Recombinant human type XVII collagen with hair growth and hair care effects and its application

Abstract

This patent application discloses a recombinant human type XVII collagen with hair growth and hair care effects and its application. The amino acid sequence of the recombinant human type XVII collagen is shown in SEQ ID NO: 1. The recombinant human type XVII collagen is composed of fragments of non-collagen domains spliced ​​together, and has strong hair growth and hair care related activities. Its activity is not only the strongest among various splicing combinations, but even stronger than the clinically commonly used drug minoxidil.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a new recombinant human type XVII collagen with hair growth and hair protection effects, and a preparation method and application thereof. Background Art

[0002] Collagen is the main extracellular matrix of the human body, accounting for about 85% of the extracellular matrix. It is the main structural protein of human tissues and organs, accounting for about 30% to 40% of the total protein in the human body. It is commonly found in the skin, blood vessels, tendons, fascia and other parts, and plays a variety of important biological functions.

[0003] Recombinant collagen is designed with the amino acid sequence of collagen in the organism as a template. It is transferred into engineered cells such as Escherichia coli and Pichia pastoris with the help of synthetic biology technology, and the collagen is prepared by using the rapid production capacity of engineered cells. Compared with animal-derived collagen, recombinant collagen has obvious advantages. For example, recombinant collagen has a single molecule and clear composition, and high-purity products can be obtained; it has no viral risks, low cytotoxicity, and extremely low immunogenicity, and is less likely to cause allergies, inflammation, fever and other problems; it has water-soluble and emulsifiable properties, strong processability, no need for cold chain transportation, easy storage, and a green and environmentally friendly production and preparation process.

[0004] Type XVII collagen (encoded by COL17A1 gene) is a transmembrane non-fibroblastic collagen, which is a homogeneous trimer composed of three identical α1 (XVII) chains, and is divided into three major structural domains: intracellular, transmembrane, and extracellular. COL17A1 protein is a component of hemidesmosomes in cells, plays an important role in the function of epithelial cells-basement membrane, regulates the adhesion, separation, development and differentiation of epithelial cells, and plays an important role in the differentiation and regeneration of hair follicle cells.

[0005] Human type XVII collagen is a transmembrane non-fibroblastic collagen composed of three identical α1(ⅩⅦ) chains, which is divided into three major structural domains: intracellular, transmembrane, and extracellular. Related studies have shown that type XVII collagen is closely related to hair follicle stem cells and skin stem cells. It is an important cytoplasmic matrix that can regulate the development and differentiation of hair follicle stem cells and skin stem cells, and has great application potential in skin repair and anti-hair loss products. Summary of the invention

[0006] The object of the present invention is to provide a recombinant human type XVII collagen which is formed by combining independent regions from the non-collagenous domain of human type XVII collagen and has strong hair growth and hair care related activities.

[0007] After in-depth research, the inventors found that by combining and splicing independent regions from the non-collagen domain of human type XVII collagen that are not connected to each other in a specific way, recombinant human type XVII collagen with strong hair growth and hair care related activities (such as the activity of promoting hair papilla cell proliferation and the activity of promoting hair growth) can be obtained.

[0008] That is, the present invention includes:

[0009] 1. A recombinant human type XVII collagen with hair growth and hair care effects, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] 2. A nucleic acid encoding the recombinant human type XVII collagen described in item 1.

[0011] 3. An expression vector comprising the nucleic acid described in item 2.

[0012] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell may be a eukaryotic or prokaryotic cell. For example, the host cell may be a bacterial host such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, or a eukaryotic host such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, or plant cells, preferably Pichia pastoris or Escherichia coli, and more preferably Pichia pastoris.

[0013] 5. A method for producing the recombinant human type XVII collagen described in item 1, comprising: culturing the host cell described in item 4 to express the recombinant human type XVII collagen described in item 1, and purifying the protein. Preferably, the host cell is induced (e.g., methanol induced) to secrete and express the recombinant human type XVII collagen.

[0014] 6. The production method according to item 5, wherein the protein is purified by a purification method selected from hydrophobic chromatography, salting out, chromatography, ion exchange chromatography, affinity chromatography, acid-base precipitation, membrane separation, or a combination of the above purification methods.

