CN117357419A - Application of anti-aging protein or recombinant protein thereof in preparation of cosmetics - Google Patents

Abstract

The invention belongs to the fields of genetic engineering technology and cosmetics, and in particular relates to application of an anti-aging protein or recombinant protein thereof in preparation of cosmetics. The present invention provides the use of an anti-aging protein or recombinant protein thereof in the preparation of at least one of the following products or compositions: a product or composition for improving the expression level of a cellular anti-wrinkle factor gene; a product or composition for improving the expression level of a cytokine gene; a product or composition that improves skin firmness in a subject; a product or composition that improves the skin elasticity value of a subject; a product or composition that improves a wrinkle-related parameter in a subject; a product or composition for delaying the natural aging of skin; a product or composition for reducing cellular senescence due to photoaging; the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1. The cosmetic prepared from the anti-aging protein can achieve the effects of tightening skin, improving skin elasticity and reducing eye wrinkles.

Description

Application of anti-aging protein or recombinant protein thereof in preparation of cosmetics

Technical Field

The invention belongs to the fields of genetic engineering technology and cosmetics, and in particular relates to application of an anti-aging protein or recombinant protein thereof in preparation of cosmetics.

Background

With the increase in average life span of humans and the increase in aging of the population, aging health problems continue to be of concern. Aging is the process of life, and "anti-aging" refers to the desire to "healthy aging" while prolonging life. For skin, the factors responsible for aging are largely divided into two categories-natural aging and photoaging. Natural aging mainly refers to aging caused by nonresistant factors such as inheritance, endocrine, immune function, etc. existing in a living body with age; photo-aging mainly means that the natural aging process is accelerated due to the effect of ultraviolet rays; climate change, environmental pollution and the like also have direct effects on the photoaging of the skin. Although aging is an unblockable natural phenomenon, people can generally slow the pace of aging by some means, and "anti-aging" cosmetics are in the list. "anti-aging" cosmetics are classified into moisturizing cosmetics, antioxidant cosmetics and bioactive cosmetics according to functions or actions (Li Xiang, hu Junjiao, li Qiong, etc.; perfume and essence cosmetics [ J ].2013,5 ].

Chinese patent application 202210715721.5 discloses an anti-aging skin care product, a preparation method and application thereof, wherein the anti-aging skin care product comprises 7-45% of active functional components by mass percent; the active functional components comprise secretin, a lysate of a fermentation product of the saccharomyces cerevisiae, hexapeptide and pentapeptide; the skin care product has effects in promoting proliferation and regeneration of epidermal cells, promoting collagen production, improving skin elasticity, and resisting skin aging.

Chinese patent application 201911286742.4 discloses an anti-aging composition which comprises the following components in parts by mass: 20-25 parts of a maritime pine bark extract; 10-15 parts of bletilla striata root extract; 8-12 parts of polygonum multiflorum extract; 17-25 parts of a solvent; the composition can promote absorption of effective components in the anti-aging composition, and scavenge free radicals, so as to delay skin aging, and has remarkable anti-aging effect.

In recent years foreign studies have found the importance of inflammatory senescence, it is believed that the senescence, pro-inflammatory cytokine network (proinflammatory cytokine network), is one of the phenomena in the senescence process, and that other senescence mechanisms, such as oxidative stress injury, can form a vicious circle, accelerating cell senescence (Jylinova J, hurme M.Gene variants as determinants of longevity: focus on the inflammatory factors [ J ]. Pflugers Arch,2010;459:239-46;Giunta S.Exploring the complex relations between inflammation and aging (Inflam-lacing): anti-inuflam-aging remodeling of Inflamm-lacing, from robustness to frailty [ J ]. Inflam Res,2008;57: 558-63). Integrated oxidation-inflammatory aging theory due to oxidative and inflammatory stress (oxidation-inflammation theory of aging, oxy-inlamam-aging) has recently been proposed as one of the main causes of aging, chronic oxidative stress affecting all cells, particularly the regulatory systems such as the nervous, endocrine and immune systems, and the interrelation of the above systems, resulting in preventing sufficient homeostasis and thus affecting health maintenance (De la funnte M, miquel j.an update of the oxidation-inflammation theory of aging: the involvement of the immune systemin oxi-inlamam-aging [ J ]. Curr Pharm Des,2009; 15:3003-26).

The anti-wrinkle factors mainly comprise Collagen I, collagen III, collagen IV, collagen VII, decorin, VCAN, BGN, MMP-1, MMP-3, smad7, c-fos, and c-jun. Each anti-wrinkle factor is involved in the aging process by a different mechanism.

Currently, there is a lack of anti-aging cosmetics in the art that can simultaneously regulate inflammatory factors and anti-wrinkle factors.

Disclosure of Invention

Epicoccum nigrum (Epicoccum nigrum) is widely found on the surface of air, soil, and senescent, freshly dead or tender plant tissue. Epicoccus nigrum belongs to the fourth pathogenic microorganism, most of which are not pathogenic, and which can not cause human or animal diseases under normal conditions, and is safe in origin.

The inventor unexpectedly separates a novel protein (code: HJ-P2) from Epicoccus nigricans, namely the anti-aging protein disclosed by the invention, and finds that the protein has excellent anti-aging effect through multiple efficacy experiments, and can remarkably regulate the expression quantity of inflammatory factors and anti-aging factors, thereby realizing the effects of tightening skin, improving skin elasticity and reducing eye wrinkles, and having important application value in the field of cosmetics.

In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:

In one aspect, the invention provides the use of an anti-aging protein or recombinant protein thereof in the preparation of at least one of the following products or compositions:

(1) A product or composition for improving the expression level of a cellular anti-wrinkle factor gene;

(2) A product or composition for improving the expression level of a cytokine gene;

(3) A product or composition that improves skin firmness in a subject;

(4) A product or composition that improves the skin elasticity value of a subject;

(5) A product or composition that improves a wrinkle-related parameter in a subject;

(6) A product or composition for delaying the natural aging of skin;

(7) A product or composition for reducing cellular senescence due to photoaging;

the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1,

the nucleotide sequence of the coded anti-aging protein is shown as SEQ ID NO. 2.

