Tyrosinase antibodies that inhibit tyrosinase and uses thereof

Abstract

The invention relates to a tyrosinase antibody for inhibiting tyrosinase and application of the tyrosinase antibody, relates to the technical field of tyrosinase inhibition, and relates to an anti-tyrosinase antibody containing a heavy chain CDR and a light chain CDR with specific sequences or an antigen binding fragment of the antibody. The anti-tyrosinase antibody inhibits tyrosinase activity, and thus it is expected that the anti-tyrosinase antibody can be effectively applied to improvement of skin whitening or treatment of skin pigmentation diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of tyrosinase inhibition, and more particularly to tyrosinase antibodies for inhibiting tyrosinase and uses thereof. Background Art

[0002] The skin color of a person is different according to many factors, and is mainly determined by the amount of melanin, carotene and hemoglobin, wherein melanin is the most decisive factor. Melanin is synthesized in melanocytes present in the skin base, and is transferred to peripheral keratinocytes to present the skin color of a person. It is known that when melanin is abnormally less, skin lesions such as vitiligo can be induced. On the contrary, when melanin is overproduced, spots and blemishes can be formed. Melanin is generated by tyrosinase acting on tyrosine as an amino acid, and tyrosinase is more activated by ultraviolet rays, therefore, if exposed to a large amount of sunlight, the skin will darken. Therefore, the basic mechanism of the medicament for developing whitening cosmetics for preventing pigmentation can include inhibiting the activity of tyrosinase, inhibiting the generation of tyrosinase, inhibiting the intermediate factor (mediator) that promotes the generation of melanin, inhibiting the reduction and photooxidation of the melanin generated, promoting the excretion of melanin and blocking ultraviolet rays, etc. Among them, the method by inhibiting the activity of tyrosinase can be said to be one of the methods that can obtain the simplest and effective skin whitening effect.

[0003] Although research on skin whitening by inhibiting the activity of tyrosinase is actively being conducted, research on antibodies that inhibit tyrosinase is still insufficient. Summary of the invention

[0004] In view of this, the present invention is proposed.

[0005] One of the purposes of the present invention is to provide an antibody protein for inhibiting tyrosinase for use in whitening skin care products and / or health care products.

[0006] A second object of the present invention is to provide an antibody that inhibits tyrosinase.

[0007] A third object of the present invention is to provide a nucleic acid molecule.

[0008] A fourth object of the present invention is to provide biological materials.

[0009] A fifth object of the present invention is to provide a composition composed of antibody proteins having skin care and whitening effects.

[0010] In one aspect, an embodiment of the present invention provides an application of an antibody protein for inhibiting tyrosinase in whitening skin care products and / or health care products, wherein the antibody protein comprises:

[0011] 1) a light chain variable region, comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO.1, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO.2, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO.3;

[0012] 2) a heavy chain variable region, comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO.4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO.5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO.6;

[0013] 3) Fc region, the sequence of the Fc region is the sequence of any FC constant region of an antibody that binds to the conserved domain of tyrosinase.

[0014] It should be noted that the Fc segment is the constant region of the antibody. The amino acid sequence of the Fc segment is the same in different antibodies of the same protein. Therefore, the Fc segment sequence in the present invention is the Fc segment sequence in any antibody that binds to the SNAP 25 protein in the prior art.

[0015] A second aspect of the embodiments of the present invention provides a tyrosinase antibody, wherein the antibody is the protein sequence.

[0016] A fusion tyrosinase antibody, wherein the fragment is a TAT peptide shown in SEQ ID NO.7 and is bound to the antibody.

[0017] The third aspect of the present invention provides a nucleic acid molecule encoding an antibody.

[0018] At the same time, a biomaterial is also provided, comprising:

[0019] 1) a recombinant expression vector comprising the nucleic acid molecule;

[0020] 2) Cells transformed with the recombinant expression vector described in 1).

[0021] A composition for whitening skin, treating or preventing skin pigmentation, wherein the active ingredient comprises the antibody.

[0022] An anti-oxidation cosmetic composition comprises the antibody.

[0023] A composition for diagnosing skin pigmentation diseases, comprising the antibody.

