JP6245688B2 - IgY-specific binding peptide and method for purifying IgY thereby - Google Patents

Description

The present invention relates to IgY-specific binding peptides.
The present invention also provides a method for purifying IgY with the peptide.

  Currently, mouse monoclonal antibodies and rabbit polyclonal antibodies are widely used as antibodies for clinical diagnosis and research and have a large market. On the other hand, chicken IgY antibody can be purified non-invasively from egg yolk in addition to the establishment of a specific antibody production method by immunization, low cost as a test animal, low cross-reactivity with animal antibodies. Is attracting attention as a merit and is expected as a new antibody as a reagent.

  Several methods have been reported as methods for purifying IgY from egg yolk (Non-patent Document 1). Egg yolk contains a lot of insoluble phospholipids and lipoproteins, and these become obstacles to affinity purification, so a protocol to remove this by precipitation using PEG, Pectin, etc. has been reported ( Non-patent document 2). Commercial IgY purification kits use the same principle, and are currently the main ones, and Eggcellent (Thermo Pierce) and EGGstract (Promega) are often used. Recently, Sock Hwee Tan et al. Reported a purification protocol that uses plant gums pectin and κ-carrageenan and is not inferior in purity or yield to commercially available ones (Non-patent Document 3). However, even in such a method, the purity of purified IgY is about 50 to 80%, and the use of an affinity column is indispensable for the preparation of IgY having a higher degree of purification.

  However, for example, specific ligands for purifying IgY, such as protein A in human IgG, have not been reported, and synthetic ligands for purification have been reported (Non-patent Document 4, or GE Healthcare's 2-mercaptopyridine immobilization column (HiTrap IgY Purification HP), from the viewpoint of the purity of the purified IgY antibody, it must be said that it is insufficient (about 70%).

The present inventors have so far used a random peptide library constructed by the T7 phage display system to isolate a peptide that specifically binds to human IgG or human IgA and use an affinity column on which the peptide is immobilized. Antibody purification methods have been reported (Patent Documents 1 and 2).
However, there have been no sufficient reports on IgY-binding peptides.

International Publication WO2008 / 054030 International publication WO2011 / 148952

Hatta, H., Kim, M., and Yamamoto, T. (1990) A novel isolation method for hen egg yolk antibody, "IgY". Agricultural and Biological Chemistry 54, 2531-2535 Ko, K. Y., and Ahn, D. U. (2007) Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. Poultry Science 86, 400-407 Tan, S. H., Mohamedali, A., Kapur, A., Lukjanenko, L., and Baker, M. S. (2012) A novel, cost-effective and efficient chicken egg IgY purification procedure.Journal of Immunological Methods 380, 73-76 Fassina, G., Verdoliva, A., Palombo, G., Ruvo, M., and Cassani, G. (1998) Immunoglobulin specificity of TG19318: a novel synthetic lIgYnd for antibody affinity purification.Journal of molecular recognition: JMR 11, 128-133

The object of the present invention is to find a peptide that specifically binds to IgY from a clone obtained by biopanning against IgY using a random peptide library.
Another object of the present invention is to construct a method for purifying IgY using this peptide.