SEQ ID NO:1：

MQFFTVASALFAAALAAPAPQTSDCPNPAHCGGSPPDPSTYENIDISDFYVRKNNGIQN AGFKLTGKNATDLSCTIGATDLPSNVVTCGDSDYRFGLTKGDTTEFGLAIYHQTSPFAGLW AIGDAPTYCHAGGNGPDDFVCQQTGPTTIVIVGQNYPGGADN。

SEQ ID NO:2：

ATGCAATTTTTCACAGTAGCTTCAGCACTATTCGCTGCTGCCTTGGCAGCGCCGGCACCGCAAACTTCGGACTGCCCGAATCCGGCGCACTGCGGAGGTAGCCCGCCGGACCCGTCCACCTACGAGAACATTGATATTAGCGACTTCTATGTTCGTAAAAACAACGGCATCCAAAACGCCGGTTTTAAACTGACCGGTAAGAACGCGACCGATCTGTCTTGTACCATTGGTGCGACCGATCTTCCGAGCAATGTTGTCACCTGCGGCGACTCCGACTACCGCTTCGGCCTGACGAAGGGTGATACCACCGAATTTGGCTTGGCGATTTATCACCAGACCAGCCCATTTGCGGGCCTGTGGGCAATCGGCGACGCTCCGACGTACTGCCATGCGGGTGGTAATGGTCCGGACGATTTCGTTTGTCAGCAGACCGGCCCTACTACGATCGTGATCGTGGGTCAGAATTATCCGGGTGGCGCGGATAAC。

In some examples, the recombinant protein further comprises a tag protein that aids in its expression and/or purification, or an active protein that enhances its activity and/or extends its half-life.

In some examples, the tag protein may be a His tag, preferably a 6 xhis tag.

In some preferred examples, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3.

In some preferred embodiments, the nucleotide encoding the recombinant protein is set forth in SEQ ID NO. 4.

SEQ ID NO:3：

MQFFTVASALFAAALAAPAPQTSDCPNPAHCGGSPPDPSTYENIDISDFYVRKNNGIQN AGFKLTGKNATDLSCTIGATDLPSNVVTCGDSDYRFGLTKGDTTEFGLAIYHQTSPFAGLW AIGDAPTYCHAGGNGPDDFVCQQTGPTTIVIVGQNYPGGADNLEHHHHHH。

SEQ ID NO:4：

ATGCAATTTTTCACAGTAGCTTCAGCACTATTCGCTGCTGCCTTGGCAGCGCCGGCACCGCAAACTTCGGACTGCCCGAATCCGGCGCACTGCGGAGGTAGCCCGCCGGACCCGTCCACCTACGAGAACATTGATATTAGCGACTTCTATGTTCGTAAAAACAACGGCATCCAAAACGCCGGTTTTAAACTGACCGGTAAGAACGCGACCGATCTGTCTTGTACCATTGGTGCGACCGATCTTCCGAGCAATGTTGTCACCTGCGGCGACTCCGACTACCGCTTCGGCCTGACGAAGGGTGATACCACCGAATTTGGCTTGGCGATTTATCACCAGACCAGCCCATTTGCGGGCCTGTGGGCAATCGGCGACGCTCCGACGTACTGCCATGCGGGTGGTAATGGTCCGGACGATTTCGTTTGTCAGCAGACCGGCCCTACTACGATCGTGATCGTGGGTCAGAATTATCCGGGTGGCGCGGATAACCTCGAGCACCACCACCACCACCACTGA。

In some examples, the subject is a human or animal, preferably a human.

In some examples, the product or composition comprises at least 1.5mg/L of an anti-aging protein or recombinant protein thereof.

In some preferred examples, the product or composition may comprise 1.5-30mg/L of an anti-aging protein or recombinant protein thereof, for example: 1.5mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, 26mg/L, 27mg/L, 28mg/L, 29mg/L, 30mg/L.

In some examples, the anti-wrinkle factors include, but are not limited to, collagen I, collagen III, collagen IV, collagen VII, decorin, VCAN, BGN, MMP-1, MMP-3, smad7, c-fos or c-jun, preferably Collagen I, collagen VII, decorin, VCAN, BGN, MMP-1, smad7 or c-fos.

In some examples, the inflammatory factor includes, but is not limited to, TNF- α, IL-1β, IL-6, IL-1α or IL-8, preferably TNF- α and/or IL-1β.

In some examples, the wrinkles include, but are not limited to, a fish tail, a head-up, an inter-eyebrow, an under-eye, a stature, a mouth Zhou Wen, a Sichuan or a neck, preferably an under-eye.

In some examples, the wrinkle-related parameters include, but are not limited to, the number of wrinkles, the perimeter of wrinkles, the area of wrinkles, the roughness of wrinkles, the average depth of wrinkles or the area of wrinkles, preferably the number of wrinkles, the perimeter of wrinkles, the area of wrinkles and the roughness of wrinkles.

In another aspect, the invention provides an application of an anti-aging protein or recombinant protein thereof in preparing cosmetics, wherein the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1,

the nucleotide sequence of the coded anti-aging protein is shown as SEQ ID NO. 2.

In some examples, the recombinant protein further comprises a tag protein that aids in its expression and/or purification, or an active protein that enhances its activity and/or extends its half-life.

In some examples, the tag protein may be a His tag, preferably a 6 xhis tag.

In some preferred examples, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3.

In some preferred embodiments, the nucleotide encoding the recombinant protein is set forth in SEQ ID NO. 4.

In some examples, the cosmetic is for:

1) Improving skin firmness of a subject; and/or

2) Improving the skin elasticity value of the subject; and/or

3) Improving wrinkle related parameters in a subject.

In some examples, the wrinkles include, but are not limited to, a fish tail, a head-up, an inter-eyebrow, an under-eye, a stature, a mouth Zhou Wen, a Sichuan or a neck, preferably an under-eye.

In some examples, the wrinkle-related parameters include, but are not limited to, the number of wrinkles, the perimeter of the wrinkles, the area of the wrinkles or the roughness of the wrinkles, the average depth of the wrinkles or the area of the wrinkles, preferably the number of wrinkles, the perimeter of the wrinkles, the area of the wrinkles and the roughness of the wrinkles.

In some examples, the 3) comprises:

(I) Reducing the number of wrinkles; and/or

(II) reducing the perimeter of the wrinkles; and/or

(III) reducing wrinkle area; and/or

(IV) reducing wrinkle roughness.

In some examples, the subject is a human.

In some examples, the cosmetic comprises at least 1.5mg/L of an anti-aging protein or recombinant protein thereof.

In some preferred examples, the cosmetic may comprise 1.5-30mg/L of anti-aging protein, for example: 1.5mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, 26mg/L, 27mg/L, 28mg/L, 29mg/L, 30mg/L.

In yet another aspect, the present invention provides a cosmetic comprising an effective amount of an anti-aging protein having the amino acid sequence shown in SEQ ID NO. 1 or a recombinant protein thereof.

