CN110078793B

CN110078793B - Polypeptide with anti-aging and repairing functions and application thereof

Info

Publication number: CN110078793B
Application number: CN201910209385.5A
Authority: CN
Inventors: 张扬; 韩卫星
Original assignee: Guangdong Peptide Normal Stem Cell Biotechnology Co ltd
Current assignee: Guangdong baioufei silk Cell Research Center Co.,Ltd.
Priority date: 2019-03-19
Filing date: 2019-03-19
Publication date: 2020-10-16
Anticipated expiration: 2039-03-19
Also published as: CN110078793A

Abstract

The invention discloses a polypeptide with anti-aging and repairing functions and application thereof. The polypeptide can promote the growth of fibroblasts and promote the fibroblasts to generate hyaluronic acid, so that the polypeptide has the effects of improving skin collagen loss, enhancing the moisturizing effect, efficiently repairing skin, brightening and whitening skin, resisting wrinkles and the like, and can be used for preparing medicines or cosmetics for resisting aging and repairing skin.

Description

Polypeptide with anti-aging and repairing functions and application thereof

The technical field is as follows:

the invention belongs to the field of medical cosmetics, and particularly relates to a polypeptide with anti-aging and repairing effects and application thereof.

Background art:

the subject of the skin is the dermal layer, which is composed primarily of fibroblasts and matrix components. Fibroblasts produce glycosaminoglycans such as collagen and hyaluronic acid to form connective tissues, and play an important role in the skin. The decrease in fibroblast function due to aging or other effects such as ultraviolet rays may cause the decrease and denaturation of matrix components such as collagen and hyaluronic acid. Oxidative stress such as ultraviolet rays causes skin damage, makes it rough, and causes other adverse reactions. Aging of the skin is accelerated due to the decrease of matrix components and oxidative stress, whereby wrinkles, spots, dullness in appearance, loss of smoothness in texture, reduction in elasticity, etc., appear to show signs of aging.

Collagen and hyaluronic acid have been the current mainstream trends as a direction to prevent skin aging. Scientific analysis hyaluronic acid is a high molecular polymer, and the method of directly applying hyaluronic acid extract to skin is not favorable for skin absorption and utilization. The hydroid body mainly comprises stem cells, and meanwhile, the content of hyaluronic acid and collagen is high, so that the ability of the hydroid stem cells to divide continuously can be maintained.

The invention content is as follows:

the invention aims to provide a polypeptide with anti-aging and repairing functions and application thereof.

The research of the invention discovers that the biological polypeptide (the amino acid sequence of the biological polypeptide is shown as SEQ ID NO. 2) extracted from the hydranth can promote the growth of fibroblasts from a cell layer and stimulate the fibroblasts to produce hyaluronic acid.

Therefore, the first object of the present invention is to provide a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 2.

The second purpose of the invention is to provide the coding gene of the polypeptide, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

The third purpose of the invention is to provide a prokaryotic expression vector containing the coding gene of the polypeptide.

The prokaryotic expression vector is preferably pGEX-4T-1.

The fourth purpose of the invention is to provide a bacterium containing the prokaryotic expression vector.

The bacterium is preferably Escherichia coli BL21(DE 3).

The fifth purpose of the invention is to provide the application of the polypeptide in preparing a preparation for promoting the growth of fibroblasts.

The sixth purpose of the invention is to provide the application of the polypeptide in preparing a preparation for promoting the fibroblasts to produce the hyaluronic acid.

The seventh purpose of the invention is to provide the application of the polypeptide in preparing drugs or cosmetics for resisting aging and repairing skin.

An eighth object of the present invention is to provide a pharmaceutical or cosmetic for anti-aging and skin-repairing comprising the polypeptide as an active ingredient.

The cosmetic can be prepared into various dosage forms such as lyophilized powder, essence, lotion, cream, etc.

The polypeptide with the novel anti-aging and efficient repairing effects is obtained from marine organism hydroids, can promote the growth of fibroblasts, and stimulates the fibroblasts to generate hyaluronic acid, so that the polypeptide has the effects of improving the loss of skin collagen, strengthening the moisturizing effect, efficiently repairing the skin, brightening and whitening the skin, resisting wrinkles and the like, and can be used for preparing medicines or cosmetics for resisting aging and repairing the skin.

