Synthesis method and application of recombinant human keratin

Abstract

The invention discloses a synthetic method and application of recombinant human keratin. The recombinant human keratin comprises an amino acid sequence as shown in SEQ ID NO: 1 or a variant of the amino acid sequence; the recombinant human keratin disclosed by the invention has a remarkable hair care effect and has a wide application value in the field of hair care; the hair quality can be remarkably improved, and the anti-static effect of the hair bundle can be remarkably improved; the water solution or shampoo formula of the recombinant human keratin synthesized by the invention has the remarkable improvement effect on combing, softening and dry combing effects of hair bundles, tensile force and frizz resistance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a synthesis method of recombinant human keratin and application thereof. Background Art

[0002] Keratin (also known as cytokeratin) is a structural protein found in epithelial cells of the skin, hair, nails and other tissues. They provide strength and elasticity to these tissues. There are at least 54 keratin genes in humans. Different keratins have specific functions and are expressed in different tissues and cell types. In 2006, the Keratin Nomenclature Committee renamed the original cytokeratin to keratin, with a molecular weight distribution between 40-68kDa. According to the isoelectric point, it can be divided into acidic keratin (also known as type I keratin) and neutral or basic keratin (also known as type II keratin). There are 28 type I keratins and 26 type II keratins. Among the 54 keratins, 17 are hair-specific keratins.

[0003] Protein is the main component of hair, accounting for 65% to 95% of its composition, most of which is keratin. Keratin provides the strength and structural support required by hair. The scale layer on the surface of hair, which is mainly composed of keratin, forms a protective covering layer to protect the protein and moisture inside. Keratin can also maintain the moisture balance of hair, making it soft, elastic and shiny. Lack of keratin may cause hair to be dry, brittle and dull. Hair is often exposed to the external environment and is susceptible to various external factors, such as light, chemical treatment, environmental pollution, mechanical damage, etc., which cause the hair cuticle scales to curl and break and a large amount of keratin to be lost. The macroscopic manifestations are rough feel, easy breakage, reduced strength, etc.

[0004] Studies have proven the protective effect of keratin on hair. One study used a reduction method to prepare a feather keratin solution to treat damaged hair, and studied the affinity and repair effect of keratin on hair. The results showed that keratin has a good affinity for hair and can form a smooth film on the surface of damaged hair, repair the damaged parts of the hair cuticle scales, and improve the gloss and elasticity of the hair. After the hair is coated with keratin, its curl retention rate is significantly improved. In addition, keratin can absorb ultraviolet rays in the range of 200 to 300nm, which can effectively improve the hair's ability to resist ultraviolet rays. In another study, L-cysteine ​​was used as a reducing agent, and keratin was extracted from rabbit hair fibers using the cysteine ​​method to prepare a new type of keratin conditioner, which was used for the care of damaged hair. The results showed that after being cared for with a conditioner containing rabbit hair keratin, the hair had good moisture absorption and styling ability. Rabbit hair keratin is a safe and effective natural hair care product additive.

[0005] As per capita disposable income increases and the public's education level improves, people's requirements for quality of life are constantly increasing, and the management of their own image is becoming more and more strict. The prevalence of the "appearance economy" has given rise to new consumer demands for shampoo and hair care products, and China's shampoo and hair care market has developed steadily.

[0006] In 2023, the scale of China's shampoo and hair care market will be 65.1 billion yuan, with an average annual compound growth rate of 3.24% from 2019 to 2023. According to this growth rate, the scale of the shampoo and hair care market is expected to reach 73.8 billion yuan by 2025. With the continuous upgrading of demand for hair care, the market application prospect of keratin in hair care products is promising. Its properties in repairing, protecting and improving hair health make it one of the most popular and important ingredients in the hair care market. Keratin is widely used in the manufacture of hair care products such as shampoo, conditioner and hair mask. It can penetrate into the hair, repair damaged protein structure, and make hair stronger and more elastic. As a natural protein, keratin meets the current market's pursuit of natural, green and sustainable products. Therefore, hair care brands tend to choose to use keratin as one of the main ingredients of their products to meet consumers' expectations for health and environmental protection.

