WO2015072786A1 - Hericium erinaceum-derived lectin specific for sialic acid linkage - Google Patents

Hericium erinaceum-derived lectin specific for sialic acid linkage Download PDF

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WO2015072786A1
WO2015072786A1 PCT/KR2014/010974 KR2014010974W WO2015072786A1 WO 2015072786 A1 WO2015072786 A1 WO 2015072786A1 KR 2014010974 W KR2014010974 W KR 2014010974W WO 2015072786 A1 WO2015072786 A1 WO 2015072786A1
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Prior art keywords
lectin
neuac
sialic acid
sialic
oligosaccharide
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PCT/KR2014/010974
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French (fr)
Korean (ko)
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김성훈
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한국생명공학연구원
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Priority to JP2016554158A priority Critical patent/JP6356256B2/en
Priority to EP14862325.9A priority patent/EP3072954B1/en
Priority claimed from KR1020140158614A external-priority patent/KR101695741B1/en
Publication of WO2015072786A1 publication Critical patent/WO2015072786A1/en
Priority to US15/158,407 priority patent/US10150799B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi

Definitions

  • the present invention is a novel helium Roe deer mushroom, Accession No .: KCTC 12499BP) NEU-1L strain, sialic acid-specific binding lectin produced therefrom, and the use of the lectin. [Background]
  • Lectins is one of the proteins capable of binding to a sugar chain that exists up to the higher animals from a microorganism, through specific binding of the sugar chain in the living organisms' protein quality control, host -pathogen interact ion, cell cell communication eu It is known to be involved in various life phenomena such as inflammation, immune response, cancer progression and development (Lam and Ng, 2011. Appl. Microbiol. Biotechnol. 89, 45).
  • Lectin is a multivalent, non-enzymatic, and non-antibody-specific protein that has been isolated from Ricinus counis by SiUmark in Russia at the end of the 19th century. Lectin has been used for research and various purposes. Recent advances in molecular biology and biochemistry have begun to classify lectins into various groups based on their sequencing and protein structure, and largely plant lectin families and invertebrate / vertebrate lectin families lectins, depending on their origin. The vertebrate lectin is classified into C-type (mannose binding lectin, MBL), S ⁇ type (galect in ' , ⁇ -galactoside-binding lectin), and P_type (mannose ⁇ 6).
  • Phosph phosphate bindg lectin Phosph phosphate bindg lectin
  • I-type select in
  • protein structurally classified as pentraxin with cyclic pentamer ic form Phosph phosphate bindg lectin
  • I-type select in
  • protein structurally classified as pentraxin with cyclic pentamer ic form Phosph phosphate bindg lectin
  • I-type select in
  • protein structurally classified as pentraxin with cyclic pentamer ic form Phosph phosphate bindg lectin
  • Plant lectins are present in almost all parts of the plant, including leaves, stems, roots, flowers, and pollen. They are also found in harvestable seeds and bulbs, making it easy to purify lectins. Was done.
  • Lectins in plants are specific for symbiotic microorganisms through immune action (i ⁇ une function), self-defense from pests, allergies, self-defense from animals, anti-molecular action, antiviral action, and intercellular interactions between plants and microorganisms. Sites are known for their colonization, transport and storage of nutrients and metal ions, protection of plant tissues from cold and cold, as a partner for increased activity of intracellular enzymes, and trafficking of glycoproteins.
  • the selective binding specificity of lectins to sugar chains can be used to recognize cells present on the surface of specific cells, viruses, and microorganisms to measure cells and to detect cancer cells early. It is used as a key element technology in image measurement to detect, locate specific proteins in cells, interact with cells, and invade viruses and microorganisms.
  • Lectins that have been developed and commercialized so far Isolated from various natural sources such as plants, animals and microorganisms, sialic acid (Neu5Ac, Neuraminic acid), galactose (Gal, galactose), N-acetylgalactosamine (N— GalNAc, N—acetylgalactosamine), fucose , Fucose), Mannose (Man, Mannose), Glucose (Glc, glucose) is a lectin capable of measuring the basic monomer (Lehmann et al., 2006, Cell.Mol.Life Sci. 63, 1331; Singh et al., 2010 Crit. Rev. Biotechnol.
  • sialic acid or its variant, N-glycolylneuraminic acid (NeuGc), which is directly or indirectly involved in microbial and viral infections and is recognized as a marker in the development of cancer cells
  • N-glycolylneuraminic acid (NeuGc)
  • MM Maackia amurensis
  • SNA Sambucus nigra
  • Limulus polyphemus lectins are known, but ⁇ (2,3)-, ⁇ (2,6) _, a (2, 8) -linkage not only accurately distinguishes, but also has a disadvantage of showing signals due to nonspecific binding because Gal5 and GalNAc to which Neu5Ac binds are recognized and bound.
  • the new lectin protein is a glycoprotein including sialic acid oligosaccharide, glycolipid, oligosaccharide and other cells having various oligosaccharide-structures, proteins, oligosaccharide compounds, microbial monitoring, and biochemical phenomena occurring through oligosaccharides.
  • the present invention has been completed by confirming that it can be used for. [Detailed Description of the Invention]
  • Still another object of the present invention is a composition or kit for measuring or detecting a glycoprotein, a glycopeptide, a glycolipid, a sugar precursor, or an oligosaccharide having a sialic acid oligosaccharide comprising a lectin specifically binding to the sialic acid oligosaccharide according to the present invention.
  • Still another object of the present invention is to provide a composition or kit for measuring or detecting a cell line, bacteria or virus having a sialic acid sugar chain using a lectin specifically binding to the sialic acid sugar chain according to the present invention.
  • the present invention provides a novel helicium erinaium (Her i cium er inaceum, roe deer mushroom, Accession No .: KCTC 12499BP) NEU-1L strain.
  • the present invention is a method for producing a lectin that specifically binds to sialic acid oligosaccharide from the fruiting body of the novel Helicium erinium (Her i cium er inaceum, Roe beetle mushroom, Accession No .: KCTC 12499BP) NEU-1L strain And lectins produced therefrom.
  • the present invention also provides a composition or kit for measuring, detecting, or detecting a glycoprotein, a glycopeptide, a glycolipid, a sugar precursor or an oligosaccharide having a sialic acid oligosaccharide including a lectin specifically binding to the sialic acid oligosaccharide according to the present invention. .
  • the present invention also provides a composition or kit for measuring, detecting, or detecting a cell line, bacteria or virus having a sialic acid oligosaccharide using a lectin specifically binding to the sialic acid oligosaccharide according to the present invention.
  • the present invention provides a use of a kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors, or oligosaccharides comprising the sialic acid oligosaccharide including the lectin.
  • the present invention also provides the use of a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having the sialic acid oligosaccharide containing the lectin on its surface.
  • the present invention provides a method for producing a lectin that specifically binds to sialic acid oligosaccharides from the fruiting body of a novel Helicium erinaceum (Rupper mushroom, Accession No .: KCTC 12499BP) NEU-1L strain and produced therefrom.
  • compositions or kits for detecting or sialic acid sugar chains By providing lectins, specific for glycoproteins, glycopeptides, glycolipids, sugar precursors or loligosaccharides having a sialic acid sugar chain containing lectins that specifically bind to sialic acid sugar chains, compositions or kits for detecting or sialic acid sugar chains It can be usefully used as an active ingredient of a cell line having a sialic acid oligosaccharide including a lectin that binds to an enemy, a composition or a kit for measuring or detecting bacteria and viruses.
  • 1 is a diagram showing the genetic correlation with a similar strain based on the 26S rDNA sequence of the Hericium er inaceum NEU-Ll strain.
  • FIG. 2 shows SDS-PAGE analysis of tectin isolated from Helicium eryniasium mushroom fruiting body using fetuin-kenjugated agarose column;
  • FT unbound pass filtrate
  • MW standard protein marker
  • Elut ion 200 fraction eluted from 200 mM galactose.
  • Figure 3 shows the separation of lectin protein from the Helicium erinasium mushroom fruiting body using DEAD-Sepharose column, Fetuin-agarose column, Superose 12 column, each step protein (left) and finally separated HEL1 and HEL2 lectin protein ( 8) is analyzed by 12% SDS-PAGE and 15% SDS-PAGE.
  • MW standard protein marker
  • Figure 4 is a purely isolated HEL1 lectin analysis results in 16% Tr icine-PAGE and MALTI-TOF MS to measure the molecular weight of the two lectin proteins HELla and HELlb contained in the HEL1 lectin;
  • HEL HEL1 lectin protein
  • Figure 5 is a diagram showing the size of the protein in the non-denaturing conditon ion of HEL1 protein purified using the Superose 12 column and the pi value of the HEL1 lectin in pi 3 ⁇ 7 IEF-gel (B) )to be;
  • HELl HEL1 lectin protein. 6 is a diagram confirming the cohort activity of the HEL1 lectin protein using human, rabbit, porcine blood red blood cells; ⁇
  • Human (B-type) human blood red blood cells (blood type B);
  • Rabbit rabbit blood red blood cells
  • Porcine porcine blood red blood cells
  • HEL1 experimental group, HEL1 lectin
  • SNA-1 positive positive control, SNA—1 lectin.
  • FIG. 8 is a diagram observed by using confocal microscopy on the surface of A549 cell line containing sialic acid oligosaccharide after fluorescent labeling of lectin isolated from the fruiting body of Helicium erythium mushroom;
  • HEL1 Heliceum erynatium Experiment using HEL1 lectin
  • SNA Positive control using Sambucus nigra (SNA) lectin
  • BSA Negative control using Bovine Serum Albumin (BSA).
  • the present invention provides a Helicium erinaceum NEU-1L strain deposited with accession number KCTC12499BP, which produces a lectin protein that specifically binds to the sialic acid oligosaccharide.
  • the strain is a 26S rDNA nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 4 It is preferred to have the ITS1-5.8S-ITS4 rDNA base sequence described, but is not limited thereto.
  • the lectin protein that specifically binds to the sialic acid oligosaccharide is preferably bound to the N-linked oligosaccharide structure or 0-linked oligosaccharide structure in the fetuin glycoprotein to which sialic acid is added, but is not limited thereto.
  • the present inventors have isolated a new strain from roe deer mushroom purchased from a mushroom cultivation farm to identify the lectin specifically binding to the sialic acid oligosaccharide produced from the mushroom, As a result of sequencing of 26S rDNA and 5.8S rDNA for molecular biological identification of the strain, it was confirmed that the 26S rDNA nucleotide sequence of the strain is the same as SEQ ID NO: 1 (see Table 1).
  • step b) separating the lectin from the cell extract by using a column containing a glycoprotein, a glycopeptide, a glycolipid, a liposaccharide, or a monosaccharide condensed with resin in the cell extract;
  • a method for preparing lectin in which sialic acid oligosaccharides specifically bind from a Helicium erythium NEU-1L strain.
  • the lectin isolated in step b) further comprises the step of selectively eluting the lectin by competitively inhibiting the binding of the sugar chain and the lectin from the resin to which the sugar chain is conjugated.
  • an extract was prepared to separate lectin from the fruit body of Hericium erinaceum, and Fetuin-agarose column chromatography was performed using the extract prepared for lectinol purification.
  • Fetuin-agarose column chromatography was performed using the extract prepared for lectinol purification.
  • a protein bound to fetuin, a glycoprotein containing sialic acid oligosaccharide was eluted.
  • the present invention also provides lectins that specifically bind to sialic acid oligosaccharides prepared by the process according to the invention.
  • the present invention has a protein N-terminal sequence produced by the strain Helicium er inaceum NEIKLL deposited with accession number KCTC12499BP, and set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, 15 Provided are lectins to which sialic acid oligosaccharides specifically bind, having a molecular weight of kDa to 20 kDa.
  • the present invention is produced in a Helicium erinaceum NEU-1L strain deposited with accession number KCTC12499BP, has a protein N-terminal sequence described in SEQ ID NO: 9, and has a molecular weight of 50 kDa to 75 kDa It provides a lectin, characterized in that the sialic acid oligosaccharide specifically binds.
  • the lectin provides a lectin characterized by a gad that binds to an oligosaccharide structure selected from the group consisting of:
  • NeuAc or NeuGc (a2 ⁇ 3) is a Gal ( ⁇ l ⁇ 4) GlcNac residue or
  • NeuAc or NeuGc (a2 ⁇ 8) is NeuAc (a 2 ⁇ 8) NeuAc ( ⁇ 2 ⁇ 3)
  • NeuAc or NeuGc (a2 ⁇ 8) is the NeuAc (a2 ⁇ 8)
  • the present invention provides a method for the determination of glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides comprising sialic acid oligosaccharides, including lectins according to the present invention, or Provide a kit for quantification.
  • the present invention provides a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having a sialic acid oligosaccharide on the surface, including the lectin according to the present invention.
  • the present invention provides a use of a kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors, or larysaccharides, including sialic acid oligosaccharides, including the lectins.
  • the present invention also provides the use of a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having the sialic acid oligosaccharide containing the lectin on its surface.
  • glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides comprising sialic acid oligosaccharides comprising the step of analyzing glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides bound to the lectins according to the invention. It provides a method for measuring or quantifying sialic oxidized sugar (conjugated) consisting of.
  • b) provides a method for measuring or quantifying a cell line having a sialic acid sugar chain, bacteria or virus, comprising analyzing a cell, bacteria or virus bound to the lectin according to the present invention.
  • the eluted protein was concentrated using Amicon Ultra Centrifugal filter (Amicon®Ultra Centr i fugal fil ter 10K).
  • HEL1 and HEL2 Lectin Proteins Ion Exchange Chromatography DEAE-Sepharose Columns, Affinity Chromatography Fetuin Columns, and Size Exclusion Chromatography Superose 1210/300
  • the HEL1 and HEL2 lectin proteins were purely separated through the GL column, and the size of each protein was about 15-20 kDa and 50-75 kDa (see FIG. 3).
  • Mass spectrometer Microflex Maldi-T0F (Bruker Daltonik GmbH. Bremen, Germany) was performed to determine the exact molecular weight of the isolated protein, and the lectin molecular weight was analyzed to be 15327.587 Da or 15536.953 Da and 73253.12 Da (FIG.
  • the HEL1 lectin was a dimer type protein of about 32 cm 5 kDa, and was identified as about pi 4.5 protein (see FIG. 5).
  • all blood red blood cells tested in the same way as SNA-I lectin used as positive control group showed the blood cells, especially porcine blood compared with rabbit blood red blood cells and human blood red blood cells. Strong pool activity was confirmed for erythrocytes (see FIG. 6).
  • lectin blot was performed using fetuin containing sialic acid oligosaccharide and bisiaalfetuin from which sialic acid was removed as a substrate. Binding to Neu5Ac ⁇ (2,3) Gal ⁇ (l, 4) GlcNAc, the basic structure of sugar chains. Neu5Ac a (2,3) Gali3 (l, 3) [GalNA,
  • a lectin produced therefrom the measurement of a glycoprotein, glycopeptide, glycolipid, glycoprecursor or oligosaccharide having a sialic acid oligosaccharide comprising a lectin that specifically binds to the sialic acid oligosaccharide, a composition or kit or sial for detection
  • Cell lines having a sialic acid oligosaccharide comprising a lectin that specifically binds an acid oligosaccharide It can be usefully used as an active ingredient of a composition or kit for measuring or detecting bacteria and viruses.
  • the present invention will be described in detail by way of examples.
  • the following method was performed to isolate a new strain from the roe deer fungus mushroom purchased from Dolsan Mushroom Farming Association (1156, Geumbong-ri, Dolsan-eup, Yeosu-si, Jeonnam).
  • spores were obtained from the spore sac of the roe deer fungus purchased from the Dolsan Mushroom Farming Association.
  • the obtained spores were plated in PDA, YDP and amino acid minimum medium prepared as a solid medium, and cultured in a stationary state at 22 ° C.
  • the mycelia of the generated single cells were separated and plated again on the same medium as above, and cultured in a stationary state at 22 ° C. Then, through morphological confirmation, the mushroom It was confirmed that the separation was pure.
  • Example ⁇ 1-1> the strain isolated in Example ⁇ 1-1> was inoculated into a liquid medium of 10 mLPDA, 10 mL YDP and 10 mL amino acid minimum medium in a 125 mL flask, and then cultured in a stationary state at 22 ° C. It was. After incubation for 15 days or more, the cells were collected and centrifuged to remove the liquid medium. The recovered cells were completely frozen using liquid nitrogen, and then '-crushed cells by physical methods. After the addition of the cell lysis buffer containing 50 mU / mL Lypticase and 50 mM TrisHCKpH 8.0) and 250 mM NaCl to the crushed cells, the reaction was carried out at 37 ° C. for more than one hour.
  • the amplified gene was cloned into pGEM-T easy vector (Promega, USA) to analyze the nucleotide sequence, and the 26S rDNA nucleotide sequence of the strain is shown in SEQ ID NO: 1 shown in Table 1 below (Table 1). Then, as a result of analyzing the flexible relationship between the 26S rDNA having the nucleotide sequence of SEQ ID NO: 1 and the similar species, the 5.8S ribosomal RNA gene and the Herculium erinaceum 18S ribosomal RNA gene Sequence identity of 98% and 98% was identified through comparison of sequences (Fig.
  • ITS1 primer (SEQ ID NO: 5) 5 '-TCCGTAGGTGAACCTGCGG-3 1
  • ITS4 primer (SEQ ID NO: 6) 5 '-TCCKXGCTTATTGATATGC— 3'
  • the following method was performed to prepare an extractol for lectin separation in a Helicium erinaceum fruiting body.
  • the frui t ing body of the Helysium eryniasium mushroom that is frozen at -80 ° C was put in a mortar, and the sample was crushed with the pestle while adding the liquid nitrogen to keep the frozen mushrooms from thawing. .
  • the mushroom fruiting body was prepared in a frozen fine powder state by continuous crushing in the mortar and then stored at -80 ° C until it was recovered and used again.
  • the cell extract was determined by the weight of the frozen fruiting body prepared by the above method, and then measured with a protease inhibitor cocktail (protease inhibi tor cocktai l, Roche, Switzer land) and ⁇ mM PMSF corresponding to three times the measured weight.