[0015] To facilitate purification, identification, quantification, etc., tags and small molecule peptides can be added without affecting the collagen structure, such as: His tag, Flag tag, c-MYC, etc.

[0016] 7. A drug, biomaterial, medical device, skin care product, or cosmetic comprising the recombinant human type XVII collagen according to item 1. Preferably, the skin care product or cosmetic is prepared in a form suitable for application to the scalp, such as shampoo, shampoo cream, or conditioner.

[0017] 8. Use of the recombinant human type XVII collagen described in item 1 in the preparation of an external medicine or cosmetic for hair growth. Preferably, the external medicine or cosmetic is prepared in a form suitable for application to the scalp.

[0018] Advantageous Effects of the Invention

[0019] (1) The recombinant human type XVII collagen of the present invention is composed of fragments of non-collagen domains spliced ​​together, and has strong hair growth and hair care related activities. Its activity is not only the strongest among various splicing combinations, but is even stronger than the clinically commonly used drug minoxidil.

[0020] (2) The constituent elements of the recombinant human type XVII collagen of the present invention are from the non-collagen domain. Its application to the human body will not cause allergic and immune rejection reactions. It has high biological activity and has the function of promoting cell proliferation, migration and cell adhesion. It can be widely used in the fields of medicine, biomaterials, medical devices, skin care products, etc.

[0021] (3) The fermentation and purification process conditions of the recombinant human type XVII collagen of the present invention are mature and have the conditions for expanding production, and can be produced on a large scale industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a schematic diagram showing the construction of the expression plasmid of recombinant human type XVII collagen Col 17.

[0023] Figure 2 The figure shows the SDS-PAGE electrophoresis results of purified Col17d, Col17e, and Col17. Figure 3 A graph showing the effects of different recombinant collagens on cell proliferation rate (* indicates comparison with the control group, *P<0.05, **P<0.01, # indicates comparison with the Col17 group, #P<0.05, ##P<0.01).

[0024] Figure 4 The figure shows the hair length of mice 28 days after administration (* indicates comparison with the normal group, *P<0.05, **P<0.01; # indicates comparison with the model group, #P<0.05, ##P<0.01).

[0025] Figure 5 The photographs show the dorsal hair growth of mice in the blank group, model group, minoxidil group, and Col17 group on days 0, 7, 14, 21, and 28. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below through specific embodiments. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the present invention.

[0027] Example 1: Expression of various recombinant human type XVII collagen

[0028] The gene of recombinant human type XVII collagen with the amino acid sequence shown in SEQ ID NO:1 was chemically synthesized. During the synthesis, EcoRI and NotI recognition sites and signal peptide recognition sites were added to the 5' and 3' ends respectively. After linearization with restriction endonuclease SacI, it was cloned into the expression vector pPICZaA. Pichia pastoris X33 was used as the expression host bacteria. The obtained pPICzaA-XVII cloning plasmid was linearized by electroporation and then transformed into X33. High-copy positive clones were selected by the bleomycin gradient method, and the Pichia pastoris genetically engineered bacteria were obtained by culturing at 30°C for 72 hours. The brief construction process of the Pichia pastoris engineered bacteria is as follows: Figure 1 shown.

[0029] The genetically engineered Pichia yeast obtained above was inoculated into YPD medium and cultured until OD600 = 25 ± 3. Then, the culture was inoculated into an NBS 415 fermenter with an initial volume of 5 L at a 10% inoculation amount. The culture temperature was 30° C., pH = 5.5, and OD was controlled at 20%-30%. When the glycerol was exhausted, glycerol fed culture was started. When the wet weight of the bacteria reached more than 200 g / L, methanol was added for induction culture. The temperature in the induction stage was 28° C. and the pH was 5.0. The fermenter was released after 48 hours of induction and the supernatant was collected by centrifugation.

[0030] Add NaCl with a final concentration of 6% to the crude protein supernatant collected by centrifugation, stir to dissolve, let stand at room temperature for 2 hours, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant. The collected supernatant is concentrated and desalted by a 30KD ultrafiltration membrane.