The present invention includes the following features.
(1) The following formula (I):
(X ^1-3 ) -C- (X ⁵ ) -W- (X ^7-11 ) -W- (X ¹³ ) -C- (X ^15-17 ) (I)
(In the formula, X ¹ and X ³ out of X ^1-3 are independently any amino acid residues other than cysteine residues, and X ² is any amino acid residue which may be a cysteine residue. And
C is a cysteine residue;
X ⁵ is an amino acid residue of T, D, V, S or E;
W is a tryptophan residue;
X ^7-11 is an amino acid sequence (S, T, R or N)-(S, A, K or R)-(I, A, V, T or G)-(V, Y, T, R or S )-(G or D),
X ¹³ is a V, L, D, S, M, T or R amino acid residue, and
3 amino acid residues of X ^15-17 are independently any amino acid residues other than cysteine residues)
It includes an amino acid sequence consisting of 17 amino acid residues, and forms a disulfide bond between the cysteine residue at position 4 and the cysteine residue at position 14, and can bind to tri-IgY. A peptide characterized by the above.
(2) X ^1-3 above is an amino acid sequence (G, R, W, E, A or V)-(V, T, C, I, W or N)-(K, R, V, S, The peptide according to (1), which is T or W).
(3) The peptide according to (2), wherein X ^1-3 is selected from the group consisting of amino acid sequences GVK, GTR, RCV, WIR, EWS, GTS, ATT, VNV or GTW .
(4) X ^15-17 is an amino acid sequence (V, Y, R, F, D, N, S, or G)-(D, N, A, R, T, S, K, or I)-( The peptide according to any one of (1) to (3), which is M, Y, D, S, P, V or G).
(5) The peptide according to (4), wherein X ^15-17 is selected from the group consisting of amino acid sequences VDM, YDY, RND, FAS, DRD, NTP, YSV, SKS, or GIG .
(6) The peptide according to any one of (1) to (5), wherein X ⁵ is T, D, or V.
(7) The above X ^7-11 is selected from the group consisting of amino acid sequences SSIVD, SAAYG, TAVTG, TKVTG, RKTTG, NRGRG, TRGSG, RSGRG or RRTSG, (1) to (6) The peptide according to any one of the above.
(8) The peptide according to any one of (1) to (7), wherein X ¹³ is V, S or R.
(9) The peptide according to any one of (1) to (8), comprising any one of the following amino acid sequences.
GVKCTWSSIVDWVCVDM (SEQ ID NO: 1)
GTRCDWSAAYGWLCYDY (SEQ ID NO: 2)
RCVCVWTAVTGWDCRND (SEQ ID NO: 3)
WIRCDWTKVTGWVCFAS (SEQ ID NO: 4)
EWSCVWRKTTGWSCDRD (SEQ ID NO: 5)
GTSCSWNRGRGWMCNTP (SEQ ID NO: 6)
ATTCTWTRGSGWSCYSV (SEQ ID NO: 7)
VNVCEWRSGRGWTCSKS (SEQ ID NO: 8)
GTWCTWRRTSGWRCGIG (SEQ ID NO: 9)
(10) The peptide according to any one of (1) to (9), to which a label is bound.
(11) A fusion protein comprising a protein linked to the peptide according to any one of (1) to (9).
(12) An immobilized peptide obtained by binding the peptide according to any one of (1) to (9) to a solid phase.
(13) A nucleic acid encoding the peptide according to any one of (1) to (9).
(14) The method includes binding the peptide according to any one of (1) to (9) or the immobilized peptide according to (12) to IgY, and recovering IgY by releasing the bound IgY. And purification method of IgY.
(15) A method for detecting IgY, comprising binding IgY in a sample to the peptide according to any one of (1) to (9) or the immobilized peptide according to (12), and detecting the bound IgY. .
(16) A kit for analyzing or purifying IgY, comprising at least one of the peptide according to any one of (1) to (9) or the immobilized peptide according to (12).
(17) An IgY separation column containing the immobilized peptide according to (12).

  According to the present invention, IgY such as tri-IgY can be purified easily and with high purity.

This figure shows the amino acid sequences (A) presented by the three IgY-binding phages obtained from the T7 phage library and the binding activity (B) by ELISA. The well of an ELISA plastic plate (Nunc Maxisorp) was coated with 50 ng of each protein, blocked with 0.5% BSA overnight, and then prepared T7 phage was added and reacted at room temperature for 1 hour. Then, after washing 5 times with PBS containing 0.1% Tween (PBST), detection was performed with biotinylated anti-T7 phage antibody and HRP-labeled streptavidin (SA). This figure shows the binding activity of biotinylated peptides to IgY antibodies. Each well of an ELISA plastic plate (Nunc Maxisorp) was coated with 50 ng of each protein, blocked with 0.5% BSA overnight, and then biotinylated peptide and HRP-labeled SA (molar ratio 4: 4). The mixture in 1) was reacted for 1 hour, washed 5 times with PBST, developed with TMB solution, and the absorbance at 450 nm was measured. This figure shows the results of affinity analysis of two IgY-binding peptides by surface plasmon resonance (SPR). (A) and (B) show the analysis results for IgY and IgY-Fc of Y4-4 peptide. The left side of the figure shows sensorgrams when BIAcore T-200 was used to inject different concentrations of peptides from 0.0024 to 10 μM for IgY and IgY-Fc immobilized on a CM5 sensor chip. The right side shows the result of the equilibrium value analysis based on the data. This figure shows the results of affinity analysis of two IgY-binding peptides by surface plasmon resonance (SPR). (C) and (D) show analysis results for IgY and IgY-Fc of the Y5-55 peptide. The left side of the figure shows sensorgrams when BIAcore T-200 was used to inject different concentrations of peptides from 0.0024 to 10 μM for IgY and IgY-Fc immobilized on a CM5 sensor chip. The right side shows the result of the equilibrium value analysis based on the data. This figure shows the results of an IgY sedimentation recovery experiment using beads immobilized with biotinylated IgY-binding peptides. This figure shows the result of an IgY binding test using a column on which an IgY antibody-binding peptide is immobilized. An IgY purification column was prepared by adding a biotinylated 4-limbed PEG (also referred to as PEG4 or (PEG) ₄ ) peptide to a HiTrap SA-HP column (1 ml, GE Healthcare) (immobilization of Y4-4 peptide) Amount of immobilized peptide: 385 nmole, Y5-55 peptide immobilized amount: 324 nmole), while flowing 25 mM phosphate buffer (pH 7.0) containing 0.25 M NaCl at a flow rate of 0.5 ml / min, the same buffer The antibody dissolved in was injected. Elution was performed by stepwise switching to 0.1M Glycine-HCl (pH 2.5), and protein elution was followed by measuring fluorescence at 340 nm with 280 nm excitation. This figure shows the purification results of IgY from chicken egg yolk extract using a column immobilized with Y4-4 peptide.

