is a result (drawing substitute photograph) of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of MGL of Example 1. It is a result of a glycoprotein detection by the enzyme immunoassay (ELISA) using MGL of Example 1. It is a result of glycoprotein detection by ELISA using SNA of Comparative Example 1. It is a result of tag glycoprotein detection by ELISA using SSA of Comparative Example 2. It is a result of glycoprotein detection by ELISA using MAM of Comparative Example 3. It is a result of glycoprotein detection by ELISA using ACG of Comparative Example 4.

  Mordicicae grosvenori is a perennial vine plant belonging to the genus Lacanca, and is mainly produced around Guilin, China. Lacanca contains sweetening ingredients and is used as a sweetener for ingredients and Rakan fruit tea. In Japan, dry products are mainly imported.

  The subunit molecular weight of the rakankalectin of the present invention by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) is 20,000 to 70,000, preferably 20,000 to 40,000. Lacanka lectin is normally expected to exist as a complex in which 2 to 4 subunits are bound. Such complexes are also within the scope of the present invention. In addition, the molecular weight by SDS-PAGE is measured according to the Laemmi method (Nature, 227, 680, 1976), for example.

  The rakankalectin of the present invention exhibits an agglutinating activity against erythrocytes of rabbits, humans (types O, A1, A2, and B), pigs, chickens, geese, and guinea pigs. In particular, rabbit and pig erythrocytes strongly aggregate.

  The rakankalectin of the present invention has affinity for 6'-sialyl lactose and 6'-sialyl lactosamine among various sugars. In particular, the minimum inhibitory concentration for 6'-sialyl lactose or 6'-sialyl lactosamine is 5 μg / ml or less.

  Lacanka lectin of the present invention includes porcine stomach mucin, bovine submandibular mucin, fetuin, α1-acid glycoprotein, thyroglobulin, lactoferrin, fibrinogen, immunoglobulin A, α2-macroglobulin, immunoglobulin among various glycoproteins. M, has affinity for prostate specific antigen. In particular, it has high affinity for immunoglobulin M (IgM) and prostate specific antigen (PSA).

  The method for producing a rakankalectin of the present invention can be isolated from an aqueous extract of Luhan fruit plant by appropriately combining known separation methods, purification methods and the like. The use parts of the raw material Rakan fruit are seeds and fruits. These may be raw or dried.

  The method for obtaining the aqueous medium extract from the Luohan fruit plant and the aqueous medium is not particularly limited as long as the aqueous medium and the Luo Han fruit can be brought into contact with each other. From the viewpoint of extraction efficiency, a method of pulverizing a dried product of Rakan fruit in an aqueous medium to form a suspension is preferable. Examples of the pulverization method include a pulverization method using a general-purpose instrument and equipment such as a mixer, a homogenizer, a blender, a (ultra) sonic device, a pressure-type breaking device, and a cracking device.

  Examples of the aqueous medium include water, a buffer solution, a mixture of an organic solvent that can be mixed with water, and water or a buffer solution. Preferably, a buffer solution or a mixture of an organic solvent and a buffer solution is used. The buffer solution is not particularly limited, and a known buffer solution can be used. Especially, what has a buffer capacity in the range of pH 3-10 is preferable, and what has a buffer capacity in the range of pH 6-8 is more preferable. Specific examples include a phosphate buffer, a citrate buffer, an acetate buffer, and a Tris buffer. Among these, a phosphate buffer is preferable from the viewpoint of extraction efficiency. Although the salt concentration of the said buffer solution does not have a restriction | limiting in particular, From the point of extraction efficiency and a buffer capacity, it is preferable that it is 1-100 mM, and it is more preferable that it is 5-20 mM.

  The buffer may further contain salts. For example, a phosphate buffered physiological saline in which sodium chloride is further added to a phosphate buffer is preferable as the aqueous medium in the present invention.

  As the organic solvent, any organic solvent that can be mixed with water can be used without particular limitation. Of these, acetone, methanol, ethanol, n-butanol, 2-propanol and acetonitrile are preferable. As content of the organic solvent in the case of mixing an organic solvent and water or a buffer solution, it is preferable that it is 5-50 mass%.