[0031] (2) The pH of the concentrated and desalted protein supernatant was adjusted to 6.5 with phosphoric acid, loaded onto a cation exchange resin, and the target protein was obtained after elution with 0.5 M NaCl;

[0032] (3) The collected target protein solution was desalted by a 30KD ultrafiltration membrane, and then pre-frozen in a -20°C refrigerator for 4 hours, and then transferred to a vacuum freeze dryer for freeze drying. After 48 hours, the freeze-dried protein was collected, which was the recombinant human type XVII collagen Col 17.

[0033] Col 17 SEQ ID NO: 1

[0034] 1 EVLNLQLPLS NSETFLSTSG SSSFGLNLQS GPSEGGSSST MYVSSSSGQE IQQYISEYMQ

[0035] 61 SDSIRSYLSG VQTTITGETF DYSELASHVV SYLRTSGYGV SLFSSSISSE DILAVLQRDD

[0036] 121 VRQYLRQYLM GPRSLLSLDY AELSSRILSY MSSSGISIYE ELLSLLRGSE FRGIVWSSIS

[0037] 181 VEDLSSYLHT AGLSFIPATY AAENSDSFRS ELISYLTSPD VRSFIVRLLS TDASHSRGSS

[0038] 241 SSSHSSSVRR GSSYSSSMST GG

[0039] The amino acid sequence of the above-mentioned Col 17 is assembled from the following 12 fragments:

[0040] SEQ ID NO:2:EVLNLQ

[0041] SEQ ID NO:3:LP

[0042] SEQ ID NO:4:LSNSETFL

[0043] SEQ ID NO:5:STSG SSSFGLNLQ

[0044] SEQ ID NO:6:S GPSEGGSSST MYV

[0045] SEQ ID NO:7:SSSSGQEIQQYISEYMQSDSIRSYLSGVQ

[0046] SEQ ID NO:8:

[0047] TTITGETFDYSELASHVVSYLRTSGYGVSLFSSSISSEDILAVLQRDDVRQYLRQYLMGPR

[0048] SEQ ID NO:9:SLLSLDY AALSSRILSY MSSSGISI

[0049] ELLSLLRGSE FRGI TO SEQ ID NO:10

[0050] SEQ ID NO:11:VWSSISVEDLSSYLHT AGLSFIP

[0051] SEQ ID NO:12:NAME AAENSDSFRS ELISYLTSPD V

[0052] SEQ ID NO:13:RSFIVRLLSTDASHSRGSS

[0053] We commissioned a commercial company to chemically synthesize the following three types of recombinant human type XVII collagen:

[0054] Col 17a: It is composed of the amino acid sequences shown in SEQ ID NO: 2, 6, and 12;

[0055] Col 17b: It is composed of the amino acid sequences shown in SEQ ID NO: 4, 5, and 11;

[0056] Col 17c: It is composed of the amino acid sequences shown in SEQ ID NO: 3, 10, and 13.

[0057] Similar to the expression and purification of the above-mentioned recombinant human type XVII collagen Col 17, the following two types of recombinant human type XVII collagen were expressed and purified:

[0058] Col 17d: It is composed of the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9;

[0059] Col 17e: It is composed of the amino acid sequences shown in SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 11, and 12.

[0060] Figure 2 This is the SDS-PAGE electrophoresis diagram of purified Col 17, Col 17d, and Col 17e.

[0061] Example 2: Effects of various recombinant human type XVII collagen on the proliferation of human dermal papilla cells

[0062] 1. Reagents and Instruments

[0063] Human dermal papilla cells (HFDPC) (ATCC), arginine (Amersco, USA), separation enzyme, trypsin, dimethyl sulfoxide (DMSO) (Sigma, USA), DMEM cell culture medium (Gibco, USA), tetramethylthiazolyl blue (MTT) (Amersco, USA), newborn fetal bovine serum (Hangzhou Sijiqing Biomaterials Co., Ltd.), and microplate reader (Boten, USA).

[0064] 2. Cell Culture

[0065] HFDPCs were cultured in DMEM medium containing 10% FBS and 1% triple antibody at 37°C in an incubator with 5% CO2. When the growth density reached 80% to 90%, the cells were digested with trypsin for 2 minutes, collected by centrifugation, and passaged at a ratio of 1:3.

