Recombinant human collagen type XVII with hair follicle growth promoting effect and its application

Abstract

This patent application discloses a recombinant human type XVII collagen with a function of promoting hair follicle growth and its application. The recombinant human type XVII collagen is composed of multiple repetitions of a dodecapeptide amino acid sequence derived from natural human type XVII collagen. The dodecapeptide and the recombinant human type XVII collagen have high activities in promoting hair follicle growth and can be used in cosmetics for preventing hair loss, promoting hair growth or promoting hair follicle cell growth, such as shampoo, hair cream, hair milk, hair lotion, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology. Specifically, it relates to a new recombinant human type XVII collagen with the function of promoting hair follicle growth, its preparation method and application. Background Art

[0002] Collagen is a biological macromolecular protein and the main component in animal connective tissues. It is widely distributed in the skin, bones, tendons, ligaments, cartilage and blood vessels of mammals. It is the main protein component of connective tissues and an important component of the extracellular matrix. Collagen is closely related to tissue formation, maturation, cell - to - cell information transmission, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotech industry and is widely used in medical materials, cosmetics and the food industry.

[0003] Currently, the collagen used industrially is mainly extracted from animal skins, bones and other tissues by methods of acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Its main source is animal tissues. However, the collagen extracted from animal tissues has risks such as animal - source diseases, and at the same time, large - scale preparation will cause excessive demand for animals, putting huge pressure on animal breeding on the supply side. There is a risk of cross - infection for animal - source diseases or human infectious diseases. With the increasing maturity of genetic engineering technology, the preparation of recombinant collagen by molecular biology methods has gradually replaced the trend of extracting collagen from animal tissues. The recombinant collagen produced in prokaryotic or eukaryotic expression systems has good cell adhesion, tissue compatibility, skin permeability, stability, can promote cell formation and growth, and has unique advantages compared with traditional animal collagen.

[0004] Compared with the traditional extraction of animal collagen, the preparation of collagen by genetic engineering recombination has the following advantages: 1. A wide variety of collagens can be produced, which can be collagens from common sources such as cows, pigs, fish, etc., and can also produce human - derived collagen, having better safety and immune superiority in fields such as medical devices. 2. It can effectively avoid animal - source diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts, and its cell pathogens cannot spread to each other due to the huge difference in cell structure from human cells. 3. The production cycle is short, the cost is saved, it can be rapidly amplified, and it is suitable for large - scale industrial production. Compared with the animal culture that often takes 3 - 5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Providing simple carbon and nitrogen sources can carry out large - scale culture, which is easy to carry out industrial - level preparation. Therefore, the preparation of recombinant collagen, especially recombinant human - derived collagen, is a hot topic in current collagen production research.

[0005] The important roles played by collagen in aspects such as wound healing, organ development, and tissue repair, for example: medical collagen injection agents for beauty and plastic surgery, collagen membranes for the treatment of burns and wounds, collagen hemostatic sponges for wound hemostasis, etc. At the same time, collagen is an important component of the extracellular matrix of the skin and has effects in aspects such as moisturizing, replenishing skin collagen, and anti-aging. Based on the above biological functions of collagen, recombinant collagen has a wide range of applications in the field of medicine and hygiene (such as hemostasis, beauty, drug delivery, wound repair, drug sustained release, etc.). Summary of the Invention

[0006] During the scientific research practice process, the inventors screened out a dodecapeptide derived from natural human type XVII collagen. The activity of this dodecapeptide and the recombinant human type XVII collagen composed of multiple repetitions of the amino acid sequence of this dodecapeptide in promoting the growth of hair follicle cells is significantly higher than that of other dodecapeptides and other recombinant human type XVII collagens also derived from natural human type XVII collagen tested by the inventors.

[0007] That is, the present invention includes:

[0008] 1. A dodecapeptide with the activity of promoting the growth of hair follicle cells, and its amino acid sequence is shown as SEQ ID No:1 (SLFSSSISSEDI).

[0009] 2. A recombinant human type XVII collagen, which is composed of multiple repetitions of a short amino acid sequence from natural human type XVII collagen as a repeating unit, wherein the short amino acid sequence is shown as SEQ ID No:1 (SLFSSSISSEDI), and the number of repetitions is more than 2 times.

[0010] 3. The recombinant human type XVII collagen according to item 2, wherein the number of repetitions is 100 times or less.

[0011] 4. The recombinant human type XVII collagen according to item 2, wherein the number of repetitions is 5 - 20 times.

[0012] 5. The recombinant human type XVII collagen according to item 2, wherein the number of repetitions is 10 - 15 times. For example, it can be 12 times, and its amino acid sequence is shown as SEQ ID No:2.

