阅读说明：本技术 生物活性多肽及其应用 (Bioactive polypeptide and application thereof ) 是由 沈陈超 濮云飞 雷相和 赵启超 郑蔚 于 2021-02-08 设计创作，主要内容包括：本发明涉及蛋白领域,特别涉及生物活性多肽及其应用。本发明提供的生物活性多肽KLQP具有较好的抗氧化、抗衰老、促进组织损伤修复的活性；一方面可以提高细胞的抗氧化能力,抵抗自由基的氧化损伤,增强机体抵抗外源刺激的能力；另一方面,能够促进细胞抵抗环境不利因素的能力,从而增强机体抵抗衰老的能力；最后,能提高组织在发生损伤后的自我修复能力；上述有益效果,对开发具有抗氧化、抗衰老、促进组织损伤修复功能的食品、保健品、药物、化妆品或医疗器械具有十分重要的意义。(The invention relates to the field of protein, in particular to bioactive polypeptide and application thereof. The bioactive polypeptide KLQP provided by the invention has good activities of resisting oxidation and aging and promoting tissue damage repair; on one hand, the antioxidant capacity of cells can be improved, the oxidative damage of free radicals can be resisted, and the capacity of resisting exogenous stimulation of an organism can be enhanced; on the other hand, the cell can be promoted to resist environmental adverse factors, so that the aging resistance of the body is enhanced; finally, the self-repairing capacity of the damaged tissues can be improved; the beneficial effects are of great significance to the development of foods, health products, medicines, cosmetics or medical appliances with the functions of resisting oxidation and aging and promoting the tissue damage repair.)

1. A polypeptide, wherein said polypeptide comprises:

(I) an amino acid sequence shown as SEQ ID No. 1; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 90% homology with the sequence of (I) or (II).

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. The nucleic acid molecule of claim 2, having the sequence shown below:

(I) a nucleotide sequence shown as SEQ ID No. 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 2; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

4. The method of claim 1, wherein the polypeptide is synthesized chemically or by genetic engineering.

5. Use of the polypeptide of claim 1 for the preparation of an antioxidant, anti-aging food, medicament or cosmetic.

6. Use of a polypeptide according to claim 1 for the preparation of a medicament, cosmetic or medical device for promoting tissue damage repair.

7. A pharmaceutical product comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient.

8. A food product comprising the polypeptide of claim 1 and a food acceptable adjuvant or adjuvant.

9. Cosmetic product comprising a polypeptide according to claim 1 and cosmetically acceptable adjuvants.

10. A medical device comprising the polypeptide of claim 1 and an acceptable carrier.

Technical Field

The invention relates to the field of protein, in particular to bioactive polypeptide and application thereof.

Background

With the development of economy and the improvement of living standard in recent years, people pay more attention to self health and skin care, and beauty and anti-aging preparations and related derivative beauty cosmetics, health care products and foods are favored by the market all the time.

Since the discovery of active polypeptide substances in life, particularly active polypeptides relevant to the physiological activities of skin tissue cells, people have been intensively researched and applied to the polypeptides, and the active polypeptides have positive effects and important values in the fields of beauty treatment, skin care and health care, aging resistance and the like.

The effectiveness of the bioactive polypeptide is obviously related to the size of molecular weight, and the protein and the polypeptide with larger molecular weight are difficult to permeate skin tissues or be directly absorbed by human bodies, so that the bioactive polypeptide cannot really play a role. The oligopeptide with the molecular weight of 100-1000Da has the characteristics of high permeability, high bioactivity, low immunogenicity and the like, can permeate human barriers such as stratum corneum, corium, mucosa and the like to be absorbed by human bodies, and has the effects of beautifying, resisting aging, promoting injury repair and the like.

Aging is a natural phenomenon and is associated with apoptosis, antioxidant levels, self-repair of organs and tissues, and the like. The anti-aging polypeptide is used as an emerging anti-aging agent in recent years, researchers utilize cell models, model organisms and the like to find that the anti-aging polypeptide has obvious effects on the aspects of anti-oxidation function, free radical elimination, apoptosis resistance, organ and tissue repair acceleration, barrier structure and function reconstruction and the like so as to delay aging.

Disclosure of Invention

In view of the above, the invention screens and improves some polypeptides in the nature, and provides a polypeptide which can significantly improve the anti-apoptosis capability and anti-oxidation function of cells and promote wound healing.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a class of polypeptides having:

(I) an amino acid sequence shown as SEQ ID No. 1; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 90% homology with the sequence of (I) or (II).