  It is preferable that the extraction step further includes a step of removing insoluble matters with respect to the aqueous medium from the mixture of the aqueous medium and the Luhan fruit plant. Examples of the method for removing insoluble materials include filtration, centrifugation, and the like, but centrifugation is preferred from the viewpoint of removal efficiency.

  The extraction step is preferably a step of obtaining an aqueous medium extract by crushing a rakanka in phosphate buffered saline (PBS) and removing insolubles by centrifugation. Alternatively, from the viewpoint of work efficiency, the step may be a step of pulverizing Rakanka, adding phosphate buffered saline, stirring well, removing insoluble matter by centrifugation, and obtaining an aqueous medium extract.

  In the method for producing the Lacanka lectin, the following purification means can be used to enable more efficient purification.

  The aqueous medium extract obtained by the above step is used to obtain a lectin-containing fraction by ammonium sulfate precipitation, and the obtained lectin fraction is further purified by affinity chromatography.

  The protein precipitation method is not limited to the ammonium sulfate precipitation method, but ammonium sulfate precipitation (salting out), trichloroacetic acid (TCA) precipitation, acetone precipitation, ethanol precipitation, TCA / acetone precipitation, ammonium acetate-containing methanol (phenol extraction pretreatment) And usual methods such as precipitation with Clean-Up Kit.

  In affinity chromatography, a column on which a sugar or glycoprotein having sialic acid 2-6 is immobilized is used. For example, a column in which sialyl lactose, fetuin, and thyroglobulin are immobilized on an agarose gel is used. For the eluate, lactose, sialyl lactose, ammonia and acetic acid are used. Among the eluted fractions, for example, a rabbit hemagglutination active fraction is collected.

  The production method of the present invention includes a step of dialyzing the fraction containing lectin obtained in the purification step, a step of concentrating the lectin solution after dialysis treatment by ultrafiltration, and the step of freeze-drying the concentrated solution. But you can. Thereby, a lectin can be isolated easily. Dialysis, concentration, and lyophilization can be performed by a commonly used known method. By the above production method, a lectin having a subunit molecular weight of 20,000 to 70,000 by SDS-PAGE is obtained.

  The present invention also provides a method for detecting a sialic acid α2-6 sugar chain using the above-mentioned rakankalectin. Since the rakankalectin specifically recognizes and binds to sialic acid α2-6 sugar chains as compared with conventional sugarcane lectins, it is specific to sugar chain compounds such as polysaccharides, glycolipids and glycoproteins containing sialic acid α2-6. Suitable for detection.

  Moreover, the ratio of the inhibitory concentration to the sialo body (for example, 6'-sialyl lactose, 6'-sialyl lactosamine) and the minimum inhibitory concentration of the asialo body (for example, lactose) is generally 10 times or more in the rakankalectin of the present invention. Preferably it is 50 times or more, Especially preferably, it is 100 times or more. Therefore, the rakankalectin of the present invention can detect not only the sialo form but also the detachment of sialic acid depending on the disease or the like.

  In addition, the rakankalectin of the present invention has an inhibitory concentration of sialic acid α2-6 type (for example, 6′-sialyllactose, 6′-sialyllactosamine) and sialic acid α2-3 type (for example, 3′-sialyllactose, 3 ′). The ratio of the minimum inhibitory concentration of (sialyllactosamine) is usually 10 times or more, preferably 50 times or more, particularly preferably 200 times or more. Therefore, the rakankalectin of the present invention can also detect differences in the binding mode of sialic acid.

  A labeling means may be incorporated in the rakankalectin used in the detection method. Hereinafter, such a lectin may be referred to as a labeled lectin. The labeled lectin of the present invention includes at least a rakankalectin and a labeling means, and is characterized by being detectably labeled.

  As the labeling means, known labeling methods can be applied without particular limitation, and examples thereof include labeling with a radioisotope, binding of a labeling compound, and the like.