  The peptide having specific or selective binding property to avian IgY found by the present inventors is a random peptide library containing one disulfide bond in the molecule constructed by the T7 phage display system (Sakamoto, K ., Ito, Y., Hatanaka, T., Soni, PB, Mori, T., and Sugimura, K. (2009) The Journal of biological chemistry 284 (15), 9986-9993) The three specific clones were isolated from the constructed library using the biopanning method, and the homology of the characteristic primary structure (sequence) common to each other was observed. Based on that sequence, the synthetic peptide showed excellent specificity and affinity for IgY. Residues essential for IgY binding of these peptides were identified, enabling an approach to affinity enhancement and application to the purification of IgY from egg yolk using the peptides. The IgY-binding peptide of the present invention consists of 17 residues, and it can be expected to construct an IgY purification system.

Hereinafter, the present invention will be described in more detail.
Specifically, the IgY-binding peptide of the present invention, a method for purifying and analyzing IgY using the peptide, and a kit for purifying or detecting such IgY are described.

<IgY binding peptide>
The peptide of the present invention has been screened as having specific or selective binding to avian IgY from a phage library containing a large number of random peptides.

That is, the peptide of the present invention has the following formula (I):
(X ^1-3 ) -C- (X ⁵ ) -W- (X ^7-11 ) -W- (X ¹³ ) -C- (X ^15-17 ) (I)
(In the formula, X ¹ and X ³ out of X ^1-3 are independently any amino acid residues other than cysteine residues, and X ² is any amino acid residue which may be a cysteine residue. And
C is a cysteine residue;
X ⁵ is an amino acid residue of T, D, V, S or E;
W is a tryptophan residue;
X ^7-11 is an amino acid sequence (S, T, R or N)-(S, A, K or R)-(I, A, V, T or G)-(V, Y, T, R or S )-(G or D),
X ¹³ is a V, L, D, S, M, T or R amino acid residue, and
3 amino acid residues of X ^15-17 are independently any amino acid residues other than cysteine residues)
It includes an amino acid sequence consisting of 17 amino acid residues, and forms a disulfide bond between the cysteine residue at position 4 and the cysteine residue at position 14, and can bind to tri-IgY. It is a peptide characterized by this.

In formula (I), for example, “1-3” in X ^1-3 is used to mean the 1st to 3rd positions in the amino acid sequence of the peptide.

  The peptide of the present invention forms a cyclic peptide by disulfide bond between two cysteine residues of formula (I) (C at position 4 and C at position 14).

According to an embodiment of the present invention, X ^1-3 of formula (I) is an amino acid sequence (G, R, W, E, A or V)-(V, T, C, I, W or N)-(K , R, V, S, T or W). Specifically, X ^1-3 is selected from the group consisting of the amino acid sequences GVK, GTR, RCV, WIR, EWS, GTS, ATT, VNV or GTW.

According to an embodiment of the present invention, X ^15-17 of formula (I) is an amino acid sequence (V, Y, R, F, D, N, S or G)-(D, N, A, R, T, S , K or I)-(M, Y, D, S, P, V or G). Specifically, X ^15-17 is selected from the group consisting of the amino acid sequences VDM, YDY, RND, FAS, DRD, NTP, YSV, SKS or GIG.