  • Fetuin-agarose column chromatography was performed to purify the lectin using the extract prepared in the method described in Example ⁇ 2-1>. Specifically, in order to purify the lectin that binds to sialic acid selective binding, lectin separation is performed using fetuin-agarose, an agarose resin to which fetuin, a glycoprotein containing sialic acid oligosaccharide, is immobilized. A column was prepared. Fetuin-agarose resin obtained by equilibrating the cell extract of the Helicium erythium mushroom fruiting body prepared by the method described in Example ⁇ 2-1> with 20 mM Tr is-HCl buffer solution (pH 8.0).
  • the dissolution rate was 1 ml per minute, and the eluted protein was concentrated using Amicon® ultra centrifugal filter (Amicon® Ul tra Centr i fugal fil ter 1 0.) ⁇ 2-3> through SDS-PAGE analysis Lectin Protein Identification
  • Example ⁇ 2-2> The coenzyme solution was isolated using SDS-PAGE, stained with Coo's assie Brill iant Blue, and purified using the Precision Plus ProteinTM Standards (Bio-Rad, USA) protein as a standard protein. Purity and size of the lectin protein were analyzed.
  • Example ⁇ 2-1> Using the extract prepared in the method described in Example ⁇ 2-1>, the following method was performed to optimize the separation of HEL1 and HEL2 lectin protein identified in Example ⁇ 2-2>.
  • the same buffer solution corresponding to 1.5 times the measured weight was added to the precipitated fruiting body again to obtain a supernatant by the same method as described above, and the obtained supernatants were combined to prepare a final cell extract.
  • Saturated concentration 80% (w / v) ammonium sulfate was added to this, the protein was precipitated while stirring slowly at 4t.
  • the precipitated protein was centrifuged at 4 ° C. at a rate of 25, 000 X g for 60 minutes to recover the precipitate, and then dissolved in 1/10 volume of 50 mM Tri s HCl (pH 7.4) buffer solution.
  • the fraction containing the lectin protein was confirmed by a red blood cell agglutination assay of 0.5% (v / v) porcine blood. After recovering the fraction showing positive reaction in the hemagglutination experiment of porcine blood, the protein fraction was concentrated using an Amicon Ultra Centrifugal filter (10K).
  • Protein fractions containing lectin activity were fetuin-agarose resin, Sigma-Aldrich, USA equilibrated with 50 mM Tris-HCl buffer (pH 7.4) by the method described in Example 2-2 above.
  • DEAE-Sepharose column fraction was injected into the column (1.5 x 17 cm) using a Pump Pl peristaltic pump (Per istat ic pump, GE-healthcare, USA) at an injection rate of 1 mL per minute. After washing with a volume of 10 times the same complete solution, the protein bound to fetuin, a glycoprotein containing sialic acid oligosaccharide, was eluted with the same buffer containing 0.2-0.5 M D-galactose. Eluted protein was concentrated using Amicon Ultra Centrifugal filter 10K.
  • Concentrated protein solution containing lectin activity was injected into a Superose 12 10/300 GL (GE healthcare) gel filtration chromatography column equilibrated with 50 mM Tris-HCl buffer (pH 7.4), followed by 0.5 AKTA purification system. The protein was eluted at a flow rate of mL / min, and the fraction containing the lectin activity was confirmed using the erythroid test of porcine blood mentioned above. Finally, after recovering the fraction showing a positive response to porcine blood red blood cells, the lectin protein fraction was concentrated using Amicon ultra centrifugal filter 10K.
  • Example of protein fraction containing the activity of the lectin obtained in each step Isolation was performed using 1 or 15% SDS-PAGE by the method described in ⁇ 2-3>, stained with Brilliant Blue in Coomassie, and purity of lectin protein purified using Precision Plus ProteinTM Standards protein as standard protein. Size was analyzed.
  • Tr icin-PAGE was transferred to PVDF membrane at 15 V for 1 hour.
  • Tr icin The protein transferred to PVDF membrane in PAGE was stained with 0.1% (w / v) Ponceau S.
  • the protein identified by the staining method was cut out from the membrane, and the protein was analyzed by Korea Mass Spectrometry (Seoul, Korea). The N-terminal sequence analysis of was performed.
  • HEL1 protein As a result, two sequences of HEL1 protein of about 15 20 kDa size identified in Example ⁇ 2-4> were analyzed with HELla and HELlb.
  • SEQ ID No. 7 As a strong signal of HELla, SEQ ID No. 7 (NH2-KEPTWGRPES-C0 2 —) and Sequence number as a weak signal for HELlb It was found to have an N-terminal protein sequence of 8 (NH2-WPVAPDYPPES-C0 2 —). 50 75 kDa protein has the sequence number
  • the lectin protein separated and purified by the method described in Example ⁇ 2-4> was eluted with distilled water using a PD-10 column (GE-Healthcare, USA) to remove salts in the protein solution, Concentrated to a concentration of at least 1 mg / ml using an Ultra Centrifugal filter (Amicon® Ultra Centr i fugal filter) 10K. Then, in a solution containing 25% acetonitrile and 0.1% tri ifo luoacetic acid in a ratio of 1: 2, sinapinic acid was added to a saturated concentration. After dissolution, the solution was mixed at a volume ratio of 1: 1 with the concentrated protein to crystallize the protein on the mass spectrometer MALDI-T0F target. The crystallized protein was analyzed by using a mass spectrometer Microf lex Maldi-T0F (Bruker Daltonik GmbH. Bremen, Germany) to analyze the size of the purified lectin.
  • the HEL1 lectin protein having the N-terminal protein sequences of HELla and HELlb identified in Example ⁇ 2′4> had a strong signal of about 5 times stronger, and the molecular weight of the HELla lectin was 15327.587 Da.
  • the molecular weight of the lectins was analyzed to be 15536.953 Da (FIG. 4).
  • the molecular weight of the HEL2 lectin was analyzed to be 73253.12 Da.
  • Example 5 HELl tweetin protein size and pi identification in non-denaturing conditions of HEL1 lectin isolated by the method described in Example ⁇ 2-4>
  • the size of HEL1 lectin was determined using a Superose 12 10/300 GL column in an AKTA purification system.
  • Molecular weight calibration kit (GE healthcare) is the standard protein of 440 kDa Fernitin, 158 kDa Aldose, 44 kDa Ovalbumin ,. 29 kDa Carbonyl anhydrase, 13.7 kDa Ribonuc lease A, 6.5 kDa Aprotinin were used.
  • the lectin activity in the fraction containing the HEL1 lectin protein eluted from the Superose 12 10/300 GL column was confirmed by the coagulation activity of porcine blood erythrocytes and compared with chromatogram standard protein.
  • HELl lectin was identified as a dimer form and protein of about 32 ⁇ 5 kDa in non-denaturing conditions ( Figure 5A).
  • pi of the HEL1 lectin protein isolated by the method ⁇ described in Example ⁇ 2-4> was confirmed through isoelectric point (Iso-ElectroFocusing, IEF) electrophoresis. Comparing with pi of standard protein through IEF-PAG gel development for 100 V 1 hour, 200 V 1 hour, 500 V 30 minutes in cathode and anode buffer using Pi 3 ⁇ 7 IEF-gel system (K0MABI0TECH)
  • the HEL1 lectin was finally identified as about pi 4.5 protein (FIG. 5B).
  • porcine blood rabbit blood, human blood (human blood B- After dilution with PBS (phosphate buffer saline) buffer solution at 10% (v / v), centrifuged at 1000 rpm at 4 ° C for 5 minutes to precipitate red blood cells (erythrocytes). The supernatant was removed and the method was repeated three times. Finally, the supernatant was diluted in PBS buffer solution to prepare a 2% (v / v) erythrocyte cell solution.
  • PBS phosphate buffer saline
  • the agglut inat ion assay used a 96-wel l plate in the V-type, and serial dilutions of 100 uL of the lmg / mL concentration of lectin protein isolated in Example ⁇ 2> were performed. After mixing with 2% erythrocytes in the same volume, 96 wel l plate was allowed to stand at room temperature for 1 hour to observe the reaction of lectins on porcine blood erythrocytes, rabbit blood erythrocytes, and human blood erythrocytes.
  • the HEL1 lectin showed a homogeneity to the red blood cells of all blood tested in the same manner as the SNA-I lectin used as a positive control group, especially in porcine blood compared to rabbit and human blood Strong fungal activity was observed for (Fig. 6).
  • fetuin containing sialic acid oligosaccharide and bisial oxidized fetuin (asialoietuin) from which sialic acid was removed were used as a substrate.
  • Lectin blot was performed. Specifically, each protein solution containing fetuin and non-silicylic acid fetuin was mixed with 5 X Lae li buffer, heated for 10 minutes, and then subjected to electrophoresis on an 83 ⁇ 4 SDS-PAGE gel to separate proteins. .
  • the membrane was washed 6 times with PBST for 10 minutes, and 0.2 mg / mL horseradish peroxidase (HRP) -conjugated anti-biotin antibody (l: 500, Sigma—Aldr ich) for 1 hour. After reaction, the membrane was washed six times with PBST for 10 minutes in the same manner as above to identify sialized proteins with ECL kit (GE Healthcare, USA). As a result, as shown in FIG.
  • HRP horseradish peroxidase
  • A549 cell line (ATCC CCL-185TM, Rockville MD, USA) containing sialic acid oligosaccharides present on animal cell surfaces.
  • the A549 cell line was obtained by culturing in serum-free medium and then centrifuging the cells.
  • the lectins isolated by the method described in Example ⁇ 2-4> were fluorescently labeled using Alexa Fluor® 488 Protein Labeling Kitdnvitrogen, USA) as known in the art.
  • 1 mg / mL bovine serum albumin (BSA) was included.
  • the cell lines were washed with PBS buffer (phosphate saline buffer) and centrifuged to obtain the cells.
  • the obtained cells were suspended in a small amount of PBS (PBSB) buffer containing bovine serum albumin.
  • PBSB PBS
  • Alexa Fluor® 488 a final concentration of 0.5 nM to 1.0 nM
  • the lectin and the reaction cell line were incubated for 1 hour on ice.
  • the cell line was centrifuged to obtain a cell line again, and then the cell line was washed three times or more times using PBSB buffer solution, and the same buffer solution was finally made to have a concentration of 1 to 5 x 10 6 cells / mL.
  • the microorganism identified under I above was accompanied by:
  • microorganism identified under I above was received by ti is International Depositary Authority on and a request to convert the original deposi to a deposit under the Budapest Treaty was received by it on

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Abstract

The present invention can be useful as an active ingredient of a composition or a kit for measuring or detecting glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides having sialic acid sugar chains, containing a lectin specifically binding to sialic acid sugar chains, or a composition or a kit for measuring or detecting cell lines, bacteria and viruses having sialic acid sugar chains, containing a lectin specifically binding to sialic acid sugar chains, by providing: a method for producing a lectin, which specifically binds to sialic acid sugar chains, from a fruiting body of a novel Hericium erinaceum (deposit number KCTC 12499BP) NEU-1L strain; and a lectin produced thereby.

Description

【명세서】  【Specification】
[발명의 명칭】  [Name of invention]
헬리시움 에리나슘 버섯 유래의 시알산 결합 특이적인 렉틴 [기술분야】  Silicic acid binding-specific lectins from Helicium erythium mushrooms
본 발명은 신규 헬리시움
Figure imgf000002_0001
노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L 균주， 이로부터 생산된 시알산 특이적으로 결합하는 렉틴， 및 상기 렉틴의 용도에 관한 것이다. [배경기술] The present invention is a novel helium
Figure imgf000002_0001
Roe deer mushroom, Accession No .: KCTC 12499BP) NEU-1L strain, sialic acid-specific binding lectin produced therefrom, and the use of the lectin. [Background]
렉틴 (Lectin)은 미생물에서 고등동물에 이르기까지 존재하는 당사슬에 결합할 수 있는 단백질의 하나로， 생명체에 있어 당사슬과의 특이적인 결합을 '통해 protein quality control , host -pathogen interact ion, cellᅳ cell communication, inflammation, immune response, cancer progression, development 등 다양한 생명현상에 관여한다고 알려져 있다 (Lam and Ng, 2011. Appl . Microbiol . Biotechnol . 89， 45). Lectins (Lectin) is one of the proteins capable of binding to a sugar chain that exists up to the higher animals from a microorganism, through specific binding of the sugar chain in the living organisms' protein quality control, host -pathogen interact ion, cell cell communication eu It is known to be involved in various life phenomena such as inflammation, immune response, cancer progression and development (Lam and Ng, 2011. Appl. Microbiol. Biotechnol. 89, 45).
렉틴은 multivalent, 효소의 활성을 포함하지 않고, 항체의 성격을 포함하지 않는 단백질로， 19 세기말 러시아의 SiUmark에 의해 최초로 Ricinus co國 unis에서 분리된 이후， 현재까지 Concanavlain A(ConA)를 비롯하여 40여종의 렉틴이 연구 및 다양한 용도로 사용되고 있다. 최근 분자생물학 및 생화학의 진보로 염기서열 및 단백질 구조에 따라 렉틴을 다양한 그룹으로 분류하기 시작했으며, 원천 (origin)에 따라 크게는 식물 (plant lectin families) 및 동물 (invertebrate/vertebrate lectin families) 렉틴으로 분류하고 있으며， vertebrate lectin은 다시 그 특성에 따라 C— type (만노즈 결합 렉틴 : mannoseᅳ binding lectin, MBL) , S~type(galect in', β -galactoside-binding lectin), P_type(mannoseᅳ 6ᅳ phosphate bindg lectin), I-type (select in) , 그리고 단백질 구조적으로 cyclic pentamer ic 형태를 갖는 Pentraxin으로 분류한다. 이렇게 다양한 렉틴이 존재하지만 세포의 생리학적 대사과정에 작용하는 invertebrate/vertebrate lectin families은 그 양이 작아서 당사슬에 선택적으로 결합하는 특성을 이용한 혈액형 진단 및 웅용에는 자연계에서 추출한 식물 렉틴이 광범위하게 사용되고 있다 (Lehmann et al .， 2006, Cell. Mol. Life Sci. 63， 1331). Lectin is a multivalent, non-enzymatic, and non-antibody-specific protein that has been isolated from Ricinus counis by SiUmark in Russia at the end of the 19th century. Lectin has been used for research and various purposes. Recent advances in molecular biology and biochemistry have begun to classify lectins into various groups based on their sequencing and protein structure, and largely plant lectin families and invertebrate / vertebrate lectin families lectins, depending on their origin. The vertebrate lectin is classified into C-type (mannose binding lectin, MBL), S ~ type (galect in ' , β -galactoside-binding lectin), and P_type (mannose ᅳ 6). Phosph phosphate bindg lectin), I-type (select in), and protein structurally classified as pentraxin with cyclic pentamer ic form. These different lectins exist but the physiology of the cells Invertebrate / vertebrate lectin families, which act on metabolic processes, are widely used in plant lectins extracted from nature for the diagnosis and use of blood types using the properties of selective binding to the sugar chains (Lehmann et al., 2006, Cell.Mol Life Sci. 63, 1331).