In some embodiments of the invention, the polypeptide has an amino acid sequence with at least 95% homology to the amino acid sequence shown in SEQ ID No. 1.

In some embodiments of the invention, the polypeptide has an amino acid sequence with at least 98% homology to the amino acid sequence shown in SEQ ID No. 1.

On the basis of the above, the invention also provides a nucleic acid molecule encoding the polypeptide.

In some embodiments of the invention, the nucleic acid molecule has a sequence as shown below:

(I) a nucleotide sequence shown as SEQ ID No. 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 2; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

The invention also provides a preparation method of the polypeptide, which comprises chemical synthesis or synthesis by a genetic engineering method.

More importantly, the invention also provides application of the polypeptide in preparing food, medicines or cosmetics with oxidation resistance and aging resistance.

The invention also provides application of the polypeptide in preparation of medicines, cosmetics or medical devices for promoting tissue damage repair.

Based on the research, the invention also provides a medicine which comprises the polypeptide and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides food, which comprises the polypeptide and acceptable auxiliary materials or auxiliary agents in the food.

The invention also provides cosmetics comprising the polypeptide and cosmetically acceptable adjuvants.

The invention also provides a medical device comprising the polypeptide and an acceptable carrier.

The bioactive polypeptide KLQP provided by the invention has good activities of resisting oxidation and aging and promoting tissue damage repair; on one hand, the antioxidant capacity of cells can be improved, the oxidative damage of free radicals can be resisted, and the capacity of resisting exogenous stimulation of an organism can be enhanced; on the other hand, the cell can be promoted to resist environmental adverse factors, so that the aging resistance of the body is enhanced; finally, the self-repairing capacity of the damaged tissues can be improved; the beneficial effects are of great significance to the development of foods, health products, medicines, cosmetics or medical appliances with the functions of resisting oxidation and aging and promoting the tissue damage repair.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the effect of different polypeptide concentrations on cell viability;

FIG. 2 shows the effect of different polypeptide concentrations on relative cell activity;

figure 3 shows the healing effect of wound injury in mice.

Detailed Description

The invention discloses a bioactive polypeptide and application thereof, and can be realized by appropriately improving process parameters by the technical personnel in the field with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In a first aspect of the invention, a biologically active polypeptide KLQP is provided, the amino acid sequence of which is Lys-Leu-Gln-Pro.

In a second aspect of the invention, there is provided a nucleotide fragment encoding the biologically active polypeptide KLQP, the sequence of which is: AAGCTGCAGCCC are provided.

In the third aspect of the invention, the preparation method of the bioactive polypeptide KLQP is provided, and the bioactive polypeptide KLQP can be directly prepared by chemical synthesis or artificially synthesized by a genetic engineering method.

In a fourth aspect of the invention, the application of the bioactive multi-day KLQP in preparing food, health products, medicines or cosmetics with antioxidant function is provided.

In the fifth aspect of the invention, the application of the bioactive polypeptide KLQP in preparing foods, health products, medicines or cosmetics with anti-aging function is provided.

In a sixth aspect, the invention provides an application of the bioactive polypeptide KLQP in preparation of medicines, cosmetics and medical instruments with the function of promoting tissue damage repair.

Specifically, the bioactive polypeptide KLQP can be used for preparing cosmetics for reducing free radical damage to skin, resisting oxidation and aging, preparing medicines for resisting oxidation and/or aging, and preparing medical instruments, cosmetics and medicines for promoting tissue-promoting injury repair; the biologically active polypeptide KLQP still has biological activity after being digested or absorbed by gastrointestinal tract, so that the biologically active polypeptide KLQP can be used for preparing food, health-care products and oral medicines with antioxidation and/or anti-aging effects.

The seventh aspect of the invention provides a bioactive polypeptide KLQP or a derivative of the bioactive polypeptide KLQP, which has the functions of resisting oxidation and aging and promoting tissue repair; the products with the functions of resisting oxidation and aging and promoting tissue damage repair comprise foods, health products, medicaments, cosmetics or medical appliances; the derivative of the bioactive polypeptide KLQP is a polypeptide derivative obtained by modifying an amino acid side chain group, an amino terminal or a carboxyl terminal of the bioactive polypeptide KLQP by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

The invention discovers a bioactive polypeptide which can obviously improve the anti-apoptosis capability and the anti-oxidation function of cells and simultaneously promote the tissue damage repair.