Description of the drawings:

FIG. 1 shows the effect of a polypeptide (the amino acid sequence of which is shown in SEQ ID NO. 2) on fibroblast growth.

FIG. 2 shows the effect of a polypeptide (the amino acid sequence of which is shown in SEQ ID NO. 2) on the production of hyaluronic acid by fibroblasts.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

1. RNA extraction

The extraction of total RNA uses Trizol (Invitrogen) and comprises the following steps:

(1) taking 50mg of green hydranth viridis whole tissue in a mortar precooled by liquid nitrogen, grinding the tissue into powder, transferring the powder into a centrifuge tube filled with 1ml of crystallized, blowing, beating and uniformly mixing;

(2) adding 200 μ L chloroform, shaking vigorously for 40s, and standing at room temperature for 5 min;

(3) centrifuging at 4 deg.C for 10min at 12,000 Xg, sucking supernatant, and transferring to new tube;

(4) adding 0.5mL of isopropanol, mixing uniformly, and precipitating at-80 ℃ overnight;

(5) centrifuging at 4 deg.C for 10min at 12,000 Xg, and discarding the supernatant;

(6) precipitating twice with 75% ethanol wash;

(7) the supernatant was discarded and 100. mu.L of DEPC was added to dissolve the RNA.

2. cDNA library construction

Construction of full-Length cDNA library Using the SMART cDNA library construction Kit (Clontech), the specific steps were as follows:

(1) synthesizing a first strand of cDNA by using MMLV by using the RNA in the step 1;

(2) amplifying the cDNA using LD PCR;

(3) digesting and purifying cDNA product by proteinase K;

(4) sfil digestion, product size separation using Chroma Spin 400;

(5) connecting the cDNA to a lambda TripEx2 vector, packaging the vector by lambda phage, and transforming the vector into host cells;

(6) and detecting the titer of the cDNA library.

3. Prokaryotic expression vector construction

(1) Designing a pair of primers covering a polypeptide sequence of a hydroid (see table 1), and adding a BamH-I site and an Xho-I site and a protective base at two ends of the primers respectively;

TABLE 1 primer sequences

(2) Performing PCR amplification by using the primer in the step (1) by using the green hydroid cDNA as a template;

(3) carrying out double enzyme digestion on the PCR product and the pGEX-4T-1 vector by using BamH-I and Xho-I respectively, and recovering and purifying enzyme digestion products;

(4) connecting the PCR product after enzyme digestion with a pGEX-4T-1 vector;

(5) and (3) transforming the ligation product into escherichia coli DH5 alpha, selecting positive clones, verifying the correctness of the vector by sequencing, and verifying the correctness of the vector by sequencing results, wherein the sequence shown as SEQ ID NO.1 is correctly inserted into the pGEX-4T-1 vector, so that the polypeptide prokaryotic expression vector is obtained for subsequent tests.

4. Prokaryotic expression and purification of recombinant proteins

(1) Extracting the polypeptide prokaryotic expression vector in the escherichia coli DH5 alpha in the step 3, and transforming the polypeptide prokaryotic expression vector into escherichia coli BL21(DE 3);

(2) selecting a single clone and culturing until OD is 0.6, adding IPTG (isopropyl thiogalactoside) to a final concentration of 0.1mM, and inducing expression under the conditions of 22 ℃ and 180 rpm;

(3) collecting bacterial liquid after 4h, centrifuging at 4 ℃ for 10min at 12,000 Xg;

(4) adding Lysozyme to a final concentration of 2mg/mL, and then carrying out ultrasonication;

(5) the recombinant protein was purified using glutaminone Sepharose 4b (ge healthcare) and the purified product was analyzed on 12% SDS-PAGE gel to give a purified polypeptide (the amino acid sequence of which is shown in SEQ ID No. 2).