[0013] 6. The nucleic acid encoding the recombinant human type XVII collagen according to item 2.

[0014] 7. An expression vector containing the nucleic acid according to item 6.

[0015] 8. A host cell into which the expression vector according to item 7 has been introduced.

[0016] 9. Use of the dodecapeptide according to item 1 or recombinant human type XVII collagen according to item 2 in the preparation of cosmetics for preventing hair loss, promoting hair growth or promoting the growth of hair follicle cells.

[0017] 10. The use according to item 9, wherein the cosmetic is in a form suitable for application to the scalp. For example, it can be shampoo, hair cream, hair lotion, hair shampoo. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SDS-PAGE electrophoresis detection diagram of recombinant human type XVII collagen prepared for Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0019] Example 1: Screening of Amino Acid Sequences with Promoting Effect on the Growth of Hair Follicle Cells

[0020] GL Biochem chemically synthesized polypeptides with 12 amino acid residues covering the entire human type XVII collagen, and finally 75 short amino acid sequences were synthesized. Each short amino acid sequence is shown in the following table.

[0021] Prepare each short amino acid sequence at a concentration of 1 mg / mL respectively, and conduct the test on the promoting proliferation effect on HFDPC. Inoculate HFDPC cells at 7000 cells / well into a sterile 96-well plate. Set three replicates for each concentration group. The blank control group adds the same volume of culture medium and is placed in an incubator at 37°C for 24 h. Discard the supernatant. Add 100 μL of culture medium and 50 μL of MTT solution to each well, shake well, and incubate in the incubator for 4 h. After 4 h, aspirate the mixture, add 150 μL of DMSO to dissolve the purple crystal formazan, shake for 10 min, and place it in an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value A of each well at a wavelength of 570 nm.

[0022] Proliferation rate = (OD of experimental group - OD of blank group) × 100% / OD of blank group.

[0023] Table 1 Promoting Proliferation Effect of 75 Dodecapeptides on HFDPC Cells

[0024]

[0025]

[0026]

[0027] As can be seen from the above results, 75 dodecapeptides showed no toxicity or inhibitory effect on HFDPC cells. Among them, the 43rd dodecapeptide had a cell proliferation promotion rate of 135.96% on HFDPC cells, and its proliferation promotion effect was significantly higher than that of other dodecapeptides. This indicates that we have screened out a short amino acid sequence from human type XVII collagen with a strong effect on promoting hair follicle cell proliferation.

[0028] Example 2: Expression and preparation of recombinant human type XVII collagen in the E. coli expression system

[0029] 1. Construction of pKK223-3-HLC expression strain

[0030] The amino acid sequence shown in SEQ ID No: 1, i.e., SLFSSSISSEDI, was repeated 12 times to obtain the amino acid sequence of recombinant human type XVII collagen shown in SEQ ID No: 2. After optimization according to the codon preference of E. coli and total gene synthesis, it was ligated onto the pKK223-3 plasmid to obtain the pKK223-3-HLC plasmid. The pKK223-3-HLC was transformed into DE3 competent cells by heat shock, and the resistant plates were spread. The single colonies on the plates were picked as the expression strains.

[0031] 2. Expression of the target protein

[0032] (1) Pick the single colonies verified to be successfully constructed by colony PCR and inoculate them into a test tube containing 3.0 ml of LB medium (added with ampicillin antibiotic), and culture at 37 °C for 10 - 12 h to obtain the seed solution.

[0033] (2) Inoculate the cultured seed solution into 100 ml of LB liquid medium at an inoculation amount of 1%,

[0034] Culture at 37 °C and 220 rpm until OD 600 = 2 - 3, add IPTG with a final concentration of 0.15 mM, cool down to 28 °C for induction. After 10 - 12 h of induction, centrifuge at 9000 rpm for 10 min to collect the bacterial cells.

[0035] (3) Prepare a bacterial suspension of 10% (g wet bacterial cells / mL PBS) with PBS buffer at pH 6.0, homogenize at 800 bar for 3 min, and centrifuge at 9000 rpm for 10 min to collect the centrifuged supernatant, which is the crude protein solution ( Figure 1 , band 1).

[0036] 3. Isolation and purification of the target protein

[0037] (1) Add NaCl with a final concentration of 60% to the centrifugally collected crude protein supernatant. After stirring and dissolving, let it stand at room temperature for 2 h, then centrifuge at 9000 rpm for 10 min and collect the supernatant. Concentrate and desalt the collected supernatant through a 10KD ultrafiltration membrane ( Figure 1 , band 2).