According to an embodiment of the present invention, X ⁵ in formula (I) is T, D or V.
According to an embodiment of the present invention, X ^{11 in} formula (I) is preferably G.
According to an embodiment of the invention, X ^7-11 of formula (I) is characterized in that it is selected from the group consisting of the amino acid sequences SSIVD, SAAYG, TAVTG, TKVTG, RKTTG, NRGRG, TRGSG, RSGRG or RRTSG.
According to an embodiment of the present invention, X ¹³ in formula (I) is V, S or R.

Specific examples of preferred peptides are peptides consisting of any of the following amino acid sequences.
GVKCTWSSIVDWVCVDM (SEQ ID NO: 1)
GTRCDWSAAYGWLCYDY (SEQ ID NO: 2)
RCVCVWTAVTGWDCRND (SEQ ID NO: 3)
WIRCDWTKVTGWVCFAS (SEQ ID NO: 4)
EWSCVWRKTTGWSCDRD (SEQ ID NO: 5)
GTSCSWNRGRGWMCNTP (SEQ ID NO: 6)
ATTCTWTRGSGWSCYSV (SEQ ID NO: 7)
VNVCEWRSGRGWTCSKS (SEQ ID NO: 8)
GTWCTWRRTSGWRCGIG (SEQ ID NO: 9)

The peptide of the present invention has a high binding affinity for chicken IgY. For example, the dissociation constant (Kd) of the peptide of the present invention can be determined by surface plasmon resonance spectrum analysis (for example, using BIACORE T-200 system (protein interaction analysis)), for example, 1 × 10 ⁻⁶ M to 1 × 10 ^-5 M or less.

  When the peptide of the present invention immobilized on a solid phase was used to actually bind IgY in avian serum, it was found to bind to IgY serotype, and IgY could be separated. Indicated.

  The peptide of the present invention can be produced by a conventional liquid phase synthesis method, a peptide synthesis method such as a solid phase synthesis method, peptide synthesis by an automatic peptide synthesizer, etc. (Kelley et al., Genetics Engineering Principles and Methods, Setlow , JK eds., Plenum Press NY. (1990) Vol. 12, p.1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989) WH Freeman Co .; Houghten, Proc. Natl. Acad. Sci USA (1985) 82: p.5132, “Seminar in Experimental Chemistry 1 Protein IV” (1992) The Biochemical Society of Japan, Tokyo Chemical Doujin, Tokyo, Japan). Alternatively, the peptide may be produced by a gene recombination method using a nucleic acid encoding the peptide of the present invention or a phage display method. For example, the target peptide can be produced by incorporating a DNA encoding the amino acid sequence of the peptide of the present invention into an expression vector, introducing it into a host cell and culturing. The produced peptide can be obtained by a conventional method, for example, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, HPLC chromatography, ammonium sulfate fractionation, ultrafiltration, immunoadsorption method. It is possible to recover or purify by, for example.

  Peptide synthesis is performed by preparing amino acids in which functional groups other than the α-amino group and α-carboxyl group to be bonded are protected, and between each α-amino group and α-carboxyl group of each amino acid. The peptide bond formation reaction is performed in Usually, the carboxyl group of the amino acid residue located at the C-terminus of the peptide is bound to the solid phase via an appropriate spacer or linker. The amino terminal protecting group of the dipeptide obtained above is selectively removed to form a peptide bond with the α-carboxyl group of the next amino acid. Such operations are continuously performed to produce a peptide having a side group protected, and finally, all the protecting groups are removed and separated from the solid phase. The details of the types of protecting groups, the protecting method, and the peptide bonding method are described in detail in the above-mentioned documents.