식물 렉틴은 식물체의 잎， 줄기, 뿌리, 꽃， 화분 등 거의 식물체의 모든 부분에 존재하며 수확이 가능한 씨와 구근에서도 발견되기 때문에 용이하게 렉틴을 정제할 수 있는 장점으로 많은 정제가 식물체를 대상으로 이루어졌다. 식물체에서의 렉틴은 면역작용 (i睡 une function), 해충으로부터 자기방어, 알레르기를 일으켜 동물로부터 자기방어， 항곰광이성 작용， 항바이러스 작용， 식물 및 미생물간의 세포간 상호작용을 통한 공생미생물의 특정부위로 colonization, 영양분 및 금속이온의 전달 및 저장, 냉온으로부터 식물 조직체의 보호， 세포내 효소의 활성 증대를 위한 파트너로써의 작용， 그리고 당단백질의 trafficking 등 다양한 기능이 알려져 있는데， 이들 모든 작용은 당사슬과의 선택적 결합을 통한 렉틴의 작용으로 이루어진다고 보고되어 있다 (Lehmann et al. , 2006， Cell. Mol. Life Sci . 63, 1331； Singh et al . , 2010 Crit. Rev. Biotechnol. 30, 99). 이러한 당사슬 특이적 결합을 하는 렉틴 특성을 이용하여， 특정 질병 발병 시 세포 표면에 존재하는 당단백질， 당지질， 을리고당의 변화를 다양한 렉틴을 이용하여 조기 측정하고， 질병을 일으키는 핵심 세포 표면의 당사슬이나 바이러스 및 미생물 표면의 당사슬올 렉틴올 이용해 aggregation 시켜 신체 내에 면역시스템에 의해 질병을 퇴치하는 방법이 현재 동물 실험을 통해 연구되고 있다. 또한 최근 광학 현미경 기술의 발전과 다양한 형광 probe 합성과 더불어 렉틴의 당사슬에 대한 선택적 결합 특이성을 이용하여, 특정 세포 및 바이러스, 미생물의 표면에 존재하는 당사슬을 인지하여 세포를 측정하여 조기에 암 세포의 발견， 세포내 특정 단백질의 위치 확인， 세포간와 상호작용， 바이러스 및 미생물의 세포내 침입 등을 관찰하는 이미지 측정의 핵심요소 기술로 사용하고 있다. 현재까지 개발되어 상용화되어 있는 렉틴은 식물， 동물， 미생물 등의 다양한 천연자원으로부터 분리되어 시알산 (Neu5Ac, Neuraminic acid), 갈락토즈 (Gal， galactose), N-아세틸갈락토사민 (N— GalNAc, N—acetylgalactosamine) , 퓨코즈 (Fuc, Fucose) , 만노즈 (Man, Mannose) , 글루코스 (Glc, glucose)의 기본 모노머를 측정할 수 있는 렉틴이 있다 (Lehmann et al. , 2006, Cell. Mol. Life Sci. 63, 1331； Singh et al. , 2010 Crit. Rev. Biotechnol . 30， 99； Kajiwara et al. , 2010， Microbes Environ. 25， 152). 하지만， 당사슬의 구조상 알파 (alpha), 베타 (beta)-결합 (linkage)의 다양성과 당사슬 체인 구성 시 모노머 간의 결합 조합 (combination)의 다양성 또한 α/β linkage 및 combinat ion으로 경우 수백， 수천종 이상의 다양한 당사슬의 구조가 이론적으로 자연계에 존재하며， 실제적으로 현재까지 개발된 렉틴의 경우 그 수가 적어서 다양한 당사슬을 측정하거나 웅용하는데 한계가 있다고 할 수 있다. 특히， 미생물， 바이러스 감염에 직， 간접적으로 관여하면서 암세포 발병시 마커로 인식되고 있는 시알산 (Neu5Ac) 또는 이의 변형체 Nᅳ글라이코릴 뉴라미닉 산 (N-glycolylneuraminic acid, NeuGc)의 경우， 현재까지 개발되어 상업화된 Maackia amurensis (MM), Sambucus nigra (SNA) , Limulus polyphemus 렉틴이 있다고 알려져 있으나， 시알산의 결합 특이성인 α(2,3)-, α(2,6)_, a (2,8)-linkage를 정확하게 구분하지 못할뿐더러 Neu5Ac가 결합하고 있는 Gal, GalNAc 등을 인지하여 결합하기 때문에 nonspecific binding으로 인한 시그널을 보이는 단점을 안고 있다. 이에， 본 발명자들은 시알산 결합이 특이적인 웩틴을 발굴하기 위하여 노력한 결과， 헬리시움 에리나슘 (Hericium erinaceum, 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP)으로부터 단백질의 정제 과정을 통하여 활성형 신규 렉틴 단백질을 획득하였으며， 상기 신규 렉틴 단백질은 시알산 당사슬을 포함한 당단백질， 당지질， 올리고 당등 다양한 당사슬-구조를 갖는 세포， 단백질， 당사슬 화합물의 측정， 미생물 모니터링 및 당사슬을 매개로 일어나는 생명현상 규명 둥 광범위한 분야에 사용될 수 있음을 확인함으로써 본 발명을 완성하였다. 【발명의 상세한 설명】 Plant lectins are present in almost all parts of the plant, including leaves, stems, roots, flowers, and pollen. They are also found in harvestable seeds and bulbs, making it easy to purify lectins. Was done. Lectins in plants are specific for symbiotic microorganisms through immune action (i 睡 une function), self-defense from pests, allergies, self-defense from animals, anti-molecular action, antiviral action, and intercellular interactions between plants and microorganisms. Sites are known for their colonization, transport and storage of nutrients and metal ions, protection of plant tissues from cold and cold, as a partner for increased activity of intracellular enzymes, and trafficking of glycoproteins. It has been reported to consist of the action of lectins through selective binding to (Lehmann et al., 2006, Cell. Mol. Life Sci. 63, 1331; Singh et al., 2010 Crit. Rev. Biotechnol. 30, 99) . By using this lectin-specific binding characteristic of the oligosaccharide, changes in glycoproteins, glycolipids and loligosaccharides present on the cell surface at the onset of a specific disease can be measured early using various lectins, The use of oligosaccharides on the surface of viruses and microorganisms to aggregate them to combat disease by the immune system in the body is currently being studied through animal experiments. In addition to the recent advances in optical microscopy technology and the synthesis of various fluorescent probes, the selective binding specificity of lectins to sugar chains can be used to recognize cells present on the surface of specific cells, viruses, and microorganisms to measure cells and to detect cancer cells early. It is used as a key element technology in image measurement to detect, locate specific proteins in cells, interact with cells, and invade viruses and microorganisms. Lectins that have been developed and commercialized so far Isolated from various natural sources such as plants, animals and microorganisms, sialic acid (Neu5Ac, Neuraminic acid), galactose (Gal, galactose), N-acetylgalactosamine (N— GalNAc, N—acetylgalactosamine), fucose , Fucose), Mannose (Man, Mannose), Glucose (Glc, glucose) is a lectin capable of measuring the basic monomer (Lehmann et al., 2006, Cell.Mol.Life Sci. 63, 1331; Singh et al., 2010 Crit. Rev. Biotechnol. 30, 99; Kajiwara et al., 2010, Microbes Environ. 25, 152). However, the diversity of alpha and beta-linkages in the oligosaccharides and the combination of the combinations between monomers in the oligosaccharide chains can also be seen in the case of α / β linkages and combinat ions. The structures of various sugar chains exist theoretically in nature, and in practice, the number of lectins that have been developed so far is limited in measuring or using various sugar chains. In particular, sialic acid (Neu5Ac) or its variant, N-glycolylneuraminic acid (NeuGc), which is directly or indirectly involved in microbial and viral infections and is recognized as a marker in the development of cancer cells, The developed and commercialized Maackia amurensis (MM), Sambucus nigra (SNA) and Limulus polyphemus lectins are known, but α (2,3)-, α (2,6) _, a (2, 8) -linkage not only accurately distinguishes, but also has a disadvantage of showing signals due to nonspecific binding because Gal5 and GalNAc to which Neu5Ac binds are recognized and bound. Therefore, the present inventors have tried to find a specific mutant sialic acid binding specific, active new lectin protein through the purification of the protein from Helicium erinaceum, roe deer mushroom, accession number: KCTC 12499BP The new lectin protein is a glycoprotein including sialic acid oligosaccharide, glycolipid, oligosaccharide and other cells having various oligosaccharide-structures, proteins, oligosaccharide compounds, microbial monitoring, and biochemical phenomena occurring through oligosaccharides. The present invention has been completed by confirming that it can be used for. [Detailed Description of the Invention]
【기술적 과제】 _  [Technical problem] _
본 발명의 목적은 신규한 헬리시움 에리나슘 (Her i cium er inaceum, 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L 균주를 제공하는 것이다. 본 발명의 또 다른 목적은 신규한 헬리시움 에리나슘 (Her i cium er inaceum , 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L 균주의 자실체로부터 시알산 당사슬에 특이적으로 결합하는 렉틴을 생산하는 방법， 및 이로부터 생산된 렉틴을 제공하는 것이다.  It is an object of the present invention to provide a novel Helicium erinasium (Her i cium er inaceum, Roe beetle mushroom, Accession No .: KCTC 12499BP) NEU-1L strain. Another object of the present invention is to produce a lectin that specifically binds to sialic acid oligosaccharides from the fruiting body of the novel Helicium er inaceum (Her i cium er inaceum, Roe beetle mushroom, Accession No .: KCTC 12499BP) NEU-1L strain To provide a lectin produced therefrom.
본 발명의 또 다른 목적은 본 발명에 따른 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 당단백질, 당펩타이드， 당지질， 당전구체 또는 을리고당의 측정 또는 검출용 조성물 또는 키트를 제공하는 것이다.  Still another object of the present invention is a composition or kit for measuring or detecting a glycoprotein, a glycopeptide, a glycolipid, a sugar precursor, or an oligosaccharide having a sialic acid oligosaccharide comprising a lectin specifically binding to the sialic acid oligosaccharide according to the present invention. To provide.
본 발명의 또 다른 목적은 본 발명에 따른 시알산 당사슬에 특이적으로 결합하는 렉틴을 이용하여 시알산 당사슬을 갖는 세포주， 박테리아 또는 바이러스의 측정 또는 검출용 조성물 또는 키트를 제공하는 것이다.  Still another object of the present invention is to provide a composition or kit for measuring or detecting a cell line, bacteria or virus having a sialic acid sugar chain using a lectin specifically binding to the sialic acid sugar chain according to the present invention.
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위해서， 본 발명은 신규한 헬리시움 에리나슘 (Her i cium er inaceum,노루궁뎅이 버섯 ,기탁번호: KCTC 12499BP) NEU-1L 균주를 제공한다.  In order to achieve the above object, the present invention provides a novel helicium erinaium (Her i cium er inaceum, roe deer mushroom, Accession No .: KCTC 12499BP) NEU-1L strain.
또한， 본 발명은 신규한 헬리시움 에리나슘 (Her i cium er inaceum , 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L균주의 자실체로부터 시알산 당사슬에 특이적으로 결합하는 렉틴을 생산하는 방법 및 이로부터 생산된 렉틴을 제공한다.  In addition, the present invention is a method for producing a lectin that specifically binds to sialic acid oligosaccharide from the fruiting body of the novel Helicium erinium (Her i cium er inaceum, Roe beetle mushroom, Accession No .: KCTC 12499BP) NEU-1L strain And lectins produced therefrom.
또한， 본 발명은 본 발명에 따른 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 당단백질， 당펩타이드, 당지질， 당전구체 또는 올리고당의 측정， 검출용 조성물 또는 키트를 제공한다. 또한, 본 발명은 본 발명에 따른 시알산 당사슬에 특이적으로 결합하는 렉틴을 이용하여 시알산 당사슬을 갖는 세포주， 박테리아 또는 바이러스의 측정， 검출용 조성물 또는 키트를 제공한다. The present invention also provides a composition or kit for measuring, detecting, or detecting a glycoprotein, a glycopeptide, a glycolipid, a sugar precursor or an oligosaccharide having a sialic acid oligosaccharide including a lectin specifically binding to the sialic acid oligosaccharide according to the present invention. . The present invention also provides a composition or kit for measuring, detecting, or detecting a cell line, bacteria or virus having a sialic acid oligosaccharide using a lectin specifically binding to the sialic acid oligosaccharide according to the present invention.
또한， 본 발명은 상기 렉틴을 포함하는 시알산 당사슬을 포함하는 당단백질, 당펩타이드， 당지질， 당전구체 또는 올리고당의 측정 또는 정량용 키트의 용도를 제공한다.  In addition, the present invention provides a use of a kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors, or oligosaccharides comprising the sialic acid oligosaccharide including the lectin.
또한， 본 발명은 상기 렉틴을 포함하는 시알산 당사슬을 표면에 갖는 세포주, 박테리아 또는 바이러스의 모니터링， 측정 또는 정량용 키트의 용도를 제공한다.  The present invention also provides the use of a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having the sialic acid oligosaccharide containing the lectin on its surface.
【유리한 효과】 Advantageous Effects
본 발명은 신규한 헬리시움 에리나슘 (Hericium erinaceum, 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L 균주의 자실체로부터 시알산 당사슬에 특이적으로 결합하는 렉틴을 생산하는 방법 및 이로부터 생산된 렉틴을 제공함으로써， 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 당단백질， 당펩타이드, 당지질， 당전구체 또는 을리고당의 측정， 검출용 조성물 또는 키트 또는 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 세포주， 박테리아 및 바이러스의 측정 또는 검출용 조성물 또는 키트의 유효성분으로 유용하게 사용될 수 있다.  The present invention provides a method for producing a lectin that specifically binds to sialic acid oligosaccharides from the fruiting body of a novel Helicium erinaceum (Rupper mushroom, Accession No .: KCTC 12499BP) NEU-1L strain and produced therefrom. By providing lectins, specific for glycoproteins, glycopeptides, glycolipids, sugar precursors or loligosaccharides having a sialic acid sugar chain containing lectins that specifically bind to sialic acid sugar chains, compositions or kits for detecting or sialic acid sugar chains It can be usefully used as an active ingredient of a cell line having a sialic acid oligosaccharide including a lectin that binds to an enemy, a composition or a kit for measuring or detecting bacteria and viruses.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 Her icium er inaceum NEU-Ll 균주의 26S rDNA 염기서열에 기초한 유사 균주와의 유전학적 상관관계를 나타낸 도이다.  1 is a diagram showing the genetic correlation with a similar strain based on the 26S rDNA sequence of the Hericium er inaceum NEU-Ll strain.
도 2는 페투인이 켄쥬게이션된 아가로즈 컬럼을 이용하여 헬리시움 에리나슘 버섯 자실체에서 분리된 텍틴의 SDS-PAGE 분석을 나타낸 도이다;  FIG. 2 shows SDS-PAGE analysis of tectin isolated from Helicium eryniasium mushroom fruiting body using fetuin-kenjugated agarose column; FIG.
CL: 세포추출액;  CL: cell extract;
FT: 비결합 통과 여액; MW: 표준 단백질 마커 ; FT: unbound pass filtrate; MW: standard protein marker;
Wash l : 컬럼 통과 여액 분획 1 ;  Wash l: column pass filtrate fraction 1;
Wash W2: 컬럼 통과 여액 분획 2 ;  Wash W2: column pass filtrate fraction 2;
Wash W3 : 컬럼 통과 여액 분획 3 ;  Wash W3: column pass filtrate fraction 3;
Elut ion 20： 20 mM 갈락토즈에서 용출된 분획;  Elut ion 20: fraction eluted at 20 mM galactose;
Elut ion 40： 40 mM 갈락토즈에서 용출된 분획; 및  Elut ion 40: fraction eluted at 40 mM galactose; And
Elut ion 200： 200 mM 갈락토즈에서 용출된 분획.  Elut ion 200: fraction eluted from 200 mM galactose.
도 3은 DEAD-Sepharose 칼럼， Fetuin-agarose 칼럼， Superose 12 칼럼을 이용하여 헬리시움 에리나슘 버섯 자실체에서 렉틴 단백질을 분리한 후， 각 단계별 단백질 (좌) 및 최종 분리된 HEL1와 HEL2 렉틴 단백질 (우)을 12% SDS-PAGE와 15% SDS-PAGE로 분석한 결과를 나타낸 도이다;  Figure 3 shows the separation of lectin protein from the Helicium erinasium mushroom fruiting body using DEAD-Sepharose column, Fetuin-agarose column, Superose 12 column, each step protein (left) and finally separated HEL1 and HEL2 lectin protein ( 8) is analyzed by 12% SDS-PAGE and 15% SDS-PAGE.
MW: 표준 단백질 마커 ;.  MW: standard protein marker;.
CL: 세포추출액;  CL: cell extract;
FT: 비결합 통과 여액;  FT: unbound pass filtrate;
W: DEAE-Sepharose 컬럼 통과 여액 분획;  W: filtrate fraction through DEAE-Sepharose column;
El : DEAE-Sepharose 컬럼에서 용출된 분획 ;  El: fraction eluted from DEAE-Sepharose column;
E2 : Fetuin-agarose 컬럼에서 용출된 분획; 및  E2: fraction eluted from Fetuin-agarose column; And
E3: Superose 12 컬럼에서 용출된 분획.  E3: fraction eluted from Superose 12 column.
도 4는 순수하게 분리된 HEL1 렉틴을 16% Tr icine-PAGE에서 분석한 결과와 MALTI-TOF MS를 이용하여 HEL1 렉틴에 포함된 두 개의 렉틴 단백질 HELla와 HELlb의 분자량을 측정한 도이다;  Figure 4 is a purely isolated HEL1 lectin analysis results in 16% Tr icine-PAGE and MALTI-TOF MS to measure the molecular weight of the two lectin proteins HELla and HELlb contained in the HEL1 lectin;
MW: 표준 단백질 마커 ; 및  MW: standard protein marker; And
HEL: HEL1 렉틴 단백질.  HEL: HEL1 lectin protein.
도 5는 Superose 12 칼럼을 이용하여 정제된 HEL1 단백질의 non-denaturing condi t ion에서 단백질의 크기를 나타낸 도 (A)이며 pi 3~7 IEF-gel에서 HEL1 렉틴의 pi 값을 분석한 도 (B)이다;  Figure 5 is a diagram showing the size of the protein in the non-denaturing conditon ion of HEL1 protein purified using the Superose 12 column and the pi value of the HEL1 lectin in pi 3 ~ 7 IEF-gel (B) )to be;
pi standard protein: 표준 단백질 마커 ; 및  pi standard protein: standard protein marker; And
HELl : HEL1 렉틴 단백질. 도 6은 인간， 토끼, 돼지 혈액 적혈구를 이용하여 HEL1 렉틴 단백질의 웅집활성을 확인한도이다; ― HELl: HEL1 lectin protein. 6 is a diagram confirming the cohort activity of the HEL1 lectin protein using human, rabbit, porcine blood red blood cells; ―
Human(B-type)： 인간 혈액 적혈구 (혈액형 B)；  Human (B-type): human blood red blood cells (blood type B);
Rabbit: 토끼 혈액 적혈구;  Rabbit: rabbit blood red blood cells;
Porcine: 돼지 혈액 적혈구;  Porcine: porcine blood red blood cells;
Blank, Control negative: 음성대조군;  Blank, Control negative: negative control group;
Test, HEL1: 실험군， HEL1 렉틴; 및  Test, HEL1: experimental group, HEL1 lectin; And
Positive, SNA-1 positive: 양성대조군， SNA—1 렉틴.  Positive, SNA-1 positive: positive control, SNA—1 lectin.
도 7은 렉틴 블롯을 이용하여 헬리시움 에리나슘 버섯 자실체에서 분리한 렉틴의 시알산 당사슬올 포함하는 페투인 (fetuin)과 시알산이 제거된 비시알산화페투인 (asialofetuin)을 기질로 이용하여, 시알화 된 단백질에 대한 결합능을 확인한도이다.  7 is a fetuin containing sialic acid oligosaccharide of lectin and sialic acid removed asialofetuin removed as a substrate, using lectin blot, isolated from Helicium erinasium mushroom fruiting body. This is the limit for binding to sialylated proteins.
도 8은 헬리시움 에리나슘 버섯 자실체에서 분리한 렉틴을 형광표지한 후， 시알산 당사슬을 포함하는 A549 세포주 표면에서 공초점현미경을 사용하여 관찰한 도이다;  FIG. 8 is a diagram observed by using confocal microscopy on the surface of A549 cell line containing sialic acid oligosaccharide after fluorescent labeling of lectin isolated from the fruiting body of Helicium erythium mushroom;
HEL1: 헬리시움 에리나슴 HEL1 렉틴을 이용한 실험군 (Experiment )； SNA: Sambucus nigra (SNA)렉틴을 이용한 양성대조군 (Posit ive control )；  HEL1: Heliceum erynatium Experiment using HEL1 lectin; SNA: Positive control using Sambucus nigra (SNA) lectin;
BSA: Bovine Serum Albumin (BSA)를 이용한 음성대조군 (Negative control). BSA: Negative control using Bovine Serum Albumin (BSA).
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
이하, 본 발명을 상세하게 설명한다. 본 발명은 시알산 당사슬에 특이적으로 결합하는 렉틴 단백질을 생산하는， 수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium erinaceum) NEU-1L 균주를 제공한다.  EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail. The present invention provides a Helicium erinaceum NEU-1L strain deposited with accession number KCTC12499BP, which produces a lectin protein that specifically binds to the sialic acid oligosaccharide.
상기 균주는 서열번호 1로 기재되는 26S rDNA 염기서열 및 서열번호 4로 기재되는 ITS1-5.8S-ITS4 rDNA 염기서열을 가지는 것이 바람직하나， 이에 한정되지 않는다. The strain is a 26S rDNA nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 4 It is preferred to have the ITS1-5.8S-ITS4 rDNA base sequence described, but is not limited thereto.
