JP2002363026A - Method for enhancing adsorption of cosmetic and cosmetic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enhancing adsorption of a cosmetic, by which the cosmetic and be firmly adsorbed on the skin, the hair, nails, or the like, for a long period of time specifically and simply and which is high in safety, and to obtain the cosmetic. SOLUTION: This cosmetic is characterized in that a fused material between a single-chain antibody having affinity for an object to be made to wear make up and an amino acid sequence to be bonded to a ligand is combined with the ligand and a cosmetic and used. The method for enhancing adsorption o a cosmetic by using this cosmetic is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for firmly adsorbing a cosmetic agent on skin, hair, nails and the like, and a cosmetic material therefor, and more particularly to hair such as head hair, eyebrows and eyelashes.
These cosmetics are applied using a method in which a protein and a cosmetic agent that bind to an object such as skin or nail are bound by metal-amino acid affinity or based on an interaction between a ligand and a corresponding binding protein. The present invention relates to a method for stably adsorbing the cosmetic agent on a subject and a cosmetic for the method.

[0002]

2. Description of the Related Art For the purpose of dyeing hair or nails, several cosmetics have been developed in which antibodies against these constituents are bound to a cosmetic agent and the binding properties of the antibodies are utilized (Japanese Patent Laid-Open Publication No. Heisei (Kokai) No. Heisei 9 (1999)). 4-29912; JP-A-6-2279
No. 55; JP-A-7-69832; and JP-A-8-1
43431 publications). Most of them apply a cosmetic / antibody complex obtained by directly binding an antibody to a pigment, a dye, or a nail agent as a cosmetic.

[0003] Only JP-A-8-143431 discloses a method utilizing a specific and strong binding force between avidin and biotin. That is, a complex of an antibody and biotin (or avidin), a complex of a cosmetic agent and avidin (or biotin) are separately prepared, the antibody complex is first applied to the skin, and then the cosmetic complex is applied. It is. Furthermore, in order to make a cosmetic agent specifically bind to hair, a protein serving as an antigen is used as a pretreatment agent.

[0004]

However, in any of these conventional methods, both the antibody and the cosmetic must be chemically modified in order to bind the antibody to the cosmetic. Therefore, there were drawbacks such as the binding site of the antibody was damaged by the chemical modification, and the desired amount of the antibody could not be bound to the cosmetic agent. In addition, the cost for chemical modification was also a problem.

[0005] In order to produce antibodies in the above-mentioned conventional techniques, a method of immunizing an animal and purifying it from serum, or a method of producing a monoclonal antibody-producing cell by a cell fusion method and producing it in an animal or a cell is used. Is an industrial production method, but its cost is high.

As described above, in the above-mentioned prior art,
(1) When an antibody is directly bound to a cosmetic agent, an attempt to bind a sufficient amount of the cosmetic agent to the antibody results in a decrease in the activity of the antibody; (2) proteins such as the antibody and avidin are unstable In addition, the method is expensive, and the yield is not stable due to chemical modification, and the cost is not favorable; and (3) the antibody must be purified as much as possible in order to chemically modify the antibody, and the cost There were problems such as increase.

[0007] In view of these drawbacks in the prior art, the present inventors do not require a special pretreatment agent for hair, skin, nails and the like, and stably and at low cost hair dyeing, makeup and skin conditioning. It is an object of the present invention to provide a method for applying cosmetics such as nails and beautiful nails, and a cosmetic therefor.

[0008]

Means for Solving the Problems The present invention has been intensively studied to achieve the above object, and as a result, it has been found that the N-terminal side of a single-chain antibody having a binding property to an object such as skin, hair, nail, etc. On the end side or inside, by a fusion that incorporates an amino acid sequence capable of binding to a ligand, and a cosmetic used in combination with a ligand and a cosmetic agent, the interaction between the ligand and the amino acid sequence capable of binding to it is The present invention has been completed by finding that a cosmetic agent can be stably and specifically adsorbed.

Here, a pair of a ligand and an amino acid sequence capable of binding to the ligand are preferably used as a metal chelate compound, an amino acid sequence capable of binding to such a compound via a metal, and cellulose. , Chitin, maltose and the like, or peptides such as glutathione and their analogs, and a pair of a ligand-binding protein or an anti-ligand antibody having affinity with these.

[0010] In the cosmetic of the present invention, a gene encoding an amino acid sequence capable of specifically affinity-binding with a ligand such as a metal, the above-mentioned polymer compound or peptide can be used as a simple substance having a binding property to an object such as skin or hair. N of chain antibody
A fusion may be prepared by preparing a vector that has been integrated into the terminal side, C-terminal side, or inside, growing host cells transformed or transfected with such a vector, and expressing the protein. On the other hand, a metal chelate functional group capable of retaining a transition metal or a ligand corresponding to each protein, that is, chitin, cellulose, amylose, glutathione, or the like is chemically or physically bonded to the surface of the cosmetic substance. Good.

Further, the present invention is characterized in that a fusion of a single-chain antibody having a binding property to a subject to be applied with an amino acid sequence capable of binding to a cosmetic, and a cosmetic are used in combination. We also provide cosmetics. Even with such a cosmetic, the cosmetic can be stably adsorbed to the target. In this case, the amino acid sequence may be a sequence constituting an anti-cosmetic agent antibody or a cosmetic-binding protein. Also, the fusion used in this cosmetic may be prepared by protein expression using the above-described genetic recombination method.

[0012] The above-mentioned single-chain antibody having a binding property to a subject to be cosmetically applied is preferably an anti-keratin antibody.
Further, such a single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, that is, the amino acid encoded by the polynucleotide shown in SEQ ID NO: 1 or an analog thereof containing a degenerate codon sequence in the polynucleotide. It is preferable to have an arrangement, and it is particularly suitably used for hair dyeing.

[0013] The present invention further provides a cosmetic comprising a combination of a ligand such as a metal chelate compound and a cosmetic agent, which has a binding property to an object to be cosmetically applied. According to the cosmetics, although the specificity and the stability are slightly inferior, it is possible to adsorb a good cosmetic agent to a target portion of interest. Further, a nickel chelate compound of nitrilotriacetic acid (Ni-NTA) is used as the metal chelate compound, and a complex of the Ni-NTA and one or more oxidases selected from the group consisting of peroxidase, uricase, glucose oxidase and lactate oxidase. Can be mentioned as preferred cosmetics, and among these oxidases, peroxidase is particularly preferred.
Hydrogen peroxide can be generated by the activity of the oxidase described above, whereby the staining substrate is oxidized and color is formed. Peroxidase can be used in combination with other oxidases as a source of hydrogen peroxide.

Further, according to the present invention, there is provided a method for enhancing the adsorption of a cosmetic agent, which comprises a fusion of a single-chain antibody having a binding property to an object to be applied with an amino acid sequence capable of binding to a ligand, and a ligand and a cosmetic agent A fusion of a single-chain antibody capable of binding to the cosmetic subject and an amino acid sequence capable of binding to the cosmetic, and a cosmetic; or a ligand and a cosmetic capable of binding to the cosmetic subject. Also provided is a method, which comprises coating an object. According to these methods, it is possible to adsorb a cosmetic agent to a target relatively stably for a long period of time, and the safety for a living body is extremely high.

Also, according to the present invention, a polynucleotide which has a nucleotide sequence shown in SEQ ID NO: 1 and which encodes a single-chain antibody that binds to skin and / or hair, which can be suitably used for formulation into cosmetics. And a single-chain antibody that binds to skin and / or hair, having an analog thereof and the amino acid sequence shown in SEQ ID NO: 2.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, makeup is applied to various parts of a body to which makeup is generally applied, such as hair, for example, hair, eyebrows, eyelashes, skin, and nails.

Accordingly, cosmetics include hair dyes, mascaras, eyebrows, foundations, pressed powders, blushers, eye shadows, eyeliners, lipsticks, cosmetic pencils, nail polish, etc., as well as sunscreen cosmetics. Are particularly limited to solids, powders (which may or may not be compressed), fluids (aqueous solutions, suspensions including emulsions, (which may include organic solvents such as ethanol)), and creams, mousses, etc. Never.

[0018] One of the main components of the cosmetic of the present invention,
A fusion comprising a single-chain antibody having a binding property to a target such as skin or hair and an amino acid sequence capable of binding to a ligand has a binding property to a target to which makeup is applied when the protein takes a tertiary structure ( (I.e., immunological binding by a single-chain antibody) and an amino acid sequence capable of binding to a ligand are designed so that they can form a corresponding binding pair without steric hindrance. Is preferred.

Antibodies, which are preferably used in the present invention and which can specifically bind to a subject to be cosmetically applied, are obtained by extracting a part of an L chain and an H chain of an antibody gene by genetic engineering, and extracting the part from the gene. It is a protein obtained by ligating and then expressing. This is referred to as a single-chain antibody (sFv) for convenience, and is also referred to herein as a single-chain antibody,
The method of ligation does not exist in natural antibodies.
Furthermore, the case where each of the L chain and the H chain is used alone is also included. The single-chain antibody does not include an antibody fragment obtained by a conventional technique such as proteolytic enzyme or reduction of the antibody. The advantages of using this single-chain antibody are that the molecular weight is as low as 1/6 or less of the original antibody and that it does not contain sugar chains, so it can be inexpensively used by using microorganisms and plants. It can be mass-produced. Furthermore, since the molecules are small, they can reach the details of the skin and hair, so that even with a small amount of use, they can be applied to the object to be applied and exhibit a desirable adsorption effect.

Next, a method for obtaining a single-chain antibody suitably used in the present invention will be described. First, human hair, skin, nails, etc. are minced and then, if necessary, hydrolyzed (acid hydrolysis, alkali hydrolysis, enzyme hydrolysis, etc.), extraction,
The antigen that has been subjected to modification (for example, carboxymethylation) or the like is administered several times to cows, horses, sheep, goats, rabbits, rats, mice, and the like to immunize. The antigen is preferably keratin or a hydrolyzate thereof, and keratin purified by a conventional method may be used. After immunization, highly proliferating cells, such as spleen cells and myeloma cells, are isolated using standard methods (Kohler G and Milstein C, Biotechnology , 2
4: 524 (1992)) to prepare a hybridoma that produces a monoclonal antibody by cell fusion. Next, for the preparation of a single-chain antibody, the genes of the V _H (variable heavy chain) and _VL (variable light chain) regions of the antibody were removed from the hybridoma immediately after cell fusion using PCR, and the V
By linking _H and _VL , a single-chain antibody (sFv) gene can be prepared. This gene is inserted into an appropriate vector, transformed into Escherichia coli (for example, E. coli TG1) by a conventional method such as electroporation, and the resulting Escherichia coli is cultured. The gene encoding the single-chain antibody may be cloned by an ELISA method, which is carried out by coating the DNA. Furthermore, a preferred clone can be selected from several clones based on the stability and binding strength of the single-chain antibody. The selected single-chain antibody clone is recovered from the culture supernatant by a conventional method using centrifugation or the like, but may be purified if necessary.

[0021] The ELISA method used for cloning, selection and immunopotency measurement of a single-chain antibody may be performed, for example, according to the following procedure. That is, ELIS
100 μl of an antigen solution (100 μg / ml) such as keratin derived from hair is added to the well of the plate for A and incubated at 4 ° C. overnight. Next, after blocking with phosphate buffered saline (PBS) containing 3% skim milk, 50 μl of an antibody-containing solution is added, and the mixture is incubated at 37 ° C. for 1 hour. After thoroughly washing the wells with PBS containing 0.05% Tween 20, a secondary antibody labeled with horseradish peroxidase (HRP) was added, followed by 3 more days.
Incubate at 7 ° C for 1 hour. 0.05% Tween20
After washing the wells with PBS containing 3,3 ′, 5,5′-tetramethylbenzidine (TM) which is a substrate for peroxidase.
B) is added and reacted at room temperature for 15 minutes, and the absorbance at 450 nm is measured.

The single-chain antibody thus obtained is used for preparing a fusion in the present invention. The molecular weight of the single-chain antibody is preferably from 300 to 80,000, more preferably from 20,000 to 40,000, of which the L chain is preferably from 0 to 23,000, more preferably from 1 to 23,000.
It corresponds to about 1,000, and the H chain is preferably 300 to 5
It is good to correspond to 0000, more preferably about 15,000.

Particularly preferred single chain antibodies include SEQ ID NO:
SEQ ID NO: encoded by the nucleotide sequence represented by 1 or an analog thereof (a sequence containing a degenerate codon)
A single-chain antibody having the amino acid sequence of 2. This single-chain antibody can selectively bind to hair and skin, provides stable adsorption of the cosmetic agent to hair and skin, and has a molecular weight of about 27,000 (including L chain: about 1
2,000, heavy chain: about 13,000), so that the hair has good penetration into the details. The outline of the vector having the nucleotide sequence encoding the single-chain antibody useful in the present invention shown in SEQ ID NO: 1 is shown in FIG.

In order to bind the single-chain antibody constituting the fusion with the cosmetic agent, specific affinity, that is, affinity binding is used instead of performing direct modification or the like by a chemical synthesis method. As a pair that provides affinity, for example,
Specific amino acid sequences include metals (chelate compounds), glutathione-binding proteins such as glutathione S-transferase and glutathione, cellulose-binding proteins and cellulose, chitin-binding proteins and chitin, maltose-binding proteins and amylose, and the like. Furthermore, a cosmetic agent and its binding protein may be used without using a metal chelate compound or a ligand as described above. Further, the binding protein may be an antibody having various ligands as antigens. The advantage of binding between a single-chain antibody and a cosmetic agent using affinity is that amino acids and proteins involved in this binding can be expressed together with the antibody by gene recombination and produced as a single protein fusion. The need to purify and chemically modify the antibody as in. Hereinafter, each affinity combination will be described.

(1) The affinity between a metal and a specific amino acid sequence forms the principle of a method for purifying an expressed recombinant protein. This purification method is based on immobilized metal ion affinity chromatography (IMAC, Immobilized metal ion chromatography).
It is called ion affinity chromatography (Bio / Te
chnology, Vol. 9, 151-156, 1991). The present invention utilizes therein the principle of IMAC represented by metal-histidine bond, that is, specific binding of a specific amino acid in a protein (polypeptide) molecule to a chelate compound via a metal. Nevertheless, there is no example in which such binding properties have been applied to cosmetics. According to this method, since no chemical modification of the antibody molecule is required, the binding to the antigen, that is, the object to be cosmeticized is not impaired, and the metal-amino acid bond is specific. The complex of the chelating compound and the cosmetic agent can be selectively bound only to the desired site of interest, and does not require any special pretreatment. Accordingly, hair can be dyed only by applying the chelating compound and the cosmetic after or simultaneously with applying the antibody to a target such as hair, and the disadvantages of the prior art can be solved. Here, the chelate compound and the cosmetic agent may be formed in advance in a complex, or may be separately applied and combined at the target site.