  In the gene recombination method, DNA encoding the peptide of the present invention is inserted into an appropriate expression vector, the vector is introduced into an appropriate host cell, the cell is cultured, and the target peptide is obtained from inside the cell or from the extracellular fluid. Recovering. The vector is not limited, but is a vector such as a plasmid, a phage, a cosmid, a phagemid, or a virus. Plasmid vectors include, but are not limited to, E. coli-derived plasmids (eg, pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.) ), Yeast-derived plasmids (eg, YEp13, YCp50, etc.). Examples of phage vectors include, but are not limited to, T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc. (Novagen)), λ phage vectors (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, animal viruses such as vaccinia viruses and Sendai viruses, and insect viruses such as baculoviruses. Examples of cosmid vectors include, but are not limited to, Lorist 6, Charomid 9-20, and Charomid 9-42. Examples of phagemid vectors include, but are not limited to, pSKAN, pBluescript, pBK, and pComb3H. The vector may include a regulatory sequence so that the target DNA can be expressed, a selection marker for selecting a vector containing the target DNA, a multi-cloning site for inserting the target DNA, and the like. Such regulatory sequences include promoters, enhancers, terminators, S-D sequences or ribosome binding sites, replication origins, poly A sites, and the like. In addition, for example, an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, a dihydrofolate reductase gene, and the like can be used as the selection marker. Host cells for introducing the vector are bacteria such as E. coli and Bacillus subtilis, yeast cells, insect cells, animal cells (for example, mammalian cells), plant cells, and the like. Transformation or transfection into these cells Includes, for example, calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method and the like. The method for culturing the transformed cells is carried out according to the usual method used for culturing host organisms. For example, culture of microorganisms such as E. coli and yeast cells contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host microorganism. In order to facilitate the recovery of the peptide of the present invention, it is preferable to secrete the peptide produced by expression outside the cell. For this purpose, DNA encoding a peptide sequence that allows secretion of the peptide from the cell is bound to the 5 ′ end of the DNA encoding the target peptide. The fusion peptide transferred to the cell membrane is cleaved by signal peptidase, and the target peptide is secreted and released into the medium. Alternatively, the target peptide accumulated in the cells can be recovered. In this case, the cells are physically or chemically destroyed, and the target peptide is recovered using protein purification techniques.

  Therefore, the present invention further relates to nucleic acids encoding the peptides of the present invention. Here, the nucleic acid includes DNA or RNA (for example, cDNA and mRNA).

  The peptides of the invention may be labeled in order to allow detection of IgY. Labels include but are not limited to, for example, fluorescent dyes, chemiluminescent dyes, enzymes, radioisotopes, fluorescent proteins, biotin and the like. Examples of preferred labels are fluorescein derivatives such as fluorescein and FITC, rhodamine derivatives such as rhodamine and tetramethylrhodamine, and fluorescent dyes such as Texas Red.

  The peptide of the present invention may be fused with any protein. If the protein is a fluorescent protein such as GFP (green fluorescent protein) or an enzyme such as peroxidase, the protein can be used as a label. In this case, the peptide of the present invention and the protein can be produced by gene recombination as a fusion protein via an appropriate linker as necessary. At this time, a fusion protein should be prepared so that the peptide of the present invention does not impair the binding property with avian IgY.

  The peptide of the present invention may be further immobilized on a solid phase that can be packed in an affinity column so that it can be used for separation and purification of IgY, analysis, and the like.

  Suitable solid phases used to immobilize peptides include, but are not limited to, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, styrene-butadiene copolymers, (meth) acrylate polymers, Examples include fluororesins, silica gels, cross-linked dextrans, polysaccharides, polysaccharides such as agarose, glasses, metals, magnetic substances, and combinations thereof. The shape of such a solid phase may be any shape such as a tray, sphere, fiber, particle, rod, flat plate, container, cell, microplate, test tube, membrane (film or membrane), gel, chip, and the like. . Specific examples include magnetic beads, glass beads, polystyrene beads, sepharose beads, silica gel beads, polysaccharide beads, polystyrene plates, glass plates, polystyrene tubes, and the like. Immobilization of the peptide of the present invention on these solid phases can be performed using methods well known to those skilled in the art, for example, physical adsorption method, covalent bond method, ion bond method and the like. Immobilization is preferably carried out by a covalent bond, and a chemical functional group (for example, hydroxy group, amino group, N-hydroxysuccinimidyl group, etc.), preferably an alkylene chain having about 4 to 20 carbon atoms, is provided on the solid surface. It has a chemical functional group as a spacer and chemically reacts with the carboxy terminus of the peptide to form an ester bond or an amide bond. The solid phase on which the peptide of the present invention is immobilized can be packed in a column such as an affinity chromatography column or HPLC column and used to detect, purify or separate IgY.

<IgY purification method>
The present invention further provides a method for purifying IgY, comprising binding the above-mentioned peptide or immobilized peptide to IgY, and releasing the bound IgY to recover IgY.

  The solid phase on which the peptide of the present invention has been immobilized is packed into a column such as an affinity chromatography column or HPLC column, equilibrated with an appropriate buffer, and cooled to room temperature to 0 ° C, preferably about 10 ° C to 0 ° C (further, A solution containing IgY is preferably applied at about 4 ° C. to bind triIgY to the peptide on the solid phase. For example, when separating IgY in serum, a binding operation can be performed by applying to a column using a buffer having a neutral pH, for example, pH 6.0 to 7.5. Elution can be carried out by passing a pH range of acid, for example, pH 2-4 buffer (for example, pH 3.5 to pH 2.5 0.2 M glycine-HCl buffer containing 0.3 M NaCl) through the column.

