CN1561960A - Method for preparing biological beauty skin-care product containing keratin cell growth factor-2 and recombined human keratin cell growth factor-2 - Google Patents
Method for preparing biological beauty skin-care product containing keratin cell growth factor-2 and recombined human keratin cell growth factor-2 Download PDFInfo
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- CN1561960A CN1561960A CN 200410030757 CN200410030757A CN1561960A CN 1561960 A CN1561960 A CN 1561960A CN 200410030757 CN200410030757 CN 200410030757 CN 200410030757 A CN200410030757 A CN 200410030757A CN 1561960 A CN1561960 A CN 1561960A
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Abstract
A biologic cosmetic for beautifying and protecting skin and treating acne, pigmentation, wound on face, etc contains the freeze-dried KGF-2 powder consisting of KGF-2 and stabilizer, and KGF-2 dedicated solvent.
Description
Technical field
The invention relates to a kind of biological beauty skin protection cosmetics, more particularly about a kind of biological beauty skin care item of body keratinized cell growth factor-2 and preparation method of recombinant human keratinized cell growth factor-2 of containing
Background technology
Body keratinized cell growth factor-2 (KGF-2) is a up-to-date member who is cloned in fibroblast growth factor (FGF) family, it is first genomic drug that utilizes the exploitation of Human Genome Project result of study, be in the body naturally existence a kind of have active soluble protein, 208 aminoacid of encoding, mainly by subcutaneous tissue emiocytosis, and the specific special receptor of surface epithelial cell that is attached to is through complicated signal transduction process, start the gene expression that participates in merisis in the epithelial cell, thereby stimulate the metabolism of epithelial tissue.
The foreign scholar did the further investigation of comparison system aspect biological function, comprising:
1, promotes epithelial hypertrophy and growth.Keratinocyte growth factor stimulation horn cell synthetic energy force rate TGF and EGF are strong 2 to 10 times; Also can promote alveolar epithelium, ductus pancreaticus, gastrointestinal tract epithelium and galactophore epithelial cell hypertrophy and seminal vesicle to grow.
2, promote wound healing, the mouse skin cutting experiment shows that keratinocyte growth factor can promote wound healing effectively fast; The intravenous injection keratinocyte growth factor can obviously reduce the ulcer level of small intestinal in the injury tolerance of small intestinal in the acute experiment and the chronic experiment.
3, stimulate the secretion of fetus pulmonary fluid.
4, the tangible Antiradiation injury effect of tool;
5, participate in the adjusting of follicular development and endometrium implantation.
Exactly because these of characteristics are arranged biology, and keratinocyte growth factor is having huge using value aspect clinical medicine and the beauty treatment.
KGF-2 is a kind of basic protein, and it belongs to fibroblast growth family (FGF), has another name called FGF-10.Used aFGF in this cell growth factor and the present beauty treatment fields, fibroblast factor differences such as bFGF, it only acts on epithelial cell, and does not act on fibroblast and endotheliocyte.And compare with FGF-7 with FGF-10, the effect of this cell growth factor is more single-minded.Prove that through correlation test it is safe that this cell growth factor is used for skin, nontoxic, can not produce side effect such as sudden change.
The mechanism of action: KGF-2 through signal transduction path, promotes epithelial growth, differentiation and migration with after the specific receptor of cell surface combines; Simultaneously also start epithelial cell hypodermic synergistic signal feed back, promote metabolism balance of each tissue of skin, improve function of each tissue of skin that comprises connective tissue, the elasticity of increase skin, the vitality that skin is shone make new advances.
Summary of the invention
The biological function characteristics that the present invention is directed to KGF-2 propose KGF-2 is applied in the beauty and skin care cosmetics, provide a kind of KGF-2 skin-care agent that is used for beauty and skin care to comprise that KGF-2 lyophilized powder and special solvent mix use according to its biological activity characteristics, avoid the long-term mixed influence KGF-2 activity of cosmetic base, very bring into play its nutrition and nursing role effectively skin.
The present invention and a kind of preparation method of new recombinant human keratinized cell growth factor-2 is provided.
These and other purpose of the present invention will and be described by following detailed description and further launch and set forth.
In the present invention, a kind of biological beauty skin care item that comprise body keratinized cell growth factor-2, form by following product::
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 1000-10000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 100-500 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler.