The single-chain antibody that binds to the skin, hair, nails, etc. corresponds to the protein molecule to be purified in IMAC, and forms a chelate bond with a metal at the N-terminal, C-terminal or inside of its amino acid sequence. The amino acid sequence to be obtained may be included so that both the immune binding site of the antibody and the amino acid sequence capable of chelating are exposed so as to be exposed on the protein surface. Its amino acid sequence is nickel, zinc,
It preferably contains amino acid residues that chelate with metals such as copper and iron, especially His, Trp, Tyr, Phe, Cys and combinations thereof. Although there is no particular rule for such an amino acid sequence, examples of amino acid sequences that can bind to metals, which have been reported in several documents, are shown below.

His-His-Leu-Gly-Gly-Ala-Lys-Glu-Ala-Gl
y-Asp-Val (SEQ ID NO: 3), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-Hi
s-Ser (SEQ ID NO: 4) Lys-Gly-Ser-Arg-Ser-His-His (SEQ ID NO: 5) His-Asn-Leu-Lys-Glu-Glu-His-Val-Pro-His (SEQ ID NO: : 6) His-Xaa-Xaa-Xaa-His (SEQ ID NO: 7) Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Ser-Gl
y-Ile-Met (SEQ ID NO: 8), His-Trp (SEQ ID NO: 9) His-Gly-His (SEQ ID NO: 10) His-Tyr-NH ₂ (SEQ ID NO: 11).

Among the above amino acid residues, His is preferable in view of affinity for metals. Therefore, an amino acid sequence containing a large number of His residues in a continuous manner provides stronger binding force and specificity. Get higher. To summarize these, the following sequence:-((Xaa) m- (His) n- (Xaa) o- (His) p) q- [where n and q are integers of 1 or more, m, o and p is 0
And Xaa represents any amino acid residue]
Is preferably used. Particularly preferably, m, o and p are 0, n is 2 to 6, and q is 1.

Of course, it is easy to incorporate these amino acids into the fusion protein by a gene recombination technique.
However, it is usually difficult to arbitrarily expose specific amino acid residues without losing the characteristics of the original protein, and in the case of antibodies, the affinity and specificity with the antigen may be impaired. Prone. Therefore, it is preferable to use a single-chain antibody in the present invention and to introduce a specific amino acid sequence at a place where loss of specificity or the like hardly occurs, that is, near the N-terminus or C-terminus of the single-chain antibody.

The obtained antibodies can be expressed and produced in microorganisms, plant bodies, animal milk, etc., purified as necessary, or used as a raw material for cosmetics by mixing with other cosmetics.

As the metal, the transition metal of the fourth period, Ni
²⁺ , Zn ²⁺ , Cu ²⁺ , and Fe ³⁺ are preferable.

The ligands forming the chelate compound include IDA (iminodiacetic acid), TED (tris (carboxymethyl) -ethylenediamine), NTA (nitrilotriacetic acid), PDTA (2-hydroxy-propylenediaminetetraacetic acid) and the like. Are known. Each of these has about 2 to 3 carboxyl groups at one end,
In these parts, it is chelated with the metal.

At the other end of the chelate compound, a cosmetic agent is bound via various residues. The method includes a chemical synthesis method and a physical adsorption method. That, -NH ₂ and -SH surfaces or molecules of each cosmetic agent, -CO
A crosslinker that selectively reacts with OH, sugar and the like to crosslink them is used. Known crosslinkers include imide ester compounds, N-hydroxysuccinimidyl ester compounds, maleimide compounds, haloacetyl compounds, pyridyldithiol compounds, carbodiimide compounds, photoreactive compounds, glyoxal compounds, hydrazide compounds, and the like. In addition, a protein having an interaction property such as a combination of avidin and biotin can also be used as a crosslinker. The chemical reaction carried out here is not performed on a biologically active substance such as an antibody or an enzyme, so that a relatively radical reaction can be performed, which is advantageous in cost. It is.

(2) Other examples of the ligand include glutathione. Representative amino acid sequences which can specifically bind to the glutathione include glutathione S-transferase in addition to anti-glutathione antibody. (GST) amino acid sequence (see SEQ ID NO: 26). There is a report that this GST was expressed as a fusion protein with another protein by a gene recombination method ( Gene 67, 31 (1988)). Therefore, the gene encoding the single-chain antibody is preferably N-terminal or C-terminal.
A fusion that can be used in the present invention can be obtained by ligating a gene encoding GST to the terminal side and expressing and producing the gene in a microorganism or a plant. The obtained single-chain antibody-GST fusion protein does not need to be particularly purified. This is because the fusion specifically binds affinity with the ligand-modified cosmetic, and the cosmetic of the present invention can be prepared by simply mixing the fusion with the cosmetic. On the other hand, glutathione, which is a ligand that can be complexed with a cosmetic agent, may be immobilized on any cosmetic agent by a chemical synthesis method as described in (1).

(3) Cellulose and cellulose-binding protein (C
BD) as well ( J. Biol. Chem.
263, 10401 (1988)). Cellulose-binding proteins are roughly classified into four types, each having a different affinity depending on the physical shape and molecular weight of the ligand cellulose. Regardless of the type of CBD (see SEQ ID NOs: 12 to 21), the antibody gene and the CBD gene are combined, expressed, and produced. On the other hand, the cosmetic agent may be bound to cellulose by a chemical synthesis method or a physical adsorption method as described in (1).

(4) In addition, similarly, in the case of chitin-binding protein (see SEQ ID NOs: 22 to 24), maltose-binding protein (see SEQ ID NO: 25), etc., each protein is a fusion protein with a single-chain antibody. And the corresponding ligands of chitin and maltose may be immobilized on the cosmetic by a chemical synthesis method or a physical adsorption method, as described in (1).

As described above, in addition to glutathione, cellulose, chitin and maltose described in (2) to (4), analogs such as carboxymethylcellulose, xylan, cellobiose, nigeran, dextran, chitosan and amylose are also suitable as ligands. Can be used.

Other than these, the ligand is not particularly limited as long as it is safe for the living body and can be blended without adversely affecting the feeling of use and color development of the cosmetic. An amino acid sequence of an antibody or binding protein capable of binding to such a ligand may be used for forming a fusion with a single-chain antibody in the present invention.

The ratio between the ligand and the cosmetic agent is not particularly limited, but if as many cosmetic agents as possible are bound to one molecule of the ligand, the efficiency of the cosmetic can be increased.

Furthermore, an amino acid sequence capable of binding to a cosmetic agent without using a ligand, for example, an amino acid sequence constituting an anti-cosmetic agent antibody or a cosmetic-binding protein, is used as a fusion component with a single-chain antibody. If it is used, the trouble of binding the cosmetic and the ligand can be omitted, and a favorable effect can be expected.

The fusion is prepared by expressing a nucleotide sequence encoding a single-chain antibody and a nucleotide sequence encoding the above amino acid sequence capable of binding to a ligand or a cosmetic agent, using both vectors so that they can be expressed. Preferably, various commonly used vectors can be used, for example, pE by Novagen.
T, pGEM from Promega, pBluescrip from Stratagene
t, pUC18 / 19, pSVL, etc., manufactured by Pharmacia.

Expression vectors usually contain a promoter operably linked to the two nucleotide sequences described above. Promoters include, for example, the phage λPL promoter, E. coli lac, trp, tac promoter, SV40 early and late promoters, T7 and T3 promoters, and retroviral LTR promoter.

The expression vector may be a marker (for example, di, hydrofolate reductase gene, neomycin resistance gene, ampicillin resistance gene, etc.) which enables selection of the transformed host, or an enhancer (SV40 enhancer, cytomegalovirus early enhancer). Promoter, adenovirus enhancer, etc.).

The construction, transformation or transfection, expression, etc. of the vectors exemplified above are described in Maniat.
is et al., Molecular Cloning, A Laboratory Manual, Cold
This may be performed by referring to the method described in Spring Harbor, second edition.

Examples of host cells for the vector include bacteria such as Escherichia coli (eg, E. coli TG1), fungi such as yeast, and plant cells such as soybean and tobacco.

For transformation or transfection, methods well known in the art such as electroporation are used.

When production in a non-human mammal is intended, the nucleotide sequence encoding the above-described fusion is ligated to a promoter, for example, to a milk protein such as casein. To create a transgenic mammal. In order to facilitate recovery of the fusion protein of interest, it is preferable to use a construct into which a nucleotide sequence corresponding to a secretion signal to milk has been inserted during production of a transgenic mammal.

When the fusion is used as a cosmetic material,
It may be mixed with basic cosmetics or with fine particles or polymer compounds.

The present invention will be further described below with reference to application examples in cosmetics.

(1) Hair dyeing of hair (a) The oxidative hair dyeing method conventionally used is a method of mixing an oxidizing dye and an oxidizing agent immediately before use, applying the mixture to hair, and performing oxidative polymerization and decolorization. It is. Oxidation dyes, various amines, phenols, etc. described in hair dye raw material standards, for example, p-aminophenol, m-aminophenol, o-aminophenol, p-phenylenediamine, m-phenylenediamine , 5-amino-o-cresol, 2,4-diaminophenol, catechol, 3,3'-iminodiphenol, pyrogallol, N-phenyl-p-phenylenediamine, 5- (2-hydroxyethylamino) 2-methyl Phenol, 2,4-diaminophenoxyethanol, 4,4-diaminodiphenylamine, o-chloro-p-phenylenediamine, p
-Methylaminophenol, toluene-3,4-diamine, toluene-2,5-diamine, p-aminophenylsulfamic acid, α-naphthol, diphenylamine, 1,5-dihydroxynaphthalene, 2,6-diaminopyridine, hydroquinone, There is phloroglucin. As the oxidizing agent, hydrogen peroxide, sodium percarbonate, sodium perborate and the like have been used for a long time. However, in recent years, peroxidase enzymes have been used to reduce the amount of skin-irritating oxidizing agents (Japanese Patent Application Laid-Open No. 47-10400), and uricase, glucose oxidase, etc. have recently been used in place of these oxidizing agents. A new method has been disclosed in which oxygen in the air is activated by an oxidizing enzyme such as an organic acid oxidase such as a sugar oxidase or a lactate oxidase to utilize the oxidizing power (JP-A-7-45385, JP-A-8-85)
No. 217652, JP-A-11-21214 and JP-A-11-21215). The group of enzymes having oxidizing power as described above is referred to as "oxidase" in the present specification, and particularly preferable oxidase in the present invention is selected from the group consisting of peroxidase, uricase, glucose oxidase, and lactate oxidase.

Although utilizing the enzymatic action as described above is a useful method, it has not been possible to avoid that expensive enzymes are washed away by hair washing in the above-mentioned conventional method. Therefore, if these enzymes are firmly immobilized on the hair using the single-chain antibody of the present invention, it is possible to reduce the outflow due to hair washing only by applying an oxidizing agent, and to maintain the hair dyeing state for a long period of time. Can be maintained.

When utilizing a metal chelate compound-amino acid affinity bond as a pair of a ligand and a binding amino acid sequence, the enzyme is modified with a chelate compound ligand,
Subsequently, the enzyme may be bound to the single-chain antibody fusion that specifically binds to the hair by forming a metal chelate compound-amino acid affinity bond by chelate-bonding the ligand with the metal. When performing hair dyeing, first, a fusion containing a single-chain antibody is applied to hair and absorbed. For this purpose, a pretreatment agent prepared by mixing a fusion containing a single-chain antibody with, for example, a rinse or a hair cream may be used. Then, the complex of the enzyme modified with the chelate compound is applied to the hair so as to bind to the single-chain antibody, but also in this case, it is necessary to use a compound formulated in a rinse, a hair cream, a spray, a shampoo, etc. preferable. Of course, it is also possible to use a mixture in which a fusion containing a single-chain antibody and a complex containing an enzyme are mixed in advance and bound. Finally, hair dyeing can be completed by applying or spraying an oxidation dye. It is necessary to mix an oxidizing agent and its substrate appropriate for the enzyme with the oxidation dye.
When peroxidase is used as the enzyme, hydrogen peroxide is used as the oxidizing agent, and when uricase is used as the enzyme, uric acid is used as the oxidizing agent. For other oxidases, the corresponding sugars and organic acids are selected.

Similarly, when a ligand other than a chelate compound is used, the cosmetic (enzyme) may be modified with each ligand.

(B) The direct hair dyeing method is a method in which a dye is directly bonded to hair. Dyes are broadly classified into organic tar dyes, inorganic pigments and natural dyes, some of which are licensed as cosmetic dyes. Methods for binding these dyes to the hair include methods such as physical adsorption of the dye and adhesion with a paste, and are commercially available as cosmetics such as hair manicure and hair mascara. However, these methods do not allow the dye to be retained on the hair for a long time, and are easily washed away by washing the hair. The invention relating to a method using an antibody to solve this drawback is disclosed in the above several publications, regardless of whether the antibody is directly bound to a pigment or a method via a polymer carrier, As described above, the chemical modification of antibodies has various problems.

In a preferred embodiment of the present invention where direct hair dyeing is carried out, pigments and lakes which are insoluble in water and oil,
The natural dye is chemically modified with the above-described chelating compound ligand to form a complex, and then is chelated to a metal. When this is applied to the hair to which the fusion containing the single-chain antibody has been applied, the molecule of the metal chelate compound bound to the dye instantaneously binds to the single-chain antibody on the hair. In order to apply the fusion containing the single-chain antibody and the chelate compound / dye complex, those which are appropriately added to rinse, spray, cream, shampoo and the like may be used as in (a) above.

Examples of pigments insoluble in water and oil include black titanium, red iron oxide, iron oxide, carbon black, squid and various oxidized polymers. Among them, especially when the dye has an organic functional group, the chelate compound can be easily bonded by various chemical reactions. When it does not have an organic functional group such as an inorganic pigment, an organic functional group such as an amino group, a carboxyl group, an aldehyde group, a hydroxyl group, or a thiol group is introduced by surface treatment such as treatment with a coupling agent or a silicon compound. do it.
Examples of the coupling agent include a silane coupling agent, a titanate coupling agent, an aluminum coupling agent, a zircoaluminate coupling agent, and the like.The silicon compound treatment includes amino-modified silicon,
Carboxy-modified silicon or the like is used. In order to bond the organic functional group to the ligand, glutaraldehyde is used when the functional group is an amino group, and 1-ethyl-3- (3) which is a water-soluble carbodiimide when the functional group is a carboxyl group.
-Dimethylaminopropyl) carbodiimide (EDC)
Is preferably used. When the organic functional group is a thiol group, N-succinimidyl-3- (2-pyridyldithio) -propionate can be used.

