CN118530338A - Recombinant human XVII type collagen with hair follicle growth promoting effect and application thereof - Google Patents

Abstract

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

Description

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

Technical Field

The present invention belongs to the technical field of synthetic biology and specifically relates to a novel recombinant human type XVII collagen with the function of promoting hair follicle growth, and a preparation method and application thereof.

Background Art

Collagen is a biopolymer protein and the main component of animal connective tissue. It is widely distributed in the skin, bones, tendons, ligaments, cartilage and blood vessels of mammals. It is the main protein component of connective tissue and an important component of the extracellular matrix. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics and food industries.

At present, the collagen used in industry is mainly extracted from animal skin, bones and other tissues by acid, alkaline hydrolysis and enzyme hydrolysis. Its main source is animal tissue, but collagen extracted from animal tissue has risks such as animal-derived diseases. At the same time, large-scale preparation will cause excessive demand for animals, and animal breeding will cause huge pressure on the supply side. There is a risk of cross-infection for animal-derived diseases or human infectious diseases. With the increasing maturity of genetic engineering technology, the use of molecular biology methods to prepare recombinant collagen has gradually replaced the trend of extracting collagen from animal tissues. Recombinant collagen produced in prokaryotic or eukaryotic expression systems has relatively good cell adhesion, tissue compatibility, skin permeability, stability, and can promote cell formation and growth. Compared with traditional animal collagen, it has its unique advantages.

Compared with traditional animal collagen extraction, genetic engineering recombinant collagen preparation has the following advantages: 1. A wide variety of collagen is produced, which can be collagen from common sources such as cattle, pigs, and fish. Human collagen can also be produced, which has better safety and immune superiority in the fields of medical devices. 2. It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Its cell pathogens cannot be transmitted to each other due to the huge difference in cell structure from humans. 3. The production cycle is short, cost-saving, can be quickly scaled up, and is suitable for industrial large-scale production. Compared with the long animal culture of 3-5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Large-scale culture can be carried out by providing simple C and N sources, and it is easy to prepare at an industrial level. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot spot in collagen production research.

Collagen plays an important role in wound healing, organ development and tissue repair, for example: medical collagen injections for cosmetic surgery, collagen membranes for burn and trauma treatment, collagen hemostatic sponges for trauma hemostasis, etc. At the same time, collagen is an important component of the extracellular matrix of the skin, and has the effects of 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 medical and health fields (such as hemostasis, beauty, drug delivery, wound repair, drug sustained release, etc.).

Summary of the invention

During the scientific research practice, the inventors screened out a dodecapeptide derived from natural human type XVII collagen. The dodecapeptide and the recombinant human type XVII collagen composed of multiple repetitions of the amino acid sequence of the dodecapeptide have significantly higher hair follicle cell growth-promoting activity than other dodecapeptides and other recombinant human type XVII collagen tested by the inventors that are also derived from natural human type XVII collagen.

That is, the present invention includes:

1. A twelve peptide having the activity of promoting hair follicle cell growth, wherein the amino acid sequence is shown in SEQ ID No: 1 (SLFSSSISSEDI).

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 as shown in SEQ ID No: 1 (SLFSSSISSEDI) and is repeated more than 2 times.

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

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

5. The recombinant human type XVII collagen according to item 2, wherein the number of repetitions is 10 to 15 times, for example, 12 times, and the amino acid sequence thereof is shown in SEQ ID No: 2.

6. A nucleic acid encoding the recombinant human type XVII collagen described in item 2.

7. An expression vector comprising the nucleic acid described in item 6.

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

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

10. The use according to item 9, wherein the cosmetic is prepared in a form suitable for application to the scalp, such as shampoo, shampoo cream, shampoo milk, or shampoo lotion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a SDS-PAGE electrophoresis detection diagram of the recombinant human type XVII collagen prepared in Example 2.

DETAILED DESCRIPTION

Example 1: Screening of amino acid sequences that promote hair follicle cell growth

A polypeptide with 12 amino acid residues covering the entire human type XVII collagen was chemically synthesized by GL Biochem, and 75 short amino acid sequences were finally synthesized. Each short amino acid sequence is shown in the following table.

Each short amino acid sequence was prepared at a concentration of 1 mg/mL to test the proliferation-promoting effect of HFDPC. HFDPC cells were inoculated in a sterile 96-well plate at 7000 cells/well. Three replicate wells were set for each concentration, and an equal volume of culture medium was added to the blank control group and placed in a 37°C incubator for 24 hours. 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 hours. After 4 hours, aspirate the mixed solution, add 150 μL of DMSO to dissolve the purple crystal formazan, shake for 10 minutes, put it in the microplate reader, detect the wavelength of 570 nm, and measure the absorbance value A of each well.

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

Table 1 Proliferation-promoting effects of 75 dodecapeptides on HFDPC cells

From the above results, it can be seen that all 75 dodecapeptides showed no toxicity or inhibitory effect on HFDPC cells. Among them, the proliferation rate of dodecapeptide No. 43 on HFDPC cells was 135.96%, which was significantly higher than that of other dodecapeptides. This shows that we have screened out a short amino acid sequence from human type XVII collagen that has a strong effect on promoting hair follicle cell proliferation.

Example 2: Expression and preparation of recombinant human type XVII collagen in the large intestine expression system

1. Construction of pKK223-3-HLC expression strain

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 Escherichia coli, the whole gene was synthesized and connected to the pKK223-3 plasmid to obtain the pKK223-3-HLC plasmid. The pKK223-3-HLC was transferred into DE3 competent cells by heat shock, coated on a resistance plate, and a single colony on the plate was picked as the expression strain.

