CN109988243A - III collagen type α of recombination human source, 1 chain and its application - Google Patents
III collagen type α of recombination human source, 1 chain and its application Download PDFInfo
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- CN109988243A CN109988243A CN201910088754.XA CN201910088754A CN109988243A CN 109988243 A CN109988243 A CN 109988243A CN 201910088754 A CN201910088754 A CN 201910088754A CN 109988243 A CN109988243 A CN 109988243A
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- type iii
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- iii collagen
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- 239000007787 solid Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 108010000998 wheylin-2 peptide Proteins 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention discloses a kind of 1 chain of recombination human source type III collagen α and its applications, the nucleotide sequence of 1 chain of recombination human source type III collagen α is encoded as shown in SEQ ID NO:1,1 chain of recombination human source type III collagen α successively includes from aminoterminal: aminoterminal affinity purification label, source of people type III collagen maturation peptide chain and c-terminus affinity purification label.Recombination human source type III collagen according to an embodiment of the present invention, there is specific affinity purification label by all designing at the both ends of source of people type III collagen maturation peptide chain, be conducive to the purifying of full-length proteins, it can also be used for the identification of collagen, and the recombination human source type III collagen according to an embodiment of the present invention is using the mature peptide chain of type III collagen, the propetide at both ends is eliminated, expression quantity is much higher than the gene comprising propetide.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly it relates to a kind of recombination human source type III collagen egg
White 1 chain of α and its application.
Background technique
Collagen is the main protein in all connective tissues including skin, bone, tendon and cartilage.For example,
It forms big fibre bundle in skin, and each fibre bundle includes many single collagenous fibrils again, so that skin texture is strong
It is strong and flexible.Content is very rich in human body for the collagen of three mature spirals, non-to a series of industry and medical application
It is often valuable.Collagen has been widely used in clinical application field, is including soft tissue expander, wound and burn
Reparation, orthopaedics and angiocarpy etc. be proved to safe and effective.
Human type III collagen's albumen (hCOL3A1) belongs to the collagen to form fiber, is distributed widely in skin, internal organ
In the extensible connective tissue such as organ or vascular system.It is in human body wound healing, and collagenous fibres are formed and angiocarpy is normal
Key effect is played in development.
There are mainly two types of the approach of kind for the acquisition of collagen, conventional method and heterologous protein including deriving from animal tissue
Expression.Conventional method mainly handles the tissue of animal origin by acid, alkali or enzymatic isolation method, and what these methods obtained is length
The mixing collagen peptide section that degree does not wait, makes troubles to purifying;Since the rejection that heterologous collagen is clinically shown is anti-
It answers, limits it as the application in terms of biomedical material and pharmaceutical carrier.The host master that heterologous protein expression uses at present
It to include mammalian cell, animal body, plant and microorganism, microbial expression has yield height, production compared to other technologies
The advantages that period is short, culture is simple, at low cost, easy acquisition high density fermentation.Currently used for recombinant expression human collagen or bright
The host strain of glue has Pichia pastoris, saccharomyces cerevisiae, Hansenula yeast, Escherichia coli and brevibacterium.Pichia pastoris fermentation expression yield
High, culture medium is simply, expression is protein stabilized and is easy to purify, and modification such as glycosylates ability after albumen has.Using finishing red ferment
Female heterogenous expression collagen has greater advantage.
In the prior art for industrial mostly gelatin, have no that collagen maturation peptide chain is mass produced.David
R. Olsen's etc. research shows that without when peptide gene, expression quantity is respectively the 18 of procollagen before both ends in the gene of type i collagen
Times, lack before N 4.6 times of peptide gene and lack peptide gene before C 3 times.And the recombinant expression source of people collagen for production, lead to
Often does not add or only add affinity tag at one end, and the mature peptide chain given expression to is easier to degrade, through traditional non-specific purifying
The purity of protein that method or the purifying of single affinity tag obtain is inadequate, it is difficult to remove Partial digestion product, be unable to satisfy to purity
Demanding application.Obtaining collagen using microorganism in the prior art, there is also following shortcomings: expressing the DNA of collagen
Sequence is the natural coding sequence (general to be obtained by reverse transcription method) in human genome, but microorganism and people are in gene
Transcription, have in translation process different on the Preference of certain difference, especially codon, use people's collagen
Natural DNA sequences encoding is expressed in microorganism, can be because of accurate translation efficiency caused by structure of codon preference, mRNA etc.
