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 recombinant human type XVII collagen is a truncated protein of human type XVII collagen, and its amino acid sequence is shown in SEQ ID NO: 1. The biological activity of the recombinant human type XVII collagen is not only higher than other truncated proteins from human type XVII collagen, but also higher than Kopyrrol, a commonly used hair growth and hair care drug in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a recombinant human type XVII collagen with the efficacy of hair growth and hair care, a preparation method thereof, and an application thereof in medicines or cosmetics. Background Art

[0002] Type XVII collagen (COL17) is a transmembrane non-fibroblastic collagen containing 1497 amino acids. It is a homogeneous trimer composed of three α1 (XVII) chains. It is divided into three structural regions: intracellular, transmembrane, and extracellular. Its N-terminus is located in the cytoplasm and its C-terminus is located in the extracellular matrix. It is the core component of the basement membrane hemidesmosomes and is mainly located in the hair follicle stem cells in the hair follicle bulb and the epidermal stem cells at the junction of the epidermis and dermis.

[0003] Type XVII collagen is expressed in a variety of tissues, including skin, colon, esophagus, placenta, small intestine, stomach and other tissues. Its expression level is highest and most concentrated in the skin. Its function is to make the epidermis and dermis tightly connected through the basement membrane zone to promote epidermal replacement and stabilize the basement membrane. Data show that the reduction of type XVII collagen expression can lead to a decrease in the adhesion of ESC to the basement membrane, causing SEC to detach from the basement membrane niche, and then affect the stemness and proliferation ability of ESC, causing changes in the thickness and stratification of the aging epidermis, resulting in a decrease in the burn and wound healing ability of aging skin. In addition, the expression of type XVII collagen can also promote the maintenance of the stemness of hair follicle stem cells, regulate the differentiation of hair follicle stem cells, and regulate the proliferation of epidermal cells between hair follicles, prevent hair follicle aging, and have a hair care effect.

[0004] In 2023, the "Recombinant Human Type XVII Collagen White Paper" jointly written by Chuangjian Medical and Frost & Sullivan Consulting Company was officially released. The report explains the structure, function, mechanism of type XVII collagen, as well as the technical advantages, value and application scenarios of recombinant human type XVII collagen. This indicates that recombinant human type XVII collagen will become a research hotspot in the future collagen market.

[0005] However, the content of type XVII collagen in the human body is extremely low, and it is a non-extracellular secretory collagen. It has a long amino acid chain and a large molecular weight, and is very difficult to extract or synthesize, and cannot be mass-produced. Therefore, this type of raw material is rarely seen on the market. Summary of the invention

[0006] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a recombinant human type XVII collagen which has good hair growth and hair care, hair growth and hair loss prevention effects, has an amino acid sequence that is completely homologous to natural human type XVII collagen and can be produced on a large scale.

[0007] In order to solve the above technical problems, the inventors conducted in-depth research. The inventors first truncated the entire amino acid sequence of human type XVII collagen and determined the active (including cell adhesion activity, cell migration activity, and tissue regeneration and hair follicle repair and regeneration biological activity) regions; then the active regions were gradually truncated, and finally a highly active truncated protein of human type XVII collagen was obtained, which has higher activity than other truncated proteins from human type XVII collagen, and even higher activity than Kopyrrol, a commonly used hair growth and hair care drug in clinical practice.

[0008] That is, the present invention includes:

[0009] 1. A recombinant human type XVII collagen, which is a truncated protein of human type XVII collagen, and its amino acid sequence is as shown in SEQ ID NO: 1 (482nd to 698th position in the COL17A1 sequence). The present patent application also discloses a recombinant human type XVII collagen having an amino acid sequence as shown in SEQ ID NO: 3 (439th to 698th position in the COL17A1 amino acid sequence) or SEQ ID NO: 5 (501st to 698th position in the COL17A1 amino acid sequence).

[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. The vectors include pPIC9, pPIC9K, pHIL-S1, pPICZαA, pYAM75P, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa (Invitrogen), pPIC3.5K, etc., but are not limited to the above plasmids. Further, pPIC9K vector is preferred.