2. Expression of Target Protein

(1) Pick a single colony that has been successfully constructed by colony PCR verification, inoculate it into a test tube containing 3.0 ml of LB medium (with ampicillin antibiotics), and culture it at 37°C for 10-12 hours to obtain seed liquid.

(2) Inoculate the cultured seed solution into 100 ml LB liquid medium at a 1% inoculum volume.

The cells were cultured at 37°C and 220 rpm until OD ₆₀₀ = 2-3, IPTG was added at a final concentration of 0.15 mM, and the temperature was lowered to 28°C for induction. After 10-12 h of induction, the cells were collected by centrifugation at 9000 rpm for 10 min.

(3) A 10% (g wet bacteria/mL PBS) bacterial suspension was prepared with PBS buffer at pH 6.0, and the mixture was homogenized at 800 bar for 3 min. The mixture was centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the crude protein solution ( FIG. 1 , lane 1).

3. Isolation and purification of target protein

(1) Add NaCl with a final concentration of 60% to the crude protein supernatant collected by centrifugation, stir to dissolve, let stand at room temperature for 2 hours, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant. The collected supernatant is concentrated and desalted by a 10KD ultrafiltration membrane (Figure 1, band 2).

(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 eluted with 0.5 M NaCl to obtain the target protein (Figure 1, band 3);

(3) The collected target protein solution is desalted by a 10KD ultrafiltration membrane, and then placed in a -20°C refrigerator for pre-freezing for 4 hours, and then transferred to a vacuum freeze dryer for freeze-drying. After 48 hours, the freeze-dried protein is collected, which is the recombinant human type XVII collagen.

Example 3: Effect of recombinant human type XVII collagen on the proliferation of HFDPC cells

The twelve peptides screened out in Example 1 and recombinant human type XVII collagen were prepared at a concentration of 1 mg/mL respectively to promote the proliferation of HFDPC. HFDPC cells were inoculated in a sterile 96-well plate at 7000 cells/well. Three replicate wells were set for each concentration, and an equal volume of culture medium was added to the blank control group and placed in a 37°C incubator for 24 hours. The supernatant was discarded, 100 μL of culture medium and 50 μL of MTT solution were added to each well, shaken well, and incubated in an incubator for 4 hours. After 4 hours, the mixed solution was aspirated, 150 μL of DMSO was added to dissolve the purple crystal formazan, shaken for 10 minutes, and placed in an ELISA instrument. The detection wavelength was 570 nm, and the absorbance value A of each well was measured.

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

Table 2 Proliferation effects of recombinant human type XVII collagen and short peptides on HFDPC cells

Sample name OD value Proliferation rate (%) blank 0.501±0.05 -- Dodecapeptide 0.666±0.03 132.93% Recombinant human type XVII collagen 0.933±0.02 ^*** 186.23%

***P＜0.001 compared with 12-peptide

As shown in 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, the recombinant human type XVII collagen has a more obvious effect on promoting the proliferation of 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.

Example 4 Comparison of the effect of recombinant human type XVII collagen on HFDPC cell proliferation

For comparison, we randomly selected two other amino acid sequences (short amino acid sequences No. 37 and No. 48 in Example 1), synthesized genes encoding recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2 by repeating them 12 times, and performed protein expression and purification as in Example 2 to obtain recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2.

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

Table 2 Proliferation effects of recombinant human type XVII collagen and short peptides on HFDPC cells

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%

***Compared with recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2, P＜0.001

As shown in 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 did not show obvious effects in promoting 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 showed a more significant effect in promoting the proliferation of HFDPC cells. In addition, unlike recombinant human type XVII collagen, recombinant human type XVII collagen-1 and recombinant human type XVII collagen-2 did not bring higher pro-HFDPC cell proliferation activity due to the repetition of short amino acid sequences.

Sequence information

SEQ ID No:1:SLFSSSISSEDI

SEQ ID No:2:SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI SLFSSSISSEDI

SLFSSSISSEDI

Claims (10)

1. A twelve peptide having the activity of promoting hair follicle cell growth, wherein the amino acid sequence is shown in SEQ ID No: 1 (SLFSSSISSEDI). 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 as shown in SEQ ID No: 1 (SLFSSSISSEDI) and is repeated more than 2 times. The recombinant human type XVII collagen according to claim 2 , wherein the number of repetitions is 100 or less. The recombinant human type XVII collagen according to claim 2, wherein the number of repetitions is 5 to 20. 5 . The recombinant human type XVII collagen according to claim 2 , wherein the number of repetitions is 10 to 15 times, for example, 12 times, and the amino acid sequence thereof is shown in SEQ ID No: 2. 6. A nucleic acid encoding the recombinant human type XVII collagen according to claim 2. 7. An expression vector comprising the nucleic acid of claim 6. 8. A host cell into which the expression vector according to claim 7 has been introduced. 9. Use of the dodecapeptide according to claim 1 or the recombinant human type XVII collagen according to claim 2 in the preparation of cosmetics for preventing hair loss, promoting hair growth or promoting hair follicle cell growth. 10. The use according to claim 9, wherein the cosmetic is prepared in a form suitable for application to the scalp, such as shampoo, shampoo cream, shampoo milk, or shampoo lotion.