Further, the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, form by following product:
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 2000-6000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 200-500 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler.
Be preferably, the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, form by following product:
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 3000-5000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 200-400 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler/cryoprotective agent.
In the biological beauty skin care item of body keratinized cell growth factor-2 of the present invention; at the buffer agent described in the KGF-2 stabilizing agent can be one of phosphate, equisetic acid, citric acid, 1,3-propanedicarboxylic acid, malic acid and succinic acid; osmotic pressure regulator can be a sodium chloride; filler/cryoprotective agent can be sucrose, glycine, mannitol, trehalose or the acceptable filler of other pharmacy; one can be used separately, also its mixture can be used.
Further, in the biological beauty skin care item of body keratinized cell growth factor-2 of the present invention, in the KGF-2 stabilizing agent, contain:
Concentration is about the buffer agent of 5-20mM,
Concentration is about the sodium chloride of 0.01-12.5mM,
Concentration is about one or more in sucrose, glycine, mannitol or the trehalose of 2-15%W/V, and water.
In the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, when as the KGF-2 skin-care agent, its solvent prescription is:
Hyaluronic acid 0.05-0.2% carbomer 0.05-0.2%
Allantoin 0.1-0.3% DC-193 0.4-0.7%
Collagen protein 0.5-2% polyquaternary ammonium salt 0.4-0.6%
The two imidazolidinyl urea 0.3-0.5% of EDTA 0.1-0.3%
Triethanolamine 1-3% vitamin E 0.15-0.35%
Surplus is a water.
In the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, when using in to skin repair, its solvent prescription is:
The two imidazolidinyl urea 0.05-0.2% of hyaluronic acid 0.015-0.04%
Azone 0.15-0.4% multivitamin 0.15-0.3%
Surplus is a water.
In the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, when using in to skin repair, its solvent prescription is:
The two imidazolidinyl urea 0.05-0.2% of hyaluronic acid 0.015-0.04%
Azone 0.15-0.4% multivitamin 0.15-0.3%
Benzophenone-4 3-7% surplus is a water.
In the biological beauty skin care item that comprise body keratinized cell growth factor-2 of the present invention, wherein the preparation method of KGF-2 lyophilized powder is: with 0.2 gram rhKGF-2 and 60 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour.
What can select is that recombinant human keratinized cell growth factor-2 of the present invention can prepare according to the following steps:
A, to select people's lung cDNA library for use be that template is carried out the pcr amplification target gene fragment;
B, each 50pmol of the positive and negative strand primer of adding, template DNA, Tap enzyme 1 μ l, contain magnesium ion Buffer3 μ l, each 2.5mmol/ml of dNTP, cumulative volume 30 μ l, cover paraffin oil, 94 ℃ of preheating 5min at first, 94 ℃ of 45s of PCR loop parameter, 59 ℃ of 45s, 72 ℃ of 45s totally 30 circulations, 72 ℃ are extended 10min then, and get 6 μ lPCR products and carry out electrophoretic examinations with 1% agarose, wherein:
Positive strand primer is 5 ｀ GGGAATTCCATATGCTTGGTCAGGACATGGTGTCACCA3 ｀
The minus strand primer is 5 ｀ TGCGCAGGATCCCTATGAGTGTACCACCATTGGAAGA3 ｀
C, by the cDNA of above-mentioned round pcr synthetic KGF-2 from people's lung cDNA library, use the DNA recombinant technique, purpose fragment sub-clone on expression vector pET21 plasmid, is used CaCl
2Change e. coli bl21 (DE3) bacterial strain over to,, obtain high expression engineering KGF-2 (BL21/pET) transforming a large amount of monoclonal bacterium colony in back by enzyme action evaluation, sequencing analysis and expression experiment;
D, centrifugal collection thalline, even oar breaking cellular wall is collected supernatant, obtains purity greater than 95% KGF-2 through SP-HP chromatography and Superdex-75 gel chromatography, has the biological activity identical with natural KGF-2.