[0007] According to statistics and forecasts from QYR (Hengzhou Bozhi), the global keratin market sales reached US$160 million in 2022 and are expected to reach US$240 million in 2029, with a compound annual growth rate (CAGR) of 6.4% (2023-2029). In 2019, the scale of my country's keratin market reached US$11.33 million, and it is expected to reach US$17.77 million in 2026. It will continue to grow in the next few years, and the market prospects are broad.

[0008] At present, the main source of keratin is extracted from feathers and animal hair. Since the different peptide chains in the keratin molecule mainly form a stable spatial structure by hydrogen bonds, salt bonds and disulfide bonds, a large number of disulfide bonds give hair stable physical and chemical properties, but make it insoluble in common solvents or mild acids and bases, making it particularly difficult to extract keratin from hair. When keratin is dissolved with stronger acids and bases, keratin with a smaller molecular weight is obtained. The method of breaking the disulfide bonds, hydrogen bonds and salt bonds in the keratin molecules, protecting the primary structure of the protein, and obtaining keratin extraction methods with higher relative molecular mass and better solubility has been explored.

[0009] With the rise of biosynthesis technology and the popularity of recombinant collagen, the synthesis of recombinant human keratin using biosynthesis technology has become a new synthesis method to replace the extraction of keratin from animal feathers in the past. There are also reports on related biosynthetic keratin, such as patent CN117430687A, which invented a recombinant keratin with a triple helix structure, and the host cell uses Escherichia coli. Compared with natural keratin, this recombinant keratin has better improvement effects on hair gloss, combing, strength and smoothness. Patent CN110117323B invented a soluble human keratin, which is mainly obtained by cloning the intermediate sequence of the human K37 gene, constructing a recombinant vector, transforming bacteria, inducing recombinant protein expression and purifying it. It has the characteristics of rapid swelling, and can promote the mitosis and migration of epidermal cells, which plays an important role in skin regeneration, repair and wound healing. Patent CN117486991A invented a recombinant keratin with the effect of preventing hair loss and promoting hair growth. Patent CN112745383B invented a recombinant human keratin mixed solution (a solution of a tetrameric structure formed by polymerizing type II alkaline cytokeratin K1 and type I acidic cytokeratin K10 in a 1:1 ratio), which can improve the protective ability of normal skin epidermis and the repair effect on the damaged stratum corneum of the skin epidermis. Summary of the invention

[0010] The purpose of the present invention is to provide a method for synthesizing recombinant human keratin and its application.

[0011] The scheme of the present invention is:

[0012] A recombinant human keratin protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or a fragment thereof, wherein the fragment is an amino acid sequence having at least 70% identity with SEQ ID NO: 1 or a mutant of the sequence of SEQ ID NO: 1.

[0013] The invention also discloses a nucleotide sequence encoding recombinant human keratin.

[0014] As a preferred technical solution, the nucleotide sequence is shown in SEQ ID NO:2.

[0015] The invention also discloses a recombinant cell, which comprises a nucleotide sequence encoding recombinant human keratin.

[0016] The recombinant cell is one of a eukaryotic cell and a prokaryotic cell.

[0017] As a preferred technical solution, the eukaryotic cell is one of human cells, mouse cells, hamster cells, insect cells, plant cells, filamentous fungi and yeast; the prokaryotic cell is one of bacteria, actinomycetes, rickettsia, chlamydia, mycoplasma, cyanobacteria and archaea.

[0018] In a further preferred embodiment, the filamentous fungus is one of Aspergillus, Mucor, Fusarium, Dermatophyte, Sporothrix, and Zygomycete;

[0019] The yeast is one of Saccharomyces cerevisiae, Pichia pastoris, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe;

[0020] The bacterium is one of the genus Bacilus, Nocardia, Microcus, Pseudomonas, Escherichia coli, red snail bacteria, green sulfur bacteria, pigmented bacteria and cyanobacteria.

[0021] The Escherichia coli includes but is not limited to wild-type Escherichia coli (e.g., K-12MG650 and K-12W3110) and commercial expression strains (e.g., BL21, BL21(DE3) and BL21 Star(DE3), BL21 AI, BL21(DE3)pLysS, BL21(DE3)pLysE, Lemo21(DE3), C41(DE3), C43(DE3) and M15 pREP4, etc.) and derivatives of the above strains.