In some examples, the nucleotide sequence encoding the anti-senescence protein is set forth in SEQ ID NO. 2.

In some examples, the recombinant protein further comprises a tag protein that aids in its expression and/or purification, or an active protein that enhances its activity and/or extends its half-life.

In some examples, the tag protein may be a His tag, preferably a 6 xhis tag.

In some preferred examples, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3.

In some preferred embodiments, the nucleotide encoding the recombinant protein is set forth in SEQ ID NO. 4.

In some examples, the cosmetic further comprises a cosmetically acceptable carrier or excipient including, but not limited to, at least one of surfactants, oils, emulsifiers, high fat ingredients, consistencies, thickeners, polymers, silicon containing compounds, fats, waxes, stabilizers, bioactive ingredients, deodorants, film forming agents, bulking agents, antioxidants, inorganic coloring pigments, water cosolvents, preservatives, solubilizing agents, right hand essence, and colorants.

In some embodiments, the cosmetically acceptable carrier or excipient is selected from, but is not limited to, one or more of deionized water, trehalose, nicotinamide, betaine, glycerol, disodium EDTA, 1, 2-hexanediol, xanthan gum, pentaerythritol tetra (ethylhexanoate), polydimethylsiloxane, glycerol stearate PEG-100, diisopropyl sebacate, myristyl myristate, cetyl ethyl hexanoate, phytosterol, behenyl alcohol, octyl dodecanol lauroyl glutamate, tocopheryl acetate, jojoba esters, tocopherols (vitamin E), sodium stearoyl glutamate, sodium polyacrylate, butylene glycol, p-hydroxyacetophenone, benzodenatonium, hyaluronic acid, glucosyl hesperidin, and fucoidan polyether-20.

In some examples, the cosmetic dosage form includes, but is not limited to, an aqueous or oily solution, an aqueous cream, an aqueous or oily gel, a facial mask or anhydrous product, and the like.

In some preferred examples, the cosmetic dosage form includes, but is not limited to, a body wash, shampoo, emulsion, microemulsion, nanoemulsion, mask, essence, lotion, ointment, foam, patch, cosmetic powder, cosmetic cotton, eye essence, eye mask, eye shadow, sun cream, eye gel, or eye cream.

In some examples, the cosmetic comprises at least 1.5mg/L of an anti-aging protein or recombinant protein thereof.

In some preferred examples, the cosmetic may comprise 1.5-30mg/L of anti-aging protein, for example: 1.5mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, 26mg/L, 27mg/L, 28mg/L, 29mg/L, 30mg/L.

In yet another aspect, the present invention provides a method of care for non-therapeutic purposes, the method of care comprising:

topical application, preferably ocular application, of an effective amount of any of the foregoing products or compositions, or any of the foregoing cosmetics, to the outer surface of the subject's skin tissue.

In yet another aspect, the present invention provides a method for preparing any one of the aforementioned cosmetics, the method comprising the steps of:

mixing the cosmetic base material with antiaging protein or its recombinant protein, and stirring to obtain cosmetic.

In some examples, the method of preparation comprises the steps of:

i) Adding the water phase material into an emulsifying pot, mixing, stirring and heating to a first temperature to obtain a water phase;

II) adding the oil phase material into an oil phase pot, stirring and heating to a second temperature, and marking as an oil phase;

III) adding the oil phase into the stirred water phase for homogenizing and emulsifying, stirring and cooling to a third temperature, adding other materials, and stirring uniformly to obtain a cosmetic base material;

IV) mixing the cosmetic base material and the anti-aging protein uniformly, and obtaining the cosmetic.

In some examples, the first and second temperatures are 70-80 ℃.

In some examples, the time of the homogenizing emulsification of step III) is 3-5min.

In some examples, the third temperature is 40-45 ℃.

Compared with the prior art, the application of the anti-aging protein or the recombinant protein thereof provided by the invention has at least the following beneficial effects:

(1) The safety is better, and the obvious cytotoxicity is not shown;

(2) Significantly reducing cellular aging due to photoaging, about a 74% decrease in beta-galactosidase activity relative to the model group;

(3) The average life of caenorhabditis elegans is prolonged significantly, and is prolonged by about 7 days at most relative to the control group;

(4) Significantly down-regulating the expression level of two inflammatory factor genes of TNF-alpha and IL-1 beta, wherein the relative expression level of TNF-alpha is down-regulated by about 33% relative to the model group, and the relative expression level of IL-1 beta is down-regulated by about 14% relative to the model group;

(5) The expression quantity of 8 anti-wrinkle factors including Collagen I, collagen VII, decorin, VCAN, BGN, MMP-1, smad7 and c-fos is obviously improved; wherein, collagen I up-regulates 19.51%, collagen VII up-regulates 31.88%, decorin up-regulates 65.79%, VCAN up-regulates 21.05%, BGN up-regulates 64.29%, MMP-1 down-regulates 31.44%, smad7 down-regulates 13.74%, c-fos down-regulates 26.85%.

(6) Remarkably improving skin tightness, skin elasticity value, and wrinkles under the eyes (number, circumference, area, roughness); wherein, after the subject used the eye essence test sample containing 3mg/L HJ-P2 protein for 4 weeks, the skin firmness was improved by about 13%, the skin elasticity value was improved by about 8%, the number of wrinkles was reduced by about 11%, the perimeter of wrinkles was reduced by about 19%, the area of wrinkles was reduced by about 15%, and the roughness of wrinkles was reduced by about 5%.

Drawings

FIG. 1 shows SDS-PAGE patterns of proteins during purification of HJ-P2 protein.

FIG. 2 is a cytomorphology diagram of the HJ-P2 protein cytotoxicity test.

FIG. 3 is a graph showing the cell viability of the HJ-P2 protein cytotoxicity test.

FIG. 4 shows the effect of HJ-P2 protein on the beta-galactosidase activity of cells.

FIG. 5 is a graph of the survival analysis of caenorhabditis elegans administered at different doses of the HJ-P2 protein.

FIG. 6 is a graph showing the survival cycle of caenorhabditis elegans administered at different doses of the HJ-P2 protein.

FIG. 7 shows the effect of HJ-P2 protein on the relative expression level of inflammatory factors.

FIG. 8 shows experimental results of an in vitro anti-wrinkle efficacy test of HJ-P2 protein.

FIG. 9 shows the improvement of HJ-P2 protein for different anti-wrinkle factors.