In the present invention, can use body keratinized cell growth factor-2 (KGF-2) and stabilizing agent and the solvent prepared by method of the present invention, also can use commercially available other body keratinized cell growth factor-2s (KGF-2) and stabilizing agent and solvent, all can use in the present invention with detection method as those disclosed body keratinized cell growth factor-2 (KGF-2) and preparation method thereof in the Chinese patent.Be preferably the product that uses U.S. Human Genome Sciences to produce, these products have all been applied for patent in China, as: body keratinized cell growth factor-2 (KGF-2) and the stabilizing agent and the solvent of CN-95197779.2, CN-00809802.6, CN-00812341.1 and the research of Shanghai neoplasm medical research Co., Ltd, the product of above-mentioned patent is listed in the present invention as a part and is used.
The biological beauty skin care item that contain body keratinized cell growth factor-2 (KGF-2) of the present invention are mainly used in: the quick reparation of damaging skin (as change skin, sunburn, sensitivity, stigma, remove nevus, stricture of vagina thorn etc.); The daily maintenance of problem skins such as skin is dark and gloomy, yellowing, tarnish, lax many wrinkles, skin matter are poor, skin sore.
Therefore, the biological beauty skin care item that contain body keratinized cell growth factor-2 (KGF-2) of the present invention have following purposes in beauty treatment fields:
1, promotes histiocyte regeneration, strengthen the cellular metabolism vigor, defying age; Promote aging skin to upgrade, strengthen wrinkle resistance.Keratinocyte growth factor promotes epithelial growth, differentiation and migration, make newborn epidermis cell replace old and feeble horn cell, keep skin thickness, prevent moisture loss, reach and make the delicate and rich glossy purpose of skin, the horn cell factor can prevent that connective tissue from replacing fatty tissue in a large number simultaneously, keeps both and is in proper proportion, avoid corium excessively to thicken, prevent the skin crumple.In addition, constantly being upgraded of aged epidermis cell can be hindered various microorganisms and pathogen to the invasion and attack of skin, prevents dermopathic generation.
2, remove cutaneous pigmentation, mottle, cicatrix and acne.Because it can epithelial differentiation and migration, make the metabolism of epidermis cell and upgrade and quicken, avoid pigment formation of deposits mottle on skin.
3, promote burn, various surgical wound and surgical wound and surgery ulcer healing.Be difficult for staying cicatrix for cosmetic surgery, reparation wound.
Problem skins such as 4,, yellowing dark and gloomy to skin, tarnish, lax many wrinkles, skin sore carry out daily reparation, especially at the quick reparation of damaging skin (as changing skin, sunburn, sensitivity, stigma, stricture of vagina thorn etc.).
All raw material of Shi Yonging and equipment and instrument and instrument etc. all are conventional uses in the present invention, can buy from market.
In the present invention, refer in particular to as non-, all amounts, percentage ratio etc. are unit of weight.
Below further specify the present invention by specific embodiment, but embodiment only be used for the explanation, can not limit the scope of the invention.
The specific embodiment
Example 1: the preparation of recombinant human horny cell growth factor-2 2
We select for use people's lung cDNA library (Clontech) to carry out the pcr amplification target gene fragment for template.Add each 50pmol of positive and negative strand primer, template DNA, Tap enzyme 1 μ l, contain magnesium ion Buffer 3 μ l, each 2.5mmol/ml of dNTP, cumulative volume 30 μ l cover paraffin oil, 94 ℃ of preheating 5min at first, 94 ℃ of 45s of PCR loop parameter, 59 ℃ of 45s, 72 ℃ of 45s totally 30 circulations, 72 ℃ are extended 10min then, get 6 μ lPCR products and carry out electrophoretic examinations with 1% agarose.
Positive strand primer is 5 ｀ GGGAATTCCATATGCTTGGTCAGGACATGGTGTCACCA3 ｀
The minus strand primer is 5 ｀ TGCGCAGGATCCCTATGAGTGTACCACCATTGGAAGA3 ｀
By above-mentioned round pcr from people's lung cDNA library the cDNA from synthetic KGF-2, use the DNA recombinant technique, with purpose fragment sub-clone on expression vector pET21 plasmid, change e. coli bl21 (DE3) bacterial strain over to CaCl2, to transforming a large amount of monoclonal bacterium colony in back by enzyme action evaluation, sequencing analysis and expression experiment, obtain high expression engineering KGF-2 (BL21/pET), and it is stable to go down to posterity, and this project bacterium induces back destination protein expression to account for expressing more than 20% and with soluble form of total protein expression.Centrifugal collection thalline, even oar breaking cellular wall is collected supernatant, obtains purity greater than 95% KGF-2 through SP-HP chromatography and Superdex-75 gel chromatography, has the biological activity identical with natural KGF-2.