[0022] The present invention also discloses a recombinant vector, which comprises a nucleotide sequence shown in SEQ ID NO: 2, and is a prokaryotic cell recombinant expression vector.

[0023] The present invention also discloses a method for synthesizing recombinant human keratin, comprising the following steps:

[0024] 1) constructing recombinant cells expressing recombinant human keratin;

[0025] 2) culturing the recombinant cell to obtain a culture;

[0026] 3) Separating and purifying from the culture to obtain recombinant human keratin.

[0027] As a preferred technical solution, the recombinant cells in 2) are cultured using one of the following methods: primary culture, secondary culture, adherent cell culture, suspension cell culture and fermentation.

[0028] As a preferred technical solution, the recombinant cells in 2) are cultured by a fermentation method, wherein the recombinant cells are cultured in a liquid culture medium in a seed shake flask. After the culture is completed, the seed liquid is inoculated into a fermenter for fermentation and induction of expression to obtain a fermentation product, i.e., a culture.

[0029] As a preferred technical solution, the method for synthesizing recombinant human keratin comprises the following steps:

[0030] 1) Construction of recombinant cells expressing recombinant human keratin

[0031] Synthesizing the nucleotide sequence encoding the recombinant human keratin as shown in SEQ ID NO: 2, and then cloning it into a recombinant expression vector to obtain an expression plasmid, transforming the expression plasmid into an Escherichia coli host cell, and collecting transformant colonies;

[0032] 2) Cultivation

[0033] The single colony of the transformant is inoculated into the liquid culture medium in the seed shake flask for cultivation. After the seed shake flask cultivation is completed, the seed liquid is inoculated into the fermentation medium in the fermenter for initial fermentation. As the fermentation proceeds, the dissolved oxygen concentration drops below the set value of dissolved oxygen, and the stirring speed, ventilation volume and tank pressure of the fermenter are adjusted in sequence to control the stability of the dissolved oxygen concentration. When the fermentation proceeds, the substrate of the fermentation medium is exhausted, the dissolved oxygen concentration rises rapidly, and fed-batch fermentation is started. At the same time, the culture temperature is lowered to room temperature. When the temperature in the fermenter is stabilized at room temperature, an ITPG solution (1 mL / L) is pumped in to induce the culture to obtain a fermentation product.

[0034] 3) Separation and purification

[0035] The fermentation product is centrifuged for solid-liquid separation, the bacterial cells are collected, the bacterial cells are homogenized, the supernatant is collected by centrifugation, the pH is adjusted, the conductivity is controlled, and the product is purified by column chromatography to obtain recombinant human keratin.

[0036] The present invention also discloses an application of recombinant human keratin in hair care and hair nourishment products, wherein the recombinant human keratin comprises an amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof.

[0037] As a preferred technical solution, the product is in the form of a preparation selected from the following: slurry, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, spray or powder.

[0038] As a preferred technical solution, the preparation is shampoo, hair care lotion, hair growth lotion, hair conditioner, hair growth essence or hair growth cream.

[0039] Advantages of the present invention:

[0040] The recombinant human keratin synthesized by the present invention has significant hair care effects, has the value of being widely used in the field of hair care, can significantly improve the quality of hair, and significantly improves the anti-static effect of hair bundles.

[0041] The present invention compares the sequences of keratin on the hair, selects a section of about 100 amino acid sequences on keratin K33B, recombinantly expresses and purifies to obtain water-soluble recombinant keratin. Experiments have shown that the recombinant human keratin prepared by the present invention has a significant effect on improving the combing smoothness, dry combing effect, tensile strength, and anti-frizziness of hair bundles using an aqueous solution or shampoo formula containing 0.1% and 0.02% of the recombinant protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a test result diagram of the friction force of a hair bundle in the test of the present invention;

[0043] Figure 2 This is a scanning electron micrograph of a hair bundle in vitro in the test of the present invention;

[0044] Figure 3 is a graph showing the rate of change of the maximum force required for dry combing a hair bundle before and after treatment in the test of the present invention;

[0045] Figure 4 is a graph showing the rate of change of the total work required for dry combing a hair bundle before and after treatment in the test of the present invention;