상기 시알산 당사슬에 특이적으로 결합하는 렉틴 단백질은 시알산이 부가되어 있는 페투인 당단백질에서 N-결합 당사슬 구조 또는 0-결합 당사슬 구조에 결합하는 것이 바람직하나, 이에 한정되지 않는다. 본 발명의 구체적인 실시예에 있어서, 본 발명자들은 버섯으로부터 생산된 시알산 당사슬에 특이적으로 결합하는 렉틴을 규명하기 위하여， 버섯재배 농가에서 구매한 노루궁뎅이 버섯에서 신규 균주를 분리하였고， 상기 분리한 균주의 분자생물학적 동정을 위하여 26S rDNA와 5.8S rDNA의 염기서열 분석한 결과， 상기 균주의 26S rDNA 염기서열은 서열번호 1과 같은 것을 확인하였다 (표 1참조) . 그런 다음，상기 서열번호 1의 염기서열을 가지는 26S rDNA와 유사종과의 유연관계를 분석한 결과， 노루궁뎅이 버섯 (Hericium er inaceum) 5.8S r ibosomal RNA유전자와 노루궁뎅이 버섯 (Her icium erinaceum) 18S r ibosomal RNA 유전자의 염기서열 유사성 비교를 통해 98%와 98 )의 서열 동일성 (sequence ident i fy)을 확인하였으며 (도 1 참조) , Her icium er inaceum NEU-L1 균주로 명명하고， 2013년 10월 4일 한국생명공학연구원 생물자원센터에 (Korean Col lect ion ' for Type Culture , KCTC)에 기탁하였다 (기탁번호 KCTC 12499BP) . 또한， 본 발명은 The lectin protein that specifically binds to the sialic acid oligosaccharide is preferably bound to the N-linked oligosaccharide structure or 0-linked oligosaccharide structure in the fetuin glycoprotein to which sialic acid is added, but is not limited thereto. In a specific embodiment of the present invention, the present inventors have isolated a new strain from roe deer mushroom purchased from a mushroom cultivation farm to identify the lectin specifically binding to the sialic acid oligosaccharide produced from the mushroom, As a result of sequencing of 26S rDNA and 5.8S rDNA for molecular biological identification of the strain, it was confirmed that the 26S rDNA nucleotide sequence of the strain is the same as SEQ ID NO: 1 (see Table 1). Then, as a result of analyzing the flexible relationship between the 26S rDNA having the nucleotide sequence of SEQ ID NO: 1 and the similar species, erectium mushroom (Hericium er inaceum) 5.8S r ibosomal RNA gene and Hericic erinaceum 18S The sequence identity of the ibosomal RNA gene was compared to confirm the sequence identity (98% and 98) of the sequence identity (see sequence 1) (see FIG. 1), and named as Her icium er inaceum NEU-L1 strain, October 2013. 4 days was deposited on the Korea Research Institute of Bioscience and biotechnology biological resource Center (Korean Col lect ion 'for Type Culture, KCTC) ( accession No. KCTC 12499BP). In addition, the present invention
(a) 수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium er inaceum) NEU-1L의 자실체로부터 세포 추출물을 수득하는 단계; 및  (a) obtaining a cell extract from a fruiting body of Helicium er inaceum NEU-1L deposited with accession no. KCTC12499BP; And
(b) 상기 세포 추출물에서 사알산화 당사슬을 포함하는 당단백질， 당펩타이드, 당지질， 을리고당 또는 단당이 컨쥬게이션되어 레진이 충진되어 있는 컬럼을 이용하여 상기의 렉틴을 분리하는 단계를 포함하는， 헬리시움 에리나슘 NEU-1L 균주로부터 시알산 당사슬이 특이적으로 결합하는 렉틴의 제조 방법을 제공한다. 상기 단계 b)에서 분리된 렉틴을 당사슬이 컨쥬게이션 되어 있는 레진으로부터 당사슬과 렉틴의 결합을 경쟁적으로 방해하여 해당 렉틴을 선택적으로 용리해 내는 단계를 더 포함하는 것이 바람직하나， 이에 한정되지 않는다. 본 발명의 구체적인 실시예에 있어서， 헬리시움 에리나슴 (Her icium erinaceum)자실체에서 렉틴을 분리하기 위하여 추출물을 제조하였으며， 렉틴올 정제하기 위하여 상기 제조된 추출물을 이용하여 Fetuin-agarose 칼럼 크로마토그래피를 수행한 결과, 시알산 당사슬을 포함하는 당단백질인 페투인에 결합한 단백질을 용출시켰다. 또한， 본 발명은 본 발명에 따른 방법으로 제조된 시알산 당사슬에 특이적으로 결합하는 렉틴을 제공한다. (b) separating the lectin from the cell extract by using a column containing a glycoprotein, a glycopeptide, a glycolipid, a liposaccharide, or a monosaccharide condensed with resin in the cell extract; Provided is a method for preparing lectin, in which sialic acid oligosaccharides specifically bind from a Helicium erythium NEU-1L strain. Preferably, the lectin isolated in step b) further comprises the step of selectively eluting the lectin by competitively inhibiting the binding of the sugar chain and the lectin from the resin to which the sugar chain is conjugated. In a specific embodiment of the present invention, an extract was prepared to separate lectin from the fruit body of Hericium erinaceum, and Fetuin-agarose column chromatography was performed using the extract prepared for lectinol purification. As a result, a protein bound to fetuin, a glycoprotein containing sialic acid oligosaccharide, was eluted. The present invention also provides lectins that specifically bind to sialic acid oligosaccharides prepared by the process according to the invention.
또한， 본 발명은 수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium er inaceum) NEIKLL균주에서 생산되고，서열번호 7，서열번호 8 또는 서열번호 9로 기재되는 단백질 N-말단 서열을 가지며， 15 kDa 내지 20 kDa의 분자량을 가지는 것을 특징으로 하는, 시알산 당사슬이 특이적으로 결합하는 렉틴을 제공한다.  In addition, the present invention has a protein N-terminal sequence produced by the strain Helicium er inaceum NEIKLL deposited with accession number KCTC12499BP, and set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, 15 Provided are lectins to which sialic acid oligosaccharides specifically bind, having a molecular weight of kDa to 20 kDa.
또한, 본 발명은 수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium erinaceum) NEU-1L 균주에서 생산되고， 서열번호 9로 기재되는 단백질 N-말단 서열을 가지며, 50 kDa내지 75 kDa의 분자량을 가지는 것을 특징으로 하는， 시알산 당사슬이 특이적으로 결합하는 렉틴을 제공한다. 상기 렉틴은 다음으로 구성된 군으로부터 선택되는 당사슬 구조에 결합하는 갓을 특징으로 하는 렉틴을 제공한다;  In addition, the present invention is produced in a Helicium erinaceum NEU-1L strain deposited with accession number KCTC12499BP, has a protein N-terminal sequence described in SEQ ID NO: 9, and has a molecular weight of 50 kDa to 75 kDa It provides a lectin, characterized in that the sialic acid oligosaccharide specifically binds. The lectin provides a lectin characterized by a gad that binds to an oligosaccharide structure selected from the group consisting of:
NeuAc 또는 NeuGc( a 2→3)가 Gal 잔기 또는 Glc 잔기에 부가된 시알산화 당사슬;  Sialic oxidized chain wherein NeuAc or NeuGc (a 2 → 3) is added to the Gal residue or Glc residue;
NeuAc 또는 NeuGc( α 2→6)가 Gal 잔기 또는 Glc 잔기에 부가된 시알산화 당사슬; NeuAc 또는 NeuGc( α2→3)가 GaK 31→4)Glc 잔기 또는 Glc(pi→4)Glc 잔기에 부가된 시알산화 당사슬; Sialylated sugar chains in which NeuAc or NeuGc (α 2 → 6) is added to the Gal residue or the Glc residue; Sialic oxidized chain wherein NeuAc or NeuGc (α2 → 3) is added to the GaK 31 → 4) Glc residue or the Glc (pi → 4) Glc residue;
NeuAc 또는 NeuGc(a2→6)가 Gal(pl→4)Glc 잔기 또는 Glc(pi→4)Glc 잔기에 부가된 시알산화 당사슬;  Sialic oxidized chain wherein NeuAc or NeuGc (a2 → 6) is added to a Gal (pl → 4) Glc residue or a Glc (pi → 4) Glc residue;
NeuAc 또는 NeuGc(a2→3)가 Gal ( β l→4)GlcNac 잔기 또는 NeuAc or NeuGc (a2 → 3) is a Gal (β l → 4) GlcNac residue or
Gal(pi→3)GlcNac 잔기에 부가된 시알산화 당사슬; Sial oxidized sugar chain added to the Gal (pi → 3) GlcNac residue;
NeuAc 또는 NeuGc(a2→6)가 Gal ( β l→4)GlcNac 잔기 또는 Gal(1 1→3)GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oligosaccharide wherein NeuAc or NeuGc (a2 → 6) is added to Gal (β 1 → 4) GlcNac residue or Gal (1 1 → 3) GlcNac residue;
NeuAc 또는 NeliGc( α2→3)가 Gal(P 1→3)[ aFuc(l→4)]GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized chain wherein NeuAc or NeliGc (α2 → 3) is added to the Gal (P 1 → 3) [aFuc (l → 4)] GlcNac residue;
NeuAc 또는 NeuGc( a2→6)가 Gal( β 1→3)[ aFuc(l→4)]GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized sugar chain wherein NeuAc or NeuGc (a2 → 6) is added to Gal (β 1 → 3) [aFuc (l → 4)] GlcNac residue;
NeuAc 또는 NeuGc( α2→3)가 GaKP 1→4)[ aFuc(l→3)]GlcNac 잔기에 부가된 시알산화 당사슬; .  Sialic oxidized chain wherein NeuAc or NeuGc (α2 → 3) is added to GaKP 1 → 4) [aFuc (l → 3)] GlcNac residue; .
NeuAc 또는 NeuGc( α 2→6)가 GaK β 1→4)[ aFuc(l→3)]GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized sugar chain wherein NeuAc or NeuGc (α 2 → 6) is added to GaK β 1 → 4) [aFuc (l → 3)] GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc 잔기에 부가된 시알산화 당사슬; Sialic oxidized sugar chain in which NeuAc or NeuGc (a2 → 8) is added to the NeuAc residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc ( a2→3) GaK β l→4)GlcNac 잔기에 부가된 시알산화 당사슬; Sialic oxidized chain wherein NeuAc or NeuGc (a2 → 8) is added to NeuAc (a2 → 3) GaK β l → 4) GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc( a 2→8)NeuAc( α2→3) NeuAc or NeuGc (a2 → 8) is NeuAc (a 2 → 8) NeuAc (α2 → 3)
Gal(pi→4)GlcNac 잔기에 부가된 시알산화 당사슬; Sialic oxidized sugar chain added to the Gal (pi → 4) GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc(a2→8) NeuAc or NeuGc (a2 → 8) is the NeuAc (a2 → 8)
[NeuAc( a2→8)]nNeuAc( a2→3) GaK β l→4)GlcNac 잔기에 부가된 시알산화 당사슬; 및 Sialylated oligosaccharide added to [NeuAc (a2 → 8)] nNeuAc (a2 → 3) GaK β 1 → 4) GlcNac residue; And
NeuAc 또는 NeuGc( α2→8)가 [NeuAdn잔기에 부가된 시알산화 당사슬. 또한， 본 발명은 본 발명에 따른 렉틴을 포함하는, 시알산 당사슬을 포함하는 당단백질, 당펩타이드, 당지질， 당전구체 또는 올리고당의 측정 또는 정량용 키트를 제공한다. Sialylated sugar chain in which NeuAc or NeuGc (α2 → 8) is added to the NeuAdn residue. In addition, the present invention provides a method for the determination of glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides comprising sialic acid oligosaccharides, including lectins according to the present invention, or Provide a kit for quantification.
또한， 본 발명은 본 발명에 따른 렉틴을 포함하는， 시알산 당사슬을 표면에 갖는 세포주， 박테리아 또는 바이러스의 모니터링， 측정 또는 정량용 키트를 제공한다.  In addition, the present invention provides a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having a sialic acid oligosaccharide on the surface, including the lectin according to the present invention.
또한， 본 발명은 상기 렉틴을 포함하는 시알산 당사슬을 포함하는 당단백질， 당펩타이드， 당지질， 당전구체 또는 을리고당의 측정 또는 정량용 키트의 용도를 제공한다.  In addition, the present invention provides a use of a kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors, or larysaccharides, including sialic acid oligosaccharides, including the lectins.
또한， 본 발명은 상기 렉틴을 포함하는 시알산 당사슬을 표면에 갖는 세포주， 박테리아 또는 바이러스의 모니터링， 측정 또는 정량용 키트의 용도를 제공한다.  The present invention also provides the use of a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having the sialic acid oligosaccharide containing the lectin on its surface.
또한， 본 발명은  In addition, the present invention
a) 본 발명에 따른 렉틴에 피검 시료를 접촉하는 단계; 및  a) contacting the test sample with the lectin according to the present invention; And
b) 본 발명에 따른 렉틴에 결합된 당단백질， 당펩타이드， 당지질， 당전구체 또는 올리고당을 분석하는 단계를 포함하는， 시알산 당사슬을 포함하는 당단백질， 당펩타이드， 당지질， 당전구체 또는 을리고당으로 구성된 시알산화 당사슬 (conjugated)의 측정 또는 정량 방법을 제공한다.  b) glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides comprising sialic acid oligosaccharides, comprising the step of analyzing glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides bound to the lectins according to the invention. It provides a method for measuring or quantifying sialic oxidized sugar (conjugated) consisting of.
또한， 본 발명은  In addition, the present invention
a) 본 발명에 따른 렉틴에 피검 시료를 접촉하는 단계 ; 및  a) contacting the test sample to the lectin according to the present invention; And
b) 본 발명에 따른 렉틴에 결합된 세포， 박테리아 또는 바이러스를 분석하는 단계를 포함하는， 시알산 당사슬을 갖는 세포주， 박테리아 또는 바이러스의 측정 또는 정량 방법을 제공한다. 본 발명의 또 다른 구체적인 실시예에 있어서， 용출된 단백질은 아미콘 울트라 센트리푸갈 필터 (Amicon®Ul tra Centr i fugal f i l ter 10K)을 이용하여 농축시켰다. 상기 방법을 사용하여 수득한 조효소 용액을 SDS-PAGE를 이용하여 분리한 후， 코마시에 브릴리언트 블루 (Coo國 assie Bri l l i ant Blue)로 염색하였으'며 Precision Plus ProteinTM Standards(Bio-Rad, USA)단백질을 표준 단백질로 사용하여 정제된 렉틴 단백질의 순도 및 크기를 분석한 결과, 시알산을 포함한 당사슬이 부가된 당단백질인 페투인에 결합한 렉틴의 크기는 두 종류의 단백질 HEL1 및 HEL2로 측정되었으며 단백질의 크기는 약 15~20 kDa 및 50~75 kDa로 확인되었다 (도 2 참조). HEL1과 HEL2 렉틴 단백질의 분리 최적화한 결과， 이온교환크로마토그래피 (ion chromatography) DEAE-Sepharose 칼럼， 친환성 크로마토그래피 (affinity chromatography) 페투인 칼럼, 그리고 크기 배제 크로마토그래피 (size exclusion chromatography) Superose 1210/300 GL 칼럼을 통해 HEL1와 HEL2 렉틴 단백질을 순수하게 분리하였으며， 각각의 단백질의 크기는 약 15~20kDa및 50 ~ 75 kDa로 확인되었다 (도 3참조). 상기 분리된 단백질의 정확한 분자량을 측정하기 위하여 질량분석기 Microflex Maldi-T0F(Bruker Daltonik GmbH. Bremen, Germany)를 수행한 결과， 각각의 렉틴 분자량은 15327.587 Da또는 15536.953 Da와 73253.12 Da으로 분석되었다 (도 4 참조). 상기 렉틴의 Non-denaturing condition에서의 HEL1 렉틴 단백질 크기 및 pi확인한 결과， HEL1렉틴은 약 32 士 5 kDa의 dimer형태의 단백질이고， 약 pi 4.5 단백질로 확인되었다 (도 5 참조). HEL1 렉틴을 이용한 혈액 적혈구의 웅집성 확인한 결과， 양성대조군으로 사용한 SNA-I 렉틴과 동일하게 테스트한 모든 혈액의 적혈구 세포에 대해 웅집성을 나타냈으며, 특히 토끼혈액 적혈구와 인간혈액 적혈구에 비해 돼지혈액 적혈구에 대해 강한 웅집 활성이 확인되었다 (도 6 참조). 또한， 렉틴의 결합능을 확인하기 위하여， 시알산 당사슬을 포함하는 페투인 (fetuin)과 시알산이 제거된 비시알산화페투인 (asialofetuin)을 기질로 이용하여， 렉틴 블랏을 수행한 결과， N-결합 당사슬의 기본 구조인 Neu5Ac α(2,3) Gal β (l,4)GlcNAc와 으결합 당사슬 . Neu5Ac a(2,3)Gali3(l,3)[GalNA,b) provides a method for measuring or quantifying a cell line having a sialic acid sugar chain, bacteria or virus, comprising analyzing a cell, bacteria or virus bound to the lectin according to the present invention. In another specific embodiment of the present invention, the eluted protein was concentrated using Amicon Ultra Centrifugal filter (Amicon®Ultra Centr i fugal fil ter 10K). Separated and the crude enzyme solution was obtained using the above method using the SDS-PAGE, Brilliant Blue (Coo國assie Bri lli ant Blue) to "said Precision Plus ProteinTM Standards (Bio-Rad , USA) hayeoteu dye upon coma The purity and size of the purified lectin protein were analyzed using the protein as a standard protein. Lectin bound to the glycoprotein added glycoprotein containing sialic acid was measured by two proteins, HEL1 and HEL2, and the size of the protein was found to be about 15-20 kDa and 50-75 kDa (see FIG. 2). ). Isolation Optimization of HEL1 and HEL2 Lectin Proteins, Ion Exchange Chromatography DEAE-Sepharose Columns, Affinity Chromatography Fetuin Columns, and Size Exclusion Chromatography Superose 1210/300 The HEL1 and HEL2 lectin proteins were purely separated through the GL column, and the size of each protein was about 15-20 kDa and 50-75 kDa (see FIG. 3). Mass spectrometer Microflex Maldi-T0F (Bruker Daltonik GmbH. Bremen, Germany) was performed to determine the exact molecular weight of the isolated protein, and the lectin molecular weight was analyzed to be 15327.587 Da or 15536.953 Da and 73253.12 Da (FIG. 4). Reference). As a result of confirming the size and pi of the HEL1 lectin protein under non-denaturing conditions of the lectin, the HEL1 lectin was a dimer type protein of about 32 cm 5 kDa, and was identified as about pi 4.5 protein (see FIG. 5). As a result of confirming the blood red blood cells using HEL1 lectin, all blood red blood cells tested in the same way as SNA-I lectin used as positive control group showed the blood cells, especially porcine blood compared with rabbit blood red blood cells and human blood red blood cells. Strong pool activity was confirmed for erythrocytes (see FIG. 6). In addition, in order to confirm the binding ability of the lectin, lectin blot was performed using fetuin containing sialic acid oligosaccharide and bisiaalfetuin from which sialic acid was removed as a substrate. Binding to Neu5Ac α (2,3) Gal β (l, 4) GlcNAc, the basic structure of sugar chains. Neu5Ac a (2,3) Gali3 (l, 3) [GalNA,
Neu5Aca(2,3)Ga 3(l,4)Galp(l,6)]의 당사슬을 포함하는 페투인에서는 렉틴 블랏의 시그널을 관찰하였으나， 음성 대조군인 비시알산화페토인의 Gal|3(l,4)GlcNAc의 비시알산화 N-당사슬과 Gali3(l,3), GlcNA, Gai (l,3)[GalNA β(1,6)]의 당사슬을 포함하는 단백질에서는 렉틴 블랏의 시그널이 확인되지 않았다 (도 7 참조). 아울러, 동물 세포 표면에 존재하는 시알산 당사슬올 포함하는 Α549 세포주에 형광표지된 렉틴을 이용하여 세포 표면을 관찰한 결과, 동물 세포 표면에 존재하는 시알산 당사슬을 포함하는In the fetuin containing the oligosaccharide of Neu5Aca (2,3) Ga 3 (l, 4) Galp (l, 6)], the lectin blot signal was observed, but the Gal | 3 (l (l) 4) Signals of lectin blots are not detected in proteins containing bisial oxidized N-glycosides of GlcNAc and oligosaccharides of Gali3 (l, 3), GlcNA, Gai (l, 3) [GalNA β (1,6)] (See Figure 7). In addition, cells using lectin fluorescently labeled on the A549 cell line containing sialic acid oligosaccharide present on the surface of animal cells The surface was observed to contain sialic acid oligosaccharides present on the animal cell surface.