In particular, when a water-soluble dye, ie, a dye, is used as a cosmetic, a large amount of the dye is added to a water-soluble polymer carrier or an insoluble polymer carrier, such as polysaccharides, proteins, polyvinyl alcohol, polyethylene glycol, and various synthetic plastics. In this case, the amount of the binding dye per single-chain antibody molecule is increased when a chelate compound is further bound, and as a result, the hair dyeing effect is improved. Thus, the method via a carrier can be applied to an insoluble dye.

Similarly, when a ligand other than the metal chelate compound is used, the cosmetic (dye) may be modified with each ligand.

Of course, these methods can also be used for dyeing eyelashes, eyebrows, nails and the like in addition to head hair.

(2) Makeup of skin Cosmetics applied to skin are basic cosmetics, foundation, pressed powder, blusher, eyeshadow, eyeliner, lipstick, cosmetic pencil, sunscreen, etc., and the component is water. In addition to oil-insoluble fine powders and polymer compounds, they are often W / O or O / W emulsion fine particles. However, these are difficult to adsorb to the skin, and the makeup breaks down over time. Therefore, the principle of the present invention can also be used to make these more firmly adsorb to the skin with antibodies. The method is described in detail below.

(A) When the cosmetic, which is the main component of the cosmetic, is a fine powder or a polymer compound insoluble in water or oil, a metal chelate compound, cellulose, chitin, or amylose is used as a ligand, and Alternatively, the cosmetic agent and the ligand are chemically bonded via a linker. Various methods are conceivable for the chemical reaction according to the method (1) above, but since the reaction substrate does not have a biological activity such as an antibody or an enzyme, a relatively radical reaction is also possible. Since the choice of chemical reaction to be performed for preparation is wide, the present invention can be said to be cost-effective.

[0062] Cosmetic agents include talc, muscovite, synthetic mica, phlogopite, which are conventionally blended in makeup cosmetics for the skin.
Red mica, biotite, lithia mica, sericite, permiculite, kaolin, titanium dioxide, titanium oxide coated mica, titanium oxide coated talc, titanium oxide coated bismuth oxychloride, bismuth oxychloride, fish scale foil, colored titanium oxide coated mica Metal powder pigments such as pearl pigments, aluminum powder, copper powder, etc .; inorganic red pigments such as iron oxide (iron oxide) and iron titanate; inorganic brown pigments such as γ-iron oxide; and inorganic yellow pigments such as yellow iron oxide and loess. , Black iron oxide, inorganic black pigments such as carbon black, mango violet, inorganic violet pigments such as cobalt violet,
Inorganic green pigments such as chromium oxide, chromium hydroxide, and cobalt titanate; inorganic blue pigments such as ultramarine and navy blue; zinc white;
Bentonite, barium sulfate, metal soap, sodium silicate, aluminum silicate, calcium silicate, barium silicate, magnesium silicate, strontium silicate, metal tungstate, calcium carbonate, magnesium carbonate, chromium oxide, chromium hydroxide, Carbon black, alumina, hydroxyapatite, boron nitride, silica,
Nylon powder, zeolite, benzoguanamine powder, ethylene tetrafluoride powder, polyamide powder,
Polyester powder, polyethylene powder, polypropylene powder, polystyrene powder, cellulose powder, Red No. 201, Red No. 202, Red 204
No., Red No. 205, Red No. 220, Red No. 226, Red No. 228, Red No. 405, Orange No. 203, Orange No. 204
Organic pigments such as No. 205, Yellow No. 205, Yellow No. 401 and Blue No. 404, Red No. 3, Red No. 104, Red No. 106,
Red 227, Red 230, Red 401, Red 50
No. 5, orange 205, yellow 4, yellow 5, yellow 202
No. 203, yellow No. 203, green No. 3 and blue No. 1 organic pigments such as zirconium, barium or aluminum lake, and natural pigments such as chlorophyll and β-carotene.

In the case of sunscreen cosmetics, in addition to titanium oxide, zinc oxide and cerium oxide, an appropriate amount of an ultraviolet absorber, for example, 2-ethylhexyl paramethoxycinnamate (octyl), 2-ethylhexyl paradimethylaminobenzoate (octyl) ), Oxybenzone (benzophenone 3), 2-ethylhexyl salicylate (octyl), 4-te
An appropriate amount of rt-butyl-4-methoxy-benzoylmethane or the like is used as a cosmetic to protect the skin from ultraviolet rays.

The fusion containing the single-chain antibody may be applied in advance by applying an appropriate amount to a makeup base such as a lotion, an emulsion, a cream, or the like, or may be mixed with the complex of the above-mentioned cosmetic agent. Is also good.

(B) In an emulsion-based cosmetic, the outer surface is covered with a hydrophilic group in an O / W emulsion and a hydrophobic group in a W / O emulsion. That is, in order to constitute the emulsion particles, the component only needs to have a hydrophilic group and a hydrophobic group. If not, it may be so modified by a surfactant.
However, many cosmetics, such as foundations, various creams, and lipsticks, are prepared as emulsions, and there are many types of cosmetics as constituents thereof. Therefore, it is not realistic to chemically bond a ligand such as a chelate compound to all the constituent components. Therefore, various ligands such as chelate compounds are prepared so as to be constituents of the emulsion particles. Hereinafter, a method of preparing an emulsion using a chelate compound as a ligand will be described.

In the case of an O / W emulsion: at one end of the ligand of the chelate compound, for example, a carboxyl group capable of chelating with a metal and having a hydrophilic property is present. At the other end of the ligand, there are amino groups, imino groups, and the like that can be bonded to the carrier, and these groups are bonded to an aliphatic polymer, various cyclic polymers, and the like, so that the chelate compound becomes a hydrophilic group. And a hydrophobic group. If this is added as one of the components during the emulsification,
Emulsion particles having a carboxyl group capable of chelating with a metal on the outer surface of the obtained particles are obtained. If the skin is coated with a fusion containing a single-chain antibody in advance, the emulsion particles will bind to the antibody. Of course, the fusion and the emulsion particles may be mixed and then applied to the skin. When a hydrophilic ligand such as cellulose, chitin, or amylose is used instead of a chelate compound, no special treatment is required, and they may be added as they are.

In the case of a W / O emulsion: an amino group or imino group contained in a ligand of a chelate compound is bonded to a polymer compound having strong hydrophilicity. Examples of the polymer compound include sugars, synthetic polyols, sugar alcohols, polysulfate compounds, polyphosphate compounds, and the like. The other end of the ligand modified with such a hydrophilic group also contains a hydrophilic carboxyl group, which becomes hydrophobic under acidic conditions. Therefore, a desired emulsion can be formed by emulsifying under acidic conditions.
The method of binding to the antibody is performed in the same manner as in the preparation of the O / W emulsion.

As the oil component for preparing the emulsion,
Squalane, liquid paraffin, vaseline, microcrystalline wax, ozokerite, ceresin, myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cetyl alcohol, hexadecyl alcohol, oleyl alcohol, cetyl-2-ethylhexanoate, 2 -Ethylhexyl palmitate, 2-octyldodecyl myristate, 2-octyldodecyl gum ester, neopentyl glycol-2-ethylhexanate, glyceride triisooctanoate, 2-octyldodecyl oleate, isopropyl myristate, glyceride triisostearate, Tricoconut oil fatty acid glyceride, olive oil, avocado oil, beeswax, myristyl myristate, mink oil, lanolin, dimethylpolysiloxane, ring Dimethyl polysiloxane, methylphenyl polysiloxane, silicone resin, polyether-modified silicone, various hydrocarbons, such as amino-modified silicones, higher fatty acids, fats and oils, esters, higher alcohols, waxes, silicone oils, and the like.

As described above, the fusion of the cosmetic of the present invention with a single-chain antibody and a ligand or an amino acid sequence capable of binding to a cosmetic agent, the ligand and the cosmetic agent are each supplied as a single composition. Or may be supplied as an appropriately formulated composition. The mixing ratio is not particularly limited, and should be appropriately adjusted in consideration of the antibody titer of the single-chain antibody, the affinity of the amino acid sequence for the ligand, the amount of the cosmetic agent bound to the ligand, and the like. is there.

The cosmetics for hair dyeing and makeup described above may further include, as optional components, a surfactant, a humectant, a lower alcohol, a thickener, an ultraviolet absorber, a fragrance, an antioxidant, and an antiseptic. An appropriate amount of a fungicide or the like may be added as necessary.

Hereinafter, examples using metal-amino acid affinity bonds will be described as typical examples, but it is needless to say that the present invention should not be construed as being limited to such examples. All percentages are based on mass / volume.

Example 1 Hair dyeing method using anti-hair single-chain antibody-peroxidase conjugate-1 Preparation of Anti-Hair Antibody First, an antibody against hair was prepared by the cell fusion method of Kohler and Milstein (supra). That is, 10 mg of human hair cut to a length of about 1 mm was administered to the peritoneal cavity of a mouse about four times every two weeks to immunize. When the antibody titer against the hair of the mouse serum increased, the spleen cells were removed,
Hybridoma populations with mouse myeloma (NS1) cells were obtained by cell fusion with PEG. This was cultured and selected to produce a monoclonal antibody. Also,
_VH and _{VL of} antibody from hybridoma immediately after cell fusion
The region gene was extracted by PCR, and VH and V
L was ligated to prepare a single chain antibody (sFv) gene. His-His-His-His-His-His (SEQ ID NO: 27) was bound to the C-terminal as an amino acid sequence that binds to a metal chelate compound as a selectable marker for examining the presence of the antibody. Next, each sFv gene was inserted into a vector as shown in FIG. 1 and transformed into E. coli TG1 by electroporation. Each of the obtained Escherichia coli was cultured in a 2 × YT-AI medium (17% bacto-tryptone, 1%).
0% Bacto-yeast extract, 5% NaC
1, 2% glucose, 100 μg / ml ampicillin,
The cells were cultured in 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and the culture solution was cloned based on the ELISA method coated with keratin derived from hair. From several clones
SFv having the gene sequence shown in SEQ ID NO: 1 was selected based on the stability and binding properties of Fv.

2. Purification of fusion containing sFv The clone selected in the above was cultured overnight in a 2 × YT-AI medium, and centrifuged at 5,000 × g for 10 minutes to remove the precipitate, thereby obtaining a supernatant containing the sFv fusion. To this supernatant, imidazole was added to a final concentration of 10 mM, and 1
50 mM phosphate buffer containing 0 mM imidazole (pH
8.0) Ni-NTA column (manufactured by QIAGEN,
Germany). After the unadsorbed protein was washed away with 50 mM phosphate buffer (pH 8.0) containing 20 mM imidazole, the sFv fusion was eluted with the same buffer containing 250 mM imidazole, and this fraction was collected.

3. Method for measuring antibody titer by ELISA 5 g of hair was finely chopped, and 0.2 M Tris-HCl buffer /
One liter of 8M urea / 0.2M β-mercaptoethanol was added, and the mixture was allowed to stand at 50 ° C. overnight. After grinding in a mortar to make hair even finer, 12,000 rpm
The mixture was centrifuged at for 30 minutes to recover the supernatant. The collected supernatant was dialyzed against pure water to obtain a keratin solution derived from hair. The obtained keratin solution was adjusted to 100 μg / ml, and ELISA was performed.
100 μl was placed in the wells of the plate and incubated at 4 ° C. overnight. After blocking the wells with PBS containing 3% skim milk, the wells were purified by the method described above.
Fv (50 μg / ml) was applied at 50 μl, and 37 ° C.
For 1 hour. Then 0.05% Tween2
After washing the plate with PBS containing 0, horseradish peroxidase (HRP) labeled Ni-NTA
(Manufactured by QIAGEN, Germany) was added, and the mixture was incubated at 37 ° C. for 1 hour. The plate was washed with PBS containing 0.05% Tween 20, then TMB, a substrate of HRP, was added, reacted at room temperature for 15 minutes, and the absorbance at 450 nm was measured.

4. Preparation of hair dye containing sFv fusion and peroxidase Fusion containing sFv purified in step (50 μg / ml)
With horseradish peroxidase and Ni-NT
A complex (Ni-NTA HRP Conjugate, QIAGEN
Germany) to a final concentration of 25 μg / ml.
After incubation at 37 ° C. for 1 hour, a conjugate containing the single-chain antibody bound to peroxidase via the chelating compound was prepared.

5. Hair dyeing test 4. Human gray hair having a length of 5 cm was added to 2 ml of each of the aqueous solutions of the antibody-peroxidase conjugates a to d shown in Table 1 with or without the antibody-peroxidase conjugate (peroxidase titer 100 mU / ml) obtained in the above. 0.5 g of the bundle was immersed for 1 hour. Then, 0.05% Tween20
After washing with PBS containing 5 times, the sample was immersed in a dye solution having the composition shown in Table 1 for 30 minutes.

The judgment of hair dye was divided into three stages according to the degree of coloring. For non-dyed hair, 3: markedly dyed; 2: clearly dyed; 1: slightly dyed;
0: No difference.

[0078]

[Table 1]

As is evident from the results shown in Table 1, good hair dyeing results were obtained even with the use of hydrogen peroxide at a concentration about 1/10 lower than usual, and the fusion of the single chain antibody of the present invention was obtained. It has also been found that hair dyes containing are excellent in terms of safety and hypoallergenicity.

The hair dyed here did not fade even after shampooing five times, suggesting that the use of a single-chain antibody would significantly improve the fixation of the dye to the hair. .

[Example 2] Direct hair dyeing method Preparation of Dye / Ni-NTA Complex A dye / Ni-NTA complex was prepared by the following method.

Ni-NTA agarose (manufactured by QIAGEN, Ger
many) 1 ml was ground in a mortar. 20 mg of Cibacron Blue (manufactured by SIGMA, USA) and 2 ml of pure water were added, shaken at 60 ° C. for 30 minutes, and 0.1 g of NaCl was further added.
The mixture was shaken at 60 ° C. for 6 minutes. After neutralization with Na ₂ CO ₃ , the mixture was left at 80 ° C. for 2 hours. It was dialyzed overnight against water containing 1% NiSO4, and further dialyzed overnight against pure water.

2. Preparation of hair dye containing sFv fusion and dye / Ni-NTA complex Cibacron Blue / Ni-NTA
1. The above [Example 1] was added to 1 ml of the complex. 1 ml of the same sFv purified as described above was added, and the mixture was incubated at 37 ° C. for 1 hour to prepare a hair dye containing a single-chain antibody bound to a dye (Cibacron Blue) via a chelate compound.