[0046] Figure 5 is a graph showing the rate of change of the force required for stretching the hair bundle before and after treatment in the test of the present invention;

[0047] Figure 6 A graph showing the rate of change of work done by stretching a hair bundle before and after treatment in the test of the present invention;

[0048] Figure 7 This is a graph showing the rate of change of the ratio of the frizzy part area of ​​the hair to the whole hair bundle area before and after the hair bundle is processed in the test of the present invention. DETAILED DESCRIPTION

[0049] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0050] Embodiment 1:

[0051] 1. Construction of keratin expression strain

[0052] The amino acid sequence (SEQ ID NO: 1) of one of the polypeptides of human keratin KRT33B (Uniprot ID: Q14525) was codon optimized to obtain its gene coding sequence (SEQ ID NO: 2) containing the start codon and the stop codon. The sequence was synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (BGI) and cloned into the expression vector pET-28a (+) provided by BGI to obtain an expression plasmid. The full sequence of the expression plasmid is SEQ ID NO: 3, named pKRT33B_18. The plasmid was transformed into BL21 (DE3) chemical transformation competent cells (CAT#: EC1002) purchased from Shanghai Weidi Biotechnology Co., Ltd. The resulting strain was named S1.

[0053] 2. Protein Fermentation Process

[0054] Prepare a fermentation medium, which includes yeast extract, glycerol, potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate heptahydrate, ammonium sulfate, citric acid, and a defoamer, sterilize at 121° C. for 30 minutes, cool to 28° C. for standby use, and add VB1 and trace elements after cooling. Magnesium sulfate heptahydrate is sterilized separately, and VB1 (concentration of 5 mg / L) and trace element solutions are filtered and sterilized.

[0055] The monoclonal S1 colony was added to more than 50 mL of liquid culture medium and cultured at 30°C and 200 rpm for 15 h as the seed for the tank.

[0056] The seeds were added into the fermentation tank containing the above fermentation medium, with the ratio of seeds to fermentation medium being 7.5%. After fermentation for 4 to 6 hours, when the dissolved oxygen rises, glycerol was added and the temperature was lowered to 24°C. After the temperature dropped to 24°C, the prepared IPTG was pumped in for induction for 20 hours.

[0057] 3. Separation and purification process

[0058] The fermentation product was centrifuged at 10000 rcf, 10 min, 20°C for solid-liquid separation, and the bacterial cells were collected. 4x 20mM PB (pH8) was added to the bacterial cells and stirred evenly at room temperature; a high-pressure homogenizer was used to homogenize twice at 800 bar at room temperature, and then 10000 rcf, centrifuged for 20 min, and the supernatant was collected at 20°C; the pH was adjusted to 8.0 with 2N NaOH, and column chromatography was performed.

[0059] Use Buffer A1 (50mM Tris-HCl, pH8.0) to balance to pH and UV baseline levels, load the sample at a flow rate of 100-200cm / h, rinse with Buffer A1 for 2-4CV to the UV baseline level after loading, at a flow rate of 150cm / h, collect the flow-through and washing liquid, target sample 1.

[0060] The chromatography column was eluted with Buffer B1 (50mM Tris-HCl+300mM NaCl, pH8.0) for 2-4CV at a flow rate of 100-200cm / h. It was washed with 1M NaCl and 1M NaOH, respectively. Then, the target sample 1 was concentrated using a 3KD ultrafiltration membrane package and ultrafiltration was replaced with pure water, and finally concentrated to the required volume. Finally, the sample concentrate was sterilized by filtration with a 0.22μm filter membrane, and the recombinant human keratin was obtained and freeze-dried for later use. 1. The recombinant human keratin in Example 1 was applied to shampoo products and participated in the following experiments: Preparation of shampoo products containing recombinant keratin (recombinant human keratin prepared in Example 1)

[0061] Shampoo formula table:

[0062]

[0063]

[0064] The preparation process of the above shampoo:

[0065] Clean the equipment thoroughly, dry it and set it aside. Weigh and dissolve items C, D and E in the above table and set it aside.

[0066] Weigh item A into a beaker, heat to 80-83°C, stir until completely dissolved, and then slowly add item B in sequence while stirring.