[0024] The present invention relates to an anti-tyrosinase antibody for inhibiting tyrosinase and its use, and more specifically, to an anti-tyrosinase antibody or an antigen-binding fragment thereof comprising a heavy chain CDR and a light chain CDR of a specific sequence. The anti-tyrosinase antibody is expected to inhibit the activity of tyrosinase, thereby being effectively used to improve skin whitening or treat skin pigmentation diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Attached Figure 1 The results of the enzyme-linked immunosorbent assay (ELISA) analysis for screening single-chain antibody (scFv) of α-Tyrosinase.

[0027] Attached Figure 2 This is the reproducibility verification result of ELISA analysis.

[0028] Attached Figure 3 Size and protein purity results are presented by SDS-PAGE after purification of cell permeable α-tyrosinase scFv protein.

[0029] Attached Figure 4 The results are for the inhibition of tyrosinase activity by the cell-permeable α-tyrosinase scFv protein.

[0030] Attached Figure 5 The results of the porcine skin penetration ability of the cell-permeable α-tyrosinase scFv protein were presented by DAB staining. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention provides a tyrosinase antibody 1) a light chain variable region, comprising a light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO.1, a light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO.2, and a light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO.3;

[0033] 2) a heavy chain variable region, comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO.4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO.5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO.6;

[0034] 3) Fc region, the sequence of the Fc region is the sequence of any FC constant region of an antibody that binds to the conserved domain of tyrosinase.

[0035] In addition, the present invention provides a fusion tyrosinase antibody, wherein the TAT peptide represented by SEQ ID NO.7 is additionally bound to the antibody fragment.

[0036] In addition, the amino acid sequence of the TAT peptide used in the present invention is YGRKKRRQRRR, SEQ ID NO.7, and the base sequence of the TAT peptide is TATGGCCGCAAAAAACGC CGCCAGCGC CGCCGC, SEQ ID NO.8.

[0037] In the present invention, the term "antibody" refers to a protein molecule used as a receptor that specifically recognizes an antigen, which includes an immunoglobulin molecule that is immunologically reactive with a specific antigen, and as examples thereof, may include all monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. In addition, the term "antibody" may include bivalent or bispecific molecules (e.g., bispecific antibodies), double antibodies, three antibodies, or four antibodies.

[0038] In the present invention, the term "monoclonal antibody" refers to an antibody molecule composed of a single molecule obtained from a substantially identical antibody group, which is different from a polyclonal antibody that can bind to multiple epitopes, and this monoclonal antibody exhibits a single binding and affinity to a specific epitope. The term "full-length antibody" in the present invention refers to a structure having two light chains of the entire length and two heavy chains of the entire length, wherein each light chain is connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), muon (μ), alpha (α), delta (δ) and epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) as subclasses. The constant region of the light chain has kappa (κ) and lambda (λ) types. IgG is a subclass, including IgG1, IgG2, IgG3 and IgG4.

[0039] The term "heavy chain" in the present invention may include all full-length heavy chains and fragments thereof, wherein the full-length heavy chains and fragments thereof include a variable region VH and three constant regions CH1, CH2 and CH3, wherein the variable region VH includes an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. And the term "light chain" in the present invention may include all full-length light chains and fragments thereof, wherein the full-length light chains and fragments thereof may include a variable region VL and a constant region CL, wherein the variable region VL includes an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen.

[0040] In addition, the present invention provides a nucleic acid molecule encoding the antibody.

[0041] The term "nucleic acid molecule" used in this specification has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and the nucleotides as the basic constituent units of nucleic acid molecules include not only natural nucleotides, but also analogs with modified sugar or base moieties. The sequence of the nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the present invention can be modified, and the modification includes the addition, deletion or non-conservative substitution or conservative substitution of nucleotides.

[0042] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0043] "Vector" in the present invention refers to a self-replicating DNA molecule used to carry cloned genes (or other fragments of cloned DNA).