Fig. 10 shows the mean change in skin firmness F4 (n=14) for each group (test and control) at different time points.

Fig. 11 shows the improvement of the skin firmness F4 mean at different time points for the different groups (test and control group) (n=14).

Fig. 12 shows the mean change in skin elasticity value Q1 (n=14) for each group (test group and control group) at different time points.

Fig. 13 shows the mean change in skin elasticity values Q1 (n=14) at different time points for different groups (test and control).

Fig. 14 shows the mean change in the number of under-eye wrinkles for each group (test and control) at different time points (n=14).

Fig. 15 shows mean changes in the circumference of the under-eye wrinkles at various time points for each group (test and control group) (n=14).

Fig. 16 shows the mean change in the area of under-eye wrinkles for each group (test and control) at different time points (n=14).

Fig. 17 shows the mean change in under-eye wrinkle roughness for each group (test and control) at different time points (n=14).

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications, such as the molecular cloning laboratory guidelines (fourth edition) (Cold spring harbor laboratory Press), ausubel, F.M et al, current Protocols in Molecular Biology, greene Publishing Assoc, and Wiley-lnterscience.

Example 1 preparation of HJ-P2 protein

The inventor creatively separates an HJ-P2 protein from Epicoccum nigrum (Epicoccum nigrum), and the amino acid sequence of the HJ-P2 protein is shown as SEQ ID NO. 1. For subsequent studies of its biological function, the inventors joined the nucleotide sequence of HJ-P2 to pET-21a expression vector (cleavage sites Nde I and Xho I) to obtain recombinant plasmid HJ-P2-pET-21a, the C-terminus of which was fused to the 6 xhis tag in the vector, and the plasmid was artificially synthesized.

1.1 Induction of expression of HJ-P2 proteins

The synthesized plasmids of HJ-P2 were each dissolved with sterile water and then transformed into E.coli over express C43 (DE 3). Recombinant E.coli was cultured in solid LB medium, at 37℃overnight in a constant temperature incubator. Taking 12mL of sterile shaking tube, adding 5mL of sterile LB culture medium (containing 100 mug/mL of ampicillin), picking up grown C43 single colony by using a sterile pipette tip, inoculating the single colony into the culture medium, placing the single colony into a constant temperature shaking table for overnight culture under the culture conditions of 37 ℃ and 200rpm for 15 hours, preserving the cultured bacterial liquid by 17.5% glycerol, and placing the bacterial liquid in a refrigerator at-80 ℃ or carrying out the next amplified culture.

Adding 75mL of liquid LB medium (sterilized at high temperature and high pressure in advance and cooled to room temperature) into a 250mL triangular flask, adding ampicillin to a final concentration of 100 μg/mL, inoculating 1.5mL of the above proliferation bacterial liquid, placing into a constant temperature shaking table, culturing at 37deg.C and 220rpm for about 2.5h until OD ₆₀₀ After about 0.7, the inducer IPTG was added to a final concentration of 0.2mmol/L and incubated overnight at 25℃for 15h.

1.2 isolation and purification of HJ-P2 protein

The buffers used for the protein isolation and purification of this example are shown below:

crushing buffer solution: 50mmol/L Tris-HCl, pH 7.8;

binding buffer: 50mmol/L Tris-HCl,300mmol/L NaCl,10mmol/L Imidazole, pH 7.8;

elution buffer: 50mmol/L Tris-HCl,300mmol/L NaCl,500mmol/L Imidazole, pH 7.8;

desalting buffer: 50mmol/L Tris-HCl, pH 7.8.

Adding a crushing buffer solution into the collected thalli according to the proportion of adding 1mL of the crushing buffer solution into each 2mL of fermentation liquid, re-suspending the thalli, crushing the thalli by using an ultrasonic cell crusher until the liquid is uniform and has good light transmission, and stopping crushing. The crushed bacterial liquid is centrifuged for 30min at 4 ℃ and 7000rpm, the supernatant (i.e. the "crude" sample) is taken, filtered through a 0.45 μm water-based filter membrane and placed on ice for standby.

Affinity chromatography: the protein was purified using AKTA protein purifier and nickel ion affinity column, the procedure was as follows:

placing the pump A and the pump B into pure water, and flushing the pumps; after the flushing is finished, setting 50% B with the flow rate of 1mL/min, and installing a nickel column; the flow rate was set at 5mL/min and the system was flushed to baseline plateau.

Pump a into the binding buffer, pump B into the elution buffer, and pump flush; after the flush was completed, 100% b was set and the system was flushed to baseline plateau.

Set 100% a, after the flush system has leveled to baseline, zero.

And (3) placing the pump A into the sample, setting the flow rate to be 2mL/min, starting loading until about 0.5mL of sample remains at the bottom, and collecting the outflow sample, namely the penetrating fluid (namely the penetrating sample).

The pump head of the pump A was rinsed with pure water, the pump A was returned to the binding buffer, the flow rate was set at 3mL/min, and the washing of the protein impurities was smoothed to baseline.

Flow rate 5mL/min, linear elution conditions were set: 0% to 50% b,60ml, and collecting the eluted peak sample (i.e. "Ni pure" sample) for subsequent desalination purification.

Set 100% b, flush the system to baseline plateau.

Pump a and pump B were both placed in pure water and pump flushing was performed, after which 50% B was set and the flushing system was stationary to baseline.

Pump a and pump B were both placed in 20% ethanol, at least 2 column volumes were flushed, the nickel column was removed and the system was shut down.

Desalting and purifying: desalting was performed using an AKTA protein purifier and desalting column, the procedure was as follows:

placing the pump A into pure water, and performing pump flushing; after the flushing is finished, setting 100% A, the flow rate is 1mL/min, installing a desalting column, setting the flow rate to 2mL/min, and flushing the system by more than 1 column volume.

Pump a into desalting buffer, pump flush, set flow rate 3mL/min after flush, flush system to baseline plateau, zero.

Pump a into the sample and begin loading until about 0.5mL of sample remains at the bottom.

Wash the a pump head with pure water, put the a pump back into the desalting buffer, collect the eluted peak protein according to UV-280 (i.e. "desalted" sample). The loading volume is not more than 1/3 of the column volume, otherwise the protein elution peak (UV-280) may overlap with the salt ion elution peak (connectivity);

pump a into purified water, pump flush, flush the system until baseline plateau.

Pump flushing by placing both pump a and pump B in 20% ethanol; the system was flushed 1 column volume above, the desalting column was removed and the system was shut down.