[0067] Cool down to 45-50℃, add item C until completely dissolved. When the temperature reaches 40-45℃, add item D and stir until completely dissolved. When the temperature reaches 35-40℃, add item F and stir until fully dissolved. When the temperature reaches 25-32℃, add item E to adjust the pH, discharge the material, and obtain shampoo.

[0068] 2. Applying the recombinant human keratin in Example 1 to an aqueous solution product and participating in the following experiment Preparation of an aqueous solution product containing recombinant keratin (recombinant human keratin prepared in Example 1)

[0069] Aqueous solution formula table:

[0070]

[0071] Aqueous solution operation process:

[0072] Clean the equipment thoroughly, dry it and set it aside. Weigh item A into a beaker, heat it to 70℃, and stir until it is completely dissolved. When the temperature reaches about 25℃, add item B.

[0073] 3. Experimental Test

[0074] 1. In vitro hair bundle friction test

[0075] Test method:

[0076] 1) Testing instruments and materials: Friction tester MTT175 (Dia-Stron Limited, UK). Black healthy hair bundles of Asian people (2.5 g, 15 cm).

[0077] 2) Test samples: Shampoo 1 with 0.1% recombinant keratin sample added and Shampoo 2 with 0.02% recombinant keratin sample added as described in the above shampoo products; negative control is control group shampoo with deionized water added; positive control is Shampoo 3 with 0.1% commercially available keratin sample added.

[0078] 3) Hair bundle selection and grouping: Select the same hair bundles and randomly group them to ensure that there are 5 bundles in the test product group and 5 bundles in the control group;

[0079] 4) Hair tress pretreatment: Apply 14% sodium lauryl ether sulfate at a dosage of 0.1 g / g hair evenly on the hair tress, rub for 30 seconds, rinse with water at a temperature of 37°C and a flow rate of 4 L / min for 30 seconds, comb the hair every 5 seconds during rinsing, and finally dry it with a narrow-tooth comb for 10 times. After pretreatment, hang the hair tress in a constant temperature and humidity chamber at a temperature of 20±1°C and a relative humidity of 50±5% to dry and balance for 24 hours.

[0080] 5) Treatment of test samples and control products: Apply the test product evenly to the hair bundle at a dosage of 0.1g / g hair, rub for 30s, rinse with water at a temperature of 37°C and a flow rate of 4L / min for 30s, and comb the water every 5 seconds during rinsing. Repeat the above steps once, treat twice in total, and finally comb dry with a narrow-tooth comb 10 times. Use the control product instead of the product in the same way as the test product group hair bundle treatment. After completion, hang in a constant temperature and humidity chamber at a temperature of 20±1°C and a relative humidity of 50±5% to dry and balance for 24 hours.

[0081] 6) Friction test: After 24 hours, the friction work parameter value of the hair bundle was measured using a friction tester MTT175 (Dia-Stron Limited, UK).

[0082] Statistical methods:

[0083] SPSS software was used to perform descriptive statistics on the test values ​​of the hair bundles treated with the test products and the control products, including quantity, mean, standard deviation, etc. Shapiro-Wilk Test was used to test the significance of normal distribution of data. If Sig. (bilateral)>0.01, it was normally distributed, and the independent sample t test was performed for parallel control, and the significant difference level α was 0.05; if Sig. (bilateral)<0.01, it was non-normally distributed, and the Mann-Whitney U test was performed for parallel control, and the significant difference level α was 0.05.

[0084] result:

[0085] Compared with the control group shampoo, shampoo 1 with 0.1% recombinant keratin sample added, shampoo 2 with 0.02% recombinant keratin sample added, and shampoo 3 with 0.1% commercial keratin sample added can significantly reduce the friction of hair bundles and improve the smoothness of hair. Moreover, shampoo 1 has a better effect on improving hair smoothness than shampoo 2, indicating that high-concentration keratin samples have a better effect on smoothing hair than low-concentration keratin samples; shampoo 1 also has a better effect on improving hair smoothness than shampoo 3, indicating that at the same concentration, the recombinant keratin sample prepared by the present invention has a better effect than the commercially available keratin sample ( Figure 1 ). Among them, shampoos containing 0.1% and 0.02% recombinant keratin have a significant effect of reducing the friction of hair strands, and it is concentration-dependent.