[0044] The term "expression vector" in the present invention refers to a recombinant DNA molecule comprising a target coding sequence and a suitable nucleic acid sequence necessary for expressing the coding sequence connected in an operable manner in a specific host organism. Preferably, the expression vector may contain one or more selective markers. The marker is generally a nucleic acid sequence having a characteristic that can be selected by a chemical method, and all genes that can distinguish cells transformed by the trait from cells transformed by the trait correspond to this. Examples thereof include antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin and chloramphenicol, but are not limited thereto and can be appropriately selected by those skilled in the art.

[0045] In order to express the DNA sequence of the present invention, any of a variety of expression control sequences can be used in the vector. Examples of useful expression control sequences can include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR of retrovirus, lac system, trp system, TAC system or TRC system, T3 promoter and T7 promoter, major operator genes and promoter regions of lambda phage, regulatory regions of fd encoding proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of the phosphatase (e.g., Pho5), promoters of yeast α-mating systems, and structures and other derived sequences known to regulate gene expression of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0046] A vector system that simultaneously expresses light chain and heavy chain in one vector or a system that expresses light chain and heavy chain on separate vectors can be a vector for expressing the antibody of the present invention. In the latter case, two vectors are introduced into the host cell by co-transformation and targeted transformation. Co-transformation is a method of simultaneously introducing each vector DNA encoding light chain and heavy chain into a host cell, and then screening cells that fully express light chain and heavy chain. Targeted transformation is a method of screening cells that transform traits using a vector containing a light chain (or heavy chain), and then transforming the screened cells expressing light chain again with a vector containing a heavy chain (or light chain), thereby finally screening cells that fully express light chain and heavy chain.

[0047] In addition, the present invention provides cells transformed with a recombinant expression vector.

[0048] The cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the relevant technical field, including, for example, Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus) and other prokaryotic host cells, but are not limited thereto.

[0049] In the method for preparing the antibody or the antigen-binding fragment of the antibody, the culture of the transformed cells can be carried out according to suitable culture media and culture conditions known in the relevant technical field. Those of ordinary skill in the art can easily adjust and use such a culture process according to the selected strain. Cell culture is divided into suspension culture and adherent culture according to the growth mode of the cells, and is divided into batch culture, fed-batch culture and continuous culture according to the culture method. The culture medium used for culture must appropriately meet the required conditions of the specific strain.

[0050] In addition, the present invention provides a composition for detecting tyrosinase antigen, which comprises the antibody or the antigen-binding fragment of the antibody as an active ingredient.

[0051] In addition, the present invention provides a cosmetic composition for skin whitening, which comprises the antibody or the antigen-binding fragment of the antibody as an active ingredient.

[0052] In addition to the active ingredients, the cosmetic composition may also contain conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

[0053] The dosage form of the cosmetic composition can be prepared as any dosage form commonly prepared in the art, and can have a dosage form selected from the group consisting of skin external ointment, cream, soft lotion, nutritious lotion, facial mask, essence, hair tonic, shampoo, conditioner, hair conditioner, hair care agent, gel, skin lotion, softener, toner, firming water, skin care water, lotion, moisturizing water, nutritious water, massage cream, nutritious cream, eye cream, moisturizing cream, hand cream, foundation, nutritious essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and shower gel, but not limited thereto. The composition of these various dosage forms can contain appropriate various basic raw materials and additives required for the formulation of the dosage form, and those skilled in the art can easily select the type and amount of these ingredients.

[0054] In case the dosage form is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc or zinc oxide etc. can be used as the carrier component.

[0055] When the dosage form is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, tricalcium silicate or polyamide powder can be used as a carrier component. In particular, when the dosage form is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether can be additionally contained.

[0056] When the dosage form is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty esters, polyethylene glycol or fatty acid esters of sorbitan anhydride.

[0057] When the dosage form is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth can be used as a carrier component.

[0058] In addition, the present invention provides a health food composition for skin whitening, which comprises the antibody or the antigen-binding fragment of the antibody as an active ingredient.

[0059] The health food composition can be provided in the form of powder, granules, tablets, capsules, syrups, beverages or pills, and the health food composition can be used together with other foods or food additives in addition to the composition according to the present invention as an effective ingredient, and can be used appropriately according to a common method. The mixed amount of the active ingredient can be appropriately determined according to its purpose of use (e.g., according to the treatment of prevention, health care or treatment).