In the bioactive polypeptide and the application thereof provided by the invention, raw materials and reagents used in the bioactive polypeptide can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 artificial synthesis of active polypeptides:

1. weighing 3g of Wang resin into a 150mL reactor, adding three times of resin volume of N-Dimethylformamide (DMF) and a certain amount of 20% piperidine (piperidine/DMF is 1:4, v: v), placing on a decolorizing shaker, shaking for 30min, removing the Fmoc protecting group on the resin, washing 3 times with 3 times of resin volume of DMF after the protection is finished, and then pumping to dry.

2. Weighing a proper amount of proline Pro and 1-Hydroxybenzotriazole (HOBT) into a centrifuge tube, adding 20mL of DMF to dissolve the proline Pro, then adding 3mL of N, N diisopropyl carbodiimide (DIC), shaking up for 1min, and reacting for 1h at 30 ℃.

3. After 1 hour, the vessel was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DIC ═ 1:1:2, v: v: v).

4. Three resin volumes of N-Dimethylformamide (DMF) and a certain amount of 20% piperidine (piperidine/DMF 1:4, v: v) were added, respectively, placed on a decolorizing shaker and shaken for 30min, the Fmoc protecting group on the branches was removed, and after the protection was removed, the branches were washed 3 times with 3 resin volumes of DMF and then drained.

5. Weighing a proper amount of the second amino acid and 1-Hydroxybenzotriazole (HOBT) into a centrifuge tube, adding 20mL of DMF to dissolve the second amino acid, adding 3mL of N, N diisopropyl carbodiimide (DIC), shaking for 1min, and reacting for 1h at 30 ℃.

6. After complete reaction, the resin was washed 3 times with DMF and drained, a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, placed on a decolorizing shaker and shaken for 30min, the Fmoc protecting group on the branches was removed, after the protection was removed, the resin was washed 3 times with DMF of 3 times the volume of the resin and drained.

7. The amino acids Gln, Leu and Lys are attached sequentially according to the steps 5-6.

8. After the last amino acid linkage was completed, the protection was removed and the column was washed with DMF three times and drained. The polypeptide was then cleaved from the resin using a cleavage solution (trifluoroacetic acid: triisopropylsilane: water: 95:2.5:2.5, v: v: v).

At this point, the artificial synthesis of the biologically active polypeptide KLQP is completed.

Example 2 antioxidant testing:

cell experiments:

1. inoculating Hacat cells into a 6cm cell culture dish containing 10% FBS 1640 culture medium, and carrying out cell passage when the cell density reaches 80%;

2. hacat cells were counted by trypsinization and diluted 2X 10 in 1640 medium with a serum concentration of 10%⁴And/ml. In 96-well plates, 90. mu.L of each well was inoculated.

3. The experiment was divided into 7 groups, 1 of which was a blank control group (without addition of phosphine oxide and bioactive peptide KLQP), and 6 groups of which were bioactive peptide KLQP concentration gradient groups, incubated with cells at 0nM, 0.125nM, 0.25nM, 0.5nM, 1nM and 2nM, respectively, in a total volume of 100. mu.L.

After 4.18h, 10ul of 1.5Mm hydrogen peroxide solution was added per well and incubation was continued for 18h in medium, after which photographing and trypan blue staining were performed by microscope and viable and dead cells were counted.

Cell viability assay: cell viability ═ number of surviving cells/total cell number × 100%. The results of the measurements are all expressed as mean ± standard deviation using the interclass t-test. All software is counted to be SPSS17.0, P is less than 0.05, and the software has significant difference.

As a result: as shown in fig. 1 and table 1.

TABLE 1 survival of cells with different polypeptide concentrations added

	Survival rate (%)
		Blank control group	96.77±1.78
0nM	2.63±1.52
		0.125nM	3.63±1.56
0.25nM	11.13±1.45**
		0.5nM	13.03±2.03**
1nM	17.70±2.56***
		2nM	27.23±4.59***

Note: t-test, x: p < 0.05, x: p < 0.01, x: p < 0.001 (compared to the 0nM group)

As can be seen from fig. 1 and table 1, when the concentration of the added bioactive polypeptide KLQP is more than 0.25nM, the survival rate of the cells is significantly different from that of the group without the added bioactive polypeptide KLQP, the survival rate of the cells in the 2 nM-added group is increased by 24.6% compared with that in the 0 nM-added group, and the survival rate of the cells is increased along with the increase of the concentration of the bioactive polypeptide KLQP.

Example 3 anti-aging test:

cell anti-apoptosis assay:

1. hacat cells were seeded into 6cm cell culture dishes containing 10% FBS 1640 medium and were changed to serum-free medium culture when the cell density reached 80%.