[0086] 2. In vitro hair bundle scanning electron microscopy (SEM) testing

[0087] Test method:

[0088] 1) Testing instruments and materials: Hitachi S-3400N scanning electron microscope, with a resolution of 1.0nm (5kV) to 1.4nm (1kV) and a vacuum degree of 1.2 to 1.6×10 -6 mPa, focusing range 300μmx200μm, accelerating voltage 5kV, magnification 1000 times. Mildly chemically damaged hair strands of Asian population (2.5g, 15cm).

[0089] 2) Test samples: an aqueous solution containing 0.1% recombinant keratin and an aqueous solution containing 0.02% recombinant keratin as described in the above aqueous solution product; a negative control is an aqueous solution without keratin (i.e., a control aqueous solution); and a positive control is an aqueous solution containing 0.1% commercially available keratin (i.e., a WLM-02 aqueous solution).

[0090] 3) Hair bundle selection and grouping: Select the same hair bundles and randomly group them to ensure that there is 1 bundle in the test product group and 1 bundle in the control group;

[0091] 4) Hair tress pretreatment: Apply 14% sodium lauryl ether sulfate at a dosage of 0.1 g / g hair evenly on the hair tress, rub for 30 seconds, rinse with water at a temperature of 37°C and a flow rate of 4 L / min for 30 seconds, comb the hair every 5 seconds during rinsing, and finally dry it with a narrow-tooth comb for 10 times. After pretreatment, hang the hair tress in a constant temperature and humidity chamber at a temperature of 20±1°C and a relative humidity of 50±5% to dry and balance for 24 hours.

[0092] 5) Treatment of test samples and control products: Apply the test product evenly to the hair bundle at a dosage of 0.01 g / g hair, place the hair bundle at a temperature of 37°C, rub for 1 minute, and comb it 20 times with a narrow-tooth comb (10 times on the front and back of the hair bundle). Use the control group product instead of the product in the same way as the test product group hair bundle.

[0093] 6) Scanning electron microscope test: Before the sample is processed, a few hairs are cut from the hair bundle and an electron microscope photo is taken (Before); after the sample is processed, a few hairs are cut from the hair bundle again and an electron microscope photo is taken (After). Results:

[0094] Compared with the negative control aqueous solution in the control group, the aqueous solution containing 0.1% recombinant keratin, the aqueous solution containing 0.02% recombinant keratin and the aqueous solution containing 0.1% commercially available keratin in the positive control can effectively protect the hair cuticle ( Figure 2 ), it can be seen that the aqueous solutions containing 0.1% and 0.02% of the recombinant keratin of the present invention have a significant protective effect on hair cuticles.

[0095] 3. In vitro hair dry combing test

[0096] Test method:

[0097] 1) Testing instruments and materials: Dia-Stron fibra.one hair multifunctional testing system --- combing test accessories Combing; Far Eastern people's chemically damaged hair bundles (15cm*3.0cm*4g).

[0098] 2) Test samples: Shampoo 1 with 0.1% recombinant keratin sample added and Shampoo 2 with 0.02% recombinant keratin sample added as described in the above shampoo products; negative control is control group shampoo with deionized water added; positive control is Shampoo 3 with 0.1% commercially available keratin sample added.

[0099] 3) Preparation and cleaning of hair bundles: The hair bundles were randomly divided into groups, with 5 hair bundles in each group; after marking, they were placed in a constant temperature and humidity room at 21±1°C and 50±10% for 24 hours. Then, 14% SDS was applied to the hair bundles for 1 minute, and the hair was combed for 2 minutes with the hand gesture like milking a cow, so that the SDS was evenly distributed on the surface of the hair bundles. The hair bundles were left to stand, and the hair bundles were rinsed with warm water for 1 minute (water temperature: 37±0.5°C; water flow rate 75±5mL / s; water hardness: 80±10ppm), and the hair bundles were combed thoroughly and dried for later use.

[0100] 4) Use the product: Apply the sample to the corresponding hair according to each group (1 min), rub the hair evenly from the roots to the ends with fingers to make foam (2 min), let it stand for 3 min, and then gently rinse it with clean water (1 min); after repeating the product use steps once, the hair is naturally dried in a constant temperature and humidity room with a temperature of 21±1°C and a humidity of 50±10%.