Ultrafiltration concentration: the desalted protein solution was subjected to ultrafiltration concentration using a 10kDa ultrafiltration tube under the conditions of: ultrafiltration at 4℃at 3000 Xg to a protein concentration above 1g/L (i.e. "concentrated" sample). Samples from the above purification steps were subjected to SDS-PAGE protein electrophoresis, and the molecular weight of the HJ-P2 protein was 17.9kDa as shown in FIG. 1.

Protein concentration determination: and (3) establishing a protein concentration standard curve according to the specification of the protein quantitative kit of the Siemens femto BCA, and simultaneously measuring the concentration of the target protein after ultrafiltration concentration. According to BCA protein concentration standard curve, the measured sample OD ₅₆₂ Substituting into standard curve equation, the concentration of HJ-P2 pure protein is calculated to be 2.45g/L (figure 1).

Example 2 cytotoxicity test

Cell seeding: fibroblast cell count of 8X 10 ³ Inoculation density of individual/wellsThe cells were seeded in 96-well plates, incubator (37 ℃, 5% CO) ₂ ) Incubate overnight.

Test group: the test set-up was zeroed, solvent control, positive control and sample. In the sample group, 8 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.

Preparing liquid: sample working solutions with different concentrations are prepared according to the following gradients: 0.0781%, 0.1563%, 0.3125%, 0.625%, 1.25%, 2.5%, 5%, 10% (v/v). The concentration of the 100% HJ-P2 protein stock solution in the test is 300mg/L.

Drug administration: administration was performed when the cell plating rate in 96 well plates reached 40% -60%. 200. Mu.L of culture solution was added to each well of the solvent control group; 200. Mu.L of culture solution containing 10% DMSO is added to each well of the positive control group; 200 mu L of culture solution containing samples with corresponding concentrations is added into each hole of the sample group; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO) ₂ ) Is cultured for 24 hours.

Detection: after the cells were cultured in a rich state for 24 hours, the supernatant was discarded, MTT working solution (0.5 mg/mL) was added, incubated at 37℃for 4 hours in the absence of light, after the incubation was completed, 150. Mu.L of DMSO was added to each well, and OD was read at 490 nm.

Cell relative viability calculation: according to the formula, the relative cell viability (%) = (sample well OD-zeroing well OD)/(solvent control well OD-zeroing well OD) ×100%.

MTT and morphological results showed that the sample HJ-P2 protein showed no significant cytotoxicity in the concentration range of 10% (v/v) based on fibroblasts, and was better in safety (FIGS. 2-3 and Table 1).

TABLE 1 MTT assay results for HJ-P2 protein of sample

Example 3 Effect of HJ-P2 protein on cell beta-galactosidase Activity

The research shows that the beta-galactosidase (beta-GAL) in the cells can be over-expressed during the aging of the cells, so the activity and the expression quantity of the beta-galactosidase can be used as the detection index of the aging of the cells. Beta-galactosidase can decompose p-nitrobenzene-beta-D-galactopyranoside and generate p-nitrophenol, and the p-nitrophenol has a maximum absorption peak at 400nm, so that the absorbance value generated by detecting the p-nitrophenol can reflect the activity of the beta-galactosidase, thereby representing the cell aging condition.

Preparation of cells: culturing NHDF cells (normal human fibroblast) with good growth state to obtain cell suspension, adding 500 μl diluted cell suspension into 24-well plate to give a concentration of 5.0X10 ⁴ cells/well。

CO is put into ₂ The incubator was cultured for 16 hours.

Add drug, incubate for 24h.

Selection of UVA ultraviolet intensity (5J/cm) ² ) The cells were irradiated with ultraviolet light.

Continue culturing for 24h.

Cell collection: cells from 24 well plates were collected separately and centrifuged at 10000rpm for 5min in two sets of centrifuge tubes.

Quantitative detection of protein: taking one group of centrifugal tube groups, discarding redundant culture medium, adding 100 mu L of cell lysate, fully and uniformly mixing, and carrying out protein quantitative detection according to the BCA protein quantitative kit.

Cell disruption: taking the rest of the centrifugal tube groups, discarding the redundant culture medium, adding 125 mu L of extracting solution into each tube, carrying out low-temperature ultrasonic disruption on cells, centrifuging the disrupted cells, 15000 Xg, 4 ℃ for 10min, and taking supernatant to be tested.

Dilution of standard solution: standard solutions (5 μmol/mL p-nitrophenol solution) were diluted to 0, 6.25, 12.5, 25, 50, 100, 200nmol/mL, respectively, according to the beta-galactosidase activity kit instructions.

Determination of samples (reagents added sequentially in 96 well plates according to table 2):

TABLE 2 amounts of reagents added in this example

The samples were prepared in Table 2, and the absorbance A was measured at 400nm and recorded as A-tube, and A-tube. Calculation Δa assay = a assay tube-a control tube, Δa standard = a standard tube-a blank tube.

Calculation of beta-galactosidase Activity:

1) Establishment of a standard curve:

a standard curve is established according to the absorbance value (x, deltaA standard) and the concentration (y, nmol/ml) of the standard tube, deltaA (x, deltaA measurement) is substituted into the standard curve, and the product amount y (nom/ml) generated by the sample is calculated.

2) Calculation of beta-GAL activity:

beta-GAL Activity (U/mg prot) = (y×V inverse total)/(V-like×Cpr)/(T)

Wherein: cpr: sample protein concentration (mg/ml); v inverse total: the total volume of the reaction system; v sample: adding the sample volume into the reaction system; t: reaction time. (production of 1nmol of p-nitrophenol per mg protein per hour is defined as one enzyme activity unit.)

The results of the repeatability verification are shown in fig. 4, and the activity of beta-galactosidase in NHDF cells subjected to UVA irradiation is obviously improved compared with that of blank groups not subjected to UVA irradiation, so that the experimental model is considered to be established. After treatment with 5mg/L of HJ-P2 sample, the activity of beta-galactosidase of NHDF cells is obviously reduced by about 74 percent relative to the activity of the model group. Thus, HJ-P2 samples at the concentrations tested can reduce cellular senescence due to photoaging.

Example 4 Effect of HJ-P2 protein on the longevity of caenorhabditis elegans

4.1 nematode synchronization experiment (hypochlorous acid cracking method)

Collecting nematodes:

taking a nematode plate, observing nematodes under a microscope, and carrying out a synchronization experiment when most nematodes are in an oviposition period. Sucking SM buffer to the nematode plate, sucking nematodes on the wash plate, collecting about the nematode solution to a centrifuge tube, centrifuging at 3500rpm for 1min, sucking off the supernatant, resuspending with SM buffer, and repeating this step 3 times.