A549 세포주에 형광 라벨된 렉틴을 사용한 결과, 상기 세포주 표면에서 형광 라벨된 렉틴을 확인함으로써， 사알산 당사슬을 확인하였다 (도 8 참조) . As a result of using a fluorescently labeled lectin in the A549 cell line, the salamic oligosaccharide was confirmed by confirming the fluorescently labeled lectin on the cell line surface (see FIG. 8).
따라서， 본 발명의 신규한 헬리시움 에리나슘 (Her i cium er inaceum , 노루궁뎅이 버섯， 기탁번호: KCTC 12499BP) NEU-1L균주의 자실체로부터 시알산 당사슬에 특이적으로 결합하는 렉틴을 생산하는 방법 및 이로부터 생산된 렉틴을 제공함으로써 , 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 당단백질， 당펩타이드， 당지질, 당전구체 또는 올리고당의 측정， 검출용 조성물 또는 키트 또는 시알산 당사슬에 특이적으로 결합하는 렉틴을 포함하는 시알산 당사슬을 갖는 세포주; 박테리아 및 바이러스꾀 측정 또는 검출용 조성물 또는 키트의 유효성분으로 유용하게 사용될 수 있다. 이하， 본 발명을 실시예에 의해 상세히 설명한다.  Therefore, the method of producing a lectin that specifically binds to sialic acid oligosaccharides from the fruiting body of the novel Helicium erinium (Her i cium er inaceum, Roe beetle mushroom, Accession No .: KCTC 12499BP) NEU-1L of the present invention And by providing a lectin produced therefrom, the measurement of a glycoprotein, glycopeptide, glycolipid, glycoprecursor or oligosaccharide having a sialic acid oligosaccharide comprising a lectin that specifically binds to the sialic acid oligosaccharide, a composition or kit or sial for detection Cell lines having a sialic acid oligosaccharide comprising a lectin that specifically binds an acid oligosaccharide; It can be usefully used as an active ingredient of a composition or kit for measuring or detecting bacteria and viruses. Hereinafter, the present invention will be described in detail by way of examples.
단， 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.  However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.
<실시예 1> 렉틴 생산 버섯 균주의 분리 및 동정 Example 1 Isolation and Identification of Lectin-producing Mushroom Strains
<1-1> 렉틴 생산 버섯 균주의 분리  <1-1> Isolation of Lectin-producing Mushroom Strains
돌산 버섯영농조합 (전남 여수시 돌산읍 금봉리 1156)에서 구매한 노루궁뎅이 버섯으로부터 신규 균주를 분리하기 위해 하기와 같은 방법을 수행하였다.  The following method was performed to isolate a new strain from the roe deer fungus mushroom purchased from Dolsan Mushroom Farming Association (1156, Geumbong-ri, Dolsan-eup, Yeosu-si, Jeonnam).
구체적으로， 돌산버섯영농조합에서 구입한 노루궁뎅이 버섯의 포자낭에서 백금이를 사용하여 포자를 획득하였다. 상기 획득한 포자를 고상배지로 제작한 PDA , YDP 및 아미노산 최소배지에 각각 도말하여 22 °C 조건에서 정치상태로 배양하였다. 10일 이상 배양 후， 생성된 단일 균체의 균사를 분리하여 상기와 같은 동일한 배지에 다시 도말하여 22°C 조건에서 정치상태로 배양하였다. 그런 다음， 형태적인 확인을 통하여 상기 버섯이 순수하게 분리된 것을 확인하였다. Specifically, spores were obtained from the spore sac of the roe deer fungus purchased from the Dolsan Mushroom Farming Association. The obtained spores were plated in PDA, YDP and amino acid minimum medium prepared as a solid medium, and cultured in a stationary state at 22 ° C. After incubation for 10 days or more, the mycelia of the generated single cells were separated and plated again on the same medium as above, and cultured in a stationary state at 22 ° C. Then, through morphological confirmation, the mushroom It was confirmed that the separation was pure.
<1-2> 렉틴 생산 버섯 균주의 동정 <1-2> Identification of Lectin-producing Mushroom Strains
상기 실시예 <1-1〉에서 분리한 균주의 분자생물학적 동정을 위하여 26S rDNA와 5.8S rDNA의 염기서열 분석을 수행하였다.  In order to identify molecular biology of the strain isolated in Example <1-1>, sequencing of 26S rDNA and 5.8S rDNA was performed.
구체적으로， 상기 실시예 <1-1>에서 분리한 균주를 125 mL의 플라스크에 10 mLPDA, 10 mL YDP및 10 mL아미노산 최소배지의 각각 액상배지에 접종한 후 , 22°C에서 정치상태로 배양하였다. 15일 이상 배양 후， 균체를 회수하여 원심분리를 하였으며， 액체 배지 성분을 제거하였다. 상기 회수된 균체는 액체질소를 이용하여 완전히 동결 시킨 후, 물리적인 방법으로 균체를 '- 파쇄하였다. 상기 파쇄된 균체에 50 mU/mL 립티케이스 (Lypticase)와 50mM TrisHCKpH 8.0) 및 250 mM NaCl이 포함된 세포 완충용액 (cell lysis buffer)을 첨가한 후， 37°C에서 한 시간 이상 반웅하였다. 반웅 후， 다시 원심분리를 통해 세포를 회수하였고， AccuPrep® Genomic DNA Extraction Kit (바이오니아， 대전， 대한민국)를 사용하여 세포내 DNA를 추출하였다. 상기 버섯 균주로부터 추출된 DNA를 주형으로 하여 26S rDNA를 증폭하기 위하여, NL1 프라이머 (서열번호 2)및 LR6프라이머 (서열번호 3)를 이용하여 94°C에서 45초， 55°C에서 1초， 72°C에서 1분 30초 30회 반복의 DNA 폴리머레이즈 중합반웅 (polymerase chain react ion)을 통해 l,146 bp의 유전자를 증폭하였다. 상기 증폭된 유전자는 pGEM-T easy 백터 (Promega, USA)에 클로닝하여 염기서열을 분석하였으며， 상기 균주의 26S rDNA 염기서열은 하기 표 1에 나타낸 서열번호 1과 같다 (표 1). 그런 다음， 상기 서열번호 1의 염기서열을 가지는 26S rDNA와 유사종과의 유연관계를 분석한 결과， 노루궁뎅이 버섯 (Hericium americanum) 5.8S ribosomal RNA 유전자와 노루궁뎅이 버섯 (Hericium erinaceum) 18S ribosomal RNA유전자의 염기서열 유사성 비교를 통해 98%와 98%의 서열 동일성 (sequence identify)을 확인하였으며 (도 1)， Hericium erinaceum NEU-L1 균주로 명명하고， 2013년 10월 4일 한국생명공학연구원 생물자원센터에 (Korean Collection for Type Culture, KCTC)에 기탁하였다 (기탁번호 KCTC 12499BP) . 또한， 상기 노루궁뎅이 버섯 (Her i cium er inaceum) NEU-L1 균주의 ITS1-5.8S-ITS4 rDNA 염기서열을 분석하기 위하여， ITS1프라이머 (서열번호 5)및 ITS4프라이머 (서열번호 6， 5 ' - TCCTCCGCTTATTGATATGC-3 '；)를 사용하여 94 °C에서 45초， 55 °C에서 30초， 72 °C에서 1분 30회 반복 DNA 폴리머레이즈 중합반웅을 통해 644 bp의 유전자를 증폭하였다. 상기 증폭된 유전자는 pGEM-T easy벡터에 클로닝하여 염기서열을 분석한 결과, 서열번호 4의 염기서열을 가지는 것을 확인하였다 (표 1) . Specifically, the strain isolated in Example <1-1> was inoculated into a liquid medium of 10 mLPDA, 10 mL YDP and 10 mL amino acid minimum medium in a 125 mL flask, and then cultured in a stationary state at 22 ° C. It was. After incubation for 15 days or more, the cells were collected and centrifuged to remove the liquid medium. The recovered cells were completely frozen using liquid nitrogen, and then '-crushed cells by physical methods. After the addition of the cell lysis buffer containing 50 mU / mL Lypticase and 50 mM TrisHCKpH 8.0) and 250 mM NaCl to the crushed cells, the reaction was carried out at 37 ° C. for more than one hour. After reaction, the cells were recovered by centrifugation and intracellular DNA was extracted using AccuPrep® Genomic DNA Extraction Kit (Bionia, Daejeon, Korea). In order to amplify the 26S rDNA and the DNA extracted from the mushroom strain as the template, using the NL1 primer (SEQ ID NO: 2) and LR6 primer (SEQ ID NO: 3) 45 seconds at 94 ° C, 1 sec at 55 ° C, The gene of l, 146 bp was amplified by DNA polymerase chain react ion of 1 minute 30 seconds 30 times at 72 ° C. The amplified gene was cloned into pGEM-T easy vector (Promega, USA) to analyze the nucleotide sequence, and the 26S rDNA nucleotide sequence of the strain is shown in SEQ ID NO: 1 shown in Table 1 below (Table 1). Then, as a result of analyzing the flexible relationship between the 26S rDNA having the nucleotide sequence of SEQ ID NO: 1 and the similar species, the 5.8S ribosomal RNA gene and the Herculium erinaceum 18S ribosomal RNA gene Sequence identity of 98% and 98% was identified through comparison of sequences (Fig. 1), named as Hericium erinaceum NEU-L1 strain, October 4, 2013 Korea Research Institute of Bioscience and Biotechnology In Korean Collection for Type Culture, KCTC) (Accession No. KCTC 12499BP). In addition, in order to analyze the ITS1-5.8S-ITS4 rDNA sequence of the strain of the Roe beetle (Her i cium er inaceum) NEU-L1, ITS1 primer (SEQ ID NO: 5) and ITS4 primer (SEQ ID NO: 6, 5 '- TCCTCCGCTTATTGATATGC-3 '; over 45 seconds at 94 ° C using a), 30 sec at 55 ° C, 1 minute 30 times repeatedly DNA polymerase raised polymerization banung at 72 ° C was amplified gene of 644 bp. The amplified gene was cloned into pGEM-T easy vector and analyzed for nucleotide sequence, and found to have the nucleotide sequence of SEQ ID NO: 4 (Table 1).
【표 1】 Table 1
Figure imgf000016_0001
CCATCTTACACCTGTGCACCOTGCGTGGGTCCGTCGGCITTGCGGTCGATG
Figure imgf000016_0001
CCATCTTACACCTGTGCACCOTGCGTGGGTCCGTCGGCITTGCGGTCGATG
GGCTOCGTTTTTCATAAACTCTTATGTATGTAACAGAATGTCAT TGCTA TAAACGCATCnATACAACTTTCAACAACGGATCTOTGGCTCTCGCATCGA TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAAnGCAGAAnCAGTGAA TCATCGMTCTTTGAACGCACOTGCGCCCCTTGGTATTCCGAGGGGCACGC CTGmGAGTGTCGTGAAAnCTCAACTCAATCCTCnGnATGAGAGGGCT GGGCTTGGACTTGGAGGTCTTGCCGGTGCTCCCTCGGGAAGTCGGCTCCTCT TGAATGCATGAGTGGATCCCTTnGTAGGGTTTGCCCnGGTGTGATAATTA TCTACGCCGCGGGTAGCCnGCGnGGTCTGOTCTAACCGTCnCGGACAA CTTTCATCTCAACTTGACCTCGAATCAGGCGGGACTACCCGCTGAAOTAAG CATATCAATAAGCGGAGGAAATC-3 '  GGCTOCGTTTTTCATAAACTCTTATGTATGTAACAGAATGTCAT TGCTA TAAACGCATCnATACAACTTTCAACAACGGATCTOTGGCTCTCGCATCGA TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAAnGCAGAAnCAGTGAA TCATCGMTCTTTGAACGCACOTGCGCCCCTTGGTATTCCGAGGGGCACGC CTGmGAGTGTCGTGAAAnCTCAACTCAATCCTCnGnATGAGAGGGCT GGGCTTGGACTTGGAGGTCTTGCCGGTGCTCCCTCGGGAAGTCGGCTCCTCT TGAATGCATGAGTGGATCCCTTnGTAGGGTTTGCCCnGGTGTGATAATTA TCTACGCCGCGGGTAGCCnGCGnGGTCTGOTCTAACCGTCnCGGACAA CTTTCATCTCAACTTGACCTCGAATCAGGCGGGACTACCCGCTGAAOTAAG CATATCAATAAGCGGAGGAAATC-3 '
ITS1 프라이머 (서열번호 5) 5 ' -TCCGTAGGTGAACCTGCGG-31 ITS1 primer (SEQ ID NO: 5) 5 '-TCCGTAGGTGAACCTGCGG-3 1
ITS4 프라이머 (서열번호 6) 5 ' -TCCKXGCTTATTGATATGC— 3 '  ITS4 primer (SEQ ID NO: 6) 5 '-TCCKXGCTTATTGATATGC— 3'
<실시예 2> 헬리시움 에리나슘 (Hericium erin ceum)으로부터 시알산 당사슬 선택적 결합을하는 렉틴 분리 정제 Example 2 Purification of Lectin Isolated from Helicium erin ceum with Selective Sialic Acid Sugar Chain
<2-1> 헬리시움 에리나슘 (Hericium erinaceum) 자실체에서 렉틴 분리를 위한추출물 제조  <2-1> Preparation of Extracts for Lectin Separation from Helicium erinaceum Fruiting Bodies
헬리시움 에리나슘 (Hericium erinaceum) 자실체에서 렉틴 분리를 위한 추출물올 제조하기 위하여 하기와 같은 방법을 수행하였다.  The following method was performed to prepare an extractol for lectin separation in a Helicium erinaceum fruiting body.
구체적으로， -80°C에서 동결보관 중인 헬리시움 에리나슘 버섯의 자실체 ( frui t ing body)를 막자 사발에 넣고 액체질소를 첨가하면서 동결된 버섯이 해동되지 않도록 유지하면서 막자로 시료를 파쇄하였다. 막자 사발 내에서의 연속적인 파쇄과정을 통해 버섯 자실체를 동결 미세 분말 상태로 제조한 후， 다시 회수하여 사용하기 전까지 -80°C에서 보관하였다. 세포추출물은 상기의 방법으로 제조된 동결 분말상태 자실체의 중량을 확인한 후， 측정 중량의 3배에 해당되는 프로테아제 억제 칵테일 (protease inhibi tor cocktai l , Roche , Switzer land)과 Γ mM PMSF가 포함된 측정 중량의 3배에 해당되는 20 mM Tris HCKpH 8.0) 완충용액을 첨가한 후， 4°C에서 6시간 이상 흔합하였다. 상기 혼합액에서 파쇄되지 않은 버섯 자실체의 입자와 비용해성 단백질을 제거하기 위해 A500S-6N 로터가 장착된 SUPRA 22K 원심분리기 (한일과학， 대한민국)를 4°C에서 7， OOO rpm으로 30분 동안 사용하여 침전물을 제거하고 상등액을 수득하였다. 상기 수득된 상등액을 A50C-6 로터가 장착된 SUPRA 22K 원심분리기를 4°C에서， 12 ,000 rpm으로 30분 동안 사용하여 침전물을 제거하고 상등액을 수득하여 렉틴 분리를 위한 최종 세포추출물로 준비하였다. Specifically, the frui t ing body of the Helysium eryniasium mushroom that is frozen at -80 ° C was put in a mortar, and the sample was crushed with the pestle while adding the liquid nitrogen to keep the frozen mushrooms from thawing. . The mushroom fruiting body was prepared in a frozen fine powder state by continuous crushing in the mortar and then stored at -80 ° C until it was recovered and used again. The cell extract was determined by the weight of the frozen fruiting body prepared by the above method, and then measured with a protease inhibitor cocktail (protease inhibi tor cocktai l, Roche, Switzer land) and Γ mM PMSF corresponding to three times the measured weight. After adding 20 mM Tris HCKpH 8.0) buffer corresponding to 3 times the weight, the mixture was mixed at 4 ° C. for 6 hours or more. SUPRA 22K centrifuge (Hanil, Korea) equipped with A500S-6N rotor (Hanil Science, South Korea) was used for 30 minutes at 4 ° C. at 7, OOO rpm to remove particles and insoluble proteins of mushroom fruit bodies that were not crushed. The precipitate was removed and a supernatant was obtained. The obtained supernatant was used as a SUPRA 22K centrifuge equipped with an A50C-6 rotor at 12,000 rpm for 30 minutes at 4 ° C. to remove the precipitate, and the supernatant was obtained as a final cell extract for lectin separation. .