[0101] 5) Dry combing test:

[0102] Including the measurement of basic values ​​and sample treatment data: Use Fibra.one to test the combability of each group of hair (total combing work, maximum combing force parameters)

[0103] Statistical methods:

[0104] SPSS statistical analysis software was used to analyze the raw data. The independent sample T test was used for normal distribution, otherwise the rank sum test was used.

[0105] result:

[0106] Compared with the negative control group shampoo, the maximum force and work required for dry combing of hair bundles treated with shampoo 1 containing 0.1% recombinant keratin, shampoo 2 containing 0.02% recombinant keratin and shampoo 3 containing 0.1% commercial keratin were significantly reduced. The three groups of experimental products can significantly improve the dry combing effect of hair bundles, and there is no significant difference between the three groups of products in terms of the maximum force required for dry combing ( Figure 3 ) and the total work required ( Figure 4 ) data, there is no significant difference among the three groups of products.

[0107] 4. In vitro hair bundle tensile test

[0108] Test method:

[0109] 1) Testing instruments and materials: Dia-Stron fibra.one hair multifunctional testing system --- combing test accessories Combing; Far Eastern people's chemically damaged hair bundle (15cm*3.0cm*4g)

[0110] 2) Test samples: an aqueous solution containing 0.1% recombinant keratin and an aqueous solution containing 0.02% recombinant keratin as described in the above aqueous solution product; a negative control is an aqueous solution without keratin (i.e., a control aqueous solution); and a positive control is an aqueous solution containing 0.1% commercially available keratin (i.e., a WLM-02 aqueous solution).

[0111] 3) Preparation and cleaning of hair bundles: The hair bundles were randomly divided into groups, with 5 hair bundles in each group; after marking, they were placed in a constant temperature and humidity room at 21±1°C and 50±10% for 24 hours. Then, 14% SDS was applied to the hair bundles for 1 minute, and the hair was combed for 2 minutes with the hand gesture like milking a cow, so that the SDS was evenly distributed on the surface of the hair bundles. The hair bundles were left to stand, and the hair bundles were rinsed with warm water for 1 minute (water temperature: 37±0.5°C; water flow rate 75±5mL / s; water hardness: 80±10ppm), and the hair bundles were combed thoroughly and dried for later use.

[0112] 4) Use the product: Apply the sample evenly in an S shape on the corresponding hair pieces according to each group (1 minute), and use your fingers to evenly apply the product from the roots to the ends of the hair (2 minutes), and place the hair bundles in a constant temperature and humidity room to dry.

[0113] 5) Single hair strand tensile test:

[0114] Measurement of basic values: Take 6 hairs from each hair bundle before treatment, put each hair on two brass tubes, flatten it with a special processing tool, and use Fibra.one to perform a single hair bundle tensile test (force required to break, work required to break)

[0115] Data measurement after sample processing: 6 hairs are taken from each hair bundle after processing, each hair is passed through two brass tubes, flattened with a special processing tool, and a single hair bundle tensile test (force required to break, work required to break) is performed using Fibra.one

[0116] Statistical methods:

[0117] SPSS statistical analysis software was used to analyze the raw data. The independent sample T test was used for normal distribution, otherwise the rank sum test was used.

[0118] result:

[0119] Compared with the negative control aqueous solution, the maximum force required for stretching and the total work used for hair treated with the aqueous solution containing 0.1% recombinant keratin, the aqueous solution containing 0.02% recombinant keratin and the aqueous solution containing 0.1% commercial keratin all increased significantly, indicating that all three groups of products can make hair stronger. And from the maximum force used for stretching before and after product treatment ( Figure 5 ) and total work ( Figure 6), there was no significant difference in the results among the three groups of products.

[0120] 5. In vitro hair anti-static test

[0121] Experimental process:

[0122] 1) Testing instruments and materials: Dia-Stron fibra.one hair multifunctional testing system --- combing test accessories Combing; Far Eastern people's chemically damaged hair bundle (15cm*3.0cm*4g)

[0123] 2) Test samples: Shampoo 1 with 0.1% recombinant keratin sample added as described in the above shampoo products; Shampoo 2 with 0.02% recombinant keratin sample added; negative control is the control group shampoo with deionized water added; positive control is Shampoo 3 with 0.1% commercial keratin sample added.