The preparation of example 2:KGF-2 lyophilized powder
With 0.2 gram rhKGF-2 and 60 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour must the KGF-2 lyophilized powder.
Wherein: the prescription of stabilizing agent is:
Concentration is about the sodium citrate of 8mM,
Concentration is about the sodium chloride of 0.1mM,
Concentration is about sucrose (7%), mannitol (5%) and trehalose (8%) and the water of 5%W/V.
Its pH value scope is: 5.5-8.0 is preferably 6.3.
The preparation of example 3:KGF-2 skin-care agent
Special solvent prescription: (%)
Hyaluronic acid 0.1
Carbomer 0.1
Allantoin 0.2
DC-193 0.6
Collagen protein 1
Polyquaternary ammonium salt 0.5
EDTA 0.2
Two imidazolidinyl urea 0.4
Triethanolamine 2
Vitamin E 0.25
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 5-10ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment 2, wiping on the face after clean sooner or later during use.
The example 4:KGF-2 after cosmetic surgery to the application in the skin repair
Special solvent prescription: (%)
Hyaluronic acid 0.025
Two imidazolidinyl urea 0.1
Azone 0.25
Multivitamin 0.25
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 10-15ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, after cosmetic surgery, be sprayed directly on on the face during use.
The application of example 5:KGF-2 in antitan agent and sunburn reparation
Special solvent prescription: (%)
Hyaluronic acid 0.025
Two imidazolidinyl urea 0.1
Azone 0.25
Multivitamin 0.25
Benzophenone-4 5
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 15-20ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, being sprayed directly on to needs sun-proof position during use.
The preparation of example 6:KGF-2 lyophilized powder
With 0.2 gram rhKGF-2 and 30 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour must the KGF-2 lyophilized powder.
Wherein: the prescription of stabilizing agent is:
Concentration is about the sodium citrate of 10mM,
Concentration is about the sodium chloride of 5mM,
Concentration is about sucrose (0.5%), glycine (2%) and the water of 8%W/V.
Its pH value scope is: 6.4.
The preparation of example 7:KGF-2 skin-care agent
Special solvent prescription: (%)
Hyaluronic acid 0.05
Carbomer 0.05
Allantoin 0.1
DC-193 0.4
Collagen protein 0.5
Polyquaternary ammonium salt 0.4
EDTA 0.1
Two imidazolidinyl urea 0.3
Triethanolamine 1
Vitamin E 0.15
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 5-10ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment 2, wiping on the face after clean sooner or later during use.
The example 8:KGF-2 after cosmetic surgery to the application in the skin repair
Special solvent prescription: (%)
Hyaluronic acid 0.015
Two imidazolidinyl urea 0.05
Azone 0.15
Multivitamin 0.15
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 10-15ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, after cosmetic surgery, be sprayed directly on on the face during use.
The application of example 9:KGF-2 in antitan agent and sunburn reparation
Special solvent prescription: (%)
Hyaluronic acid 0.015
Two imidazolidinyl urea 0.05
Azone 0.15
Multivitamin 0.15
Benzophenone-4 3
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 15-20ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, being sprayed directly on to needs sun-proof position during use.
The preparation of example 10:KGF-2 lyophilized powder
With 0.2 gram rhKGF-2 and 80 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour must the KGF-2 lyophilized powder.
Wherein: the prescription of stabilizing agent is:
Concentration is about the sodium phosphate of 20mM,
Concentration is about the sodium chloride of 12.5mM,
Concentration is about sucrose (5%), mannitol (5%) and trehalose (5%) and the water of 15%W/V.
Its pH value scope is: 6.2.
The preparation of example 11:KGF-2 skin-care agent
Special solvent prescription: (%)
Hyaluronic acid 0.2
Carbomer 0.2
Allantoin 0.3
DC-193 0.7
Collagen protein 2
Polyquaternary ammonium salt 0.6
EDTA 0.3
Two imidazolidinyl urea 0.5
Triethanolamine 3
Vitamin E 0.35
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 5-10ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment 2, wiping on the face after clean sooner or later during use.