[0012] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell includes a bacterial cell, a fungal cell, a plant cell, and an animal cell. The bacteria are selected from Escherichia coli, Lactobacillus, Bacillus licheniformis, Bacillus subtilis, or Streptomyces; or, the fungal cell is selected from Saccharomyces cerevisiae, Candida, Torulopsis, Rhodotorula, Hansenula, Pichia pastoris, or Kluyveromyces; or, the plant cell is selected from tobacco cells or Arabidopsis cells; or, the animal cell is selected from Sf9 cells, CHO cells, NS0 cells, Sp2 / 0 cells, HEK293 cells, HKB11 cells, PER.C6 cells, HeLa cells, or CAP cells. Further, Pichia pastoris is preferred.

[0013] 5. A method for producing the recombinant human type XVII collagen described in item 1, comprising the steps of culturing the host cell described in item 4 to express the recombinant human type XVII collagen described in item 1, and purifying the recombinant human type XVII collagen.

[0014] 6. The production method according to item 5, wherein the host cell is caused to secrete the recombinant human type XVII collagen into a culture medium.

[0015] 7. Use of the recombinant human type XVII collagen described in item 1 in the preparation of medicines or cosmetics for hair growth and care, repairing hair follicles, and / or promoting hair follicle tissue regeneration.

[0016] 8. The use according to item 7, wherein the drug is an external medicine. The external medicine is preferably prepared in a form suitable for application to the scalp.

[0017] 9. The use according to item 7, wherein the cosmetic is prepared in a form suitable for application to the scalp, such as shampoo, shampoo liquid, shampoo cream.

[0018] The recombinant human type XVII collagen provided by the present invention has remarkable effects on the proliferation, migration, adhesion and other aspects of hair follicle cells, and has the functions of increasing hair volume, protecting hair, repairing hair follicles and promoting the regeneration of hair follicle tissue.

[0019] As described above, the recombinant collagen of the present invention has the following beneficial effects:

[0020] 1. The amino acid sequence is completely homologous to natural human type XVII collagen and is basically non-immunogenic.

[0021] 2. It has a small molecular weight and is not easily degraded, and can be expressed efficiently, making it suitable for large-scale production.

[0022] 3. It has strong biological activities such as cell adhesion activity, cell migration promotion activity, hair follicle tissue regeneration and hair follicle repair promotion activity. Its activity is not only higher than other truncated proteins derived from human type XVII collagen, but even higher than Kopyrrol, a commonly used hair growth and hair care drug in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Electrophoresis diagram of three types of recombinant human type XVII collagen expression.

[0024] Figure 2 Electrophoresis of three types of recombinant human type XVII collagen purification Specific implementation plan

[0025] 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.

[0026] Example 1 Preparation of recombinant human type XVII collagen in a yeast expression system

[0027] 1) Preparation of shuttle plasmid

[0028] According to the full-length nucleotide sequence of human type XVII collagen, the codon optimization of the yeast expression system was performed to obtain the target gene sequence of recombinant human type XVII collagen NT-1 (SEQ ID NO: 2), the target gene sequence of NT-2 (SEQ ID NO: 4), and the target gene sequence of NT-3 (SEQ ID NO: 6), wherein the amino acid sequences of recombinant human type XVII collagen NT-1, NT-2, and NT-3 are shown in SEQ ID NOs: 1, 3, and 5, respectively. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis, and the synthesized gene was connected to the pPIC9K plasmid to obtain pPIC9K-XVII-NT-1, pPIC9K-XVII-NT-2, and pPIC9K-XVII-NT-3 plasmids.

[0029] 2) Preparation of yeast expression strains

[0030] The three plasmids pPIC9K--XVII-NT-1, pPIC9K-XVII-NT-2, and pPIC9K-XVII-NT-3 were linearized with Pme I and transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a screening marker to screen transformants to obtain three yeast expression strains.

[0031] Inducible expression of target protein

[0032] XVII-NT-1 was used as an example to induce the expression of the target protein.

[0033] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium, and culture it at 30°C and 200 rpm for 48 h for activation;

[0034] Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at 1% of the inoculum, and culture at 30°C, 200 rpm for 24 h as the seed for the upper tank;

[0035] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0036] (4) When the OD600 reaches 70, 50% glycerol is added. When the OD600 reaches about 120, the addition is stopped. When the dissolved oxygen rebounds to 100%, methanol is added for induction.