  The labeling compound is not particularly limited as long as it is usually used for this purpose, and examples thereof include a direct or indirect labeling compound, an enzyme, and a fluorescent compound. Specific examples include biotin, digoxigenin, horseradish-derived peroxidase, fluorescein isothiocyanate, and CyDye. These labeling compounds can be bound to lectins by a conventional method. In particular, an avidin-biotin or streptavidin-biotin system is preferable in terms of high sensitivity.

  Detection or fractionation of the sialic acid α2-6 sugar chain can be carried out, for example, by lectin chromatography using the immobilized Rakanka lectin. Lectin chromatography is affinity chromatography that utilizes the property that lectins bind specifically to sugar chains. When combined with HPLC (HPLAC), high throughput can be expected.

  As a carrier for immobilizing a rakankalectin, gel materials such as agarose, dextran, cellulose, starch, polyacrylamide and the like are generally used. For these, commercially available products can be used without particular limitation, and examples thereof include Sepharose 4B and Sepharose 6B (both manufactured by GE Healthcare Bioscience).

  Materials used for lectin chromatography include those in which lectins are immobilized on microplates or nanowells.

The concentration of the rakankalectin to be immobilized is usually 0.001 to 100 mg / ml, preferably 0.01 to 20 mg / ml. When the carrier is an agarose gel, it is activated with CNBr or the like and then coupled with the lectin. A lectin may be immobilized on a gel into which an activation spacer has been introduced. Further, the lectin may be immobilized on a gel into which a formyl group has been introduced and then reduced with NaCNBH ₃ . A commercially available activated gel such as NHS-Sepharose (manufactured by GE Healthcare Bioscience) may also be used.

A sample suspected of the presence of sialic acid α2-6 sugar chain is applied to the column, and then a buffer is passed for washing and equilibration purposes. An example of the buffer is a molar concentration of 5 to 500 mM, preferably 10 to 500 mM, a pH of 4.0 to 10.0, preferably 6.0 to 9.0, and a NaCl content of 0 to 0.0. 5M, preferably _0.1~0.2M, CaCl _2, MgCl _2, or, MnCl ₂ content 0 to 10 mm, a buffer preferably 0-5 mM.

  After washing the affinity column, elution of the sialic acid α2-6 sugar chain is performed using a desorbing agent such as sodium chloride or hapten sugar in a neutral non-denaturing buffer solution that can effectively elute the sugar chain. This buffer may be the same as described above. The concentration of the desorbing agent is preferably 1 to 400 mM, particularly preferably 10 to 200 mM.

  For the detection of sugar chains, chromatography, enzyme immunoassay (ELISA), Western blot, lectin chip, lectin staining, immunoprecipitation, aggregation method, surface plasmon resonance method, electrophoresis, etc. other than the above are well known to those skilled in the art. Can be used in the way.

  The specimen containing the sugar chain is not particularly limited. Examples thereof include blood, plasma, serum, tears, saliva, body fluid, milk, urine, cell culture supernatant, and secretions from transformed animals.

  Specific examples of the substance having a sialic acid α2-6 sugar chain that is a target of the detection method include 6′-sialyl lactose, 6′-sialyl lactosamine, immunoglobulin M (IgM), prostate specific antigen (PSA), and the like. Can be mentioned. The sugar chain that acts on the detection method is preferably a tumor marker.

  The target of the detection method of the present invention is preferably a disease in which the involvement of sialic acid α2-6 sugar chain is pointed out. Specific examples include Alzheimer's disease, cardiovascular disease, alcoholism, IgA nephropathy, liver cancer, prostate cancer, ovarian cancer, myocardial infarction, fibrosis, pancreatitis and diabetes.