The example 12:KGF-2 after cosmetic surgery to the application in the skin repair
Special solvent prescription: (%)
Hyaluronic acid 0.04
Two imidazolidinyl urea 0.2
Azone 0.4
Multivitamin 0.3
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 10-15ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, after cosmetic surgery, be sprayed directly on on the face during use.
The application of example 13:KGF-2 in antitan agent and sunburn reparation
Special solvent prescription: (%)
Hyaluronic acid 0.04
Two imidazolidinyl urea 0.2
Azone 0.4
Multivitamin 0.4
Benzophenone-4 7
Excess water.
The bottled special solvent of the aseptic subpackaged one-tenth of above-mentioned prescription 15-20ml.With 1 of the lyophilized powder among 1 bottle of solvent dissolving embodiment two, being sprayed directly on to needs sun-proof position during use.
The preparation of example 14:KGF-2 lyophilized powder
With 0.2 gram rhKGF-2 and 90 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour must the KGF-2 lyophilized powder.
Wherein: the prescription of stabilizing agent is:
Concentration is about the sodium citrate of 10mM,
Concentration is about the sodium chloride of 20mM,
The PH of 1mM is 6.2 EDTA disodium.
7% sucrose.
Its pH value scope is: 6.2.
Positive strand primer is 5 ｀ GGGAATTCCATATGCTTGGTCAGGACATGGTGTCACCA3 ｀
The minus strand primer is 5 ｀ TGCGCAGGATCCCTATGAGTGTACCACCATTGGAAGA3 ｀
Claims (10)
1, a kind of biological beauty skin care item that comprise body keratinized cell growth factor-2 is characterized in that being made up of following product:
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 1000-10000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 100-500 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler.
2, the biological beauty skin care item that comprise body keratinized cell growth factor-2 according to claim 1 is characterized in that being made up of following product::
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 2000-6000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 200-500 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler.
3, the biological beauty skin care item that comprise body keratinized cell growth factor-2 according to claim 1 is characterized in that being made up of following product::
The KGF-2 lyophilized powder: 1 weight portion,
The KGF-2 special solvent: the 3000-5000 weight portion,
Wherein: comprise 1 weight portion KGF-2 and 200-400 weight portion KGF-2 stabilizing agent in the KGF-2 lyophilized powder, described stabilizing agent comprises buffer agent, osmotic pressure regulator and filler/cryoprotective agent.
4, according to the described biological beauty skin care item that comprise body keratinized cell growth factor-2 of claim 1-3; it is characterized in that at the buffer agent described in the KGF-2 stabilizing agent can be one of phosphate, equisetic acid, citric acid, 1,3-propanedicarboxylic acid, malic acid and succinic acid; osmotic pressure regulator can be a sodium chloride; filler/cryoprotective agent can be sucrose, glycine, mannitol, trehalose or the acceptable filler of other pharmacy; one can be used separately, also its mixture can be used.
5, the biological beauty skin care item that comprise body keratinized cell growth factor-2 according to claim 4 is characterized in that KGF-2 suddenly decides to contain in the agent:
Concentration is about the buffer agent of 5-20mM,
Concentration is about the sodium chloride of 0.01-125mM,
Concentration is about one or more in sucrose, glycine, mannitol or the trehalose of 2-15%W/V, and water.
6,, it is characterized in that when as the KGF-2 skin-care agent, its solvent prescription is according to the described biological beauty skin care item that comprise body keratinized cell growth factor-2 of claim 1-3:
Hyaluronic acid 0.05-0.2% carbomer 0.05-0.2%
Allantoin 0.1-0.3% DC-193 0.4-0.7%
Collagen protein 0.5-2% polyquaternary ammonium salt 0.4-0.6%
The two imidazolidinyl urea 0.3-0.5% of EDTA 0.1-0.3%
Triethanolamine 1-3% vitamin E 0.15-0.35%
Surplus is a water.
7, according to the described biological beauty skin care item that comprise body keratinized cell growth factor-2 of claim 1-3, it is characterized in that when in to skin repair, using its solvent prescription is:
The two imidazolidinyl urea 0.05-0.2% of hyaluronic acid 0.015-0.04%
Azone 0.15-0.4% multivitamin 0.15-0.3%
Surplus is a water.