[0060] The effective dose of the antibody or the antigen-binding fragment of the antibody contained in the health food composition can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or for the purpose of health conditioning, it can be used in an amount below the above range, but since there is no problem with the safety of the active ingredient, it can indeed be used in an amount above the above range.

[0061] The type of health food is not particularly limited, and examples thereof may include meat, sausage, bread, chocolate, candy, snacks, biscuits, pizza, ramen, other noodles, chewing gum, dairy products (including ice cream), various soups, beverages, tea, functional drinks, alcoholic beverages, and vitamin complexes, etc.

[0062] In addition, the present invention provides a pharmaceutical composition for preventing or treating skin pigmentation diseases, comprising the antibody or the antigen-binding fragment of the antibody as an active ingredient.

[0063] Specifically, the skin pigmentation disease may be chloasma, freckles, dark spots, birthmarks, drug-induced pigmentation, post-inflammatory pigmentation, or hyperpigmentation caused by dermatitis, but is not limited thereto.

[0064] The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier, which is a carrier commonly used in preparation, including lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. In addition to the above ingredients, the pharmaceutical composition of the present invention may also additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0065] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenterally administered, the pharmaceutical composition can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In the case of oral administration, since proteins or peptides are digested, oral compositions can be formulated in a manner that coats the active agent or prevents decomposition in the stomach. The composition of the present invention can be administered by any device that can move the active substance to the target cells.

[0066] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity, and an ordinarily skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0067] The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily implemented by a person skilled in the art to which the present invention belongs, so as to be prepared into a unit volume form or prepared to be loaded into a multi-volume container. In this case, the dosage form can be a solution, suspension or emulsion in an oil or aqueous medium, or can also be a concentrate, powder, suppository, powder, granule, tablet or capsule form, and can also contain a dispersant or stabilizer.

[0068] The compositions of the invention may be administered as the sole therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with existing therapeutic agents.

[0069] In addition, the present invention also provides a composition for diagnosing skin pigmentation diseases, which comprises the antibody or the antigen-binding fragment of the antibody as an active ingredient.

[0070] Specifically, the skin pigmentation disease may be chloasma, freckles, dark spots, birthmarks, drug-induced pigmentation, post-inflammatory pigmentation, or hyperpigmentation caused by dermatitis, but is not limited thereto.

[0071] In addition, the present invention provides a method for providing information required for diagnosing a skin pigmentation disease, the method comprising the steps of: contacting the antibody or the antigen-binding fragment of the antibody with a sample isolated from an individual suspected of having a skin pigmentation disease; and detecting tyrosinase protein from the sample through an antigen-antibody reaction.

[0072] Specifically, the skin pigmentation disease may be chloasma, freckles, dark spots, birthmarks, drug-induced pigmentation, post-inflammatory pigmentation, or hyperpigmentation caused by dermatitis, but is not limited thereto.

[0073] As long as the method can detect tyrosinase protein by measuring the binding between antigen and antibody, it can be included in the above-mentioned method for detecting tyrosinase protein by antigen-antibody reaction. These methods are well known in the art, and examples thereof include protein immunoblotting (Western blot), ELISA, radioimmunoassay, radioimmunodiffusion, fluorescent immunoassay, immunoblotting, octoclot immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, immunochromatography, FACS, protein chip, etc., and are not limited thereto.

[0074] In addition, the tools or reagents used for the immunological analysis of the antigen-antibody reaction may include a suitable carrier or support, a label that can generate a detectable signal, a solubilizing agent, a detergent, a stabilizer, etc. As for the suitable carrier, although not limited thereto, when the labeling substance is an enzyme, it may also include a substrate that can measure the enzyme activity, a suitable buffer solution, a secondary antibody labeled with a colorimetric enzyme or a fluorescent substance, a colorimetric substrate, a reaction terminator, etc.

[0075] The present invention will be described in more detail below by way of examples. These examples are only used to more specifically describe the present invention, and according to the gist of the present invention, the scope of the present invention is not limited to these examples, which is obvious to those of ordinary skill in the art to which the present invention belongs.