  The aforementioned Lacanka lectin is a novel lectin that is different from conventionally known lectins in biochemical properties such as physicochemical properties and binding specificity to sugar chains. In particular, since it specifically recognizes the sialic acid α2-6 sugar chain, a diagnostic agent or a test reagent for a disease caused by the sialic acid α2-6 sugar chain, a specific binding agent for separation and analysis of the sialic acid α2-6 sugar chain It can be used as an immunomodulator and the like. Therefore, the present invention provides a diagnostic agent or diagnostic kit for detecting a sialic acid α2-6 sugar chain comprising the above-mentioned lacanka lectin as an active ingredient, and a sialic acid α2-6 sugar chain comprising a rakankalectin as an active ingredient. A specific binding agent for is provided.

  The diagnostic agent or diagnostic agent kit diagnoses Alzheimer's disease, cardiovascular disease, alcoholism, IgA nephropathy, liver cancer, prostate cancer, ovarian cancer, myocardial infarction, fibrosis, pancreatitis, diabetes, etc. Useful for.

  In the diagnostic agent or diagnostic agent kit, a normal diagnostic agent such as a label, an enzyme and its chromogenic substrate, a radioisotope, a luminescent substance, a fluorescent substance, a colored substance, a buffer solution, a standard protein, a reaction stop solution, a plate, etc. Includes known materials used in kits.

  The present invention also provides a sialic acid comprising separating a sialic acid α2-6 sugar chain and a non-sialic acid α2-6 sugar chain by using the rakankalectin as a binding medium for the sialic acid α2-6 sugar chain. A method for fractionating α2-6 sugar chains is provided.

  The fractionation method of the present invention is, for example, lectin chromatography using the immobilized Lacanka lectin. The details are the same as those described in the detection method. When purifying the sialic acid α2-6 sugar chain, an affinity column is prepared by covalently binding rakankalectin to a column carrier made of agarose or cellulose via a functional group, and the sugar chain sample is passed through. After passing through the column, the adsorbed sialic acid α2-6 sugar chain is recovered. When purifying the non-sialic acid α2-6 sugar chain, when the sugar chain sample is passed through the column, the sugar chain sample passing without adsorption is collected.

  The target to be purified by the fractionation method of the present invention can be of two types: sialic acid α2-6 sugar chain and non-sialic acid α2-6 sugar chain.

  The present invention provides a pharmaceutical composition for preventing / treating a disease caused by a sialic acid α2-6 sugar chain, which contains a rakankalectin. Examples of pharmaceutical uses include pathogen antagonists, infection inhibitors, preventives, traditional Chinese medicines, and traditional medicines that have binding properties to sialic acid α2-6 sugar chains. Examples of pathogens capable of binding to the sialic acid α2-6 sugar chain include influenza virus and adenovirus. For example, these viruses and rakankalectins act antagonistically on the human throat and nasal mucosa to prevent the virus from entering.

  The rakankalectin of the present invention is processed into a liquid, powder, granule, tablet, capsule, syrup and the like for use as an oral medicine.

  In the pharmaceutical composition of the present invention, pharmacologically usable carriers, excipients, auxiliaries and the like can be added as long as the effects of the present invention are not inhibited. Specifically, lactose, sucrose, fructose, glucose, glucose hydrate, sucrose, purified sucrose, erythritol, xylitol, sorbitol, mannitol, palatinose, reduced palatinose, powdered maltose, water candy, carmellose, dextrin, corn starch , Pregelatinized starch, partially pregelatinized starch, potato starch, corn starch, hydroxypropyl starch, amino acid, kaolin, silicic anhydride, silicic acid, aluminum silicate, sodium bicarbonate, calcium phosphate, calcium dihydrogen phosphate, calcium carbonate, oxidation Magnesium, aluminum hydroxide, fatty acid or salt thereof, fatty acid monoglyceride and diglyceride, alcohol, vegetable oil, olive oil, soybean oil, corn oil, fatty oil, oil and fat, viscous paraffin Carriers or excipients such as propylene glycol, ethylene glycol, polyethylene glycol, glycerin; crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, Hydroxypropyl methylcellulose acetate succinate, carmellose sodium, ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, dextrin, pullulan, polyvinylvin Pyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl methacrylate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, macrogol, etc. Binders: wheat starch, rice starch, corn starch, synthetic aluminum silicate, dry aluminum hydroxide gel, magnesium metasilicate magnesium phosphate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, waxes, hydrogenated vegetable oil, polyethylene glycol , Light anhydrous silicic acid, synthetic aluminum silicate, stearic acid, macrogol, talc, magnesium stearate, calcium stearate Lubricants such as calcium, hydrous silicon dioxide, sucrose fatty acid ester; lubricant; crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice Disintegrants such as starch, corn starch, potato starch, partially pregelatinized starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth; soy lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene Polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate, sorbitan monopalmitate Surfactants such as sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol; emulsifiers; solubilizers such as sodium phosphate; absorption enhancers; hydrochloric acid, citric acid, sodium citrate, PH adjusters such as acetic acid, tartaric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, lactic acid; brighteners such as natural resins; stabilizers; antioxidants; preservatives; wetting agents; Fragrances; soothing agents and the like.