3. Hair dyeing test 2 above. 0.5 g of a human gray hair bundle having a length of 5 cm was immersed in 2 ml of the aqueous solution a shown in Table 2 containing the dye-Ni-NTA antibody complex obtained in the above for 1 hour. Then 0.05
Washed 5 times with PBS containing% Tween20. As a control, the same hair dyeing test was performed on the solutions having the compositions shown in b to d in Table 2.

The determination of hair dye was divided into three stages according to the degree of coloring. For non-dyed hair, 3: markedly dyed; 2: clearly dyed; 1: slightly dyed;
0: No difference.

[0086]

[Table 2]

As shown in the results of Table 2, good dyeing results were obtained with the hair dye of the present invention.

[Example 3] Hair dyeing method 2 using anti-hair single-chain antibody-peroxidase conjugate-2 According to the composition shown in Tables 3a to 3g, 4. SFv fusion and peroxidase Ni as described in
Using the conjugate with the NTA complex, hair dyes of various compositions were further prepared.

At this time, the sFv fusion was prepared according to 1. And 2. The one prepared by the procedure described in the paragraph and the one which was further concentrated 200 times by ultrafiltration (filtration membrane: manufactured by Millipore, nitrocellulose membrane) were used.

About 2 g of a 3 cm long human gray hair bundle was immersed in a mixed solution of the prepared sFv fusion and peroxidase / Ni-NTA complex for 1 hour at room temperature. At this time, only the sample “g” was treated at room temperature for 10 minutes. Then
After washing 5 times with PBS containing 0.05% Tween 20, each was immersed in the dye solution shown in the lower part at room temperature for 30 minutes.
Thereafter, the plate was washed five times with PBS containing 0.05% Tween20.

Next, Example 1 was applied to all hair dye samples.
The determination was performed in the same manner as described in (1). The results are shown in Table 3 below.

[0092]

[Table 3]

As a result, in the hair dyeing method using the single-chain antibody fusion of the present invention, a complex of Ni-NTA and peroxidase was used in an amount of 0.02 to 2% by volume, preferably p-phenylenediamine as a substrate. It became clear that a good hair dyeing state was obtained when 1-2% by volume was blended.

The hair dyed here did not fade even after shampooing five times, suggesting good fixation of the dye to the hair.

[Example 4] Hair dyeing method using only Ni-NTA / peroxidase complex According to the compositions shown in Tables a to i, Ni-NTA, which is a ligand having a binding property to a subject to be applied with makeup, is used. A hair dye to be used in combination with peroxidase was prepared. In advance, about 3 cm was added to the solution shown in Table 4
Approximately 2 g of a human gray hair bundle having a length of was soaked at room temperature for 1 hour.
At this time, only the sample “g” was performed at 37 ° C. Then, after washing 5 times with PBS containing 0.05% Tween 20,
Each was immersed in the dye solution shown in the lower part at room temperature for 30 minutes. Thereafter, the plate was washed five times with PBS containing 0.05% Tween20.

Next, Example 1 was applied to all the hair dye samples.
The determination was performed in the same manner as described in (1). The results are shown in Table 4 below.

[0097]

[Table 4]

As a result, in the hair dyeing method utilizing the oxidation of p-phenylenediamine, the hair dye was treated with the Ni-NTA / peroxidase complex in advance with a smaller amount of hydrogen peroxide than conventionally used. It turned out that this could be achieved.

The hair dyed here did not fade even after shampooing five times, indicating good fixability of the pigment to the hair.

[Example 5] Moisture lotion Preparation of IDA · agarose complex agarose 20g and 2M NaOH 20 ml, of NaBH ₄ epichlorohydrin and 75mg of 2ml were mixed and stirred at room temperature for 2 hours. Then add 2M NaOH to 2
0 ml and 10 ml of epichlorohydrin were added and allowed to react overnight. After washing the obtained gel with water, 2M Na ₂ CO ₃
Dissolved in 50 ml. 5 g IDA and 0.06 g Na
BH ₄ was added and stirred at 60 ° C. overnight. After washing with water and washing with dilute acetic acid, wash with water until the pH becomes neutral.
An agarose complex was obtained.

2. Preparation of IDA-agarose-sFv complex [Example 1] 2. 2 ml of the same sFv fusion as purified in 1. IDA agarose 2m prepared in
was added and incubated at 37 ° C. for 1 hour to prepare a single-chain antibody-agarose complex.

3. Measurement of the water content of the stratum corneum In order to verify the water retention by the single-chain antibody-agarose complex, the water content was determined by measuring the water content of the stratum corneum.

The water content of the stratum corneum was measured by a capacitance conductance meter (IBS) according to the method of Tagami et al. (Fragrance Journal Extra Edition, No. 9 pp. 19-24 (1988)).
(SKICOS301). At the time of measurement, the measurement site was washed with warm water of 37 ° C. for 30 seconds, and then
Five measurements were made at 0 ° C. and 50% relative humidity, and the average was taken as the measured value. The result is shown in FIG.

Here, Sample 1 is the result using the single-chain antibody-agarose complex, Sample 2 is the result using only IDA-agarose, and Sample 3 is the result using only water.

As shown in FIG. 2, when the single-chain antibody-agarose complex was applied, the water retention was superior to the other cases, and it was clear that this effect could be maintained for a long time.

[0106]

As described above, according to the present invention,
Cosmetic can be stably adsorbed on the skin, hair, nails, etc., and is extremely safe for the living body. It can be supplied at a low cost because it does not waste expensive antibodies etc. Is done.

Further, according to the present invention, it is possible to reduce the amount of reagents that may be harmful to the living body, such as a coloring substrate such as hydrogen peroxide and phenylenediamine in hair dyes, Can be reduced to minimize any adverse effects that may be caused.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a vector containing a nucleotide sequence A7H encoding a single-chain antibody suitably used in the present invention.

FIG. 2 is a graph showing the effect of the moisture lotion according to one embodiment of the present invention on the water content of the stratum corneum, as a function of the skin conductance over time.

[Procedure amendment]

[Submission Date] July 19, 2002 (2002.7.1)
9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

[Document Name] Statement

Patent application title: Method for enhancing adsorption of cosmetic agent and cosmetic

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for firmly adsorbing a cosmetic agent on skin, hair, nails and the like, and a cosmetic material therefor, and more particularly to hair such as head hair, eyebrows and eyelashes.
These cosmetics are applied using a method in which a protein and a cosmetic agent that bind to an object such as skin or nail are bound by metal-amino acid affinity or based on an interaction between a ligand and a corresponding binding protein. The present invention relates to a method for stably adsorbing the cosmetic agent on a subject and a cosmetic for the method.

[0002]

2. Description of the Related Art For the purpose of dyeing hair or nails, several cosmetics have been developed in which antibodies against these constituents are bound to a cosmetic agent and the binding properties of the antibodies are utilized (Japanese Patent Laid-Open Publication No. Heisei (Kokai) No. Heisei 9 (1999)). 4-29912; JP-A-6-2279
No. 55; JP-A-7-69832; and JP-A-8-1
43431 publications). Most of them apply a cosmetic / antibody complex obtained by directly binding an antibody to a pigment, a dye, or a nail agent as a cosmetic.

[0003] Only JP-A-8-143431 discloses a method utilizing a specific and strong binding force between avidin and biotin. That is, a complex of an antibody and biotin (or avidin), a complex of a cosmetic agent and avidin (or biotin) are separately prepared, the antibody complex is first applied to the skin, and then the cosmetic complex is applied. It is. Furthermore, in order to make a cosmetic agent specifically bind to hair, a protein serving as an antigen is used as a pretreatment agent.

[0004]

However, in any of these conventional methods, both the antibody and the cosmetic must be chemically modified in order to bind the antibody to the cosmetic. Therefore, there were drawbacks such as the binding site of the antibody was damaged by the chemical modification, and the desired amount of the antibody could not be bound to the cosmetic agent. In addition, the cost for chemical modification was also a problem.

[0005] In order to produce antibodies in the above-mentioned conventional techniques, a method of immunizing an animal and purifying it from serum, or a method of producing a monoclonal antibody-producing cell by a cell fusion method and producing it in an animal or a cell is used. Is an industrial production method, but its cost is high.

As described above, in the above-mentioned prior art,
(1) When an antibody is directly bound to a cosmetic agent, an attempt to bind a sufficient amount of the cosmetic agent to the antibody results in a decrease in the activity of the antibody; (2) proteins such as the antibody and avidin are unstable In addition, the method is expensive, and the yield is not stable due to chemical modification, and the cost is not favorable; and (3) the antibody must be purified as much as possible in order to chemically modify the antibody, and the cost There were problems such as increase.

[0007] In view of these drawbacks in the prior art, the present inventors do not require a special pretreatment agent for hair, skin, nails and the like, and stably and at low cost hair dyeing, makeup and skin conditioning. It is an object of the present invention to provide a method for applying cosmetics such as nails and beautiful nails, and a cosmetic therefor.

[0008]

Means for Solving the Problems The present invention has been intensively studied to achieve the above object, and as a result, it has been found that the N-terminal side of a single-chain antibody having a binding property to an object such as skin, hair, nail, etc. On the end side or inside, by a fusion that incorporates an amino acid sequence capable of binding to a ligand, and a cosmetic used in combination with a ligand and a cosmetic agent, the interaction between the ligand and the amino acid sequence capable of binding to it is The present invention has been completed by finding that a cosmetic agent can be stably and specifically adsorbed.

Here, a pair of a ligand and an amino acid sequence capable of binding to the ligand are preferably used as a metal chelate compound, an amino acid sequence capable of binding to such a compound via a metal, and cellulose. , Chitin, maltose and the like, or peptides such as glutathione and their analogs, and a pair of a ligand-binding protein or an anti-ligand antibody having affinity with these.

[0010] In the cosmetic of the present invention, a gene encoding an amino acid sequence capable of specifically affinity-binding with a ligand such as a metal, the above-mentioned polymer compound or peptide can be used as a simple substance having a binding property to an object such as skin or hair. N of chain antibody
A fusion may be prepared by preparing a vector that has been integrated at the terminal side, C-terminal side, or inside, growing a host cell transformed or transfected with such a vector, and expressing the protein. On the other hand, a metal chelate functional group capable of holding a transition metal or a ligand corresponding to each protein, that is, chitin, cellulose, amylose, glutathione, or the like is chemically or physically bonded to the surface of the cosmetic substance. Good.

Further, the present invention is characterized in that a fusion of a single-chain antibody having a binding property to a subject to be applied with an amino acid sequence capable of binding to a cosmetic, and a cosmetic are used in combination. We also provide cosmetics. Even with such a cosmetic, the cosmetic can be stably adsorbed to the target. In this case, the amino acid sequence may be a sequence constituting an anti-cosmetic agent antibody or a cosmetic-binding protein. Also, the fusion used in this cosmetic may be prepared by protein expression using the above-described genetic recombination method.

[0012] The above-mentioned single-chain antibody having a binding property to a subject to be cosmetically applied is preferably an anti-keratin antibody.
Further, such a single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, that is, the amino acid encoded by the polynucleotide shown in SEQ ID NO: 1 or an analog thereof containing a degenerate codon sequence in the polynucleotide. It is preferable to have an arrangement, and it is particularly suitably used for hair dyeing.

[0013] The present invention further provides a cosmetic comprising a combination of a ligand such as a metal chelate compound and a cosmetic agent, which has a binding property to an object to be cosmetically applied. According to the cosmetics, although the specificity and the stability are somewhat inferior, it is possible to adsorb a good cosmetic agent to a target site of interest. Further, a nickel chelate compound of nitrilotriacetic acid (Ni-NTA) is used as the metal chelate compound, and a complex of the Ni-NTA and one or more oxidases selected from the group consisting of peroxidase, uricase, glucose oxidase and lactate oxidase. Can be mentioned as preferred cosmetics, and among these oxidases, peroxidase is particularly preferred.
Hydrogen peroxide can be generated by the activity of the oxidase described above, whereby the dye substrate is oxidized and color is formed. Peroxidase can be used in combination with other oxidases as a source of hydrogen peroxide.

Further, according to the present invention, there is provided a method for enhancing the adsorption of a cosmetic agent, which comprises a fusion of a single-chain antibody having a binding property to an object to be applied with an amino acid sequence capable of binding to a ligand, and a ligand and a cosmetic agent A fusion of a single-chain antibody capable of binding to the cosmetic subject and an amino acid sequence capable of binding to the cosmetic, and a cosmetic; or a ligand and a cosmetic capable of binding to the cosmetic subject. Also provided is a method, which comprises coating an object. According to these methods, it is possible to adsorb a cosmetic agent to a target relatively stably for a long period of time, and the safety for a living body is extremely high.

Also, according to the present invention, a polynucleotide which has a nucleotide sequence shown in SEQ ID NO: 1 and which encodes a single-chain antibody that binds to skin and / or hair, which can be suitably used for formulation into cosmetics. And a single-chain antibody that binds to skin and / or hair, having an analog thereof and the amino acid sequence shown in SEQ ID NO: 2.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, makeup is applied to various parts of a body to which makeup is generally applied, such as hair, for example, hair, eyebrows, eyelashes, skin, and nails.

Accordingly, cosmetics include hair dyes, mascaras, eyebrows, foundations, pressed powders, blushers, eye shadows, eyeliners, lipsticks, cosmetic pencils, nail polish, etc., as well as sunscreen cosmetics. Are particularly limited to solids, powders (which may or may not be compressed), fluids (aqueous solutions, suspensions including emulsions, (which may include organic solvents such as ethanol)), and creams, mousses, etc. Never.

[0018] One of the main components of the cosmetic of the present invention,
A fusion comprising a single-chain antibody having a binding property to a target such as skin or hair and an amino acid sequence capable of binding to a ligand has a binding property to a target to which makeup is applied when the protein takes a tertiary structure ( (I.e., immunological binding by a single-chain antibody) and an amino acid sequence capable of binding to a ligand are designed so that they can form a corresponding binding pair without steric hindrance. Is preferred.

Antibodies, which are preferably used in the present invention and which can specifically bind to a subject to be cosmetically applied, are obtained by extracting a part of an L chain and an H chain of an antibody gene by genetic engineering, and extracting the part from the gene. It is a protein obtained by ligating and then expressing. This is referred to as a single-chain antibody (sFv) for convenience, and is also referred to herein as a single-chain antibody,
The method of ligation does not exist in natural antibodies.
Furthermore, the case where each of the L chain and the H chain is used alone is also included. The single-chain antibody does not include an antibody fragment obtained by a conventional technique such as proteolytic enzyme or reduction of the antibody. The advantages of using this single-chain antibody are that the molecular weight is as low as 1/6 or less of the original antibody and that it does not contain sugar chains, so it can be inexpensively used by using microorganisms and plants. It can be mass-produced. Furthermore, since the molecules are small, they can reach the details of the skin and hair, so that even with a small amount of use, they can be applied to the object to be applied and exhibit a desirable adsorption effect.