[0037] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 h. The fermentation was terminated and the culture medium was centrifuged at 12000 rpm for 2 min. The supernatant was taken and the protein yield and purity were tested by BCA method and SDS-PAGE method. The protein electrophoresis diagram is shown in Figure 1 The BCA result showed that the protein concentration in each liter of fermentation broth was 8.3 g / L.

[0038] The same induction expression method was used to obtain 6 g / L of XVII-NT-2 fermentation broth and 5.4 g / L of XVII-NT-3 fermentation broth.

[0039] 4) Purification of target protein

[0040] (1) The fermentation liquid is centrifuged to remove the bacterial cells to obtain a semi-clear liquid;

[0041] (2) salting out the fermentation supernatant, collecting the precipitate, and obtaining a crude purified sample;

[0042] (3) After the crude purified sample is redissolved, it is subjected to ion exchange chromatography to obtain a pure sample.

[0043] (4) The protein solution was freeze-dried to obtain three types of recombinant human type XVII collagen freeze-dried sponges (XVII-NT-1, XVII-NT-2, and XVII-NT-3).

[0044] Example 2: Characterization of recombinant human type XVII collagen

[0045] The protein electrophoresis diagram of the fermented and purified samples was detected by SDS-PAGE. The results were as follows: Figure 2 As shown, the predicted molecular weight is about 20 kDa, which is consistent with the theoretical molecular weight. The protein sequence coverage determined by mass spectrometry is 100%, and the electrophoretic purity is ≥95%.

[0046] Example 3 Recombinant human type XVII collagen human hair follicle dermal papilla cell proliferation experiment

[0047] (1) Collect human hair follicle dermal papilla cells (HFDPCs) in the logarithmic growth phase and remove serum components on the cell membrane by trypsin digestion. Count the cells and adjust the cell concentration to 10 per well. 3 -10 4 Continue to culture overnight.

[0048] (2) According to the experimental design, the recombinant human type XVII collagen XVII-NT-1, XVII-NT-2, and XVII-NT-3 of the present invention were prepared into 0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml, respectively, and filtered through a 0.22 um sterile filter membrane for later use. In addition, a comparable drop (0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml) was used as a positive control;

[0049] (3) The above three raw materials and the positive control were added to each well of the 96-well plate prepared with cells according to the above concentrations. A blank control group was also set up and the cells were cultured for another 24 hours.

[0050] (4) At the end of cell culture, aspirate the culture medium and wash the plate three times with PBS to remove non-adherent cells and residual culture medium. Then add 20ul of MTT solution (5mg / ml in PBS) to each well and continue incubation for 4 hours. Terminate the culture and carefully aspirate the culture supernatant in the well. For suspended cells, centrifuge before aspirating the culture supernatant in the well. Add 150ul of DMSO to each well and shake for 10 minutes to fully dissolve the crystals.

[0051] Colorimetry: Select 490nm wavelength, measure the light absorbance of each well on an enzyme-linked immunosorbent assay, record the results, and draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.

[0052] The cell proliferation of each group is shown in Table 1 below. When the concentration of the three samples was 0.1 mg / ml-0.5 mg / ml, the proliferation rate gradually increased with the increase of concentration. When the concentration increased to above 0.5 mg / ml, the proliferation rate tended to be stable. The recombinant human type XVII collagen XVII-NT-1 of the present invention has a significant difference in the proliferation effect on HFDPC cells compared with XVII-NT-2, XVII-NT-3 and comparable results (P<0.01). This shows that the recombinant human type XVII collagen XVII-NT-1 of the present invention can more significantly promote the proliferation of human hair follicle dermal papilla cells.

[0053] Table 1: Proliferation rate of HFDPC cells promoted by three collagens (x±s, n=3)

[0054]

[0055] Example 4 Inhibition test of recombinant human type XVII collagen on 5α-reductase in SZ95 cells

[0056] Studies have shown that excessive secretion of 5a-reductase will react with testosterone in the body to produce another male hormone, dihydrotestosterone (DHT). DHT will block nutrients to hair follicles and cause hair follicle atrophy. Therefore, if the activity of 5a-reductase can be inhibited, hair follicle atrophy will be blocked and hair loss can be treated.