  In the present invention, when a rakankalectin is used as a nasal drug, it takes the form of a solution, a gel, an aerosol or the like. As the carrier, higher alcohols such as octyldodecanol, fatty acid esters such as isopropyl myristate, suspending agents and the like can be used.

  In the present invention, when rakankalectin is used as an ointment or cream, petrolatum, liquid paraffin, squalane, cetanol, stearyl alcohol and the like can be used as a carrier.

  The dose of the rakankalectin of the present invention depends on the use of an antagonist of a pathogen having a binding property to the sialic acid α2-6 sugar chain, the condition of the patient to be administered, the administration method, etc. Can be optimized.

  The present invention also provides a sanitary article carrying a lacan lectin. By carrying the air purifier filter, mask case, etc. in a form that retains its activity, it antagonizes viruses in the air and prevents infection of the human body. The rakankalectin of the present invention may be used as a spray as a sanitary product.

  In order to use the rakankalectin of the present invention as a functional food, it is processed into a liquid, powder, granule, tablet, capsule, syrup form and the like.

The lacanka lectin of the present invention may be added directly to the raw material during the production of general processed foods such as troches, rice cakes, drops, gums, tea, bread, cooked rice, soups, side dishes, confectionery and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the present invention is not limited to the following examples.
[Example 1] Production of Lacanka lectin (1) Production of Lacanka lectin Lacanka lectin (MGL) was isolated and purified from Lacanka according to the purification step shown in FIG.

(Degreasing and extraction)
About 34 g of dried Rakanka fruit was crushed with a pulverizer, the pulverized product was transferred to a beaker, and 1 L of acetone was added and stirred. Subsequently, the mixed liquid of the pulverized product was filtered with a filter paper to perform acetone degreasing. The crushed material remaining on the filter paper was spread on a vat and dried in a draft overnight. Transfer the dried pulverized product after degreasing with acetone to a beaker, add 160 ml of 10 mM phosphate buffered saline (pH 7.4) (hereinafter abbreviated as PBS), and then stir overnight (7 ° C.) with a stirrer. The extraction was performed. This solution was centrifuged (15,000 rpm, 20 min, 4 ° C.), and the supernatant was filtered to obtain a Lacanca extract.

(Precipitation with ammonium sulfate)
51.04 g of ammonium sulfate (a ratio of 176 g of ammonium sulfate to 1 L of the solution) was added to 290 ml of the extract. After thoroughly stirring and dissolving, the mixture was allowed to stand at 4 ° C. for 3 hours. The mixture was centrifuged (10,000 rpm, 20 min, 4 ° C.), and the precipitate was collected by adding pure water to obtain a 30% saturated ammonium sulfate precipitate fraction. The 30% saturated ammonium sulfate supernatant fraction after centrifugation was used in the next step.