Next, a method for obtaining a single-chain antibody suitably used in the present invention will be described. First, human hair, skin, nails, etc. are minced and then, if necessary, hydrolyzed (acid hydrolysis, alkali hydrolysis, enzyme hydrolysis, etc.), extraction,
The antigen that has been subjected to modification (for example, carboxymethylation) or the like is administered several times to cows, horses, sheep, goats, rabbits, rats, mice, and the like to immunize. The antigen is preferably keratin or a hydrolyzate thereof, and keratin purified by a conventional method may be used. After immunization, highly proliferating cells, such as spleen cells and myeloma cells, are isolated using standard methods (Kohler G and Milstein C, Biotechnology , 2
4: 524 (1992)) to prepare a hybridoma that produces a monoclonal antibody by cell fusion. Next, for the preparation of a single-chain antibody, the genes of the V _H (variable heavy chain) and _VL (variable light chain) regions of the antibody were removed from the hybridoma immediately after cell fusion using PCR, and the V
By linking _H and _VL , a single-chain antibody (sFv) gene can be prepared. This gene is inserted into an appropriate vector, transformed into Escherichia coli (for example, E. coli TG1) by a conventional method such as electroporation, and the resulting Escherichia coli is cultured. The gene encoding the single-chain antibody may be cloned by an ELISA method, which is carried out by coating the DNA. Furthermore, a preferred clone can be selected from several clones based on the stability and binding strength of the single-chain antibody. The selected single-chain antibody clone is recovered from the culture supernatant by a conventional method using centrifugation or the like, but may be purified if necessary.

[0021] The ELISA method used for cloning, selection and immunopotency measurement of a single-chain antibody may be performed, for example, according to the following procedure. That is, ELIS
100 μl of an antigen solution (100 μg / ml) such as keratin derived from hair is added to the well of the plate for A and incubated at 4 ° C. overnight. Next, after blocking with phosphate buffered saline (PBS) containing 3% skim milk, 50 μl of an antibody-containing solution is added, and the mixture is incubated at 37 ° C. for 1 hour. After thoroughly washing the wells with PBS containing 0.05% Tween 20, a secondary antibody labeled with horseradish peroxidase (HRP) was added, followed by 3 more days.
Incubate at 7 ° C for 1 hour. 0.05% Tween20
After washing the wells with PBS containing 3,3 ′, 5,5′-tetramethylbenzidine (TM) which is a substrate for peroxidase.
B) is added and reacted at room temperature for 15 minutes, and the absorbance at 450 nm is measured.

The single-chain antibody thus obtained is used for preparing a fusion in the present invention. The molecular weight of the single-chain antibody is preferably from 300 to 80,000, more preferably from 20,000 to 40,000, of which the L chain is preferably from 0 to 23,000, more preferably from 1 to 23,000.
It corresponds to about 1,000, and the H chain is preferably 300 to 5
It is good to correspond to 0000, more preferably about 15,000.

Particularly preferred single chain antibodies include SEQ ID NO:
SEQ ID NO: encoded by the nucleotide sequence represented by 1 or an analog thereof (a sequence containing a degenerate codon)
A single-chain antibody having the amino acid sequence of 2. This single-chain antibody can selectively bind to hair and skin, provides stable adsorption of the cosmetic agent to hair and skin, and has a molecular weight of about 27,000 (including L chain: about 1
2,000, heavy chain: about 13,000), so that the hair has good penetration into the details. The outline of the vector having the nucleotide sequence encoding the single-chain antibody useful in the present invention shown in SEQ ID NO: 1 is shown in FIG.

In order to bind the single-chain antibody constituting the fusion with the cosmetic agent, specific affinity, that is, affinity binding is used instead of performing direct modification or the like by a chemical synthesis method. As a pair that provides affinity, for example,
Specific amino acid sequences include metals (chelate compounds), glutathione-binding proteins such as glutathione S-transferase and glutathione, cellulose-binding proteins and cellulose, chitin-binding proteins and chitin, maltose-binding proteins and amylose, and the like. Furthermore, a cosmetic agent and its binding protein may be used without using a metal chelate compound or a ligand as described above. Further, the binding protein may be an antibody having various ligands as antigens. The advantage of binding between a single-chain antibody and a cosmetic agent using affinity is that amino acids and proteins involved in this binding can be expressed together with the antibody by gene recombination and produced as a single protein fusion. The need to purify and chemically modify the antibody as in. Hereinafter, each affinity combination will be described.

(1) The affinity between a metal and a specific amino acid sequence forms the principle of a method for purifying an expressed recombinant protein. This purification method is based on immobilized metal ion affinity chromatography (IMAC, Immobilized metal ion chromatography).
It is called ion affinity chromatography (Bio / Te
chnology, Vol. 9, 151-156, 1991). The present invention utilizes therein the principle of IMAC represented by metal-histidine bond, that is, specific binding of a specific amino acid in a protein (polypeptide) molecule to a chelate compound via a metal. Nevertheless, there is no example in which such binding properties have been applied to cosmetics. According to this method, since no chemical modification of the antibody molecule is required, the binding to the antigen, that is, the object to be cosmeticized is not impaired, and the metal-amino acid bond is specific. The complex of the chelating compound and the cosmetic agent can be selectively bound only to the desired site of interest, and does not require any special pretreatment. Accordingly, hair can be dyed only by applying the chelating compound and the cosmetic after or simultaneously with applying the antibody to a target such as hair, and the disadvantages of the prior art can be solved. Here, the chelate compound and the cosmetic agent may be formed in advance in a complex, or may be separately applied and combined at the target site.

The single-chain antibody that binds to the skin, hair, nails, etc. corresponds to the protein molecule to be purified in IMAC, and forms a chelate bond with a metal at the N-terminal, C-terminal or inside of its amino acid sequence. The amino acid sequence to be obtained may be included so that both the immune binding site of the antibody and the amino acid sequence capable of chelating are exposed so as to be exposed on the protein surface. Its amino acid sequence is nickel, zinc,
It preferably contains amino acid residues that chelate with metals such as copper and iron, especially His, Trp, Tyr, Phe, Cys and combinations thereof. Although there is no particular rule for such an amino acid sequence, examples of amino acid sequences that can bind to metals, which have been reported in several documents, are shown below.

His-His-Leu-Gly-Gly-Ala-Lys-Glu-Ala-Gl
y-Asp-Val (SEQ ID NO: 3), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-Hi
s-Ser (SEQ ID NO: 4) Lys-Gly-Ser-Arg-Ser-His-His (SEQ ID NO: 5) His-Asn-Leu-Lys-Glu-Glu-His-Val-Pro-His (SEQ ID NO: : 6) His-Xaa-Xaa-Xaa-His (SEQ ID NO: 7) Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Ser-Gl
y-Ile-Met (SEQ ID NO: 8), His-Trp (SEQ ID NO: 9) His-Gly-His (SEQ ID NO: 10) His-Tyr-NH ₂ (SEQ ID NO: 11).

Among the above amino acid residues, His is preferable in view of affinity for metals. Therefore, an amino acid sequence containing a large number of His residues in a continuous manner provides stronger binding force and specificity. Get higher. To summarize these, the following sequence:-((Xaa) m- (His) n- (Xaa) o- (His) p) q- [where n and q are integers of 1 or more, m, o and p is 0
And Xaa represents any amino acid residue]
Is preferably used. Particularly preferably, m, o and p are 0, n is 2 to 6, and q is 1.

Of course, it is easy to incorporate these amino acids into the fusion protein by a gene recombination technique.
However, it is usually difficult to arbitrarily expose specific amino acid residues without losing the characteristics of the original protein, and in the case of antibodies, the affinity and specificity with the antigen may be impaired. Prone. Therefore, it is preferable to use a single-chain antibody in the present invention and to introduce a specific amino acid sequence at a place where loss of specificity or the like hardly occurs, that is, near the N-terminus or C-terminus of the single-chain antibody.

The obtained antibodies can be expressed and produced in microorganisms, plant bodies, animal milk, etc., purified as necessary, or used as a raw material for cosmetics by mixing with other cosmetics.

As the metal, the transition metal of the fourth period, Ni
²⁺ , Zn ²⁺ , Cu ²⁺ , and Fe ³⁺ are preferable.

The ligands forming the chelate compound include IDA (iminodiacetic acid), TED (tris (carboxymethyl) -ethylenediamine), NTA (nitrilotriacetic acid), PDTA (2-hydroxy-propylenediaminetetraacetic acid) and the like. Are known. Each of these has about 2 to 3 carboxyl groups at one end,
In these parts, it is chelated with the metal.

At the other end of the chelate compound, a cosmetic agent is bound via various residues. The method includes a chemical synthesis method and a physical adsorption method. That, -NH ₂ and -SH surfaces or molecules of each cosmetic agent, -CO
A crosslinker that selectively reacts with OH, sugar and the like to crosslink them is used. Known crosslinkers include imide ester compounds, N-hydroxysuccinimidyl ester compounds, maleimide compounds, haloacetyl compounds, pyridyldithiol compounds, carbodiimide compounds, photoreactive compounds, glyoxal compounds, hydrazide compounds, and the like. In addition, a protein having an interaction property such as a combination of avidin and biotin can also be used as a crosslinker. The chemical reaction carried out here is not performed on a biologically active substance such as an antibody or an enzyme, so that a relatively radical reaction can be performed, which is advantageous in cost. It is.

(2) Other examples of the ligand include glutathione. Representative amino acid sequences which can specifically bind to the glutathione include glutathione S-transferase in addition to anti-glutathione antibody. (GST) amino acid sequence (see SEQ ID NO: 26). There is a report that this GST was expressed as a fusion protein with another protein by a gene recombination method ( Gene 67, 31 (1988)). Therefore, the gene encoding the single-chain antibody is preferably N-terminal or C-terminal.
A fusion that can be used in the present invention can be obtained by ligating a gene encoding GST to the terminal side and expressing and producing the gene in a microorganism or a plant. The obtained single-chain antibody-GST fusion protein does not need to be particularly purified. This is because the fusion specifically binds affinity with the ligand-modified cosmetic, and the cosmetic of the present invention can be prepared by simply mixing the fusion with the cosmetic. On the other hand, glutathione, which is a ligand that can be complexed with a cosmetic agent, may be immobilized on any cosmetic agent by a chemical synthesis method as described in (1).

(3) Cellulose and cellulose-binding protein (C
BD) as well ( J. Biol. Chem.
263, 10401 (1988)). Cellulose-binding proteins are roughly classified into four types, each having a different affinity depending on the physical shape and molecular weight of the ligand cellulose. Regardless of the type of CBD (see SEQ ID NOs: 12 to 21), the antibody gene and the CBD gene are combined, expressed, and produced. On the other hand, the cosmetic agent may be bound to cellulose by a chemical synthesis method or a physical adsorption method as described in (1).

(4) In addition, similarly, in the case of chitin-binding protein (see SEQ ID NOs: 22 to 24), maltose-binding protein (see SEQ ID NO: 25), etc., each protein is a fusion protein with a single-chain antibody. And the corresponding ligands of chitin and maltose may be immobilized on the cosmetic by a chemical synthesis method or a physical adsorption method, as described in (1).

As described above, in addition to glutathione, cellulose, chitin and maltose described in (2) to (4), analogs such as carboxymethylcellulose, xylan, cellobiose, nigeran, dextran, chitosan and amylose are also suitable as ligands. Can be used.

Other than these, the ligand is not particularly limited as long as it is safe for the living body and can be blended without adversely affecting the feeling of use and color development of the cosmetic. An amino acid sequence of an antibody or binding protein capable of binding to such a ligand may be used for forming a fusion with a single-chain antibody in the present invention.

The ratio between the ligand and the cosmetic agent is not particularly limited, but if as many cosmetic agents as possible are bound to one molecule of the ligand, the efficiency of the cosmetic can be increased.

Furthermore, an amino acid sequence capable of binding to a cosmetic agent without using a ligand, for example, an amino acid sequence constituting an anti-cosmetic agent antibody or a cosmetic-binding protein, is used as a fusion component with a single-chain antibody. If it is used, the trouble of binding the cosmetic and the ligand can be omitted, and a favorable effect can be expected.

The fusion is prepared by expressing a nucleotide sequence encoding a single-chain antibody and a nucleotide sequence encoding the above amino acid sequence capable of binding to a ligand or a cosmetic agent, using both vectors so that they can be expressed. Preferably, various commonly used vectors can be used, for example, pE by Novagen.
T, pGEM from Promega, pBluescrip from Stratagene
t, pUC18 / 19, pSVL, etc., manufactured by Pharmacia.

Expression vectors usually contain a promoter operably linked to the two nucleotide sequences described above. Promoters include, for example, the phage λPL promoter, E. coli lac, trp, tac promoter, SV40 early and late promoters, T7 and T3 promoters, and retroviral LTR promoter.

The expression vector may be a marker (for example, di, hydrofolate reductase gene, neomycin resistance gene, ampicillin resistance gene, etc.) which enables selection of the transformed host, or an enhancer (SV40 enhancer, cytomegalovirus early enhancer). Promoter, adenovirus enhancer, etc.).

The construction, transformation or transfection, expression, etc. of the vectors exemplified above are described in Maniat.
is et al., Molecular Cloning, A Laboratory Manual, Cold
This may be performed by referring to the method described in Spring Harbor, second edition.

Examples of host cells for the vector include bacteria such as Escherichia coli (eg, E. coli TG1), fungi such as yeast, and plant cells such as soybean and tobacco.

For transformation or transfection, methods well known in the art such as electroporation are used.

When production in a non-human mammal is intended, the nucleotide sequence encoding the above-described fusion is ligated to a promoter, for example, to a milk protein such as casein. To create a transgenic mammal. In order to facilitate recovery of the fusion protein of interest, it is preferable to use a construct into which a nucleotide sequence corresponding to a secretion signal to milk has been inserted during production of a transgenic mammal.

When the fusion is used as a cosmetic material,
It may be mixed with basic cosmetics or with fine particles or polymer compounds.

The present invention will be further described below with reference to application examples in cosmetics.