8, according to the described biological beauty skin care item that comprise body keratinized cell growth factor-2 of claim 1-3, it is characterized in that when in to skin repair, using its solvent prescription is:
The two imidazolidinyl urea 0.05-0.2% of hyaluronic acid 0.015-0.04%
Azone 0.15-0.4% multivitamin 0.15-0.3%
Benzophenone-4 3-7% surplus is a water.
9, according to the described biological beauty skin care item that comprise body keratinized cell growth factor-2 of claim 1-3, it is characterized in that wherein the preparation method of KGF-2 lyophilized powder is: with 0.2 gram rhKGF-2 and 60 gram stabilizing agents, add the 1000ml deionized water, filtration sterilization, aseptic subpackaged 1000 bottles, lyophilization 20-40 hour.
10, a kind of preparation method of recombinant human keratinized cell growth factor-2, its feature comprises the steps:
A, to select people's lung cDNA library for use be that template is carried out the pcr amplification target gene fragment;
B, each 50pmol of the positive and negative strand primer of adding, template DNA, Tap enzyme 1 μ l, contain magnesium ion Buffer3 μ l, each 2.5mmol/ml of dNTP, cumulative volume 30 μ l, cover paraffin oil, 94 ℃ of preheating 5min at first, 94 ℃ of 45s of PCR loop parameter, 59 ℃ of 45s, 72 ℃ of 45s totally 30 circulations, 72 ℃ are extended 10min then, and get 6 μ lPCR products and carry out electrophoretic examinations with 1% agarose, wherein:
Positive strand primer is 5`GGGAATTCCATATGCTTGGTCAGGACATGGTGTCACCA 3`
The minus strand primer is 5`TGCGCAGGATCCCTATGAGTGTACCACCATTGGAAGA 3`
C, by the cDNA of above-mentioned round pcr synthetic KGF-2 from people's lung cDNA library, use the DNA recombinant technique, purpose fragment sub-clone on expression vector pET21 plasmid, is used CaCl
2Change e. coli bl21 (DE3) bacterial strain over to,, obtain high expression engineering KGF-2 (BL21/pET) transforming a large amount of monoclonal bacterium colony in back by enzyme action evaluation, sequencing analysis and expression experiment;
D, centrifugal collection thalline, even oar breaking cellular wall is collected supernatant, obtains purity greater than 95% KGF-2 through SP-HP chromatography and Superdex-75 gel chromatography, has the biological activity identical with natural KGF-2.
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| CNB2004100307571A CN100562306C (en) | 2004-04-02 | 2004-04-02 | Contain the biological beauty skin care item of body keratinized cell growth factor-2 and the preparation method of recombinant human keratinized cell growth factor-2 |
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| CNB2004100307571A CN100562306C (en) | 2004-04-02 | 2004-04-02 | Contain the biological beauty skin care item of body keratinized cell growth factor-2 and the preparation method of recombinant human keratinized cell growth factor-2 |
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| CN105708722A (en) * | 2016-03-15 | 2016-06-29 | 深圳市真兴病毒技术有限公司 | Factor composition and composite freeze-dried powder for promoting skin cell growth and skin care product |
| CN107510633A (en) * | 2017-09-29 | 2017-12-26 | 上海雨淑缇化妆品有限公司 | A kind of ocular cell assigns anti-ageing Essence living and preparation method thereof |
| CN107693443A (en) * | 2017-09-29 | 2018-02-16 | 上海雨淑缇化妆品有限公司 | A kind of energy activated Essence of eye and preparation method thereof |
| CN112043625A (en) * | 2020-09-16 | 2020-12-08 | 广州因码生物科技有限公司 | Spray capable of entering eyes and containing growth factor activity protection technology |
| CN112175064A (en) * | 2020-10-12 | 2021-01-05 | 珠海乐维再生医学科技有限公司 | Preparation method of KGF protein exosome, KGF protein exosome dry powder, application and hair care agent |
| CN112175064B (en) * | 2020-10-12 | 2022-02-11 | 珠海乐维再生医学科技有限公司 | Preparation method of KGF protein exosome, KGF protein exosome dry powder, application and hair care agent |
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| CN100562306C (en) | 2009-11-25 |
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