<2-2> 페투인-아가로즈 (Fetuin-agarose) 칼럼 크로마토그래피를 이용한 렉틴 정제 <2-2> Lectin Purification Using Fetuin-agarose Column Chromatography
상기 실시예 <2ᅳ1>에서 기재된 방법으로 제조한 추출물을 사용하여 렉틴을 정제하기 위하여 Fetuin-agarose 칼럼 크로마토그래피를 수행하였다. 구체적으로， 시알산 선택적 결합을 하는 렉틴을 정제하기 위해서 시알산 당사슬을 포함하고 있는 당단백질인 페투인이 고정되어 있는 아가로즈 레진인 페투인-아가로즈 ( fetuin-agarose)을 이용하여 렉틴 분리를 위한 칼럼을 제작하였다. 상기 실시예 <2-1〉에 기재된 방법으로 제조한 헬리시움 에리나슘 버섯 자실체의 세포추출물을 20 mM Tr is-HCl 완충용액 (pH 8.0)으로 평형화시킨 페투인-아가로즈 레진 (Fetuin-agarose resin, Sigma-Aldr ich, USA) 칼럼 ( 1.0 x 10cm)에 상기 세포추출물을 Pump Pᅳ 1 튜브연동식 펌프 (Per i stat ic pump, GE-heal thcare , USA)를 이용하여 주입속도를 분당 1 mL로 하여 주입하였다. 등일 완층용액 10 배 부피로 세척한 다음， 20 mM, 40 mM, 200 mM D-갈락토즈 (D-galactose)가 포함되어 있는 동일 완층용액으로 2배， 4배 및 10배 부피로 갈락토즈의 농도를 증가시키면서 시알산 당사슬을 포함하는 당단백질인 페투인에 결합한 단백질을 용출시켰다. 이때 용출 속도는 분당 1 ml로 하였으며, 용출된 단백질은 아미콘 울트라 센트리푸갈 필터 (Amicon® Ul tra Centr i fugal f i l ter 1이0을 이용하여 농축시켰다. <2-3> SDS-PAGE분석을 통한 렉틴 단백질 확인  Fetuin-agarose column chromatography was performed to purify the lectin using the extract prepared in the method described in Example <2-1>. Specifically, in order to purify the lectin that binds to sialic acid selective binding, lectin separation is performed using fetuin-agarose, an agarose resin to which fetuin, a glycoprotein containing sialic acid oligosaccharide, is immobilized. A column was prepared. Fetuin-agarose resin obtained by equilibrating the cell extract of the Helicium erythium mushroom fruiting body prepared by the method described in Example <2-1> with 20 mM Tr is-HCl buffer solution (pH 8.0). resin, Sigma-Aldr ich, USA) column extract (1.0 x 10 cm) using the pump P 을 1 tube peristaltic pump (Per i stat ic pump, GE-heal thcare, USA) infusion rate per minute It was injected into mL. After washing with 10-fold volume of equiaxed complete layer, the concentration of galactose in 2-fold, 4-fold and 10-fold volumes of the same complete solution containing 20 mM, 40 mM, 200 mM D-galactose. The protein bound to fetuin, a glycoprotein containing sialic acid oligosaccharide, was eluted with increasing. At this time, the dissolution rate was 1 ml per minute, and the eluted protein was concentrated using Amicon® ultra centrifugal filter (Amicon® Ul tra Centr i fugal fil ter 1 0.) <2-3> through SDS-PAGE analysis Lectin Protein Identification
상기 실시예 <2-2>에 기재된 방법으로 정제하여 수득한 조효소 용액의 단백질을 확인하기 위하여 하기와 같은 방법을 수행하였다.  The following method was carried out to confirm the protein of the coenzyme solution obtained by purification by the method described in Example <2-2>.
구체적으로 상기 실시예 <2-2>에 기재된 방법으로 정제하여 수득한 조효소 용액을 SDS-PAGE를 이용하여 분리한 후， 코마시에 브릴리언트 블루 (Coo画 assie Bri l l iant Blue)로 염색하였으며 Precision Plus ProteinTM Standards(Bio-Rad, USA) 단백질을 표준 단백질로 사용하여 정제된 렉틴 단백질의 순도 및 크기를 분석하였다. Specifically obtained by purification by the method described in Example <2-2> The coenzyme solution was isolated using SDS-PAGE, stained with Coo's assie Brill iant Blue, and purified using the Precision Plus ProteinTM Standards (Bio-Rad, USA) protein as a standard protein. Purity and size of the lectin protein were analyzed.
그 결과， 도 2에 나타낸 바와 같이， 시알산을 포함한 당사슬이 부가된 당단백질인 페투인에 결합한 렉틴의 크기는 두 종류의 단백질 HEL1 및 HEL2로 측정되었으며 단백질의 크기는 약 15 20 kDa 및 50 75 kDa로 확인되었다 (도 2) . <2-4>헬리시움 에리나슘 자실체에서 HEL1 렉틴 분리 최적화  As a result, as shown in Fig. 2, the size of the lectin bound to fetuin, the glycoprotein to which oligosaccharide including sialic acid was added, was measured by two kinds of proteins, HEL1 and HEL2, and the size of the protein was about 15 20 kDa and 50 75 It was confirmed by kDa (FIG. 2). <2-4> Optimization of HEL1 Lectin Separation in Helicium erinanium Fruiting Body
상기 실시예 <2-1〉에 기재된 방법으로 제조한 추출물을 사용하여, 상기 실시예 <2-2>에서 확인된 HEL1과 HEL2 렉틴 단백질의 분리 최적화를 위하여 하기와 같은 방법을 수행하였다.  Using the extract prepared in the method described in Example <2-1>, the following method was performed to optimize the separation of HEL1 and HEL2 lectin protein identified in Example <2-2>.
구체적으로 상가 실시예 <2-1>에 기재된 방법으로 제조된 동결 분말상태 자실체를 중량 확인한 후， 측정 중량의 2.5배에 해당되는 50 mM Tr i s HCKpH 7.4) 완충용액을 첨가한 후， 4°C에서 12시간 이상 흔합하였다. 상기 흔합액에서 파쇄되지 않은 버섯 자실체의 입자와 비용해성 단백질을 제거하기 위해 4°C에서 20 ,000 X g의 속도로 30분 동안 침전물을 제거하고 상등액올 수득하였다. 미 제거된 입자를 제거하기 위해 4°C에서 25,000 X g의 속도로 45분 동안 침전물을 제거하고 상등액을 재수득하였다. 침전된 자실체에 다시 측정 중량의 1.5배에 해당되는 동일한 완충용액을 넣고 상기의 동일한 방법으로 상등액을 수득한 후， 각각의 수득한 상등액을 합쳐서 최종 세포추출물을 제작하였다. 여기에 포화농도 80%(w/v) 황산암모늄을 넣은 후， 4t에서 천천히 교반하면서 단백질을 침전시켰다. 침전된 단백질은 4°C에서 25, 000 X g의 속도로 60분 동안 원심분리를 통하여 , 침전물을 회수한 후， 1/10 부피의 50 mM Tri s HCl (pH 7.4)완충용액으로 용해시킨 후，동일 완층용액에 투석 또는 PD-10 desalt ing칼럼 (GE heal thcare)을 통과시켜 , 단백질 조효소 액 내에 황산암모늄을 제거하였다. Desalting 된 단백질 조효소 액을 DEAD_Sepharose 칼럼 (1.5 x 17 cm)에 주밉하였으며,조효소 액을 주입한 칼럼을 5배의 컬럼 부피의 50mMTrisHCl(pH 7.4) 완층용액으로 평형화 (equilibrium)시켰다. AKTA purification system(GE healthcare)을 이용하여 CK~0.5 M NaCl이 포함된 50 mM Tris HCKpH 7.4) 완충용액을 step gradient ¾는 linear gradient로 완층용액을 홀려주어 DEAD-Sepharose 칼럼으로부터 렉틴 단백질을 용리시켰다. 렉틴 단백질이 포함된 분획은 0.5%(v/v) 돼지혈액의 적혈구 웅집실험 (agglutination assay)을 통해 확인하였다. 돼지혈액의 적혈구 응집실험에서 양성반웅을 나타내는 분획을 회수한 후， 아미콘 울트라 센트리푸갈 필터 (Amicon Ultra Centrifugal filter 10K)을 이용하여 단백질 분획을 농축시켰다. Specifically, after weighing the frozen powdered fruiting body prepared by the method described in Example <2-1>, after adding 50 mM Tr is HCKpH 7.4) buffer solution corresponding to 2.5 times the measured weight, 4 ° C More than 12 hours. The precipitate was removed for 30 minutes at a rate of 20,000 × g at 4 ° C. to remove particles and insoluble proteins of the mushroom fruiting body which were not crushed in the mixture, and a supernatant was obtained. The precipitate was removed for 45 minutes at a rate of 25,000 X g at 4 ° C. to remove unremoved particles and the supernatant was reacquired. The same buffer solution corresponding to 1.5 times the measured weight was added to the precipitated fruiting body again to obtain a supernatant by the same method as described above, and the obtained supernatants were combined to prepare a final cell extract. Saturated concentration 80% (w / v) ammonium sulfate was added to this, the protein was precipitated while stirring slowly at 4t. The precipitated protein was centrifuged at 4 ° C. at a rate of 25, 000 X g for 60 minutes to recover the precipitate, and then dissolved in 1/10 volume of 50 mM Tri s HCl (pH 7.4) buffer solution. ， The same complete solution was passed through dialysis or PD-10 desalt ing column (GE heal thcare) to remove ammonium sulfate in the protein coenzyme solution. The desalted protein coenzyme solution was pressed onto a DEAD_Sepharose column (1.5 × 17 cm), and the column injected with the coenzyme solution was equilibrated with a 5-fold column volume of 50m MTrisHCl (pH 7.4) complete solution. Using the AKTA purification system (GE healthcare), 50 mM Tris HCKpH 7.4) buffer containing CK ~ 0.5 M NaCl was used to elute the lectin protein from the DEAD-Sepharose column. The fraction containing the lectin protein was confirmed by a red blood cell agglutination assay of 0.5% (v / v) porcine blood. After recovering the fraction showing positive reaction in the hemagglutination experiment of porcine blood, the protein fraction was concentrated using an Amicon Ultra Centrifugal filter (10K).
렉틴 활성을 포함한 단백질 분획은 상기 실시예 <2— 2〉에서 기재된 방법으로 50 mM Tris-HCl 완충용액 (pH 7.4)으로 평형화시킨 페투인-아가로즈 레진 (Fetuin—agarose resin, Sigma-Aldrich, USA) 칼럼 (1.5 x 17cm)에 DEAE-Sepharose 칼럼 분획을 Pump P-l 튜브연동식 펌프 (Per istat ic pump, GE-healthcare, USA)를 이용하여 주입속도를 분당 1 mL로 하여 주입하였다. 동일 완층용액 10 배 부피로 세척한 다음， 0.2~0.5 M D-갈락토즈 (D-galactose)가 포함되어 있는 동일 완충용액으로 시알산 당사슬을 포함하는 당단백질인 페투인에 결합한 단백질을 용출시켰다. 용출된 단백질은 아미콘 울트라 센트리푸갈 필터 (Amicon Ultra Centrifugal filter 10K)을 이용하여 농축시켰다.  Protein fractions containing lectin activity were fetuin-agarose resin, Sigma-Aldrich, USA equilibrated with 50 mM Tris-HCl buffer (pH 7.4) by the method described in Example 2-2 above. ) DEAE-Sepharose column fraction was injected into the column (1.5 x 17 cm) using a Pump Pl peristaltic pump (Per istat ic pump, GE-healthcare, USA) at an injection rate of 1 mL per minute. After washing with a volume of 10 times the same complete solution, the protein bound to fetuin, a glycoprotein containing sialic acid oligosaccharide, was eluted with the same buffer containing 0.2-0.5 M D-galactose. Eluted protein was concentrated using Amicon Ultra Centrifugal filter 10K.
렉틴 활성을 포함한 농축된 단백질 용액을 50 mM Tris-HCl 완충용액 (pH 7.4)으로 평형화시킨 Superose 12 10/300 GL(GE healthcare) 젤 여과 크로마토그래피 칼럼에 주입한 후, AKTA purification system올 이용하여 0.5 mL/min유속으로 단백질을 용출 시켰으며 , 렉틴 활성이 포함된 분획을 상기에서 언급한 돼지혈액의 적혈구 웅집실험을 이용하여 확인하였다. 최종적으로 돼지혈액 적혈구에 대해 양성반웅을 나타내는 분획을 회수한 후， 아미콘 울트라 센트리푸갈 필터 10K을 이용하여 렉틴 단백질 분획을 농축시켰다.  Concentrated protein solution containing lectin activity was injected into a Superose 12 10/300 GL (GE healthcare) gel filtration chromatography column equilibrated with 50 mM Tris-HCl buffer (pH 7.4), followed by 0.5 AKTA purification system. The protein was eluted at a flow rate of mL / min, and the fraction containing the lectin activity was confirmed using the erythroid test of porcine blood mentioned above. Finally, after recovering the fraction showing a positive response to porcine blood red blood cells, the lectin protein fraction was concentrated using Amicon ultra centrifugal filter 10K.
각 단계별에서 수득한 렉틴의 활성이 포함한 단백질 분획올 상기 실시예 <2-3>에서 기재된 방법으로 1 또는 15% SDS-PAGE를 이용하여 분리한 후， 코마시에 브릴리언트 블루로 염색하였으며 Preci sion Plus ProteinTM Standards 단백질을 표준 단백질로 사용하여 정제된 렉틴 단백질의 순도 및 크기를 분석하였다. Example of protein fraction containing the activity of the lectin obtained in each step Isolation was performed using 1 or 15% SDS-PAGE by the method described in <2-3>, stained with Brilliant Blue in Coomassie, and purity of lectin protein purified using Precision Plus ProteinTM Standards protein as standard protein. Size was analyzed.
그 결과， 도 3에 나타낸 바와 같이， 이온교환크로마토그래피 ( ion chromatography) DEAE-Sepharose 칼럼， 친환성 크로마토그래피 (af f ini ty chromatography) 페투인 칼럼, 그리고 크기 배제 크로마토그래피 (si ze exclusion chromatography) Superose 12 10/300 GL칼럼을 통해 HELl와 HEL2렉틴 단백질을 순수하게 분리하였으며， 각각의 단백질의 크기는 약 15~20 kDa 및 50~75 kDa로 확인되었다 (도 3) .  As a result, as shown in FIG. 3, ion chromatography DEAE-Sepharose column, af ini ty chromatography petouin column, and size zeolite exclusion chromatography Superose The HELl and HEL2 lectin proteins were purely separated through 12 10/300 GL columns, and the size of each protein was about 15-20 kDa and 50-75 kDa (FIG. 3).
<실시예 3> 헬리시움 에리나슘 (Hericium erinaceum) 자실체에서 분리된 렉틴 단백질의 N-말단서열 분석 Example 3 Analysis of N-terminal Sequences of Lectin Proteins Isolated from Helicium erinaceum Fruiting Bodies
헬라시움 에리나슴 버섯 자실체에서 분리된 시알산에 선택적 결합을 하는 렉틴 단백질의 아미노산 서열을 구명하기 위하여， 상기 실시예 <2-4>에 기재된 방법으로 분리된 렉틴 단백질의 N-말단 아미노산 서열을 하기와 같은 방법으로 분석하였다.  In order to determine the amino acid sequence of the lectin protein that selectively binds to sialic acid isolated from the fruiting body of the Hellasium erythima, the N-terminal amino acid sequence of the lectin protein isolated by the method described in Example <2-4> was used. The analysis was carried out as follows.
구체적으로， 상시 실시예 <2-4>에 기재된 방법으로 분리된 렉틴 단백질을 16% Tr icin-PAGE를 이용하여 단백질을 전개한 후, Trans-Blot SD Semi -Dry Transfer Cel KBio-Rad, USA)를 이용하여 15 V에서 1시간 동안 Tr icin-PAGE상의 단백질을 PVDF멤브레인으로 이동을 시켰다. Tr icin— PAGE에서 PVDF 멤브레인으로 옮겨진 단백질은 0.1% (w/v) 퐁슈 에스 (Ponceau S)로 염색하였으며， 상기 염색방법으로 확인 된 단백질을 멤브레인으로부터 잘라내어 한국질량분석기술 (서울， 대한민국)에서 단백질의 N—말단서열 분석을 수행하였다.  Specifically, after lectin protein isolated by the method described in Example <2-4> was developed using 16% Tr icin-PAGE, Trans-Blot SD Semi-Dry Transfer Cel KBio-Rad, USA) The protein on Tr icin-PAGE was transferred to PVDF membrane at 15 V for 1 hour. Tr icin—The protein transferred to PVDF membrane in PAGE was stained with 0.1% (w / v) Ponceau S. The protein identified by the staining method was cut out from the membrane, and the protein was analyzed by Korea Mass Spectrometry (Seoul, Korea). The N-terminal sequence analysis of was performed.