[0124] 3) Preparation and cleaning of hair bundles: Select the same hair bundles and randomly group them to ensure that there are 5 bundles in the test product group and 5 bundles in the control group; after marking, place them in a constant temperature and humidity room at 21±1℃ and 50±10% for 24 hours. Then apply 14% SDS to the hair bundles for 1 minute, comb the hair with a comb for 2 minutes like milking a cow, so that the SDS is evenly distributed on the surface of the hair bundles, let it stand, rinse the hair bundles with warm water for 1 minute (water temperature: 37±0.5℃; water flow rate 75±5mL / s; water hardness: 80±10ppm), comb the hair bundles thoroughly, and dry them for later use.

[0125] 4) Use the product: Apply the sample to the corresponding hair according to each group (1 min), rub the hair evenly from the roots to the ends with fingers to make foam (2 min), let it stand for 3 min, and then gently rinse it with clean water (1 min); after repeating the product use steps once, the hair is naturally dried in a constant temperature and humidity room with a temperature of 21±1°C and a humidity of 50±10%.

[0126] 5) Anti-static test:

[0127] Measurement of basic value: Before using the product, comb each group of hair bundles at least 10 times at a fixed speed and procedure to cause friction static electricity, use image camera VISIA to take pictures of the corresponding hair bundles, export the pictures, analyze and record the proportion of the frizzy hair area to the whole hair bundle area.

[0128] Data measurement after sample processing: After using the product according to step 4), comb each group of hair bundles at least 10 times at a fixed speed and procedure to cause friction static electricity, use image camera VISIA to photograph the corresponding hair bundles, export the photos, analyze and record the proportion of the frizzy hair area to the entire hair bundle area.

[0129] Compare the improvement rates of hair area ratios of different product groups and evaluate the antistatic effects of the products on hair.

[0130] Statistical methods:

[0131] SPSS statistical analysis software was used to analyze the raw data. The independent sample T test was used for normal distribution, otherwise the rank sum test was used.

[0132] result:

[0133] Compared with the control group, the improvement rate of hair area ratio of the hair bundles treated with shampoo 1 containing 0.1% recombinant keratin, shampoo 2 containing 0.02% recombinant keratin and shampoo 3 containing 0.1% commercial keratin was significantly improved, indicating that the three products can significantly improve the anti-static effect of hair bundles, but there is no significant difference between the three products ( Figure 7 ).

[0134] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A recombinant human keratin, characterized in that: The recombinant human keratin comprises the amino acid sequence shown in SEQ ID NO: 1 or a fragment comprising the sequence shown in SEQ ID NO: 1, wherein the fragment has an amino acid sequence at least 70% identical to SEQ ID NO: 1 or a mutant of the sequence of SEQ ID NO:

1.

2. A nucleotide sequence encoding the recombinant human keratin as claimed in claim 1.

3. The nucleotide sequence encoding recombinant human keratin according to claim 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant cell, characterized in that: The recombinant cell comprises a nucleotide sequence encoding the recombinant human keratin according to claim 2, and the recombinant cell is one of a eukaryotic cell and a prokaryotic cell.

5. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide sequence according to claim 2 or 3.

6. A method for synthesizing recombinant human keratin as claimed in claim 1, characterized in that: The following steps are involved: 1) constructing a recombinant cell expressing recombinant human keratin as claimed in claim 4; 2) culturing the recombinant cell to obtain a culture; 3) Separating and purifying from the culture to obtain recombinant human keratin.

7. A method for synthesizing recombinant human keratin according to claim 6, characterized in that: The recombinant cells in 2) are cultured by one of the following methods: primary culture, secondary culture, adherent cell culture, suspension cell culture and fermentation.

8. A method for synthesizing recombinant human keratin according to claim 6 or 7, characterized in that: The recombinant cells in 2) are cultured by a fermentation method, wherein the recombinant cells are cultured in a liquid culture medium in a seed shake flask. After the culture is completed, the seed liquid is inoculated into a fermentation tank for fermentation and induction of expression to obtain a fermentation product, i.e., a culture.

9. Use of the recombinant human keratin according to claim 1 in hair care and hair nourishment products.

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