  Whether or not IgY has been recovered can be measured, for example, by electrophoresis followed by Western blotting using an anti-tri-IgY antibody. Electrophoresis was performed by SDS-PAGE using a 5-20% acrylamide gradient gel. Western blot transferred the protein after migration to a PVDF membrane, blocked with skim milk, and then used an appropriate labeled antibody. Can be detected.

  The method of the present invention is useful for obtaining an IgY-rich fraction in a process of purifying IgY from an IgY-containing product produced by various methods. Therefore, it is preferable to use the method of the present invention in column chromatography such as affinity chromatography and HPLC. In the purification of IgY, in addition to such chromatographic methods, conventional protein purification techniques such as gel filtration chromatography, ion exchange column chromatography, reverse phase column chromatography, etc., ammonium sulfate fractionation, Ultrafiltration and the like can be combined as appropriate.

<Analysis method of IgY>
The present invention further provides a method for detecting IgY, which comprises binding IgY in a sample to the above-described peptide of the present invention or immobilized peptide, and detecting the bound IgY. Here, detection shall include either qualitative or quantitative analysis.

  For detection of IgY, the sample is bound to a membrane or polystyrene well plate using a suitable buffer during the operation, and the labeled peptide of the present invention is contacted with this. After washing as necessary, the level of the label is qualitatively determined. Or it can carry out by quantifying.

  Alternatively, when using an HPLC column on which the peptide of the present invention is immobilized as described above, a sample containing tri-IgY is injected into the column, and a binding buffer is passed to bind tri-IgY to the peptide. For example, detect and record protein with absorbance at 280 nm or 450 nm, or 350 nm fluorescence with 280 nm excitation light, and elution in elution buffer (eg, 0.1 M glycine hydrochloride buffer pH 2.5 containing 0.15 M NaCl) ), And qualitative and quantitative determination of IgY can be performed by the peak and peak area that are eluted from the column.

<Kit and column>
The present invention further provides a kit for analysis (qualitative, quantitative, etc.) or purification of IgY, comprising at least one of the above-mentioned peptides or immobilized peptides of the present invention.

  Individual peptides or immobilized peptides contained in the kit of the present invention are accommodated in individual containers. Further, if necessary, the kit may be provided with instructions for use that describe the analysis procedure and purification procedure of IgY. Furthermore, the kit may contain reagents and buffers necessary for analysis, an immobilized peptide packed column, and the like.

The present invention further provides an IgY separation column containing the above-described immobilized peptide of the present invention.
Immobilized peptides can be produced by covalently or non-covalently binding peptides to a chromatographic support (packing material). Examples of such carriers are polysaccharide-based carriers such as agarose and sepharose, silica gel-based carriers, resin-based or polymer-based carriers. When the peptide is bound to the carrier, it may be bound via a spacer such as a hydrocarbon chain (for example, C4 to C16).

  The above-mentioned column for separating IgY is a column for separating IgY, and specifically, a column such as a chromatography column or a high performance liquid chromatography (HPLC) column for analyzing or purifying / sorting IgY. Include. The size of the column is not particularly limited, and can be changed according to the use for analysis, purification and fractionation, the amount to be applied (loaded) or injected. The material of the column may be a metal, plastic, glass, or the like that is usually used as a column.

  The above-mentioned column can be produced by closely packing the immobilized peptide (dried or wet state) of the present invention prepared according to the above-described method into the column.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

[Example 1]
<Search for IgY-binding peptides>
Three phage clones that bind to chicken IgY were obtained by biopanning on chicken IgY using a random peptide library having a cyclic structure with two Cys constructed by the T7 phage display method.

  FIG. 1 shows the sequences of peptides displayed by the three phages obtained and the binding activity by ELISA. All of the obtained phages showed binding activity only to chicken IgY or its Fc fragment (IgY-Fc), and did not show binding activity to other proteins. In the comparison of the amino acid sequences of the peptides presented by the obtained clones, amino acids with high conservability including two Trps were found inside the two Cys derived from the library (FIG. 1A).

Among the three clones, Y4-4 and Y5-55 were synthesized with peptides and their binding activities were evaluated. Peptide synthesis was performed by adding biotinylated (PEG) ₄ to the N terminus and amidating the C terminus, and forming an SS bond in the molecule by air oxidation, followed by purification on a reverse phase column. A thing was used.