[0077] Example 1 Screening of α-Tyrosinase scFv Antibodies

[0078] 1. Perform Biopanning

[0079] Using a 7.6×10 9Biopanning was performed on the OPAL database of diversity. 5 μg of tyrosinase antigen was immobilized on epoxy magnetic beads and reacted with input phage. Phage reacted with antigen were eluted and the output titer was measured. Input and output titers were measured each time to obtain information on biopanning and to confirm whether biopanning was performed normally. 2×10 12 The phage with colony count / ml ≥ was taken as the input phage. It can be seen that the output increased with the progress of bio-panning from 1.7×10 6 Colony count / ml, 2nd time 3.65×10 6 Colony count / ml, 3rd time 4.68×10 8 Therefore, it can be confirmed that bio-panning is proceeding normally.

[0080] 2.ELISA analysis

[0081] ELISA analysis was performed to screen for highly sensitive and highly specific antibodies. The obtained phage was used to infect E. coli and smeared on an LB plate supplemented with antibiotics. After culturing in an incubator at 30°C for 16 hours, the generated colonies were randomly collected. After each colony was cultured in LB medium, it was treated with isopropyl-β-D-thiogalactoside (IPTG) to express scFv, and the E. coli was lysed to extract the water-soluble fraction, and ELISA was performed. First, 30 ng of tyrosinase antigen was fixed on a 96-well ELISA plate and blocked with phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA). After 1 hour, it was treated with the cell lysate obtained above and reacted at 37°C for 2 hours. After washing the plate three times with PBS containing 0.1% Tween 20, HRP conjugated anti-HA antibody was diluted 1:1000 in blocking solution and reacted at 37°C for 1 hour. The plate was washed five times with PBS containing 0.1% Tween 20, then developed with TMB substrate, and the scFv antibody bound to the antigen was measured using an ELISA reader. 96 antibodies were analyzed, and 11 antibodies (OD 0.4 and OD 0.1) were arbitrarily selected as the positive guideline and the negative guideline, respectively. Figure 1 ).

[0082] Example 2 Sequencing of α-tyrosinase scFv antibody

[0083] For the 11 tyrosinase scFv positive clones screened by ELISA analysis, individual clones were screened by sequencing. Since there may be duplicate clones in the positive clones screened previously, sequence analysis is required. According to the sequence analysis results, 2 of the 11 positive clones were confirmed to be individual clones.

[0084] Example 3 Purification and sensitivity analysis of the screened α-tyrosinase antibodies

[0085] 1. Purification of α-tyrosinase scFv antibody

[0086] A previously screened α-tyrosinase scFv antibody was purified (Table 1). A cloned transformation ER2738 E. coli cell was cultured in 500 ml of SB medium, and then lysed using 1X TES buffer. The cell lysate was reacted with 0.5 ml of Ni beads and eluted using imidazole.

[0087] Table 1

[0088]

[0089] 2. Sensitivity analysis of purified α-tyrosinase scFv antibody

[0090] Sensitivity analysis was performed using purified α-tyrosinase scFv antibody. The sensitivity analysis utilized ELISA analysis. First, various concentrations of tyrosinase antigen were fixed to the ELISA plate, and then 10μg / ml of α-tyrosinase scFv antibody was treated. The analysis was also performed using HRP-labeled anti-HA antibody diluted at a ratio of 1:1000. The analysis results were EC50-1368ng / ml, LOD-12.5ng / ml ( Figure 2 ).