5. Determination of the Activity of a polypeptide to promote the growth of fibroblasts

(1) Inoculating human skin fibroblast line BJ cells into a 96-well plate (100 uL/well, cell inoculation amount is 1 × 10)⁴One/well), 5% CO at 37 ℃₂And (4) plating in an incubator (the culture medium is an L-15 basal culture medium containing bovine serum and without adding a growth agent).

(2) After culturing for 6h, adding 100 μ L of the purified polypeptide solution obtained in step 4 into the culture medium, and making the final polypeptide concentration 5, 10, 20, 30 μ M (polypeptide group), taking a group of 3 repeated additions of PBS with equal volume as a control, and making the control be 0(PBS group), and adding no cell single addition culture medium as a negative control.

(3) The cells were cultured in an incubator for 48h, 10. mu.L of MTT solution (5mg/mL) was added to each well, the incubation in the incubator was continued for 3h, the medium was discarded, and 100. mu.L of DMSO was added to each well and the mixture was shaken until the crystals were completely dissolved (MTT cell viability assay kit).

(4) And detecting the absorbances at 450nm and 625nm by using a microplate reader, and calculating the growth rate of the fibroblast.

(5) The results show that the polypeptide has a tendency of promoting the growth of fibroblasts in the concentration range of 5-30 μ M, and the cell growth rate of the polypeptide group (the final concentration of the polypeptide is 10 μ M) is greater than that of the PBS group, as shown in FIG. 1.

6. Determination of the Effect of the polypeptide on promoting hyaluronic acid production

(1) The amount of hyaluronic acid produced by fibroblasts was determined by ELISA.

(2) Human dermal fibroblast cell line BJ cells were seeded in 48-well plates (200. mu.L/well, cell inoculum size 1 × 10)⁵Culture medium is L-15 basic culture medium containing bovine serum but no growth agent), and the culture medium is treated at 37 ℃ and 5% CO₂The culture was carried out overnight in an incubator.

(3) After 24h, 20. mu.L of the polypeptide solution was added to each group to give final polypeptide concentrations of 0, 100, 200, 300, 400, and 500. mu.M, respectively, and the mixture was incubated in an incubator for 48 h.

(4) Each group of cell cultures was centrifuged to collect the supernatant.

(5) And (3) measuring the content of the hyaluronic acid in the supernatant by using an ELISA method of a hyaluronic acid measuring kit.

(6) The results show that the content of hyaluronic acid shows an ascending trend and then becomes stable in a certain concentration range of the polypeptide (see figure 2).

Sequence listing

<110> Guangdong peptide Noro Stem cell Biotech Co., Ltd

<120> a polypeptide with anti-aging and repairing functions and uses thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>60

<212>DNA

<213> Green hydroid (Hydra viridis)

<400>1

gaatatttct ccgaattctc caaagaattc gtgtccgaag aatcctcctt ccttgaattc 60

<210>2

<211>20

<212>PRT

<213> Green hydroid (Hydra viridis)

<400>2

Leu Ile Lys Arg Leu Lys Arg Phe Leu Lys His Arg Leu Leu Arg Arg

1 5 10 15

Lys Glu Leu Lys

20

Claims

1. A polypeptide, characterized in that the amino acid sequence thereof is shown as SEQ ID NO. 2.

2. A gene encoding the polypeptide of claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID No. 1.

4. A prokaryotic expression vector comprising a gene encoding the polypeptide of claim 2 or 3.

5. The prokaryotic expression vector according to claim 4, wherein the prokaryotic expression vector is pGEX-4T-1.

6. A bacterium comprising the prokaryotic expression vector of claim 4.

7. The bacterium according to claim 6, wherein said bacterium is Escherichia coli BL21(DE 3).

8. Use of the polypeptide of claim 1 for the preparation of a formulation for promoting the growth of fibroblasts or for promoting the production of hyaluronic acid by fibroblasts.

9. The use of the polypeptide of claim 1 for the preparation of a medicament or cosmetic for anti-aging and skin repair.

10. A drug or cosmetic for anti-aging and skin repair comprising the polypeptide of claim 1 as an active ingredient.