Lysis of nematodes:

nematode lysate was prepared with 5M NaOH and 5% naclo, in proportion of 5M NaOH:5% naclo=1: 2, preparing the nematode suspension and the nematode lysate according to the following steps of 2:1, and rapidly and continuously shaking, and observing the cracking state of the nematodes in the tube until the complete morphology of the nematodes is invisible to naked eyes, wherein the process is not more than 5min. Immediately, centrifugation was carried out at 3500rpm for 2min, and the supernatant was carefully aspirated off. The eggs were lightly resuspended in SM buffer and washed repeatedly more than 3 times.

Starvation culture:

the eggs of the wire worm washed with the lysate are resuspended with SM buffer, then transferred to a centrifuge tube, added with a proper amount of SM buffer, and placed in a shaking table at 20 ℃ for shaking culture for 24 hours.

Transfer:

the synchronized nematodes were collected by centrifugation at 3500rpm for 2min, transferred to 90mm NGM plates, and incubated at 20℃for two days to L4 for subsequent experiments.

4.2 nematode longevity experiments

Preparation of NGM Medium plates for Life experiments

Preparing NGM solid culture medium, sterilizing, cooling to 50deg.C, adding 5-Fu, mixing, sucking 4mL of culture medium with a pipette, placing on 35mm diameter plate, uniformly distributing culture medium on the whole plate, pouring about 50, sealing the redundant plates, and storing at 4deg.C.

Preparing HJ-P2 protein aqueous solutions with the concentration of 3mg/L, 1.5mg/L and 0.75mg/L respectively, taking 400 mu L of the aqueous solution, adding the aqueous solution into a prepared NGM plate, taking chlorogenic acid as a positive control and sterile water as a blank control, adding 80 mu L of inactivated OP50 bacterial heavy suspension into the NGM plate after the sample working solution is immersed into the NGM plate and dried, and airing.

Insect pick

2 duplicate plates were set, and each working plate picked 20 nematodes in the post-L4 stage after synchronization and incubated at 20 ℃.

Nematode plate change and life observation

The following day of plating, the record is observed, the head or tail of the nematode is lightly touched by an inoculating needle every other day, if the nematode does not respond, the nematode is picked up as dead, the nematode is recorded, all the nematodes die, statistical data are obtained, and a nematode life curve graph is produced. In the life test process, the plate is replaced according to actual conditions, and if the pollution condition of the plate appears, the plate is replaced in time.

Test results

In the experiment, chlorogenic acid is used as a positive control, a blank control is added, and the test concentrations of HJ-P2 protein samples are respectively as follows: 3mg/L, 1.5mg/L, 0.75mg/L, 5 treatment groups total.

Experimental results show that 3mg/L HJ-P2 can prolong the average life of nematodes for about 7 days, 1.5mg/L HJ-P2 can prolong the average life of nematodes for about 3 days, and 0.75mg/L HJ-P2 has no remarkable prolonging capability on the life of nematodes. Thus, it was demonstrated that 3mg/L and 1.5mg/L HJ-P2 significantly prolonged nematode life, and both had significant anti-aging effects (FIGS. 5 and 6).

Example 5 influence of HJ-P2 protein on the relative expression level of inflammatory factors

Plank: THP-1 was seeded in 24-well plates, 5X 10 per well ⁵ Individual cells.

Sample preparation: determining the test final concentration of each sample according to the maximum safe concentration of the sample measured in the pre-experiment; the samples were pre-diluted with the appropriate solvents to give a sample dilution concentration of 100 times the final concentration tested.

Sample addition: adding 10 mu L of solvent into the negative control group (NC) and the model group (M); 10 mu L of corresponding sample diluent is added to each sample group (Sn) corresponding hole, and 10 mu L of 1mM dexamethasone mother liquor (final concentration is 10 mu M) is added to the positive control group (PC) corresponding hole; culturing in incubator for 24 hr.

LPS treatment: 10 mu L of 100 mu g/mL LPS (final concentration is 1 mu g/mL) is added to corresponding holes of a model group (M), a positive control group (PC) and each sample group (Sn), and 10 mu L of culture solution is added to corresponding holes of a negative control group (NC); culturing in incubator for 24 hr.

RNA extraction and reverse transcription: collecting each group of cells, and extracting total RNA of each group of cells by using RaPure Total RNA Kit; each group of total RNAs was reverse transcribed into cDNAs using HiScript IIQ RT SuperMix for qPCR (+gDNA wind), respectively

qPCR detection

The relative expression levels of TNF-. Alpha.and IL-1β (GAPDH gene as an internal reference gene) were detected by qPCR using each set of cDNA templates.

In this example, 3 independent replicates were performed, and the Mean (Mean) and Standard Deviation (SD) of the relative expression levels of each group were calculated, and the results were expressed as mean±sd. Comparisons between groups used t-test statistical analysis (double-tailed), P <0.05 considered the difference significant, and P <0.01 considered the difference extremely significant.

The test result shows that in the test, the relative expression quantity of each inflammatory factor of the negative control group is obviously lower than that of the model group (LPS treatment), which indicates that the inflammatory cell model is established; the relative expression quantity of each inflammatory factor in the positive control group (dexamethasone treatment) is obviously lower than that in the model group, which indicates that the positive control group is established; in conclusion, the quality control index of the experiment meets the requirements, and the detection system is established.

Repeated verification shows that the relative expression amount of the two inflammatory factors of TNF-alpha and IL-1 beta in the 5mg/L HJ-P2 sample group is lower than that in the model group, and thus, the 5mg/L HJ-P2 can inhibit the up-regulation of the expression of the two inflammatory factors of TNF-alpha and IL-1 beta caused by LPS (figure 7). Wherein the relative expression of TNF- α was reduced by about 33% relative to the model group following administration of 5mg/L HJ-P2. The relative expression of IL-1β was down-regulated by about 14% relative to the model group.

Example 6 influence of HJ-P2 protein on anti-wrinkle factor

In this example, after the fibroblast is irradiated with UVA, the anti-wrinkle effect of the sample to be measured is evaluated by detecting the change in gene expression of 13 anti-wrinkle factors. The mechanism of action of 13 anti-wrinkle factors is shown in Table 3.

TABLE 3 mechanism of action of anti-wrinkle factors

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Test method

Inoculation: according to 2X 10 ⁵ Cell/well seeding Density cells were seeded into 6-well plates, incubator (37 ℃, 5% CO) ₂ ) Incubate overnight.