  To the 30% saturated ammonium sulfate supernatant fraction, 113.92 g of ammonium sulfate (a ratio of 356 g of ammonium sulfate to 1 L of the solution) was added. After thoroughly stirring and dissolving, the mixture was allowed to stand at 4 ° C. overnight. After the mixture is centrifuged (10,000 rpm, 20 min, 4 ° C.), the precipitate is recovered by adding pure water, put into a dialysis membrane (Spectra Pore 132 650 MWCO: 6-8,000), and purified water. And dialyzed. It was recovered from the dialysis membrane and used as a 30-80% saturated ammonium sulfate precipitate fraction.

(Affinity chromatography)
The 30-80% saturated ammonium sulfate precipitate fraction was subjected to fetuin-immobilized agarose equilibrated with PBS. The column was washed with PBS and then eluted with 0.2M lactose / PBS and 0.2M ammonia. Fractions showing hemagglutination activity (HA) (← → part of FIG. 2) were combined, replaced with pure water by ultrafiltration, and lyophilized. 0.03 mg and 0.3 mg of MGL lactose-eluted fraction and ammonia fraction were obtained, respectively.

(SDS-polyacrylamide electrophoresis)
SDS-polyacrylamide electrophoresis of MGL was performed using an electrophoresis apparatus (product name: Phassystem, manufactured by GE Healthcare Bioscience). The following MGL and molecular weight markers were prepared, and electrophoresis was performed according to the product protocol and conventional methods.
Lane 1: Molecular weight marker (APRO)
Lane 2: MGL (Lactose elution fraction) Reducing agent (+)
Lane 3: MGL (Lactose elution fraction) Reducing agent (-)
Lane 4: MGL (ammonia elution fraction) reducing agent (+)
Lane 5: MGL (ammonia elution fraction) Reducing agent (-)
Lane 6: Molecular weight marker (APRO)
Gel: Gradient 10-15 (manufactured by GE Healthcare Bioscience)
Sample: 1 μL / lane
Stain: Silver stain

  FIG. 3 shows the results of SDS-polyacrylamide electrophoresis (SDS-PAGE) of MGL. By SDS-PAGE using the above 10-15% gel, it was confirmed that the subunit molecular weight was about 60,000 for the reducing agent (+) and the subunit molecular weight was about 30,000 for the reducing agent (-).

(Hemagglutination activity test)
A hemagglutination activity test was performed on MGL using the erythrocytes of animals shown in Table 1. The results are shown in Table 1.

  As shown in Table 1, MGL was found to have high agglutination activity on rabbit and porcine erythrocytes, but low aggregation activity on bovine, horse and sheep erythrocytes.

(Sugar binding specificity)
The sugar binding specificity of MGL was evaluated by a hemagglutination inhibition test using various monosaccharides, oligosaccharides and polysaccharides shown in Table 2 and glycoproteins shown in Table 3.

  A 2-fold dilution series of 10 μL of monosaccharide, oligosaccharide, polysaccharide and glycoprotein solution was prepared on a 96-well U-bottom microtiter plate. 10 μL of a lectin solution prepared to a titer of 4 in advance was added to each well. After sensitizing by allowing to stand at room temperature for 1 hour, 10 μL of 4% rabbit erythrocyte suspension was added to each well, and further allowed to stand at room temperature for 1 hour. Thereafter, the dilution rate of the sugar and glycoprotein solution that completely inhibits red blood cell aggregation was determined with the naked eye. The lowest concentration showing inhibition was taken as the minimum inhibitory concentration. The smaller the minimum inhibitory concentration, the higher the affinity for lectins. The results are shown in Tables 2 and 3.

For comparison, the following commercial lectins, referred to as sialic acid specific lectins:
Comparative Example 1: SNA (prepared by J-Oil Mills, Inc.),
Comparative Example 2: SSA (Seikagaku Biobusiness Co., Ltd.-J-Oil Mills),
Comparative Example 3: MAM (Seikagaku Biobusiness Co., Ltd.-J-Oil Mills Co., Ltd.) and Comparative Example 4: ACG (J-Oil Mills Co., Ltd. in-house preparation)
Was used to evaluate the sugar binding specificity. The results are shown in Tables 2 and 3.