[0038] (2) Adjust the pH of the concentrated and desalted protein supernatant to 6.5 with phosphoric acid, load it onto a cation exchange resin, and obtain the target protein after elution with 0.5 M NaCl ( Figure 1 , band 3);

[0039] (3) After desalting the collected target protein solution through a 10KD ultrafiltration membrane, place it in a -20°C refrigerator for pre-freezing for 4 h, then transfer it to a vacuum freeze dryer for freeze-drying. After 48 h, collect the freeze-dried protein, which is recombinant human type XVII collagen.

[0040] Example 3: Proliferation-promoting effect of recombinant human type XVII collagen on HFDPC cells

[0041] Prepare the dodecapeptide screened in Example 1 and recombinant human type XVII collagen with a concentration of 1 mg / mL respectively, and conduct the proliferation-promoting effect on HFDPC. Seed HFDPC cells at 7000 cells / well in a sterile 96-well plate. Set three replicates for each concentration group. The blank control group adds an equal volume of culture medium and is placed in an incubator at 37°C for 24 h. Discard the supernatant, add 100 μL of culture medium and 50 μL of MTT solution to each well, shake well, incubate in the incubator for 4 h. After 4 h, aspirate the mixture, add 150 μL of DMSO to dissolve the purple crystal formazan, shake for 10 min, place it in an enzyme-linked immunosorbent assay (ELISA) reader, detect at a wavelength of 570 nm, and measure the absorbance value A of each well.

[0042] Proliferation rate = (OD of experimental group - OD of blank group) × 100% / OD of blank group.

[0043] Table 2 Proliferation-promoting effects of recombinant human type XVII collagen and short peptide on HFDPC cells

[0044] Sample Name OD Value Proliferation Rate (%) Blank 0.501±0.05 -- Dodecapeptide 0.666±0.03 132.93％ Recombinant Human Type XVII Collagen <![CDATA[0.933±0.02 *** > 186.23％

[0045] *** P < 0.001 compared with the dodecapeptide

[0046] As can be seen from Table 2, compared with the blank group, the dodecapeptide and recombinant human type XVII collagen can significantly promote the proliferation of HFDPC cells. Moreover, compared with the dodecapeptide, recombinant human type XVII collagen has a more obvious proliferation-promoting effect on HFDPC cells (P < 0.001). This shows that the functional amino acid sequence SLFSSSISSEDI has a better effect on promoting the growth of hair follicle cells after multiple times.

[0047] Example 4 Comparison of the effect of recombinant human type XVII collagen on the proliferation of HFDPC cells

[0048] As a comparison, we randomly selected two other amino acid sequences (the 37th and 48th short amino acid sequences in Example 1), synthesized genes encoding recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2, which were the repeats of these sequences 12 times, and carried out protein expression and purification as in Example 2 to obtain recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2.

[0049] The test of the effect of recombinant human type XVII collagen, recombinant human type XVII collagen-1, and recombinant human type XVII collagen-2 on the proliferation of HFDPC cells was carried out as in Example 3. The test results are shown in Table 3.

[0050] Table 2 Promoting proliferation effects of recombinant human type XVII collagen and short peptides on HFDPC cells

[0051] Sample Name OD Value Proliferation Rate (%) Blank 0.569±0.01 -- Recombinant Human Type XVII Collagen 1.027±0.07*** 180.39％ Recombinant Human Type XVII Collagen-1 0.622±0.03 109.19％ Recombinant Human Type XVII Collagen-2 0.602±0.03 105.68％

[0052] *** P < 0.001 compared with recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2

[0053] As can be seen from Table 3, compared with the blank group, recombinant human type XVII collagen can significantly promote the proliferation of HFDPC cells, while recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2 do not show an obvious promoting effect on the proliferation of HFDPC cells. Moreover, compared with recombinant human type XVII collagen-1 and recombinant human type XVII collagen-1, recombinant human type XVII collagen shows a more significant promoting effect on the proliferation of HFDPC cells. In addition, different from recombinant human type XVII collagen, recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2 do not bring higher promoting activities for the proliferation of HFDPC cells due to the repetition of short amino acid sequences.

[0054] Sequence information

[0055] SEQ ID No:1:SLFSSSISSEDI

[0056] SEQ ID No:2:SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

[0057] SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

[0058] SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

[0059] SLFSSSISSEDI

Claims

1. A twelve peptide having the activity of promoting HFDPC cell proliferation, wherein the amino acid sequence thereof is shown in SEQ ID No:

1.

2. A recombinant human type XVII collagen, which is composed of a short amino acid sequence from natural human type XVII collagen as a repeating unit repeated multiple times, wherein: The short amino acid sequence is shown in SEQ ID No: 1, and is repeated 12 times; the amino acid sequence of the recombinant human type XVII collagen is shown in SEQ ID No:

2.

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

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

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

Images