Preparing liquid: test working fluids were prepared according to the test packets of table 4.

Drug administration: according to the test group of Table 3, when the cell plating rate in the 6-well plate reaches 40% -60%, group administration is carried out, the administration amount of each well is 2mL, 3 compound wells are arranged in each group, and the incubator (37 ℃, 5% CO) ₂ ) And incubated for 24h.

UVA irradiation: according to the experimental group, the group with UVA irradiation was subjected to 30J/cm ² Is placed in an incubator (37 ℃ C., 5% CO) ₂ ) The culture was continued for 24 hours.

Collecting cells: after the incubation, the old solution was aspirated, washed twice with PBS, 1mL RNAiso Plus was added to each well, and after the lysed cells were blown down, the samples were collected.

Gene expression detection: RNA was extracted, reverse transcribed to cDNA, and then subjected to fluorescent quantitative PCR detection, and the result was calculated by a 2-DeltaCT method.

Statistical analysis of results: graphPad Prism was used to map and the results were expressed as mean±sd. Comparisons between groups were performed using t-test statistical analysis. Statistical analysis was double tailed. P < 0.05 was considered to have a significant difference and P < 0.01 was considered to have a very significant difference.

Table 4 test packets of this embodiment

The results show (FIG. 8 and FIG. 9) that, based on fibroblasts, the HJ-P2 protein samples have significantly up-regulated gene expression levels of Collagen I, collagen VII and Decorin, VCAN, BGN and significantly down-regulated gene expression levels of MMP-1, smad7 and c-fos at a concentration of 3mg/L compared with the control group; wherein Collagen I up-regulates 19.51%, collagen VII up-regulates 31.88%, decorin up-regulates 65.79%, VCAN up-regulates 21.05%, BGN up-regulates 64.29%, MMP-1 down-regulates 31.44%, smad7 down-regulates 13.74%, and c-fos down-regulates 26.85%. Based on the above, the anti-wrinkle effect can be achieved by up-regulating the expression level of the genes of Collagen I, collagen VII and Decorin, VCAN, BGN and down-regulating the expression level of the genes of MMP-1, smad7 and c-fos at the concentration of 3 mg/L.

Example 7 application of HJ-P2 protein in preparing eye essence

The materials used in this example:

aqueous phase material: deionized water, trehalose, nicotinamide, betaine, glycerol, disodium EDTA, 1, 2-hexanediol, xanthan gum;

oil phase materials: pentaerythritol tetrakis (ethylhexanoate), polydimethylsiloxane, glycerol stearate PEG-100, diisopropyl sebacate, myristyl myristate, cetyl ethylhexanoate, phytosterol, behenyl alcohol, octyldodecanol lauroyl glutamate, tocopheryl acetate, jojoba esters, tocopherols (vitamin E), sodium stearoyl glutamate, sodium polyacrylate;

other materials: butanediol, p-hydroxyacetophenone, benidiammonium, hyaluronic acid, glucosyl hesperidin, fuchsin steareth-20.

Preparation method

(1) Weighing water phase materials according to the proportion, adding the water phase materials into an emulsifying pot, mixing, stirring and heating to 70-80 ℃, and recording as a water phase;

(2) Weighing oil phase materials according to the proportion, adding the oil phase materials into an oil phase pot, stirring and heating to 70-80 ℃, and recording as an oil phase;

(3) Adding the oil phase into the stirred water phase, homogenizing and emulsifying for 3-5 minutes, stirring and cooling to 40-45 ℃, adding other materials weighed according to the proportion, and uniformly stirring to obtain an eye essence base material;

(4) Half of the eye essence base material is taken, HJ-P2 protein with the final concentration of 3mg/L is added, and the mixture is stirred uniformly, thus obtaining the eye essence test sample.

Example 8 evaluation of safety of eye essence

Experimental technical basic information

Test article: the "eye essence base" and "eye essence test sample" prepared in example 6 "

Number of human subjects: 30 people

Age: between 23 and 45 years of age

Gender: female 28, male 2

Health status: the skin of the subject is healthy, no skin disease allergy history exists, and the volunteer selection standard of the subject is met.

Experimental methods

Selecting a qualified spot tester, dripping about 0.020mL-0.025mL of a test object into the spot tester in a closed spot pasting test mode, pasting a special adhesive tape on the inner side of the arm of a subject, respectively observing skin reactions at 0.5, 24 and 48 hours, and recording the results according to skin reaction grading standards in cosmetic hygiene standards.

Experimental results

The test results of the human skin patch show that: 30 subjects observe skin reactions at 0.5 h, 24 h and 48h through the eye essence base material and eye essence test sample patch test, wherein 0 cases show skin adverse reactions, which indicates that the eye essence base material and the eye essence test sample are safe to use.

Example 9 test of anti-aging Effect of eye essence

The peri-ocular region of asian adult subjects was selected and two different test samples were used for random distribution around the left and right eyes. The efficacy of tightening, improving elasticity, and anti-wrinkle of the samples was evaluated by measuring the numerical changes of the pre-use sample (W0), the post-use sample 1 week (W1), the post-use sample 2 weeks (W2), the tightening (F4) of the post-use sample 4 weeks (W4), the elasticity value (Q1), the parameters of the under-eye wrinkles (number, circumference, area, roughness Rz) of the subject's periocular region.

9.1 testing basic information

Study object: 14 healthy adult females

Age: 18-60 years old

Inclusion primary criteria:

for those with wrinkles around the eyes, the level of the under-eye lines is 2-6 (wrinkle level evaluation is referred to "skin aging Spectrum (Asian person"));

the basal value of the periocular skin elasticity Q1 is 0.35-0.55;

periocular skin firmness F4 base >5

Sample information:

test group: eye essence test sample "

Control group: "eye essence base material"

The method of use: after the face is cleaned in the morning and evening every day, a proper amount of samples are taken at random from one side of the eye periphery, are uniformly smeared on the skin around the eye, and are gently massaged until being absorbed

Main instrument: skin elasticity tester (MPA 580, germany), skin rapid optical imaging system (VISIO 4D, france)

9.2 test flow

(1) W0 first visit: the technician carries out explanation and signing of the informed consent form to the subject and completes the primary screening;

(2) the face of the test subject is not smeared with any substance on the same day, and after the test subject cleans the face, the test subject sits still in a constant temperature and humidity room for 30min; after 30min, the technician takes measurements on the subject: photographing by a skin rapid optical imaging system and measuring by a skin elasticity tester;

(3) after the W0 measurement is completed by the included subject, a technician distributes the sample to the subject, explains the using method of the sample and tries the sample once on site;

(4) w1, W2, W4 return visit test: the testing process is the same as the steps (2) - (3);

(5) each return visit test point technician needs to confirm the sample recovery, weighing and service conditions;

(6) and finishing the test, sorting the data and giving a report.