(1) Hair dyeing of hair (a) The oxidative hair dyeing method conventionally used is a method of mixing an oxidizing dye and an oxidizing agent immediately before use, applying the mixture to hair, and performing oxidative polymerization and decolorization. It is. Oxidation dyes, various amines, phenols, etc. described in hair dye raw material standards, for example, p-aminophenol, m-aminophenol, o-aminophenol, p-phenylenediamine, m-phenylenediamine , 5-amino-o-cresol, 2,4-diaminophenol, catechol, 3,3'-iminodiphenol, pyrogallol, N-phenyl-p-phenylenediamine, 5- (2-hydroxyethylamino) 2-methyl Phenol, 2,4-diaminophenoxyethanol, 4,4-diaminodiphenylamine, o-chloro-p-phenylenediamine, p
-Methylaminophenol, toluene-3,4-diamine, toluene-2,5-diamine, p-aminophenylsulfamic acid, α-naphthol, diphenylamine, 1,5-dihydroxynaphthalene, 2,6-diaminopyridine, hydroquinone, There is phloroglucin. As the oxidizing agent, hydrogen peroxide, sodium percarbonate, sodium perborate and the like have been used for a long time. However, in recent years, peroxidase enzymes have been used to reduce the amount of skin-irritating oxidizing agents (Japanese Patent Application Laid-Open No. 47-10400), and uricase, glucose oxidase, etc. have recently been used in place of these oxidizing agents. A new method has been disclosed in which oxygen in the air is activated by an oxidizing enzyme such as an organic acid oxidase such as a sugar oxidase or a lactate oxidase to utilize the oxidizing power (JP-A-7-45385, JP-A-8-85)
No. 217652, JP-A-11-21214 and JP-A-11-21215). The group of enzymes having oxidizing power as described above is referred to as "oxidase" in the present specification, and particularly preferable oxidase in the present invention is selected from the group consisting of peroxidase, uricase, glucose oxidase, and lactate oxidase.

Although utilizing the enzymatic action as described above is a useful method, it has not been possible to avoid that expensive enzymes are washed away by hair washing in the above-mentioned conventional method. Therefore, if these enzymes are firmly immobilized on the hair using the single-chain antibody of the present invention, it is possible to reduce the outflow due to hair washing only by applying an oxidizing agent, and to maintain the hair dyeing state for a long period of time. Can be maintained.

When utilizing a metal chelate compound-amino acid affinity bond as a pair of a ligand and a binding amino acid sequence, the enzyme is modified with a chelate compound ligand,
Subsequently, the enzyme may be bound to the single-chain antibody fusion that specifically binds to the hair by forming a metal chelate compound-amino acid affinity bond by chelate-bonding the ligand with the metal. When performing hair dyeing, first, a fusion containing a single-chain antibody is applied to hair and absorbed. For this purpose, a pretreatment agent prepared by mixing a fusion containing a single-chain antibody with, for example, a rinse or a hair cream may be used. Then, the complex of the enzyme modified with the chelate compound is applied to the hair so as to bind to the single-chain antibody, but also in this case, it is necessary to use a compound formulated in a rinse, a hair cream, a spray, a shampoo, etc. preferable. Of course, it is also possible to use a mixture in which a fusion containing a single-chain antibody and a complex containing an enzyme are mixed in advance and bound. Finally, hair dyeing can be completed by applying or spraying an oxidation dye. It is necessary to mix an oxidizing agent and its substrate appropriate for the enzyme with the oxidation dye.
When peroxidase is used as the enzyme, hydrogen peroxide is used as the oxidizing agent, and when uricase is used as the enzyme, uric acid is used as the oxidizing agent. For other oxidases, the corresponding sugars and organic acids are selected.

Similarly, when a ligand other than a chelate compound is used, the cosmetic (enzyme) may be modified with each ligand.

(B) The direct hair dyeing method is a method in which a dye is directly bonded to hair. Dyes are broadly classified into organic tar dyes, inorganic pigments and natural dyes, some of which are licensed as cosmetic dyes. Methods for binding these dyes to the hair include methods such as physical adsorption of the dye and adhesion with a paste, and are commercially available as cosmetics such as hair manicure and hair mascara. However, these methods do not allow the dye to be retained on the hair for a long time, and are easily washed away by washing the hair. The invention relating to a method using an antibody to solve this drawback is disclosed in the above several publications, regardless of whether the antibody is directly bound to a pigment or a method via a polymer carrier, As described above, the chemical modification of antibodies has various problems.

In a preferred embodiment of the present invention where direct hair dyeing is carried out, pigments and lakes which are insoluble in water and oil,
The natural dye is chemically modified with the above-described chelating compound ligand to form a complex, and then is chelated to a metal. When this is applied to the hair to which the fusion containing the single-chain antibody has been applied, the molecule of the metal chelate compound bound to the dye instantaneously binds to the single-chain antibody on the hair. In order to apply the fusion containing the single-chain antibody and the chelate compound / dye complex, those which are appropriately added to rinse, spray, cream, shampoo and the like may be used as in (a) above.

Examples of pigments insoluble in water and oil include black titanium, red iron oxide, iron oxide, carbon black, squid and various oxidized polymers. Among them, especially when the dye has an organic functional group, the chelate compound can be easily bonded by various chemical reactions. When it does not have an organic functional group such as an inorganic pigment, an organic functional group such as an amino group, a carboxyl group, an aldehyde group, a hydroxyl group, or a thiol group is introduced by surface treatment such as treatment with a coupling agent or a silicon compound. do it.
Examples of the coupling agent include a silane coupling agent, a titanate coupling agent, an aluminum coupling agent, a zircoaluminate coupling agent, and the like.The silicon compound treatment includes amino-modified silicon,
Carboxy-modified silicon or the like is used. In order to bond the organic functional group to the ligand, glutaraldehyde is used when the functional group is an amino group, and 1-ethyl-3- (3) which is a water-soluble carbodiimide when the functional group is a carboxyl group.
-Dimethylaminopropyl) carbodiimide (EDC)
Is preferably used. When the organic functional group is a thiol group, N-succinimidyl-3- (2-pyridyldithio) -propionate can be used.

In particular, when a water-soluble dye, ie, a dye, is used as a cosmetic, a large amount of the dye is added to a water-soluble polymer carrier or an insoluble polymer carrier, such as polysaccharides, proteins, polyvinyl alcohol, polyethylene glycol, and various synthetic plastics. In this case, the amount of the binding dye per single-chain antibody molecule is increased when a chelate compound is further bound, and as a result, the hair dyeing effect is improved. Thus, the method via a carrier can be applied to an insoluble dye.

Similarly, when a ligand other than the metal chelate compound is used, the cosmetic (dye) may be modified with each ligand.

Of course, these methods can also be used for dyeing eyelashes, eyebrows, nails and the like in addition to head hair.

(2) Makeup of skin Cosmetics applied to skin are basic cosmetics, foundation, pressed powder, blusher, eyeshadow, eyeliner, lipstick, cosmetic pencil, sunscreen, etc., and the component is water. In addition to oil-insoluble fine powders and polymer compounds, they are often W / O or O / W emulsion fine particles. However, these are difficult to adsorb to the skin, and the makeup breaks down over time. Therefore, the principle of the present invention can also be used to make these more firmly adsorb to the skin with antibodies. The method is described in detail below.

(A) When the cosmetic, which is the main component of the cosmetic, is a fine powder or a polymer compound insoluble in water or oil, a metal chelate compound, cellulose, chitin, or amylose is used as a ligand, and Alternatively, the cosmetic agent and the ligand are chemically bonded via a linker. Various methods are conceivable for the chemical reaction according to the method (1) above, but since the reaction substrate does not have a biological activity such as an antibody or an enzyme, a relatively radical reaction is also possible. Since the choice of chemical reaction to be performed for preparation is wide, the present invention can be said to be cost-effective.

[0062] Cosmetic agents include talc, muscovite, synthetic mica, phlogopite, which are conventionally blended in makeup cosmetics for the skin.
Red mica, biotite, lithia mica, sericite, permiculite, kaolin, titanium dioxide, titanium oxide coated mica, titanium oxide coated talc, titanium oxide coated bismuth oxychloride, bismuth oxychloride, fish scale foil, colored titanium oxide coated mica Metal powder pigments such as pearl pigments, aluminum powder, copper powder, etc .; inorganic red pigments such as iron oxide (iron oxide) and iron titanate; inorganic brown pigments such as γ-iron oxide; and inorganic yellow pigments such as yellow iron oxide and loess. , Black iron oxide, inorganic black pigments such as carbon black, mango violet, inorganic violet pigments such as cobalt violet,
Inorganic green pigments such as chromium oxide, chromium hydroxide, and cobalt titanate; inorganic blue pigments such as ultramarine and navy blue; zinc white;
Bentonite, barium sulfate, metal soap, sodium silicate, aluminum silicate, calcium silicate, barium silicate, magnesium silicate, strontium silicate, metal tungstate, calcium carbonate, magnesium carbonate, chromium oxide, chromium hydroxide, Carbon black, alumina, hydroxyapatite, boron nitride, silica,
Nylon powder, zeolite, benzoguanamine powder, ethylene tetrafluoride powder, polyamide powder,
Polyester powder, polyethylene powder, polypropylene powder, polystyrene powder, cellulose powder, Red No. 201, Red No. 202, Red 204
No., Red No. 205, Red No. 220, Red No. 226, Red No. 228, Red No. 405, Orange No. 203, Orange No. 204
Organic pigments such as No. 205, Yellow No. 205, Yellow No. 401 and Blue No. 404, Red No. 3, Red No. 104, Red No. 106,
Red 227, Red 230, Red 401, Red 50
No. 5, orange 205, yellow 4, yellow 5, yellow 202
No. 203, yellow No. 203, green No. 3 and blue No. 1 organic pigments such as zirconium, barium or aluminum lake, and natural pigments such as chlorophyll and β-carotene.

In the case of sunscreen cosmetics, in addition to titanium oxide, zinc oxide and cerium oxide, an appropriate amount of an ultraviolet absorber, for example, 2-ethylhexyl paramethoxycinnamate (octyl), 2-ethylhexyl paradimethylaminobenzoate (octyl) ), Oxybenzone (benzophenone 3), 2-ethylhexyl salicylate (octyl), 4-te
An appropriate amount of rt-butyl-4-methoxy-benzoylmethane or the like is used as a cosmetic to protect the skin from ultraviolet rays.

The fusion containing the single-chain antibody may be applied in advance by applying an appropriate amount to a makeup base such as a lotion, an emulsion, a cream, or the like, or may be mixed with the complex of the above-mentioned cosmetic agent. Is also good.

(B) In an emulsion-based cosmetic, the outer surface is covered with a hydrophilic group in an O / W emulsion and a hydrophobic group in a W / O emulsion. That is, in order to constitute the emulsion particles, the component only needs to have a hydrophilic group and a hydrophobic group. If not, it may be so modified by a surfactant.
However, many cosmetics, such as foundations, various creams, and lipsticks, are prepared as emulsions, and there are many types of cosmetics as constituents thereof. Therefore, it is not realistic to chemically bond a ligand such as a chelate compound to all the constituent components. Therefore, various ligands such as chelate compounds are prepared so as to be constituents of the emulsion particles. Hereinafter, a method of preparing an emulsion using a chelate compound as a ligand will be described.

In the case of an O / W emulsion: at one end of the ligand of the chelate compound, for example, a carboxyl group capable of chelating with a metal and having a hydrophilic property is present. At the other end of the ligand, there are amino groups, imino groups, and the like that can be bonded to the carrier, and these groups are bonded to an aliphatic polymer, various cyclic polymers, and the like, so that the chelate compound becomes a hydrophilic group. And a hydrophobic group. If this is added as one of the components during the emulsification,
Emulsion particles having a carboxyl group capable of chelating with a metal on the outer surface of the obtained particles are obtained. If the skin is coated with a fusion containing a single-chain antibody in advance, the emulsion particles will bind to the antibody. Of course, the fusion and the emulsion particles may be mixed and then applied to the skin. When a hydrophilic ligand such as cellulose, chitin, or amylose is used instead of a chelate compound, no special treatment is required, and they may be added as they are.

In the case of a W / O emulsion: an amino group or imino group contained in a ligand of a chelate compound is bonded to a polymer compound having strong hydrophilicity. Examples of the polymer compound include sugars, synthetic polyols, sugar alcohols, polysulfate compounds, polyphosphate compounds, and the like. The other end of the ligand modified with such a hydrophilic group also contains a hydrophilic carboxyl group, which becomes hydrophobic under acidic conditions. Therefore, a desired emulsion can be formed by emulsifying under acidic conditions.
The method of binding to the antibody is performed in the same manner as in the preparation of the O / W emulsion.

As the oil component for preparing the emulsion,
Squalane, liquid paraffin, vaseline, microcrystalline wax, ozokerite, ceresin, myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cetyl alcohol, hexadecyl alcohol, oleyl alcohol, cetyl-2-ethylhexanoate, 2 -Ethylhexyl palmitate, 2-octyldodecyl myristate, 2-octyldodecyl gum ester, neopentyl glycol-2-ethylhexanate, glyceride triisooctanoate, 2-octyldodecyl oleate, isopropyl myristate, glyceride triisostearate, Tricoconut oil fatty acid glyceride, olive oil, avocado oil, beeswax, myristyl myristate, mink oil, lanolin, dimethylpolysiloxane, ring Dimethyl polysiloxane, methylphenyl polysiloxane, silicone resin, polyether-modified silicone, various hydrocarbons, such as amino-modified silicones, higher fatty acids, fats and oils, esters, higher alcohols, waxes, silicone oils, and the like.

As described above, the fusion of the cosmetic of the present invention with a single-chain antibody and a ligand or an amino acid sequence capable of binding to a cosmetic agent, the ligand and the cosmetic agent are each supplied as a single composition. Or may be supplied as an appropriately formulated composition. The mixing ratio is not particularly limited, and should be appropriately adjusted in consideration of the antibody titer of the single-chain antibody, the affinity of the amino acid sequence for the ligand, the amount of the cosmetic agent bound to the ligand, and the like. is there.

The cosmetics for hair dyeing and makeup described above may further include, as optional components, a surfactant, a humectant, a lower alcohol, a thickener, an ultraviolet absorber, a fragrance, an antioxidant, and an antiseptic. An appropriate amount of a fungicide or the like may be added as necessary.

Hereinafter, examples using metal-amino acid affinity bonds will be described as typical examples, but it is needless to say that the present invention should not be construed as being limited to such examples. All percentages are based on mass / volume.