그 결과， 상기 실시예 <2-4>에서 확인된 약 15 20 kDa 크기의 HEL1 단백질 두 개의 서열 HELla와 HELlb로 분석되었으며， HELla의 강한 시그널로 서열번호 7(NH2-KEPTWGRPES-C02— )과 HELlb의 약한 시그널로 서열번호 8(NH2-WPVAPDYPPES-C02— )의 N-말단 단백질 서열을 가지는 것으로 확인되었다. 50 75 kDa 크기의 단백질은 서열번호As a result, two sequences of HEL1 protein of about 15 20 kDa size identified in Example <2-4> were analyzed with HELla and HELlb. As a strong signal of HELla, SEQ ID No. 7 (NH2-KEPTWGRPES-C0 2 —) and Sequence number as a weak signal for HELlb It was found to have an N-terminal protein sequence of 8 (NH2-WPVAPDYPPES-C0 2 —). 50 75 kDa protein has the sequence number
9(NH2-GGHSVPLTNF丽 AQYFTEISLGSPPQQFKV-aV )의 N-말단 단백질 서열을 가지는 것을 확인하였다. It was confirmed that it had the N-terminal protein sequence of 9 (NH2-GGHSVPLTNFlli AQYFTEISLGSPPQQFKV-aV).
<실시예 4> 질량분석기를 이용한분리 정제된 렉틴 단백질 크기 분석 Example 4 Analysis of Purified Lectin Protein Size by Mass Spectrometry
상기 실시예 <2-4〉에 기재된 방법으로 분리된 단백질의 정확한 분자량을 측정하기 위하여 질량분석기 Microf lex Maldi-TOF(Bruker Daltonik GmbH. Bremen Germany)를 수행하였다.  A mass spectrometer Microf lex Maldi-TOF (Bruker Daltonik GmbH. Bremen Germany) was performed to determine the exact molecular weight of the protein isolated by the method described in Example <2-4>.
구체적으로， 상기 실시예 <2-4>에 기재된 방법으로 분리 정제된 렉틴 단백질을 PD-10 컬럼 (GE-Healthcare, USA)을 이용하여 증류수로 용출시켜 단백질 용액 내에 있는 염을 제거한 후, 아미콘 울트라 센트리푸갈 필터 (Amicon®Ultra Centr i fugal f i lter ) 10K를 이용하여 1 mg/ml 이상의 농도로 농축시켰다. 그런 다음， 25% 아세토니트릴 (acetoni tr i le)과 0.1% 트리폴루오아세테트산 (tr i f luoroacet ic acid)을 1: 2 비율로 혼합한 용액에 시나피닉산 (sinapinic acid)을 포화농도로 용해시킨 후, 상기 용액을 농축된 단백질과 부피비로 1 : 1로 흔합하여 질량분석기 MALDI-T0F 타깃 위에서 단백질올 결정화시켰다. 상기 결정화된 단백질을 질량분석기 Microf lex Maldi-T0F(Bruker Daltonik GmbH. Bremen, Germany)을 이용하여 분리 정제된 렉틴의 크기를 분석하였다.  Specifically, the lectin protein separated and purified by the method described in Example <2-4> was eluted with distilled water using a PD-10 column (GE-Healthcare, USA) to remove salts in the protein solution, Concentrated to a concentration of at least 1 mg / ml using an Ultra Centrifugal filter (Amicon® Ultra Centr i fugal filter) 10K. Then, in a solution containing 25% acetonitrile and 0.1% tri ifo luoacetic acid in a ratio of 1: 2, sinapinic acid was added to a saturated concentration. After dissolution, the solution was mixed at a volume ratio of 1: 1 with the concentrated protein to crystallize the protein on the mass spectrometer MALDI-T0F target. The crystallized protein was analyzed by using a mass spectrometer Microf lex Maldi-T0F (Bruker Daltonik GmbH. Bremen, Germany) to analyze the size of the purified lectin.
그 결과， 상기 실시예 <2ᅳ4>에서 확인된 HELla와 HELlb의 N-말단 단백질 서열을 갖는 HEL1 렉틴 단백질은 5배 정도의 강한 시그널을 나타내는 HELla 렉틴의 분자량은 15327.587 Da으로 약한 시그널을 나타내는 HELlb 렉틴의 분자량은 15536.953 Da으로 분석되었다 (도 4) . HEL2렉틴의 분자량은 73253.12 Da으로 분석되었다.  As a result, the HEL1 lectin protein having the N-terminal protein sequences of HELla and HELlb identified in Example <2′4> had a strong signal of about 5 times stronger, and the molecular weight of the HELla lectin was 15327.587 Da. The molecular weight of the lectins was analyzed to be 15536.953 Da (FIG. 4). The molecular weight of the HEL2 lectin was analyzed to be 73253.12 Da.
<실시예 5> Non-denaturing condition에서의 HELl짹틴 단백질 크기 및 pi 확인 상기 실시예 <2-4>에 기재된 방법으로 분리한 HEL1 렉틴의 non-denaturing condition에서의 HELl 렉틴 단백질 크기를 확인하기 위하여， AKTA purification system에서 Superose 12 10/300 GL 칼럼을 이용하여 HEL1 렉틴의 크기를 확인하였다. 분자 질량 교정 키트 (Molecular weight calibration kit) (GE healthcare)의 표준 단백질로 440 kDa페르니틴 (Ferrtin), 158 kDa 알도즈 (Aldose), 44 kDa 오발부민 (Ovalbumin),. 29 kDa 카르보닐 언하드라아제 (Carbonic anhydrase), 13.7 kDa리보뉴클레이즈 에이 (Ribonuc lease A), 6.5 kDa 아프로티닌 (Aprotinin)을 사용하였다. Superose 12 10/300 GL 칼럼에서 용출되는 HEL1 렉틴 단백질을 포함하는 분획에 존재하는 렉틴의 활성을 돼지혈액 적혈구의 웅집활성으로 확인하였으며 크로마토그램올 표준단백질과 비교하여 상대적인 크기를 확인하였다. <Example 5> HELl tweetin protein size and pi identification in non-denaturing conditions of HEL1 lectin isolated by the method described in Example <2-4> To determine the size of HELl lectin protein in non-denaturing conditions, the size of HEL1 lectin was determined using a Superose 12 10/300 GL column in an AKTA purification system. Molecular weight calibration kit (GE healthcare) is the standard protein of 440 kDa Fernitin, 158 kDa Aldose, 44 kDa Ovalbumin ,. 29 kDa Carbonyl anhydrase, 13.7 kDa Ribonuc lease A, 6.5 kDa Aprotinin were used. The lectin activity in the fraction containing the HEL1 lectin protein eluted from the Superose 12 10/300 GL column was confirmed by the coagulation activity of porcine blood erythrocytes and compared with chromatogram standard protein.
그 결과， 도 5A에 나타낸 바와 같이， non-denaturing condition에서 HELl 렉틴은 약 32 ± 5 kDa의 dimer 형태와 단백질로 확인되었다 (도 5A). 또한， 상기 실시예 <2-4>에 기재된 방법^로 분리한 HEL1 렉틴 단백질의 pi는 등전점 (Iso-ElectroFocusing, IEF) 전기영동 (electrophoresis)을 통하여 확인하였다. Pi 3~7 IEF-gel system (K0MABI0TECH)을 이용하여 음극과 양극 완충용액에서 100 V 1시간， 200 V 1시간， 500 V 30분 간의 IEF-PAG 젤 전개를 통하여 표준단백질의 pi와 비교한 결과， 도 5B에 나타낸 바와 같이， 최종적으로 HEL1 렉틴은 약 pi 4.5 단백질로 확인되었다 (도 5B).  As a result, as shown in Figure 5A, HELl lectin was identified as a dimer form and protein of about 32 ± 5 kDa in non-denaturing conditions (Figure 5A). In addition, pi of the HEL1 lectin protein isolated by the method ^ described in Example <2-4> was confirmed through isoelectric point (Iso-ElectroFocusing, IEF) electrophoresis. Comparing with pi of standard protein through IEF-PAG gel development for 100 V 1 hour, 200 V 1 hour, 500 V 30 minutes in cathode and anode buffer using Pi 3 ~ 7 IEF-gel system (K0MABI0TECH) As shown in FIG. 5B, the HEL1 lectin was finally identified as about pi 4.5 protein (FIG. 5B).
<실시예 6> HEL1 렉틴을 이용한 적혈구의 웅집성 확인 Example 6 Confirmation of Magnitude of Erythrocytes Using HEL1 Lectin
상기 실시예 <2-4〉에 기재된 방법으로 분리한 HEL1렉틴의 혈액 적혈구에 대한 웅집성 활성을 확인하기 위하여， 돼지혈액 (porcine blood), 토끼혈액 (rabbit blood), 인간혈액 (human blood B-type)올 10% (v/v)로 PBS (phosphate buffer saline) 완충용액에 희석한 후， 4°C에서 1000 rpm의 속도로 5분 동안 원심분리하여 적혈구 (red blood cells, erythrocytes)를 침전 시켰으며, 상등액을 제거하고 상기의 방법을 세 번 반복하여, 최종적으로 PBS 완충 용액에 희석하여 2%(v/v) 적혈구 세포 용액을 제작하였다. 웅집실험 (agglut inat ion assay)은 V-형태의 96 wel l plate를 사용하였으며， 실시예 <2ᅳ4>에서 분리한 O. l mg/mL농도의 렉틴 단백질 100 uL을 연속적인 1/2 희석을 하여 동일 부피의 2% 적혈구 세포와 흔합을 한 후， 상온에서 96 wel l plate을 1 시간 정치하여 돼지 혈액 적혈구, 토끼 혈액 적혈구, 인간 혈액 적혈구에 대한 렉틴의 웅집 반웅을 관찰하였다. 이때 양성 대조군으로 시알산 당사슬에 선택적 결합을 하는 SNA-I 렉틴을 동일 농도로 사용하였으며， 음성 대조군으로는 렉틴이 포함되지 않는 PBS 완층용액을 사용하였다. In order to confirm the coarse activity of blood erythrocytes of HEL1 lectin isolated by the method described in Example <2-4>, porcine blood, rabbit blood, human blood (human blood B- After dilution with PBS (phosphate buffer saline) buffer solution at 10% (v / v), centrifuged at 1000 rpm at 4 ° C for 5 minutes to precipitate red blood cells (erythrocytes). The supernatant was removed and the method was repeated three times. Finally, the supernatant was diluted in PBS buffer solution to prepare a 2% (v / v) erythrocyte cell solution. The agglut inat ion assay used a 96-wel l plate in the V-type, and serial dilutions of 100 uL of the lmg / mL concentration of lectin protein isolated in Example <2> were performed. After mixing with 2% erythrocytes in the same volume, 96 wel l plate was allowed to stand at room temperature for 1 hour to observe the reaction of lectins on porcine blood erythrocytes, rabbit blood erythrocytes, and human blood erythrocytes. At this time, the same concentration of SNA-I lectin, which selectively binds to the sialic acid oligosaccharide, was used as a positive control, and a complete PBS solution containing no lectin was used as a negative control.
그 결과， 도 6에 나타낸 바와 같이， HEL1 렉틴은 양성대조군으로 사용한 SNA-I 렉틴과 동일하게 테스트한 모든 혈액의 적혈구 세포에 대해 웅집성을 나타냈으며， 특히 토끼혈액과 인간혈액에 비해 돼지혈액에 대해 강한 웅집 활성이 확인되었다 (도 6) .  As a result, as shown in Figure 6, the HEL1 lectin showed a homogeneity to the red blood cells of all blood tested in the same manner as the SNA-I lectin used as a positive control group, especially in porcine blood compared to rabbit and human blood Strong fungal activity was observed for (Fig. 6).
<실시예 7>렉틴 블랏을 이용한시알산당사슬포함당단백질의 측정 <Example 7> Determination of sialic acid sugar chain-containing glycoprotein using lectin blot
상기 실시예 <2-4>에 기재된 방법으로 분리한 렉틴의 결합능을 확인하기 위하여 시알산 당사슬을 포함하는 페투인 ( fetuin)과 시알산이 제거된 비시알산화페투인 (asialoietuin)을 기질로 이용하여， 렉틴 블랏을 수행하였다. 구체적으로， 페투인과 비실알산 페투인이 포함된 각각의 단백질 용액을 5 X 램리 버퍼 (Lae瞧 l i buffer)와 흔합하여 10분 동안 가열한 후， 8¾ SDS-PAGE 겔로 전기영동하여 단백질을 분리하였다. 상기 분리된 SDS-PAGE 겔 상의 단백질을 Trans-Blot SD Semi -Dry Transfer Cel KBio-Rad , USA)를 이용하여 15 V에서 1시간 동안 니트로셀를로스 (ni trocel lulose) 멤브레인에 트랜스퍼 (transfer) 하고, 이를 3%소혈청알부민 (bovine serum albumin; BSA)이 포함된 포스페이트 식염수 완충액 (phosphate sal ine buffer) , PBST 및 0.5% 트원에 첨가하여 1시간 동안 블로킹 (blocking) 하였다. 그런 다음， 비오틴 (biot in)이 컨쥬게이션된 상기 렉틴 1 mg/mL이 포함되어 있는 3% BSA-PBS에 막을 넣어 상온에서 4 시간 동안 배양하였다. 이때 사용한 상기 실시예 <2-3>에 기재된 방법으로 분리한 렉틴의 바이오틴화 (biot inylat ion) 컨쥬게이션은 항 바이오틴화 키트 (Ant ibody Biot inylat ion Kit , Genomine 대한민국)올 사용하여 수행하였다. 렉틴과 멤브레인을 인큐베이션 한 후， 멤브레인을 PBST로 10 분 동안 6회 세척하였고, 0.2 mg/mL 호스라디시 페록시다제 (horseradish peroxidase, HRP)- 컨쥬게이션된 항-비오틴 항체 (conjugated anti-biotin antibody)(l:500, Sigma— Aldr ich)과 한 시간 동안 반웅시켰다. 반웅 후, 상기와 같은 방법으로 막을 PBST로 10 분 동안 6회 세척하여 , ECL 키트 (GE Healthcare, USA)로 시알화된 단백질을 확인하였다. 그 결과， 도 7에 나타낸 바와 같이 N-결합 당사슬의 기본 구조인 Neu5Ac α(2,3) Gali3(l,4)GlcNAc와 0-결합 당사슬 Neu5Ac a (2,3)Gal 13 (1,3) [GalNA, Neu5Aca(2,3)GaU3(l,4)Gaip(l,6)]의 당사슬을 포함하는 페투인에서는 렉틴 블랏의 사그널을 관찰하였으나， 음성 대조군인 비시알산화페토인의 Ga 3(l,4)GlcNAc의 비시알산화 N-당사슬과 GaU3(l,3), GlcNA, Gal β( 1,3) [GalNA β(1,6)]의 당사슬을 포함하는 단백질에서는 렉틴 블랏의 시그널이 확인되지 않았다 (도 7). <실시예 8> 형광표지된 렉틴을 이용하여 시알산 당사슬을 포함하는 동물 세포 표면 관찰확인 In order to confirm the binding ability of the lectins separated by the method described in Example <2-4>, fetuin containing sialic acid oligosaccharide and bisial oxidized fetuin (asialoietuin) from which sialic acid was removed were used as a substrate. , Lectin blot was performed. Specifically, each protein solution containing fetuin and non-silicylic acid fetuin was mixed with 5 X Lae li buffer, heated for 10 minutes, and then subjected to electrophoresis on an 8¾ SDS-PAGE gel to separate proteins. . Transfer the protein on the isolated SDS-PAGE gel to a nitrocel lulose membrane at 15 V using Trans-Blot SD Semi-Dry Transfer Cel KBio-Rad, USA for 1 hour, It was added to phosphate saline buffer containing 3% bovine serum albumin (BSA), PBST and 0.5% TWN and blocked for 1 hour. Then, the membrane was placed in 3% BSA-PBS containing 1 mg / mL of the biotin conjugated biotin incubated at room temperature for 4 hours. The biotin inylat ion conjugation of the lectins isolated by the method described in Example <2-3> used at this time was carried out using an anti-body biotylation kit (Genomine). Korea). After incubating the membrane with lectin, the membrane was washed 6 times with PBST for 10 minutes, and 0.2 mg / mL horseradish peroxidase (HRP) -conjugated anti-biotin antibody (l: 500, Sigma—Aldr ich) for 1 hour. After reaction, the membrane was washed six times with PBST for 10 minutes in the same manner as above to identify sialized proteins with ECL kit (GE Healthcare, USA). As a result, as shown in FIG. 7, Neu5Ac α (2,3) Gali3 (l, 4) GlcNAc, which is the basic structure of the N-linked sugar chain, and the 0-linked sugar chain Neu5Ac a (2,3) Gal 13 (1,3) In fetuin containing the sugar chain of [GalNA, Neu5Aca (2,3) GaU3 (l, 4) Gaip (l, 6)], the signal of lectin blot was observed, but Ga 3 of bisial oxidized fetoin, a negative control, was observed. Signals of lectin blot in proteins containing bisial oxidized N-sugar chains of (l, 4) GlcNAc and oligosaccharides of GaU3 (l, 3), GlcNA, Gal β (1,3) [GalNA β (1,6)] This was not confirmed (Figure 7). <Example 8> Observation and observation of the surface of the animal cell containing sialic acid oligosaccharide using fluorescently labeled lectin
동물 세포 표면에 존재하는 시알산 당사슬을 포함하는 Α549 세포주 (ATCC CCL-185TM, Rockville MD, USA)에 형광표지된 렉틴을 이용하여 세포 표면을 관찰하였다.  Cell surface was observed using lectin fluorescently labeled on A549 cell line (ATCC CCL-185TM, Rockville MD, USA) containing sialic acid oligosaccharides present on animal cell surfaces.