  From Tables 2 and 3, MGL binds to sialic acid α2-6 sugar chains such as 6′-sialyl lactose and 6′-sialyl lactosamine, but 3′-sialyl lactose and 3′-sialyl lactosamine. Such a sialic acid α2-3 sugar chain did not bind. In addition, glycoprotein did not bind to asialo form from which sialic acid was eliminated.

(3) Detection of glycoproteins by ELISA Enzyme immunoassay using biotin-labeled MGL of Example 1, SNA of Comparative Example 1, SSA of Comparative Example 2, MAM of Comparative Example 3 and ACG of Comparative Example 4 The glycoprotein was detected and evaluated by the method.

  After dissolving 1 mg / ml of the glycoprotein shown in Table 4 and albumin, which is a protein having no sugar chain as a control, with 0.1 M carbonate buffer (pH 9.5) and adding it to a microtiter plate (Nunc439454), 4 Allowed to stand overnight at ° C. After washing 3 times with 0.05% Tween / PBS (hereinafter abbreviated as PBS-T), PBS containing 1% bovine serum albumin (BSA) was added to the wells and incubated at 37 ° C. for 1 hour. After washing 3 times with PBS, a biotin-labeled lectin solution appropriately diluted with PBS-T containing 1% BSA was added to the wells and allowed to stand at 37 ° C. for 1 hour. After washing three times with PBS-T, an HRP-labeled streptavidin solution diluted with PBS-T containing 1% BSA was added to the wells and incubated at 37 ° C. for 30 minutes. After washing 3 times with PBS-T, TMB Peroxidase substrate system (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 10 minutes in the dark. The reaction was stopped with 1M phosphoric acid, and the absorbance at 450 nm was measured with a microplate reader (MPR-A4i manufactured by Tosoh Corporation).

From the measured value, the reaction value ([absorbance at 450 nm] − [value of well (blank) reacted without solid phase on the plate)) was calculated. Affinity ranking was performed by classifying the relative values according to the following criteria. The calculation results are shown in Table 4 and FIGS.
◎: 0.4 or more ○: 0.2 or more and less than 0.4 Δ: 0.1 or more and less than 0.2 ×: 0 to less than 0.1

  From Table 4, MGL bound strongly to immunoglobulin M (IgM) and prostate antigen (PSA).

Claims (10)

It can be extracted from the Mordicae grosvenori plant and has the following physicochemical properties:
(1) The subunit molecular weight by SDS-polyacrylamide electrophoresis is 20,000 to 70,000.
(2) Strong agglutination activity on rabbit and pig erythrocytes,
(3) A rakankalectin characterized by having affinity for sialic acid α2-6 sugar chain.   The rakankalectin according to claim 1, which has sugar-binding specificity for 6'-sialyllactose and 6'-sialyllactosamine.   The rakankalectin according to claim 1 or 2, which has a sugar-binding specificity for immunoglobulin M and prostate specific antigen.   It is a manufacturing method of the Lacanka lectin as described in any one of Claims 1-3, Comprising: It collect | recovers from the aqueous medium extract of a Rakan fruit plant, The manufacturing method of the Lacanka lectin characterized by the above-mentioned.   The method for producing a rakanka lectin according to claim 4, comprising subjecting the aqueous medium extract to ammonium sulfate precipitation and affinity chromatography.   A method for detecting a sialic acid α2-6 sugar chain, which comprises causing a sugar chain to act on the rakankalectin according to any one of claims 1 to 3.   A research reagent or a research determination kit for detecting a sialic acid α2-6 sugar chain, wherein the active ingredient is the rakankalectin according to any one of claims 1 to 3.   A diagnostic agent or diagnostic kit for detecting a sialic acid α2-6 sugar chain, comprising the kankan lectin according to any one of claims 1 to 3 as an active ingredient.   The specific binding agent with respect to the sialic acid (alpha) 2-6 sugar chain which uses the lacanka lectin as described in any one of Claims 1-3 as an active ingredient.   A method for fractionating a sialic acid α2-6 sugar chain, wherein the lacanka lectin according to any one of claims 1 to 3 is allowed to act.