[0057] The present invention conducts the following experiment to evaluate the inhibition of recombinant human type XVII collagen on SZ95 cell 5α reductase:

[0058] (1) SZ95 cells with good growth status were digested with trypsin and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 4×10 4 / mL. Cell culture 24h;

[0059] (2) Cell modeling: The experiment set up blank group, model group, and sample group. The model group and sample group were induced with 20 ng / mL IGF-1;

[0060] (3) In the sample group, recombinant human type XVII collagen XVII-NT-1, XVII-NT-2, and XVII-NT-3 of the present invention were added to the cells on the basis of modeling, and the positive control was comparable, and the concentrations were set to (0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml). In the model group, culture medium was added on the basis of modeling, and then the cells were placed in a 37° C., 5% CO2 incubator for incubation for 48 h.

[0061] (4) The cell supernatant of each group was collected and the 5α-dihydrotestosterone (5α-DHT) content in the cell supernatant was detected using a kit as shown in Table 2.

[0062] Table 2: 5α-dihydrotestosterone (5α-DHT) content in the supernatant of cells treated with three collagens (x±s, n=3)

[0063]

[0064] As shown in Table 2, compared with the model group, the 5α-DHT content in the cell supernatant after treatment of the recombinant human type XVII collagen XVII-NT-1, XVII-NT-2, XVII-NT-3, and cocomplex of the present invention was reduced, among which XVII-NT-1 had a significant difference compared with the model group and cocomplex group (P<0.01). Therefore, the recombinant human type XVII collagen XVII-NT-1 of the present invention has a more significant inhibitory effect on 5α-DHT, that is, it has a more significant effect of increasing hair growth and preventing hair loss.

[0065] Sequence information

[0066] SEQ ID NO: 1 Amino acid sequence of recombinant type XVII collagen (XVII-NT-1)

[0067] 1TTITGETFD YSELASHVVS YLRTSGYGVS LFSSSISSED ILAVLQRDDV RQYLRQYLMMGPRGPPGPPGA

[0068] 71SGDGSLLSLD YAELSSRILS YMSSSGISIG LPGPPGPPGL PGTSYEELLS LLRGSEFRGIVGPPGPPGPP

[0069] 141GIPGNVWSSISVEDLSSYLH TAGLSFIPGPPGPPGPPGPRGPPGVSGALATYAAENSDSFRSELISYLTS

[0070] 211PDVRSFIVSEQ ID NO: 2 Nucleotide sequence of recombinant human type XVII collagen (XVII-NT-1)

[0071]

[0072]

[0073] SEQ ID NO: 3 Amino acid sequence of recombinant type XVII collagen (XVII-NT-2)

[0074] SEQ ID NO: 4 Nucleotide sequence of recombinant type XVII collagen (XVII-NT-2)

[0075]

[0076]

[0077] SEQ ID NO: 5 Amino acid sequence of recombinant type XVII collagen (XVII-NT-3)

[0078] SEQ ID NO: 6 Nucleotide sequence of recombinant type XVII collagen (XVII-NT-3)

[0079]

Claims

1. A recombinant human type XVII collagen, which is a truncated protein of human type XVII collagen, and its amino acid sequence 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 method for producing recombinant human type XVII collagen according to claim 1, comprising the steps of culturing a host cell into which the expression vector according to claim 3 has been introduced to express the recombinant human type XVII collagen according to claim 1, and purifying the recombinant human type XVII collagen. The production method according to claim 4 , wherein the host cell is caused to secrete the recombinant human type XVII collagen into a culture medium.

6. Use of the recombinant human type XVII collagen according to claim 1 in the preparation of medicines or cosmetics for hair growth and care, repairing hair follicles, and / or promoting hair follicle tissue regeneration.

7. The use according to claim 6, wherein The medicine is for external use.

8. The use according to claim 6, wherein The cosmetic is formulated in a form suitable for application to the scalp.

Images