구체적으로， 상기 A549 세포주는 무혈청배지에서 배양한 후, 세포를 원심분리하여 수득하였다. 상기 실시예 <2-4>에 기재된 방법으로 분리된 렉틴은 당업계의 알려진 방법으로 Alexa Fluor ®488 Protein Labeling Kitdnvitrogen, USA)을 이용하여 형광 표지를 하였다. 상기 실시예 <2-4>에 기재된 방법으로 분리 정제한 렉틴이 상기 세포 표면에 존재하는 시알산 당사슬에 대한 결합능을 확인하기 위하여 1 mg/mL 소혈청알부민 (bovine serum albumin, BSA)이 포함된 PBS 완충용액 (phosphate saline buffer)을 사용하여 상기 세포주를 세척한 후 원심분리를 하여 상기 세포를 다사 수득하였다. 수득된 상기 세포를 소혈청알부민이 포함된 PBS(PBSB) 완충용액 소량에 현탁을 한 후， Alexa Fluor ®488이 컨쥬게이션된 상기의 렉틴을 최종 농도가 0.5 nM 내지 1.0 nM가 되도록 첨가한 후， 렉틴과 반웅하는 세포주를 얼음상에서 1시간 동안 배양하였다. 배양 1시간 후, 상기 세포주를 원심분리하여 다시 세포주를 수득한 후， PBSB 완충용액을 이용하여 해당 세포주를 3회 이상 세척하고， 최종적으로 1내지 5 x l06세포 /mL농도가 되도록 동일 완충용액에 현탁 하였다. 현탁된 세포 내의 형광 라벨링 된 렉틴과 결합하는 세포 표면 시알산 당사슬을 Zei ss LSM510 공초점현口]경 (Confocal Laser Scanning Mi croscope , Car 1 Zei ss Germany)을 이용하여 확인하였다. Specifically, the A549 cell line was obtained by culturing in serum-free medium and then centrifuging the cells. The lectins isolated by the method described in Example <2-4> were fluorescently labeled using Alexa Fluor® 488 Protein Labeling Kitdnvitrogen, USA) as known in the art. In order to confirm the binding ability of the lectin separated and purified by the method described in Example <2-4> to sialic acid oligosaccharide present on the cell surface, 1 mg / mL bovine serum albumin (BSA) was included. The cell lines were washed with PBS buffer (phosphate saline buffer) and centrifuged to obtain the cells. The obtained cells were suspended in a small amount of PBS (PBSB) buffer containing bovine serum albumin. After the addition of the lectin conjugated with Alexa Fluor® 488 to a final concentration of 0.5 nM to 1.0 nM, the lectin and the reaction cell line were incubated for 1 hour on ice. After 1 hour of incubation, the cell line was centrifuged to obtain a cell line again, and then the cell line was washed three times or more times using PBSB buffer solution, and the same buffer solution was finally made to have a concentration of 1 to 5 x 10 6 cells / mL. Suspended in Cell surface sialic acid oligosaccharides that bind fluorescently labeled lectins in suspended cells were identified using a Zei ss LSM510 confocal microscope (Confocal Laser Scanning Mi croscope, Car 1 Zei ss Germany).
그 결과， 도 8에 나타낸 바와 같이 동물 세포 표면에 존재하는 시알산 당사슬을 포함하는 A549 세포주에 형광 라벨된 렉틴을 사용한 결과， 상기 세포주 표면에서 형광 라벨된 렉틴을 확인함으로써， 시알산 당사슬을 확인하였다 (도 8) .  As a result, as shown in Figure 8 using a fluorescent labeled lectin in the A549 cell line containing sialic acid oligosaccharide present on the surface of the animal cell, the sialic acid oligosaccharide was confirmed by confirming the fluorescent labeled lectin on the cell line surface (FIG. 8).
【수탁번호】 [Accession number]
기탁기관명 ： 한국생명공학연구원  Depositary Name ： Korea Research Institute of Bioscience and Biotechnology
수탁번호 ： KCTC12499BP  Accession number ： KCTC12499BP
수탁일자 ： 20131004 Trusted date ： 20131004
BUDAPEST TREATY ON THE IKTERNATiONAl. RECOGNiTION OF THE DEPOSIT BUDAPEST TREATY ON THE IKTERNATiONAl. RECOGNiTION OF THE DEPOSIT
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INTERNATIONAL FORMINTERNATIONAL FORM
RECEIPT EN THE CASE OF AN ORIGINAL DEPOSIT RECEIPT EN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 issued pursuant to Rule 7.1
O： KHVl Seong hun O ： KHVl Seong hun
Korea Research Institute of Bioscience and Biotechnology  Korea Research Institute of Bioscience and Biotechnology
125 Gwahak-ro, Yuseong-gu, Daejeon 305-806  125 Gwahak-ro, Yuseong-gu, Daejeon 305-806
Republic of Korea  Republic of Korea
I . IDENTIFICATION OF THE MICROORGANISM I. IDENTIFICATION OF THE MICROORGANISM
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This International Depositary Authority accepts the microorganism identified under I above, which was received by it on October 04, 2013.  This International Depositary Authority accepts the microorganism identified under I above, which was received by it on October 04, 2013.
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The microorganism identified under I above was received by ti is International Depositary Authority on and a request to convert the original deposi to a deposit under the Budapest Treaty was received by it on  The microorganism identified under I above was received by ti is International Depositary Authority on and a request to convert the original deposi to a deposit under the Budapest Treaty was received by it on
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125 Gwahak-ro, Yuseong-gu,  125 Gwahak-ro, Yuseong-gu,
Daejeon 305—806 Director  Daejeon 305—806 Director
Republic of Korea
Figure imgf000027_0001
2013 Republic of Korea
Figure imgf000027_0001
2013
Form BP/4 (KCTC Fonn 17) so!e page 번 역 문 특허질 ^상비생클기탁의 국쳬격 ^인에 한 부다페스트 조악 국제 서식 Form BP / 4 (KCTC Fonn 17) so! E page Budapest Zodiac International Form in the National Territory
규칙 7.1에 의한  According to rule 7.1
원기탁에 대한수탁증  Receipt of Won Deposit
TO； 김성 ΐ TO; Kim Sung ΐ
대한 ¾국* 대전시 유성구 과학 ¾ 125한국생 공학: ¾구원 305-806 Korea ¾ Country * Yuseong-gu, Daejeon Science ¾ 125 Korean Biotechnology: ¾ Salvation 305-806
Figure imgf000028_0001
Figure imgf000028_0001
BP/4형식 (KCTC 제 17형식 )  BP / 4 type (KCTC type 17)

Claims

【청구의 범위]  [Claim]
【청구항 1】  [Claim 1]
시알산 당사슬에 특이적으로 결합하는 렉틴 단백질을 생산하는， 수탁번호Accession No., which produces a lectin protein that specifically binds to sialic acid oligosaccharides
KCTC12499BP로 기탁된 헬리시움.에리나슘 (Her icium er inaceum) NEU-1L 균주. Helicium. Hernia er inaceum NEU-1L strain deposited with KCTC12499BP.
【청구항 2】 [Claim 2]
제 1항에 있어서, 상기 균주는 서열번호 1로 기재되는 26S rDNA 염기서열 및 서열번호 4로 기재되는 ITS1-5.8S-ITS4 rDNA 염기서열을 가지는 것을 특징으로 하는 헬리시움 에리나슘 NEU-1L 균주. [Claim 2] The Helicium erythium NEU-1L strain according to claim 1, wherein the strain has 26S rDNA nucleotide sequence shown in SEQ ID NO: 1 and ITS1-5.8S-ITS4 rDNA nucleotide sequence shown in SEQ ID NO: 4. .
【청구항 3】 [Claim 3]
제 1항에 있어서， 상기 시알산 당사슬에 특이적으로 결합하는 렉틴 단백질은 시알산이 부가되어 있는 페투인 당단백질쎄서 N-결합 당사슬 구조 또는 0-결합 당사슬 구조에 결합하는 것을 특징으로 하는 헬리시움 에리나슘 NEU-1L 균주. According to claim 1, wherein the lectin protein that specifically binds to the sialic acid oligosaccharide is a helicium characterized in that it binds to the N-linked oligosaccharide structure or 0-linked oligosaccharide structure to which the sialic acid is added fetuin glycoprotein Erinanium NEU-1L strain.
【청구항 4】 [Claim 4]
(a) 수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Her icium erinaceum) NEU-1L의 자실체로부터 세포 추출물을 수득하는 단계; 및  (a) obtaining a cell extract from a fruiting body of Hericium erinaceum NEU-1L deposited with accession no. KCTC12499BP; And
(b) 상기 세포 추출물에서 시알산화 당사슬을 포함하는 당단백질， 당펩타이드， 당지질， 을리고당 또는 단당이 컨쥬게이션되어 레진이 충진되어 있는 컬럼을 이용하여 상기의 렉틴을 분리하는 단계를 포함하는， 헬리시움 에리나슘 NEU-1L 균주로부터 시알산 당사슬이 특이적으로 결합하는 렉틴의 제조 방법.  (b) separating the lectin from the cell extract by using a column in which a glycoprotein, glycopeptide, glycolipid, loligosaccharide or monosaccharide, including sialic oligosaccharide, is conjugated and filled with a resin; A method for producing lectin, in which sialic acid oligosaccharides specifically bind from Helicium erinasium NEU-1L strain.
【청구항 5】 [Claim 5]
제 4항에 있어서， 상기 단계 b)에서 분리된 렉틴을 당사슬이 컨쥬게이션 되어 있는 레진으로부터 당사슬과 렉틴의 결합을 경쟁적으로 방해하여 해당 렉틴을 선택적으로 용리해 내는 단계를 더 포함하는 것을 특징으로 하는, 헬리시움 에리나슘 NEU-1L 균주로부터 시알산 당사슬이 특이적으로 결합하는 렉틴의 제조 방법 The method of claim 4, further comprising the step of selectively eluting the lectin by competitively inhibiting the binding of the sugar chain and the lectin from the resin to which the sugar chain is conjugated to the lectin isolated in step b). Of Lectin with Sialic Acid Sugar Chain Specific Binding from Helicium Erinadium NEU-1L Strain Manufacturing method
【청구항 6】 [Claim 6]
제 4항 또는 제 5항의 방법으로 제조된， 시알산 당사슬에 특이적으로 결합하는 렉틴. Lectin that is specifically bound to sialic acid oligosaccharides prepared by the method of claim 4 or 5.
[청구항 Π Claim Port Π
수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium erinaceum) NEU-1L 균주에서 생산되고， 서열번호 7 또는 서열번호 8로 기재되는 단백질 N-말단 서열을 가지며， 15 kDa 내지 20 kDa의 분자량을 가지는 것을 특징으로 하는， 시알산 당사슬이 특이적으로 결합하는 렉틴. Produced in the Helicium erinaceum NEU-1L strain deposited with accession no. Lectin, characterized in that the sialic acid oligosaccharide specifically binds.
【청구항 8】 [Claim 8]
수탁번호 KCTC12499BP로 기탁된 헬리시움 에리나슘 (Hericium erinaceum) NEU-1L 균주에서 생산되고， 서열번호 9로 기재되는 단백질 N-말단 서열올 가지며， 50 kDa 내지 75 kDa의 분자량을 가지는 것을 특징으로 하는， 시알산 당사슬이 특이적으로 결합하는 렉틴 It is produced in the Helicium erinaceum NEU-1L strain deposited with accession no. Lectin to which sialic acid oligosaccharides specifically bind
【청구항 9】 [Claim 9]
제 7항 또는 제 8항에 있어서， 상기 렉틴은 다음으로 구성된 군으로부터 선택되는 당사슬 구조에 결합하는 것을 특징으로 하는 렉틴; The lectin according to claim 7 or 8, wherein the lectin binds to an oligosaccharide structure selected from the group consisting of: lectins;
NeuAc 또는 NeuGc(a2→3)가 Gal 잔기 또는 Glc 잔기에 부가된 시알산화 당사슬;  Sialic oligosaccharide wherein NeuAc or NeuGc (a2 → 3) is added to the Gal residue or Glc residue;
NeuAc 또는 NeuGc(a2→6)가 Gal 잔기 또는 Glc 잔기에 부가된 시알산화 당사슬;  Sialic oligosaccharide wherein NeuAc or NeuGc (a2 → 6) is added to the Gal residue or Glc residue;
NeuAc 또는 NeuGc( α2→3)가 Gal(pi→4)Glc 잔기 또는 Glc( β l→4)Glc 잔기에 부가된 시알산화 당사슬; ' Sialic oxidized chain wherein NeuAc or NeuGc (α2 → 3) is added to the Gal (pi → 4) Glc residue or the Glc (β 1 → 4) Glc residue; '
NeuAc 또는 NeuGc(a2→6)가 Gal(pl→4)Glc 잔기 또는 Glc( β l→4)Glc 잔기에 부가된 시알산화 당사슬 ; NeuAc or NeuGc (a2 → 6) is present in the Gal (pl → 4) Glc residue or in the Glc (β l → 4) Glc residue. Added sialic oxidized sugar chain;
NeuAc 또는 NeuGc( α2→3)가 Gal( |3 l→4)GlcNac 잔기 또는 Gal( β l→3)GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized sugar chain wherein NeuAc or NeuGc (α2 → 3) is added to the Gal (| 3 l → 4) GlcNac residue or the Gal (β l → 3) GlcNac residue;
NeuAc 또는 NeuGc( a2→6)가 Gal( β l→4)GlcNac 잔기 또는 Gal( β l→3)GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized chain wherein NeuAc or NeuGc (a2 → 6) is added to the Gal (β 1 → 4) GlcNac residue or the Gal (β 1 → 3) GlcNac residue;
NeuAc 또는 NeuGc( α2→3)가 Gal( β 1→3)[ aFuc(l→4)]GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized chain wherein NeuAc or NeuGc (α2 → 3) is added to Gal (β 1 → 3) [aFuc (l → 4)] GlcNac residue;
NeuAc 또는 NeuGc( a2→6)가 Gal(pi→3)[ aFuc(l→4)]GlcNac 잔기에 부가된 시알산화 당사슬 ;  Sialic oxidized chain wherein NeuAc or NeuGc (a2 → 6) is added to Gal (pi → 3) [aFuc (l → 4)] GlcNac residue;
NeuAc 또는 NeuGc(a2→3)가 GaK β 1→4)[ aFuc(l→3)]GlcNac 잔기에 부가돤 시알산화 당사슬; Sialic oxidized sugar chain, wherein NeuAc or NeuGc (a2 → 3) is added to GaK β 1 → 4) [aFuc (l → 3)] GlcNac residue;
NeuAc 또는 NeuGc( a2→6)가 GaK β 1→4)[ aFuc(l→3)]GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oxidized sugar chain wherein NeuAc or NeuGc (a2 → 6) is added to GaK β 1 → 4) [aFuc (l → 3)] GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc 잔기에 부가된 시알산화 당사슬;  Sialic oxidized sugar chain in which NeuAc or NeuGc (a2 → 8) is added to the NeuAc residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc(a2→3) GaK β l→4)GlcNac 잔기에 부가된 시알산화 당사슬 ; Sialylated oligosaccharide wherein NeuAc or NeuGc (a2 → 8) is added to NeuAc (a2 → 3) GaK β 1 → 4) GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc( a 2→8)NeuAc( a 2→3) GaK l→4)GlcNac 잔기에 부가된 시알산화 당사슬;  Sialic oligosaccharide wherein NeuAc or NeuGc (a2 → 8) is added to NeuAc (a 2 → 8) NeuAc (a 2 → 3) GaK 1 → 4) GlcNac residue;
NeuAc 또는 NeuGc(a2→8)가 NeuAc(a2→8) [NeuAc( a2→8)]nNeuAc( a2→3) Gal(131→4)GlcNac 잔기에 부가된 시알산화 당사슬; 및  Sialic oxidized chain wherein NeuAc or NeuGc (a2 → 8) is added to NeuAc (a2 → 8) [NeuAc (a2 → 8)] nNeuAc (a2 → 3) Gal (131 → 4) GlcNac residue; And
NeuAc 또는 NeuGc(a2→8)가 [NeuAc ]n 잔기에 부가된 시알산화 당사슬.  Sialylated sugar chain in which NeuAc or NeuGc (a2 → 8) is added to the [NeuAc] n residue.
【청구항 10] [Claim 10]
제 7항 또는 제 8항의 렉틴을 포함하는， 시알산 당사슬을 포함하는 당단백질, 당펩타이드， 당지질， 당전구체 또는 을리고당의 측정 또는 정량용 키트. A kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors, or oligosaccharides, including sialic acid oligosaccharides, comprising the lectins of claim 7 or 8.
【청구항 11】 [Claim 11]
제 7항 또는 제 8항의 렉틴을 포함하는， 시알산 당사슬을 표면에 갖는 세포주， 박테리아 또는 바이러스의 모니터링， 측정 또는 정량용 키트. Cell line having a sialic acid oligosaccharide on the surface, comprising the lectin of claim 7 or 8, Kit for monitoring, measuring or quantifying bacteria or viruses.
【청구항 12】 [Claim 12]
a) 제 7항 또는 제 8항의 렉틴에 피검 시료를 접촉하는 단계 ; 및  a) contacting the test sample with the lectin of claim 7; And
b) 상기 렉틴에 결합된 당단백질， 당펩타이드, 당지질， 당전구체 또는 을리고당을 분석하는 단계를 포함하는， 시알산 당사슬을 포함하는 당단백질, 당펩타이드， 당지질， 당전구체 또는 올리고당으로 구성된 시알산화 '당사슬 (conjugated)와측정 또는 정량 방법 . 【청구항 13】 b) a sial composed of a glycoprotein, a glycopeptide, a glycolipid, a sugar precursor, or an oligosaccharide comprising a sialic acid oligosaccharide, comprising analyzing the glycoprotein, glycopeptide, glycolipid, glycoprecursor or oligosaccharide bound to the lectin Oxidized ' conjugated and measured or quantified method. [Claim 13]
a) 제 7항 또는 제 8항의 롁틴에 피검 시료를 접촉하는 단계; 및  a) contacting the test sample with the quentin of claim 7 or 8; And
b) 상기 렉틴에 결합된 세포， 박테리아 또는 바이러스를 분석하는 단계를 포함하는， 시알산 당사슬을 갖는 세포주， 박테리아 또는 바이러스의 측정 또는 정량 방법 .  b) measuring or quantifying a cell line, bacterium or virus with sialic acid oligosaccharide, comprising analyzing the cells, bacteria or virus bound to the lectin.
[청구항 14】 [Claim 14]
제 7항 또는 제 8항의 렉틴을 포함하는 시알산 당사슬을 포함하는 당단백질， 당펩타이드， 당지질， 당전구체 또는 올리고당의 측정 또는 정량용 키트의 용도. 【청구항 15】  Use of a kit for measuring or quantifying glycoproteins, glycopeptides, glycolipids, sugar precursors or oligosaccharides comprising sialic acid oligosaccharides comprising the lectins of claim 7 or 8. [Claim 15]
제 7항 또는 제 8항의 렉틴을 포함하는 시알산 당사슬을 표면에 갖는 세포주， 박테리아 또는 바이러스의 모니터링, 측정 또는 정량용 키트의 용도.  Use of a kit for monitoring, measuring or quantifying a cell line, bacteria or virus having a sialic acid oligosaccharide comprising the lectin of claim 7 or 8 on its surface.
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