[0091] Example 4 Construction of cell-permeable α-tyrosinase scFv

[0092] 1. Construction of TAT-α-tyrosinase scFv construct

[0093] The polymerase chain reaction (PCR) product of the previously screened scFv insertion restriction enzyme site and TAT was produced using primers. 30 μl of each PCR product was treated with 4 μl buffer 3.1, 1 μl EcoRI, 1 μl XhoI, and 4 μl distilled water, and after reacting at 37°C for 1 hour, the DNA was separated to ensure the insert to be inserted into the vector. For the pET 28a (+) vector, after transformation to DH5α competent cells, it was smeared on an LB plate supplemented with kanamycin, cultured at 37°C for 16 hours, and colonies were extracted the next day and inoculated in LB medium supplemented with kanamycin, and then cultured at 37°C for 16 hours. The next day, after isolating the vector using a mini-prep kit, 30 μl of the vector was treated with 4 μl of buffer 3.1, 1 μl of EcoRI, 1 μl of Xho I, 1 μl of CIP, and 3 μl of distilled water, and purified after reacting at 37°C for 1 hour. The concentrations of the purified vector and insert were measured by a spectrophotometer (nanodrop), and T4 ligase was used to connect at room temperature for 16 hours in the ratio of vector and insert. After completing the connection (ligation), after transformation to DH5α, it was smeared on an LB plate supplemented with kanamycin and cultured at 37°C for 16 hours. The colony was extracted the next day and inoculated into an LB medium supplemented with kanamycin and cultured at 37°C for 16 hours. The next day, in order to confirm whether the insert was inserted, the plasmid was isolated and restricted with EcoRI and XhoI, and the band was confirmed by electrophoresis on 1% agarose gel.

[0094] 2. Purification of TAT-α-tyrosinase scFv antibody

[0095] The cloned TAT-α-tyrosinase scFv antibody clone was transformed into a BL21 (DE3) E. coli host, and the transformant was induced with 0.5 mM IPTG. The scFv antibody was suspended in a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), crushed using an ultrasonic pulverizer, and then centrifuged to obtain a supernatant. The protein was purified using a resin with affinity for Ni-NTA. Good expression was confirmed by SDS-PAGE analysis. The protein size was confirmed to be about 30 kDa ( Figure 3 ).

[0096] Example 5 Cytotoxicity assay of cell permeable anti-tyrosinase scFv

[0097] In order to understand the cytotoxicity of TAT-α-tyrosinase scFv antibody, mouse-derived malignant melanoma cells B16F10 were cultured in a CO2 incubator at 37°C. After culture, TAT-α-tyrosinase scFv antibody was treated with different concentrations (0.1ppm, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm), and then further cultured under the same culture conditions. After the culture was completed, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added for culture, and then the culture medium was removed, dimethyl sulfoxide (DMSO: Dimethyl sulfoxide) was added, and after appropriate shaking, the absorbance was measured at 570nm using an ELISA reader system. The values ​​are shown in Table 2. As shown in Table 2, when TAT-α-tyrosinase scFv was treated at concentrations of 0.1ppm, 1ppm, 5ppm, 10ppm, 50ppm, and 100ppm, it was confirmed that the cell shape and cell survival rate of malignant melanoma cells were almost unchanged up to 100ppm. Therefore, it can be seen that TAT-α-tyrosinase scFv is a substance with no cytotoxicity up to a concentration of 100ppm.

[0098] Table 2

[0099]

[0100] Example 6 Experiment on the inhibitory effect of cell-permeable anti-tyrosinase scFv on tyrosinase activity

[0101] The effect of TAT-α-tyrosinase scFv on tyrosinase activity related to melanin production was studied. 100 μl of 0.1 M phosphate buffer (pH 6.8) was added to 5 μg of TAT-α-tyrosinase scFv, and 10 μl of 300 units / ml mushroom tyrosinase was added. Then, 20 μl of 1.5 mM L-tyrosine solution was added. After reacting at 37°C for 20 minutes, the absorbance at 490 nm was measured using an ELISA Reader system. The values ​​are shown in Figure 4 .like Figure 4 As shown, it was confirmed that TAT-α-tyrosinase scFv inhibited tyrosinase activity and showed an effect similar to that of arbutin at the same concentration.

[0102] Example 7 Experiment on the inhibitory effect of cell-permeable anti-tyrosinase scFv on melanin production

[0103] In order to confirm the inhibitory effect of TAT-α-tyrosinase scFv antibody on melanin production in cells, B16F10 cell line was treated with TAT-α-tyrosinase scFv antibody, and then the content of melanin production in cells was measured. Malignant melanoma cells B16F10 cells were cultured in a CO 2 incubator at 37°C. After that, 10ppm of TAT-α-tyrosinase scFv antibody was treated, and 100ppm of arbutin was treated as a positive control area, and then further cultured under the same culture conditions. After culture, each well was washed with PBS, 130μl of 1N NaOH solution was added thereto, and dissolved at 60°C for 1 hour, and then the absorbance was measured at 475nm with an enzyme-linked immunosorbent assay system (ELISA Reader system), and the values ​​are shown in Table 3. As shown in Table 3, it was confirmed that TAT-α-tyrosinase scFv inhibited the production of melanin and showed an effect similar to arbutin.