  FIG. 2 shows the binding activity of these peptides by ELISA. Both types of peptides showed specific binding activity to IgY and IgY-Fc.

  Furthermore, FIG. 3 shows the results of affinity analysis by SPR with BIAcore T-200 using this peptide.

  The Y4-4 peptide binds to IgY and IgY-Fc with approximately the same affinity (7.0, Kd values of 7.8 μM), whereas the Y5-55 peptide binds slightly stronger (4.2, 4.1 μM Kd) These peptides were expected to function satisfactorily as purification ligands (Table 1).

  Furthermore, in the same manner as described above, six phage clones that bind to chicken IgY were obtained by biopanning on chicken IgY using a random peptide library having a cyclic structure with two Cys constructed by the T7 phage display method (Table 1). 2).

All of the peptides in the sequenced table have the structure C ⁴ -X ⁵ -W ⁶ -X ⁷ X ⁸ X ⁹ X ¹⁰ G ¹¹ -W ¹² -X ¹³ -C ¹⁴ and position 11 The basic skeleton shown in FIG. 1 was the same except that all amino acid residues (X ¹¹ ) were Gly.

<Purification of IgY>
In order to examine the usefulness of the obtained IgY-binding peptide, it was examined whether or not IgY could be recovered using beads to which the IgY-binding peptide was bound.

  Specifically, biotinylated Y4-4 and Y5-55 peptides (10 nmol / 100 μl) were added to SA (streptavidin) -immobilized beads (SA-Agarose: 1 nmol SA / 10 μl) and bound, and the excess peptide was removed by washing. It was. An IgY solution (25 mMPB containing 30 μg / 100 ul of 0.25 NaCl) was added to the beads, and after reacting at room temperature for 1 hour, the beads were collected by centrifugation and washed twice with 25 mMPB containing 0.25 NaCl. Thereafter, the beads were dispersed in 90 μl of SDS sample solution, subjected to SDS-PAGE using 1/3 (30 μl), and the results of CBB staining of the gel after electrophoresis are shown in FIG.

  With beads immobilized with Y4-4 and Y5-55 peptides, IgY was successfully recovered from the solution (FIG. 4, lanes 1 and 3). On the other hand, human IgG used as a control was hardly recovered by this bead (FIG. 4, lanes 2 and 4). Also, IgY could not be recovered even when beads without immobilized peptide were used (FIG. 4, lane 5). Thus, it was found that the IgY-binding peptide identified this time functions as an affinity ligand for recovering IgY from solution by bead precipitation.

  Furthermore, in order to examine whether IgY-binding peptides function as affinity ligands for IgY purification columns, columns with biotinylated peptides immobilized on SA immobilized columns (GE Healthcare, HiTrap-SA, 1 ml) And the binding behavior of IgY to this column was examined (Fig. 5).

  As a control experiment, biotin was added to a HiTrap-SA column (1 ml), and IgY, IgY-Fc or human IgG was injected into a column saturated with SA. As shown in FIG. Were eluted in the fractions. Next, when an experiment was performed using a column on which Y4-4 (FIG. 5B) or Y5-55 peptide (FIG. 5C) was immobilized, human IgG was eluted in the flow-through fraction, but IgY and IgY -Fc was almost completely adsorbed on the column. In addition, by switching to an eluent of 0.1 M Glycine-HCl (pH 2.5) after 5 minutes, IgY and IgY-Fc bound to about 11 minutes were eluted as peaks (however, the peak at about 17 minutes is , The protein eluted when switching from 0.1 M Glycine-HCl to PBS). The above shows that the column on which Y4-4 or Y5-55 is immobilized functions sufficiently as an affinity column for purifying chicken IgY.

  Finally, purification of IgY from chicken egg yolk extract was examined using the column immobilized with Y4-4 peptide used in FIG. 1 ml of egg yolk was diluted 5-fold with 50 mM PB (pH 7.0) containing 0.25 M NaCl, and then slowly stirred at 4 ° C. overnight. The insoluble fraction was precipitated by centrifugation (10000 g × 15 minutes), and the supernatant (5 ml) was applied to the column. After washing the column with 50 mM PB (pH 7.0) containing 0.25 M NaCl, the column was eluted with 0.1 M glycine hydrochloride buffer (pH 6A) or pH 3.0 (FIG. 6B). SDS-PAGE was performed on the yolk extract fraction, the flow through purification by elution at pH 2.5 and 3.0 (about 18 minutes) and the eluted fraction (about 43 minutes) (FIG. 6C). In the purification using the peptide-immobilized column, proteins other than IgY contained in the egg yolk were collected in the flow-through fraction, and the fraction adsorbed on the column and eluted at acidic pH showed a band almost exclusively of IgY. The results of this family show that this peptide-immobilized column actually functions as a specific and efficient purification column for IgY from egg yolk.