2. Hacat cells were cultured in serum-free medium for 24h, trypsinized, counted, diluted to 2X 10 using 0.25% FBS 1640 medium⁴And/ml. In 96-well plates, 90uL of each well was inoculated.

3. The polypeptide concentration gradients were set at 0nM, 0.125nM, 0.25nM, 0.5nM, 1nM and 2nM, respectively, for incubation with cells in a total volume of 100. mu.L.

4. The CCK-8 kit is adopted in the test, and the specific references can be found in methods of Hao Y (Hao Y, 2019), Cai Y (Cai Y, 2019) and the like; after 48h, 10ul of CCK-8 reaction was added to each well and incubation continued for 3h in medium, and the absorbance of each well was measured at a wavelength of 450 nm.

5. The absorbance values at 0nM were set as 100% and calculated for each of the other concentration treatment conditions. The results of the measurements are all expressed as mean ± standard deviation using the interclass t-test. All software is counted to be SPSS17.0, P is less than 0.05, and the software has significant difference.

TABLE 2 relative cellular Activity at different polypeptide concentrations

	Relative cell Activity (%)
		0nM	99.99±11.77
0.125nM	107.84±7.46
		0.25nM	114.57±6.96
0.5nM	118.86±4.56
		1nM	122.01±2.04**
2nM	119.85±0.79**

Note: t-test, x: p < 0.05, x: p < 0.01, x: p < 0.001 (compared to the 0nM group)

As can be seen from FIG. 2, when the concentration of KLQP reaches 1nM or more, the relative activity of the cells is 122%, which is 22.02% higher than that of the 0nM group, and the relative activity of the cells is increased with the increase of the concentration of KLQP.

Example 4 testing of repair function on skin:

wound healing test:

animal experiments: 90 healthy female Kunming mice with the weight of 25-30g are selected and divided into 3 groups, each group has 30 mice, and the groups have no statistical difference until the experiment is finished, and no mice die.

The comparison group is 2 groups, and the comparison group is 1: a blank comparison group (without molding); control group 2: normal saline-convalescent liquid control group (molding); experimental group 3: 1nM polypeptide repair solution (KLQP) was used.

Mouse wound injury: mice were anesthetized with 1% pentobarbital sodium (0.1ml/20g) intraperitoneally. The mice were then shaved clean of their back hairs and sterilised with iodophor, and two holes of symmetrical size, approximately 8mm x 8mm, were drilled on both sides of the mouse back with a hole drill. After operation, the mice are placed beside the heater and placed back to the breeding room for normal breeding after waking up.

And (3) effect testing: treatment was performed on the day of total skin excision. Applied twice daily, approximately 20 μ L per well spot. Experiment group 3 mice were coated with physiological saline on the left side of their backs and polypeptide repair solution (1nM) on the right side; in control group 2, the left side of the back of the mouse was coated with physiological saline, and the right side of the back was treated with a new healing fluid. The back wounds of the mice were photographed every 3 days, and the wound repair rate was calculated.

Determination of wound healing rate: the size of the mouse wound was measured every other day with a vernier caliper, and the wound healing rate was calculated, which is (wound size before treatment-wound size after treatment)/wound size before treatment × 100%.

The results of the measurements are all expressed as mean ± standard deviation using the interclass t-test. All software is counted to be SPSS17.0, P is less than 0.05, and the software has significant difference.

As a result: the wound area of the treated group was reduced to different degrees on the 3 rd and 7 th days of application compared with the normal saline control group. The results are shown in Table 3:

TABLE 3 wound healing rates at different times for each group

	Wound healing Rate (%) on day 3	Wound healing Rate (%) on day 7
			Control group 1	N.D	N.D
Control group 2 (left side-physiological saline)	18±1.23	41±1.12
			Control group 2 (Right side-recovery new liquid)	25±0.77*	68±0.96*
Experimental group 3 (left side-physiological saline)	19±1.12	39±1.07
			Experimental group 3 (Right side-polypeptide repair liquid)	29±0.77*	79±0.96**

Note: t-test, x: p < 0.05, x: p < 0.01 (compared to saline treatment); N.D: not detect

Therefore, compared with the normal saline treatment group, the polypeptide repair liquid in the experimental group 3 has an obvious effect of promoting wound healing, and the repair effect is equivalent to that of the new rehabilitation liquid.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Zhejiang Huazhi Dai pharmaceutical industry group medicine development Co., Ltd

<120> bioactive polypeptide and application thereof

<130> MP2037232

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Leu Gln Pro

1

<210> 2

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aagctgcagc cc 12