Example 1 Hair dyeing method using anti-hair single-chain antibody-peroxidase conjugate-1 Preparation of Anti-Hair Antibody First, an antibody against hair was prepared by the cell fusion method of Kohler and Milstein (supra). That is, 10 mg of human hair cut to a length of about 1 mm was administered to the peritoneal cavity of a mouse about four times every two weeks to immunize. When the antibody titer against the hair of the mouse serum increased, the spleen cells were removed,
Hybridoma populations with mouse myeloma (NS1) cells were obtained by cell fusion with PEG. This was cultured and selected to produce a monoclonal antibody. Also,
_VH and _{VL of} antibody from hybridoma immediately after cell fusion
The region gene was extracted by PCR, and VH and V
L was ligated to prepare a single chain antibody (sFv) gene. His-His-His-His-His-His (SEQ ID NO: 27) was bound to the C-terminal as an amino acid sequence that binds to a metal chelate compound as a selectable marker for examining the presence of the antibody. Next, each sFv gene was inserted into a vector as shown in FIG. 1 and transformed into E. coli TG1 by electroporation. Each of the obtained Escherichia coli was cultured in a 2 × YT-AI medium (17% bacto-tryptone, 1%).
0% Bacto-yeast extract, 5% NaC
1, 2% glucose, 100 μg / ml ampicillin,
The cells were cultured in 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside), and the culture solution was cloned based on the ELISA method coated with keratin derived from hair. From several clones
SFv having the gene sequence shown in SEQ ID NO: 1 was selected based on the stability and binding properties of Fv.

2. Purification of fusion containing sFv The clone selected in the above was cultured overnight in a 2 × YT-AI medium, and centrifuged at 5,000 × g for 10 minutes to remove the precipitate, thereby obtaining a supernatant containing the sFv fusion. To this supernatant, imidazole was added to a final concentration of 10 mM, and 1
50 mM phosphate buffer containing 0 mM imidazole (pH
8.0) Ni-NTA column (manufactured by QIAGEN,
Germany). After the unadsorbed protein was washed away with 50 mM phosphate buffer (pH 8.0) containing 20 mM imidazole, the sFv fusion was eluted with the same buffer containing 250 mM imidazole, and this fraction was collected.

3. Method for measuring antibody titer by ELISA 5 g of hair was finely chopped, and 0.2 M Tris-HCl buffer /
One liter of 8M urea / 0.2M β-mercaptoethanol was added, and the mixture was allowed to stand at 50 ° C. overnight. After grinding in a mortar to make hair even finer, 12,000 rpm
The mixture was centrifuged at for 30 minutes to recover the supernatant. The collected supernatant was dialyzed against pure water to obtain a keratin solution derived from hair. The obtained keratin solution was adjusted to 100 μg / ml, and ELISA was performed.
100 μl was placed in the wells of the plate and incubated at 4 ° C. overnight. After blocking the wells with PBS containing 3% skim milk, the wells were purified by the method described above.
Fv (50 μg / ml) was applied at 50 μl, and 37 ° C.
For 1 hour. Then 0.05% Tween2
After washing the plate with PBS containing 0, horseradish peroxidase (HRP) labeled Ni-NTA
(Manufactured by QIAGEN, Germany) was added, and the mixture was incubated at 37 ° C. for 1 hour. The plate was washed with PBS containing 0.05% Tween 20, then TMB, a substrate of HRP, was added, reacted at room temperature for 15 minutes, and the absorbance at 450 nm was measured.

4. Preparation of hair dye containing sFv fusion and peroxidase Fusion containing sFv purified in step (50 μg / ml)
With horseradish peroxidase and Ni-NT
A complex (Ni-NTA HRP Conjugate, QIAGEN
Germany) to a final concentration of 25 μg / ml.
After incubation at 37 ° C. for 1 hour, a conjugate containing the single-chain antibody bound to peroxidase via the chelating compound was prepared.

5. Hair dyeing test 4. Human gray hair having a length of 5 cm was added to 2 ml of each of the aqueous solutions of the antibody-peroxidase conjugates a to d shown in Table 1 with or without the antibody-peroxidase conjugate (peroxidase titer 100 mU / ml) obtained in the above. 0.5 g of the bundle was immersed for 1 hour. Then, 0.05% Tween20
After washing with PBS containing 5 times, the sample was immersed in a dye solution having the composition shown in Table 1 for 30 minutes.

The judgment of hair dye was divided into three stages according to the degree of coloring. For non-dyed hair, 3: markedly dyed; 2: clearly dyed; 1: slightly dyed;
0: No difference.

[0078]

[Table 1]

As is evident from the results shown in Table 1, good hair dyeing results were obtained even with the use of hydrogen peroxide at a concentration about 1/10 lower than usual, and the fusion of the single chain antibody of the present invention was obtained. It has also been found that hair dyes containing are excellent in terms of safety and hypoallergenicity.

The hair dyed here did not fade even after shampooing five times, suggesting that the use of a single-chain antibody would significantly improve the fixation of the dye to the hair. .

[Example 2] Direct hair dyeing method Preparation of Dye / Ni-NTA Complex A dye / Ni-NTA complex was prepared by the following method.

Ni-NTA agarose (manufactured by QIAGEN, Ger
many) 1 ml was ground in a mortar. 20 mg of Cibacron Blue (manufactured by SIGMA, USA) and 2 ml of pure water were added, shaken at 60 ° C. for 30 minutes, and 0.1 g of NaCl was further added.
The mixture was shaken at 60 ° C. for 6 minutes. After neutralization with Na ₂ CO ₃ , the mixture was left at 80 ° C. for 2 hours. It was dialyzed overnight against water containing 1% NiSO4, and further dialyzed overnight against pure water.

2. Preparation of hair dye containing sFv fusion and dye / Ni-NTA complex Cibacron Blue / Ni-NTA
1. The above [Example 1] was added to 1 ml of the complex. 1 ml of the same sFv purified as described above was added, and the mixture was incubated at 37 ° C. for 1 hour to prepare a hair dye containing a single-chain antibody bound to a dye (Cibacron Blue) via a chelate compound.

3. Hair dyeing test 2 above. 0.5 g of a human gray hair bundle having a length of 5 cm was immersed in 2 ml of the aqueous solution a shown in Table 2 containing the dye-Ni-NTA antibody complex obtained in the above for 1 hour. Then 0.05
Washed 5 times with PBS containing% Tween20. As a control, the same hair dyeing test was performed on the solutions having the compositions shown in b to d in Table 2.

The determination of hair dye was divided into three stages according to the degree of coloring. For non-dyed hair, 3: markedly dyed; 2: clearly dyed; 1: slightly dyed;
0: No difference.

[0086]

[Table 2]

As shown in the results of Table 2, good dyeing results were obtained with the hair dye of the present invention.

[Example 3] Hair dyeing method 2 using anti-hair single-chain antibody-peroxidase conjugate-2 According to the composition shown in Tables 3a to 3g, 4. SFv fusion and peroxidase Ni as described in
Using the conjugate with the NTA complex, hair dyes of various compositions were further prepared.

At this time, the sFv fusion was prepared according to 1. And 2. The one prepared by the procedure described in the paragraph and the one which was further concentrated 200 times by ultrafiltration (filtration membrane: manufactured by Millipore, nitrocellulose membrane) were used.

About 2 g of a 3 cm long human gray hair bundle was immersed in a mixed solution of the prepared sFv fusion and peroxidase / Ni-NTA complex for 1 hour at room temperature. At this time, only the sample “g” was treated at room temperature for 10 minutes. Then
After washing 5 times with PBS containing 0.05% Tween 20, each was immersed in the dye solution shown in the lower part at room temperature for 30 minutes.
Thereafter, the plate was washed five times with PBS containing 0.05% Tween20.

Next, Example 1 was applied to all hair dye samples.
The determination was performed in the same manner as described in (1). The results are shown in Table 3 below.

[0092]

[Table 3]

As a result, in the hair dyeing method using the single-chain antibody fusion of the present invention, a complex of Ni-NTA and peroxidase was used in an amount of 0.02 to 2% by volume, preferably p-phenylenediamine as a substrate. It became clear that a good hair dyeing state was obtained when 1-2% by volume was blended.

The hair dyed here did not fade even after shampooing five times, suggesting good fixation of the dye to the hair.

[Example 4] Hair dyeing method using only Ni-NTA / peroxidase complex According to the compositions shown in Tables a to i, Ni-NTA, which is a ligand having a binding property to a subject to be applied with makeup, is used. A hair dye to be used in combination with peroxidase was prepared. In advance, about 3 cm was added to the solution shown in Table 4
Approximately 2 g of a human gray hair bundle having a length of was soaked at room temperature for 1 hour.
At this time, only the sample “g” was performed at 37 ° C. Then, after washing 5 times with PBS containing 0.05% Tween 20,
Each was immersed in the dye solution shown in the lower part at room temperature for 30 minutes. Thereafter, the plate was washed five times with PBS containing 0.05% Tween20.

Next, Example 1 was applied to all the hair dye samples.
The determination was performed in the same manner as described in (1). The results are shown in Table 4 below.

[0097]

[Table 4]

As a result, in the hair dyeing method utilizing the oxidation of p-phenylenediamine, the hair dye was treated with the Ni-NTA / peroxidase complex in advance with a smaller amount of hydrogen peroxide than conventionally used. It turned out that this could be achieved.

The hair dyed here did not fade even after shampooing five times, indicating good fixability of the pigment to the hair.

[Example 5] Moisture lotion Preparation of IDA · agarose complex agarose 20g and 2M NaOH 20 ml, of NaBH ₄ epichlorohydrin and 75mg of 2ml were mixed and stirred at room temperature for 2 hours. Then add 2M NaOH to 2
0 ml and 10 ml of epichlorohydrin were added and allowed to react overnight. After washing the obtained gel with water, 2M Na ₂ CO ₃
Dissolved in 50 ml. 5 g IDA and 0.06 g Na
BH ₄ was added and stirred at 60 ° C. overnight. After washing with water and washing with dilute acetic acid, wash with water until the pH becomes neutral.
An agarose complex was obtained.

2. Preparation of IDA-agarose-sFv complex [Example 1] 2. 2 ml of the same sFv fusion as purified in 1. IDA agarose 2m prepared in
was added and incubated at 37 ° C. for 1 hour to prepare a single-chain antibody-agarose complex.

3. Measurement of the water content of the stratum corneum In order to verify the water retention by the single-chain antibody-agarose complex, the water content was determined by measuring the water content of the stratum corneum.

The water content of the stratum corneum was measured by a capacitance conductance meter (IBS) according to the method of Tagami et al. (Fragrance Journal Extra Edition, No. 9 pp. 19-24 (1988)).
(SKICOS301). At the time of measurement, the measurement site was washed with warm water of 37 ° C. for 30 seconds, and then
Five measurements were made at 0 ° C. and 50% relative humidity, and the average was taken as the measured value. The result is shown in FIG.

Here, Sample 1 is the result using the single-chain antibody-agarose complex, Sample 2 is the result using only IDA-agarose, and Sample 3 is the result using only water.

As shown in FIG. 2, when the single-chain antibody-agarose complex was applied, the water retention was superior to the other cases, and it was clear that this effect could be maintained for a long time.

[0106]

As described above, according to the present invention,
Cosmetic can be stably adsorbed on the skin, hair, nails, etc., and is extremely safe for the living body. It can be supplied at a low cost because it does not waste expensive antibodies etc. Is done.

Further, according to the present invention, it is possible to reduce the amount of reagents that may be harmful to the living body, such as a coloring substrate such as hydrogen peroxide and phenylenediamine in hair dyes, Can be reduced to minimize any adverse effects that may be caused.

[0108]