[0104] Table 3

[0105]

[0106] Example 8 Experiment on the inhibitory effect of cell-permeable anti-tyrosinase scFv on active oxygen species

[0107] In order to confirm the inhibitory effect of TAT-α-tyrosinase scFv antibody on reactive oxygen species in cells, B16F10 cell line was treated with TAT-α-tyrosinase scFv antibody, and then the intracellular reactive oxygen content was measured using fluorescence staining. B16F10 cells, which are malignant melanoma cells, were cultured in a CO 2 incubator at 37°C. After that, 100 ppm of TAT-α-tyrosinase scFv antibody was treated, and 100 ppm of arbutin was treated as a positive control area, and then further cultured under the same culture conditions. After culture, 0.2 mM hydrogen peroxide was added to each well, and then cultured for a further 2 hours. Carboxyl-H 2DCF-DA was added and reacted at 37°C for 30 minutes. After washing with PBS, PBS was added, and fluorescence was measured at 475 / 535 nm using an ELISA Reader system, and the values ​​are shown in Table 4. As shown in Table 4, it was confirmed that TAT-α-tyrosinase scFv inhibited the generation of ROS and exhibited a superior effect to arbutin at the same concentration.

[0108] Table 4

[0109]

[0110] Example 9 Skin penetration analysis of cell permeable anti-tyrosinase scFv

[0111] In order to confirm the skin penetration of TAT-α-tyrosinase scFv, it was confirmed by tissue staining using pig skin. Pig skin was treated with 10μg of TAT-α-tyrosinase scFv antibody and incubated in a CO2 incubator at 37°C for 16h. After fixing the pig skin with 4% formaldehyde, paraffin blocks were prepared and the tissue was cut 5μm using a microtome. After that, the tissue was mounted on a slide, the paraffin was removed and after a hydration process, the tissue section was blocked for 30min. The blocked tissue was reacted with anti-His HRP antibody at room temperature for 1 hour. After washing with PBST, it was reacted with DAB chromogen solution for 5min, then stained by H&E staining method and observed with an optical microscope. Figure 5 The results of comparison of the skin permeability of α-tyrosinase scFv and TAT-α-tyrosinase scFv are shown. Figure 5 As shown, it was confirmed that α-tyrosinase scFv was not delivered to pig skin tissue at all, while TAT-α-tyrosinase scFv had effective skin delivery ability.

[0112] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0113] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of antibody protein for inhibiting tyrosinase in whitening skin care products and / or health care products, characterized in that: The antibody protein comprises: 1) a light chain variable region, comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO.1, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO.2, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO.3; 2) a heavy chain variable region, comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO.4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO.5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO.6; 3) Fc region, the sequence of the Fc region is the sequence of any FC constant region of an antibody that binds to the conserved domain of tyrosinase.

2. A tyrosinase antibody, characterized in that: The antibody is the protein sequence described in claim 1.

3. A fusion tyrosinase antibody, wherein: A fragment in which the TAT peptide represented by SEQ ID NO. 7 binds to the antibody according to claim 2.

4. A nucleic acid molecule encoding the antibody of claim 2.

5. A biomaterial comprising: 1) A recombinant expression vector comprising the nucleic acid molecule of claim 3; 2) Cells transformed with the recombinant expression vector described in 1).

6. A composition for whitening skin, treating or preventing skin pigmentation, characterized in that: The active ingredient comprises the antibody according to claim 2.

7. An antioxidant cosmetic composition, characterized in that: Comprising the antibody according to claim 2 or 3.

8. A composition for diagnosing skin pigmentation diseases, characterized in that: Comprising the antibody according to claim 2 or 3.

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