9.3 test items

Tightness F4: the F4 values were measured 3 times in parallel on the test area using a skin elasticity tester, and averaged. The smaller this value, the tighter the skin.

Elasticity value Q1: the Q1 value was measured 3 times in parallel on the test area using a skin elasticity tester, and the average value was taken. This value is close to 1, indicating a better skin elasticity.

Wrinkle parameters:

number of wrinkles: the smaller the value, the less wrinkles are indicated

Perimeter of wrinkles: the smaller the value, the less wrinkles are indicated

Area of wrinkles: the smaller the value, the less wrinkles are indicated

Wrinkle roughness Rz: the smaller the value, the less wrinkles are indicated

The results show that compared with the prior samples, the skin compactness F4, the skin elasticity value Q1 and the wrinkles under the eyes (quantity, perimeter, area and roughness) of the test group are obviously improved; compared with the control group, the skin compactness F4, the skin elasticity Q1 and the wrinkles under the eyes (number, perimeter, area and roughness) of the test group are obviously improved. Compared to prior to use, the subjects had an improvement in skin firmness of about 13%, an improvement in skin elasticity value of about 8%, a reduction in the number of wrinkles of about 11%, a reduction in the perimeter of wrinkles of about 19%, a reduction in the area of wrinkles of about 15%, and a reduction in the roughness of wrinkles of about 5% after 4 weeks of use of the ocular essence test specimen. Thus, it was demonstrated that the "eye essence test sample" containing HJ-P2 protein had effects of tightening skin, improving skin elasticity, and improving under-eye wrinkles (fig. 10 to 17).

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims (14)

1. Use of an anti-aging protein or recombinant protein thereof in the preparation of at least one of the following products or compositions:

(1) A product or composition for improving the expression level of a cellular anti-wrinkle factor gene;

(2) A product or composition for improving the expression level of a cytokine gene;

(3) A product or composition that improves skin firmness in a subject;

(4) A product or composition that improves the skin elasticity value of a subject;

(5) A product or composition that improves a wrinkle-related parameter in a subject;

(6) A product or composition for delaying the natural aging of skin;

(7) A product or composition for reducing cellular senescence due to photoaging;

the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding anti-aging protein is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the recombinant protein further comprises a tag protein that aids its expression and/or purification, or an active protein that enhances its activity and/or extends its half-life;

preferably, the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 3.

3. Use according to claim 1 or 2, characterized in that the product or composition comprises at least 1.5mg/L of anti-ageing protein or recombinant protein thereof.

4. Use according to claim 3, wherein the anti-wrinkle factor comprises Collagen i, collagen iii, collagen iv, collagen VII, decorin, VCAN, BGN, MMP-1, MMP-3, smad7, c-fos or c-jun, preferably Collagen i, collagen VII, decorin, VCAN, BGN, MMP-1, smad7 or c-fos; and/or

The inflammatory factor comprises TNF-alpha, IL-1 beta, IL-6, IL-1 alpha or IL-8, preferably TNF-alpha and/or IL-1 beta; and/or

The wrinkles include fish tail lines, head lines, intereyebrow lines, under eye lines, stature lines, mouth Zhou Wen, chinese character lines or neck lines, preferably under eye lines; and/or

The wrinkle-related parameters include the number of wrinkles, the circumference of wrinkles, the area of wrinkles, the roughness of wrinkles, the average depth of wrinkles or the area ratio of wrinkles, preferably the number of wrinkles, the circumference of wrinkles, the area of wrinkles and the roughness of wrinkles.

5. The application of the anti-aging protein or the recombinant protein thereof in preparing cosmetics is characterized in that the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding anti-aging protein is shown as SEQ ID NO. 2.

6. The use according to claim 5, wherein the recombinant protein further comprises a tag protein that aids its expression and/or purification, or an active protein that enhances its activity and/or extends its half-life;

Preferably, the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 3.

7. Use according to claim 5 or 6, characterized in that the cosmetic is used for:

1) Improving skin firmness of a subject; and/or

2) Improving the skin elasticity value of the subject; and/or

3) Improving a wrinkle-related parameter in a subject;

the subject is a human.

8. The use according to claim 7, wherein the wrinkle-related parameters comprise the number of wrinkles, the perimeter of wrinkles, the area of wrinkles, the roughness of wrinkles, the average depth of wrinkles or the area of wrinkles, preferably the number of wrinkles, the perimeter of wrinkles, the area of wrinkles and the roughness of wrinkles.

9. A cosmetic is characterized by comprising an anti-aging protein or recombinant protein thereof, wherein the amino acid sequence of the anti-aging protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding anti-aging protein is shown as SEQ ID NO. 2.

10. The cosmetic according to claim 9, characterized in that said recombinant protein further comprises a tag protein that aids its expression and/or purification, or an active protein that enhances its activity and/or prolongs its half-life;

preferably, the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 3.

11. Cosmetic according to claim 9 or 10, characterized in that it comprises at least 1.5mg/L of anti-ageing protein or recombinant protein thereof;

preferably, the cosmetic comprises 1.5-30mg/L of the anti-aging protein or recombinant protein thereof.

12. The cosmetic product according to claim 11, wherein the cosmetic product further comprises a cosmetically acceptable carrier or excipient selected from at least one of surfactants, oils, emulsifiers, high fat ingredients, consistence agents, thickeners, polymers, silicon containing compounds, fats, waxes, stabilizers, bioactive ingredients, deodorants, film formers, bulking agents, antioxidants, inorganic coloring pigments, water co-solvents, preservatives, solubilizers, rightward concentrates and colorants.

13. The cosmetic according to claim 12, wherein the cosmetic is in a dosage form selected from at least one of a body wash, a shampoo, an emulsion, a microemulsion, a nanoemulsion, a mask, an essence, a lotion, an ointment, a foam, a patch, a cosmetic powder, a cosmetic cotton, an eye essence, an eye mask, an eye shadow, a sun cream, an eye gel, and an eye cream.

14. A method for preparing a cosmetic according to any one of claims 9 to 13, characterized in that it comprises the steps of:

Mixing the cosmetic base material with antiaging protein or its recombinant protein, and stirring to obtain cosmetic.