[Sequence List] SEQUENCE LISTING <110> NIHON KOLMAR CO., LTD. <120> Method for enhancing adsorption of makeup, and cosmetics. <130> 2000PA0439 <160> 27 <210> 1 <211> 798 <212> DNA < 213> Artificial Sequence <220> <221> CDS <222> (1) .. (771) <220> <223> Nucleotide Sequence of Novel Single Chain Antibody. <400> 1 gag gtg caa ctg cag gag tca gga cct gag ctg gtg aag cct ggg gct 48 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag atg tcc tgc aag gct tct gga tac aca ttc act gac tat 96 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gtt ata agc tgg gtg aag cag aga act gga cag ggc ctt gag tgg att 144 Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gag att tat cct gga agt ggt agt act tac tac aat gag aag ttc 192 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 aag ggc aag gcc aca ctg act gca gac aaa tcc tcc aac aca gcc tac 240 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Al a Tyr 65 70 75 80 atg cag ctc agc agc ctg aca tct gag gac tct gcg gtc tat ttc tgt 288 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 gca aga aga aga agc cct tac tac ggc tac ttt gac tac tgg ggc caa 336 Ala Arg Arg Arg Ser Pro Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 ggg acc acg gtc acc gtc tcc tca ggt gga ggc ggt tca ggc gga ggt 384 Gly Thr Thr Val Thr Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 ggc tct ggc ggt ggc gga tcg gac att gtg atg act cag tct cct gct 432 Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala 130 135 140 tcc tta gct gta tct ctg ggg cag agg gcc acc atc tct tgc agg gcc 480 Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala 145 150 155 160 agc caa agt gtc agt aca tct agg ttt agt tat atg cac tgg tac caa 528 Ser Gln Ser Val Ser Thr Ser Arg Phe Ser Tyr Met His Trp Tyr Gln 165 170 175 cag aaa cca gga cag cca ccc aaa ctc ctc atc aag tat gca tcc aac 576 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys T yr Ala Ser Asn 180 185 190 cta gaa tct ggg gtc cct gcc agg ttc agt ggc agt ggg tct ggg aca 624 Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 gac ttc acc ctc aac atc cat cct gtg gag gag gag gat act gca aga 672 Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Thr Ala Arg 210 215 220 tat cac tgt cag cac agt tgg gag att ccg tac acg ttc gga ggg ggc 720 Tyr His Cys Gln His Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 acc aag ctg gaa atc aaa cgg gcg gcc gca ggt cac cac cac cac cac 768 Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly His His His His 245 250 255 cac tga 774 His <210> 2 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Sequence of Novel Single Chain Antibody. <400> 2 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Arg Ser Pro Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala 130 135 140 Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala 145 150 155 160 Ser Gln Ser Val Ser Thr Ser Arg Phe Ser Tyr Met His Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser Asn 180 185 190 Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Thr Ala Arg 210 215 220 Tyr His Cys Gln His Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly His His His His His 245 250 255 His <210> 3 <211> 12 <212> PRT <213> Arti ficial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 3 His His Leu Gly Gly Ala Lys Glu Ala Gly Asp Val 1 5 10 <210> 4 <211 > 14 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 4 Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 5 Lys Gly Ser Arg Ser His His 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400 > 6 His Asn Leu Lys Glu Glu His Val Pro His 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 7 His Xaa Xaa Xaa His 1 5 <210> 8 <211> 15 <212> PRT <213> Artificial Sequ ence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 8 Met Arg Gly Ser His His His His His His Gly Ser Gly Ile Met 1 5 10 15 <210> 9 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 9 His Trp 1 <210> 10 <211 > 3 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 10 His Gly His 1 <210> 11 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 11 His Tyr 1 <210> 12 <211> 104 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 12 Gln Pro Pro Ala Gly Arg Ala Cys Glu Ala Thr Tyr Ala Leu Val Asn 1 5 10 15 Gln Trp Pro Gly Gly Phe Gln Ala Glu Val Thr Val Lys Asn Thr Gly 20 25 30 Ser Ser Pro Ile Asn Gly Trp Thr Val Gln Trp Thr Leu P ro Ser Gly 35 40 45 Gln Ser Ile Thr Gln Leu Trp Asn Gly Asp Leu Ser Thr Ser Gly Ser 50 55 60 Asn Val Thr Val Arg Asn Val Ser Trp Asn Gly Asn Val Pro Ala Gly 65 70 75 80 Gly Ser Thr Ser Phe Gly Phe Leu Gly Ser Gly Thr Gly Gln Leu Ser 85 90 95 Ser Ser Ile Thr Cys Ser Ala Ser 100 <210> 13 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 13 Ala Pro Gly Cys Arg Val Asp Tyr Ala Val Thr Asn Gln Trp Pro Gly 1 5 10 15 Gly Phe Gly Ala Asn Val Thr Ile Thr Asn Leu Gly Asp Pro Val Ser 20 25 30 Ser Trp Lys Leu Asp Trp Thr Tyr Thr Ala Gly Gln Arg Ile Gln Gln 35 40 45 Leu Trp Asn Gly Thr Ala Ser Thr Asn Gly Gly Gln Val Ser Val Thr 50 55 60 Ser Leu Pro Trp Asn Gly Ser Ile Pro Thr Gly Gly Thr Ala Ser Phe 65 70 75 80 Gly Phe Asn Gly Ser Trp Ala Gly Ser Asn Pro Thr Pro Ala Ser Phe 85 90 95 Ser Leu Asn Gly Thr Thr Cys Thr Gly Thr Val Pro 100 105 <210> 14 <211> 107 <212 > PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellul ose binding protein. <400> 14 Pro Val Thr Ser Thr Pro Ser Cys Thr Val Val Tyr Ser Thr Asn Ser 1 5 10 15 Trp Asn Val Gly Phe Thr Gly Ser Val Lys Ile Thr Asn Thr Gly Thr 20 25 30 Thr Pro Leu Thr Trp Thr Leu Gly Phe Ala Phe Pro Ser Gly Gln Gln 35 40 45 Val Thr Gln Gly Trp Ser Ala Thr Trp Ser Gln Thr Gly Thr Thr Val 50 55 60 Thr Ala Thr Gly Leu Ser Trp Asn Ala Thr Leu Gln Pro Gly Gln Ser 65 70 75 80 Thr Asp Ile Gly Phe Asn Gly Ser His Pro Gly Thr Asn Thr Asn Pro 85 90 95 Ala Ser Phe Thr Val Asn Gly Glu Val Cys Gly 100 105 <210> 15 <211> 108 <212> PRT < 213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 15 Ser Gly Pro Ala Gly Cys Gln Val Leu Trp Gly Val Asn Gln Trp Asn 1 5 10 15 Thr Gly Phe Thr Ala Asn Val Thr Val Lys Asn Thr Ser Ser Ala Pro 20 25 30 Val Asp Gly Trp Thr Leu Thr Phe Ser Phe Pro Ser Gly Gln Gln Val 35 40 45 Thr Gln Ala Trp Ser Ser Thr Val Thr Gln Ser Gly Ser Ala Val Thr 50 55 60 Val Arg Asn Ala Pro Trp Asn Gly Ser Ile Pro Ala Gly Gly Thr Ala 65 70 75 80 Gln Phe Gly Phe Asn Gly Ser His Thr Gly Thr Asn Ala Ala Pro Thr 85 90 95 Ala Phe Ser Leu Asn Gly Thr Pro Cys Thr Val Gly 100 105 <210> 16 <211> 106 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 16 Thr Gly Ser Cys Lys Val Glu Tyr Asn Ala Ser Ser Trp Asn Thr Gly 1 5 10 15 Phe Thr Ala Ser Val Arg Val Thr Asn Thr Gly Thr Thr Ala Leu Asn 20 25 30 Gly Trp Thr Leu Thr Phe Pro Phe Ala Asn Gly Gln Thr Val Gln Gln 35 40 45 Gly Trp Ser Ala Asp Trp Ser Gln Ser Gly Thr Thr Val Thr Ala Lys 50 55 60 Asn Ala Ala Trp Asn Gly Ser Leu Ala Ala Gly Gln Thr Val Asp Ile 65 70 75 80 Gly Phe Asn Gly Ala His Asn Gly Thr Asn Asn Lys Pro Ala Ser Phe 85 90 95 Thr Leu Asn Gly Ala Thr Cys Thr Val Gly 100 105 <210> 17 <211> 103 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 17 Ala Ala Ser Gly Gly Asn Cys Gln Tyr Val Val Thr Asn Gln Trp Asn 1 5 10 15 Asn Gln Phe Thr Ala Val Ile Arg V al Arg Asn Asn Gly Ser Ser Ala 20 25 30 Ile Asn Arg Trp Ser Val Asn Trp Ser Tyr Ser Asp Gly Ser Arg Ile 35 40 45 Thr Asn Ser Trp Asn Ala Asn Val Thr Gly Asn Asn Pro Trp Ala Ala 50 55 60 Ser Ala Leu Gly Trp Asn Ala Asn Ile Gln Pro Gly Gln Thr Ala Glu 65 70 75 80 Phe Gly Phe Gln Gly Thr Lys Gly Ala Gly Ser Arg Gln Val Pro Ala 85 90 95 Val Thr Gly Ser Val Cys Gln 100 <210> 18 <211> 102 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 18 Ala Val Cys Glu Tyr Arg Val Thr Asn Glu Trp Gly Ser Gly Phe Thr 1 5 10 15 Ala Ser Ile Arg Ile Thr Asn Asn Gly Ser Ser Thr Ile Asn Gly Trp 20 25 30 Ser Val Ser Trp Asn Tyr Thr Asp Gly Ser Arg Val Thr Ser Ser Trp 35 40 45 Asn Ala Gly Leu Ser Gly Ala Asn Pro Tyr Ser Ala Thr Pro Val Gly 50 55 60 Trp Asn Thr Ser Ile Pro Ile Gly Ser Ser Val Glu Phe Gly Val Gln 65 70 75 80 Gly Asn Asn Gly Ser Ser Arg Ala Gln Val Pro Ala Val Thr Gly Ala 85 90 95 Ile Cys Gly Gly Gln Gly 100 <210> 19 <211> 104 <212> PRT <213> H omo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 19 Gln Thr Ala Thr Cys Ser Tyr Asn Ile Thr Asn Glu Trp Asn Thr Gly 1 5 10 15 Tyr Thr Gly Asp Ile Thr Ile Thr Asn Arg Gly Ser Ser Ala Ile Asn 20 25 30 Gly Trp Ser Val Asn Trp Gln Tyr Ala Thr Asn Arg Leu Ser Ser Ser 35 40 45 Trp Asn Ala Asn Val Ser Gly Ser Asn Pro Tyr Ser Ala Ser Asn Leu 50 55 60 Ser Trp Asn Gly Asn Ile Gln Pro Gly Gln Ser Val Ser Phe Gly Phe 65 70 75 80 Gln Val Asn Lys Asn Gly Gly Ser Ala Glu Arg Pro Ser Val Gly Gly 85 90 95 Ser Ile Cys Ser Gly Ser Val Ala 100 <210> 20 <211> 97 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 20 Ala Cys Thr Tyr Thr Ile Asp Ser Glu Trp Ser Thr Gly Phe Thr Ala 1 5 10 15 Asn Ile Thr Leu Lys Asn Asp Thr Gly Ala Ala Ile Asn Asn Trp Asn 20 25 30 Val Asn Trp Gln Tyr Ser Ser Asn Arg Met Thr Ser Gly Trp Asn Ala 35 40 45 Asn Phe Ser Gly Thr Asn Pro Tyr Asn Ala Thr Asn Met Ser Trp Asn 50 55 60 Gly Ser Ile Ala Pro Gly Gln Ser Ile Ser Phe Gly Leu Gln Gly Glu 65 70 75 80 Lys Asn Gly Ser Thr Ala Glu Arg Pro Thr Val Thr Gly Ala Ala Cys 85 90 95 Asn <210> 21 <211> 97 <212> PRT < 213> Homo sapiens <220> <223> Known amino acid sequence of a cellulose binding protein. <400> 21 Val Ser Gly Ala Leu Lys Ala Glu Tyr Thr Ile Asn Asn Trp Gly Ser 1 5 10 15 Gly Tyr Gln Val Leu Ile Lys Val Lys Asn Asp Ser Ala Ser Arg Val 20 25 30 Asp Gly Trp Thr Leu Lys Ile Ser Lys Ser Glu Val Lys Ile Asp Ser 35 40 45 Ser Val Cys Val Asn Ile Ala Glu Glu Gly Gly Tyr Tyr Val Ile Thr 50 55 60 Pro Met Ser Trp Asn Ser Ser Leu Glu Pro Ser Ala Ser Val Asp Phe 65 70 75 80 Gly Ile Gln Gly Ser Gly Ser Ile Ser Thr Ser Val Asn Ile Ser Val 85 90 95 Gln <210> 22 <211> 51 < 212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a chitin binding protein. <400> 22 Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr 1 5 10 15 Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln 20 25 30 Pro His Thr Ser L eu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Trp 35 40 45 Gln Leu Gln 50 <210> 23 <211> 55 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a chitin binding protein. <400> 23 Ala Ala Gln Trp Gln Ala Gly Thr Ala Tyr Lys Gln Gly Asp Leu Val 1 5 10 15 Thr Tyr Leu Asn Lys Asp Tyr Glu Cys Ile Gln Pro His Thr Ala Leu 20 25 30 Thr Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp Lys Tyr Val Gly 35 40 45 Glu Gly Thr Gly Gly Gly Thr 50 55 <210> 24 <211> 52 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a chitin binding protein. <400> 24 Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr 1 5 10 15 Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln 20 25 30 Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu 35 40 45 Trp Gln Leu Gln 50 <210> 25 <211> 385 <212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a maltose binding protein. <400> 25 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thrl Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp 370 375 380 Glu 385 <210> 26 <211> 220 < 212> PRT <213> Homo sapiens <220> <223> Known amino acid sequence of a glutathione binding protein (GST). <400> 26 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Ty r Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Plu Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp 210 215 220 <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <22 3> Amino acid sequence of a peptide which can bind to a metal of chelate compound. <400> 27 His His His His His His 1 5

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a vector containing a nucleotide sequence A7H encoding a single-chain antibody suitably used in the present invention.

FIG. 2 is a graph showing the effect of the moisture lotion according to one embodiment of the present invention on the water content of the stratum corneum, as a function of the skin conductance over time.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 7/13 A61K 7/13 (72) Inventor Takeo Yoneda 4-4-1 Fushimicho, Chuo-ku, Osaka-shi, Osaka No. 1 F-term in Nippon Colmer Co., Ltd. (reference) 4C083 AC771 AC931 AC932 AD261 AD321 AD411 AD412 CC02 CC28 CC36 EE07

Claims (18)

[Claims] 1. A cosmetic comprising a combination of a single-chain antibody having a binding property to a subject to be applied with a makeup of an amino acid sequence capable of binding to a ligand, and a combination of a ligand and a cosmetic. . 2. The amino acid sequence capable of binding to a ligand has the following sequence:-((Xaa) m- (His) n- (Xaa) o- (His) p)
q- [where n and q are integers of 1 or more, m, o and p are 0
And Xaa represents any amino acid residue]
The cosmetic according to claim 1, which is: 3. The cosmetic according to claim 1, wherein the ligand is a metal chelate compound. 4. The cosmetic according to claim 1, wherein the amino acid sequence is a sequence constituting an anti-ligand antibody or a ligand-binding protein. 5. The method according to claim 1, wherein the ligand is cellulose, chitin,
The cosmetic according to claim 1 or 4, wherein the cosmetic is selected from the group consisting of maltose and glutathione and analogs thereof. 6. A cosmetic comprising a combination of a fusion of a single-chain antibody having a binding property to an object to be applied with an amino acid sequence capable of binding to the cosmetic, and a cosmetic. 7. The cosmetic according to claim 6, wherein the amino acid sequence is a sequence constituting an anti-cosmetic agent antibody or a cosmetic-binding protein. 8. A host cell transformed or transfected with a vector in which the fusion contains both a nucleotide sequence encoding the single-chain antibody and a nucleotide sequence encoding the amino acid sequence in an expressible manner. 8. A method according to claim 1, wherein said gene is obtained by a genetic recombination method.
The cosmetic according to any one of the above. 9. The cosmetic according to claim 1, wherein the single-chain antibody is an anti-keratin antibody. 10. The cosmetic according to claim 1, wherein the single-chain antibody has an amino acid sequence represented by SEQ ID NO: 2. 11. The method according to claim 9, wherein the single-chain antibody has an amino acid sequence encoded by the polynucleotide shown in SEQ ID NO: 1 or an analog thereof, which encodes the amino acid sequence shown in SEQ ID NO: 2. Cosmetics. 12. A cosmetic comprising a combination of a ligand having a binding property to a subject to be applied with a cosmetic and a cosmetic agent. 13. The cosmetic according to claim 12, wherein the ligand is a metal chelate compound. 14. The metal chelate compound is a nickel chelate compound of nitrilotriacetic acid (Ni-NTA), and the Ni-NTA and one or more oxidases selected from the group consisting of peroxidase, uricase, glucose oxidase, and lactate oxidase. The cosmetic according to claim 13, which is a hair dye used in combination with the cosmetic. 15. A method for enhancing the adsorption of a cosmetic agent, comprising: a fusion of a single-chain antibody having a binding property to a subject to be applied with an amino acid sequence capable of binding to a ligand; A fusion of a single-chain antibody capable of binding to a subject to be applied and an amino acid sequence capable of binding to a cosmetic, and a cosmetic; or a ligand and a cosmetic capable of binding to the subject to be applied are coated on the subject A method comprising: 16. A cosmetic method using the cosmetic according to claim 1. Description: 17. A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and encoding a single-chain antibody that binds to skin and / or hair, and an analog thereof. 18. A single-chain antibody having the amino acid sequence shown in SEQ ID NO: 2 and binding to skin and / or hair.