[0066] 3. MTT assay to determine cell proliferation rate

[0067] Take HFDPC in the logarithmic growth phase for digestion, count the live cells, and inoculate the cells in a 96-well cell culture plate at a density of 5x10 per well. 3 cells, 200 μL per well, discard the culture medium after the cells adhere to the wall overnight, add 200 μL of culture medium containing 0.1 mg / mL Col 17, Col 17a, Col 17b, Col 17c, Col 17d and Col 17e respectively, and add only culture medium to the blank control group, and continue to culture for 24 hours. At the end of drug treatment, add 20 μL of 5g / L MTT to each well, continue to culture for 4 hours, discard the waste liquid, add 200 μL DMSO to each well, shake the culture plate thoroughly, and use a microplate reader to measure the absorbance (A) value of each well at a wavelength of 550 nm to calculate the cell proliferation rate.

[0068] 4. Results

[0069] The results are as follows Figure 3 As shown. It can be seen that Col 17 significantly promotes the proliferation of HFDPC (compared with the blank control group, P<0.01), Col 17a, Col 17b, Col 17c, and Col 17e also promote the proliferation of HFDPC (compared with the blank control group, P<0.05), and Col 17d has no promoting effect on the proliferation of HFDPC (no significant difference compared with the blank control group). Moreover, the promoting effect of Col17 on the proliferation of HFDPC is significantly better than Col 17a, Col 17b, Col 17c, and Col 17d (P<0.01 or P<0.05).

[0070] Example 3: Effect of recombinant human type XVII collagen on androgenic alopecia mouse model

[0071] 1. Experimental drugs and reagents

[0072] The recombinant human type XVII collagen Col 17, 5% minoxidil (Dafeixin), depilatory cream, and testosterone (Sinopharm Chemical Reagent Co., Ltd.) prepared in Example 1.

[0073] 2. Test methods

[0074] Male C57BL / 6 mice aged 6-8 weeks were adapted to feeding for one week. Before the experiment, the mice were randomly divided into 4 groups. The hair on the back of the mice was removed, with an area of ​​about 3cm*3cm. The experiment was divided into 4 groups, namely blank group, testosterone group, minoxidil group, and recombinant human type XVII collagen aqueous solution group. Except for the blank control group, 100μL of testosterone (mass fraction 0.05%) was applied to the back of the mice in the other groups to prepare a mouse androgenic alopecia model. At the same time as the modeling, 100μL of the drug was evenly applied to the selected depilatory experimental observation area. The blank control group and testosterone group were smeared with the same volume of normal saline, and the drug was administered once a day.

[0075] 3. Detection indicators

[0076] The back skin of the mice was observed and photographed daily. After 28 days, 30 hairs were randomly plucked from three different parts of the shaved area, and the length was measured and recorded.

[0077] 4. Test results

[0078] pass Figure 4 and Figure 5 , the hair growth of mice after 28 days and the photos of hair growth of mice show that the hair growth of mice in the recombinant human type XVII collagen group has exceeded the hair length of mice in the blank control group on the 28th day, indicating that the recombinant human type XVII collagen group has the effect of promoting hair growth in androgen model mice. Compared with minoxidil, most of the skin of mice in the Col 17 group has begun to grow hair and turn black on the 14th day, while the mice in the minoxidil group only began to grow hair in the local area of ​​the back on the 21st day, indicating that Col 17 has a faster and stronger effect of promoting hair growth than minoxidil.

Claims

1. A recombinant human type XVII collagen with hair growth and hair care effects, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the recombinant human type XVII collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced.

5. The method for producing recombinant human type XVII collagen according to claim 1, comprising: The host cell of claim 4 is cultured to express the recombinant human type XVII collagen of claim 1, and the protein is purified.

6. The production method according to claim 5, wherein: The protein purification adopts a purification method selected from hydrophobic chromatography, salting out, chromatographic chromatography, ion exchange chromatography, affinity chromatography, acid-base precipitation, membrane separation, or a combination of the above purification methods.

7. A medicine, medical device or cosmetic comprising the recombinant human type XVII collagen according to claim 1.

8. Use of the recombinant human type XVII collagen according to claim 1 in the preparation of external medicines or cosmetics for hair growth.

9. The use according to claim 8, wherein The external medicine or cosmetic is prepared in a form suitable for application to the scalp.

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