  According to the present invention, a peptide capable of specifically binding to an IgY antibody is provided. By using an affinity column on which this peptide is immobilized, IgY can be highly purified.

Sequence number 1-9: Artificial sequence

Claims (16)

Formula (I) below:
(X ^1-3 ) -C- (X ⁵ ) -W- (X ^7-11 ) -W- (X ¹³ ) -C- (X ^15-17 ) (I)
( Wherein X ^1-3 is selected from the group consisting of amino acid sequences GVK, GTR, RCV, WIR, EWS, GTS, ATT, VNV and GTW from the N-terminal side to the C-terminal side ,
C is a cysteine residue;
X ⁵ is an amino acid residue of T, D, V, S or E;
W is a tryptophan residue;
X ^7-11 is selected from the group consisting of the amino acid sequence SSIVD, SAAYG, TAVTG, TKVTG, RKTTG, NRGRG, TRGSG, RSGRG and RRTSG from the N-terminal side to the C-terminal side ,
X ¹³ is a V, L, D, S, M, T or R amino acid residue, and
X ^15-17 is selected from the group consisting of the amino acid sequence VDM, YDY, RND, FAS, DRD, NTP, YSV, SKS, and GIG from the N-terminal side to the C-terminal side. A peptide comprising an amino acid sequence comprising a group, having a disulfide bond formed between a cysteine residue at position 4 and a cysteine residue at position 14, and capable of binding to triIgY. The peptide according to claim 1 , wherein X ^1-3 is selected from the group consisting of amino acid sequences GVK, GTR and RCV from the N-terminal side to the C-terminal side . The peptide according to claim 1 or 2 , wherein X ^15-17 is selected from the group consisting of amino acid sequences VDM, YDY and RND from the N-terminal side to the C-terminal side . X ⁵ above, T, characterized in that it is a D or V, peptide according to any one of claims 1-3. The X ^7-11 is selected from the group consisting of the amino acid sequences SSIVD, SAAYG, TAVTG and TKVTG from the N-terminal side to the C-terminal side, according to any one of claims 1 to 4. The described peptides. The peptide according to any one of claims 1 to 5 , wherein X ¹³ is an amino acid residue of V, L, or D. The peptide according to any one of claims 1 to 6 , which comprises any one of the following amino acid sequences.
GVKCTWSSIVDWVCVDM (SEQ ID NO: 1)
GTRCDWSAAYGWLCYDY (SEQ ID NO: 2)
RCVCVWTAVTGWDCRND (SEQ ID NO: 3)
WIRCDWTKVTGWVCFAS (SEQ ID NO: 4)
EWSCVWRKTTGWSCDRD (SEQ ID NO: 5)
GTSCSWNRGRGWMCNTP (SEQ ID NO: 6)
ATTCTWTRGSGWSCYSV (SEQ ID NO: 7)
VNVCEWRSGRGWTCSKS (SEQ ID NO: 8)
GTWCTWRRTSGWRCGIG (SEQ ID NO: 9) The peptide according to claim 7, comprising any one of the following amino acid sequences.
GVKCTWSSIVDWVCVDM (SEQ ID NO: 1)
GTRCDWSAAYGWLCYDY (SEQ ID NO: 2)
RCVCVWTAVTGWDCRND (SEQ ID NO: 3) The peptide according to any one of claims 1 to 8 , wherein a label is bound thereto. A fusion protein comprising a protein linked to the peptide according to any one of claims 1 to 8 . An immobilized peptide obtained by binding the peptide according to any one of claims 1 to 8 to a solid phase. A nucleic acid encoding the peptide according to any one of claims 1 to 8 . Claim 1 can be coupled with avian IgY immobilized peptide according to the peptide or claim 11 according to any one of 8, and, recovering the IgY by releasing the bound IgY, tri IgY purification method. A method for detecting avian IgY, comprising: binding a tri- IgY in a sample to the peptide according to any one of claims 1 to 8 or the immobilized peptide according to claim 11 , and detecting the bound IgY. A kit for analysis or purification of avian IgY, comprising at least one of the peptide according to any one of claims 1 to 8 or the immobilized peptide according to claim 11 . A tri- IgY separation column containing the immobilized peptide according to claim 11 .

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