Lowly.
Summary of the invention
The present invention is directed at least solve one of above-mentioned technical problem.
For this purpose, the present invention proposes a kind of 1 chain of recombination human source type III collagen α, recombination human source type III collagen α
The purifying of 1 chain and identification are convenient.
The present invention also proposes a kind of application of 1 chain of recombination human source type III collagen α.
1 chain of recombination human source type III collagen α of embodiment according to a first aspect of the present invention, encodes the recombined human
The nucleotide sequence of 1 chain of source type III collagen α is as shown in SEQ ID NO:1, the recombination human source type III collagen α 1
Chain successively includes from aminoterminal: aminoterminal affinity purification label, source of people type III collagen maturation peptide chain and c-terminus are affine pure
Change label.
1 chain of recombination human source type III collagen α according to the above embodiment of the present invention can also have following add
Technical characteristic
According to one embodiment of present invention, the aminoterminal affinity purification label is 6His label, the c-terminus parent
It is Strep label with purification tag.
According to one embodiment of present invention, the source of people type III collagen maturation peptide chain include human III type collagen N-terminal peptide,
People III Collagen Type VI is at helical region, human III type collagen C-terminal peptide, and the nucleotide sequence of the source of people type III collagen maturation peptide chain is such as
Shown in SEQ ID NO:2.
According to one embodiment of present invention, the nucleotide sequence of the source of people type III collagen maturation peptide chain is by natural gum
Its codon is adjusted to obtain by protogene sequence according to the frequency of use of host's codon.
According to one embodiment of present invention, the host is Pichia pastoris, described to be adjusted to the natural gum former base
Because the rare codon optimization in sequence in Pichia pastoris is its preferred codons.
According to one embodiment of present invention, the amplification of the nucleotide sequence of the source of people type III collagen maturation peptide chain is drawn
Object F (SEQ ID NO:3) and R (SEQ ID NO:4) is as follows:
F:CGGAATTCCATCATCATCATCATCATCAATACGACTCTTATGACGTGA, restriction enzyme site are EcoR I;
R:CGGAATTCTTACTTCTCGAATTGTGGGTGAGACCATCCATAATATGGTGCGAAT CCAC, restriction enzyme site
EcoR I。
According to one embodiment of present invention, the N-terminal increase of the amplimer can express the nucleotide sequence of 6His,
The end C is expanded after increasing the nucleotide sequence that can express Strep.
The application of 1 chain of recombination human source type III collagen α of embodiment according to a second aspect of the present invention, the recombined human
Source type III collagen is applied to medical dressing, the mass fraction of the medical dressing each component are as follows: according to above-described embodiment
The recombination human source type III collagen 0.1%~0.5%;Moisturizer 1%~10%;Carbomer 0.1%~1%;Three
Ethanol amine 0.1%~10%;Remaining is purified water.
According to one embodiment of present invention, the recombination human source type III collagen is applied to collagen dressing patch,
The collagen dressing patch is made of non-woven fabrics base material and the collagen dressing being located on the non-woven fabrics base material, described
The mass fraction of collagen dressing each component are as follows: the recombination human source type III collagen according to above-described embodiment
0.1%~0.5%;Moisturizer 1%~10%;Thickener 0.1%~2%;Remaining is purified water.
According to one embodiment of present invention, the skin that the recombination human source type III collagen is applied to micro-shaping sticks
Film protective agent, the mass fraction of the skin and mucosa protecting group each component are as follows: the recombination human source according to above-described embodiment
Type III collagen 0.1%~1%;Sodium Hyaluronate 0.1%~1%;Moisturizer 0~40%;Remaining is purified water.
According to one embodiment of present invention, the recombination human source type III collagen is applied to external use skin care, described
The mass fraction of external use skin care each component are as follows: the recombination human source type III collagen 0.01% according to above-described embodiment
~0.1%;Moisturizer 1%~5%;Butanediol 1%~5%;Sodium Hyaluronate 0.01%~0.1%;Carbomer 0.1%~
1%;Glycine betaine 1%~5%;Silk peptide 0.01%~0.1%;Beta glucan 0.1%~1%;Jojoba ester 0.01%~
0.1%;Dipotassium glycyrrhizinate 0.1%~0.5%;Triethanolamine 0.1%~1%;Remaining is purified water.
According to one embodiment of present invention, the recombination human source type III collagen is applied to importing property skin care item, institute
State the mass fraction of importing property skin care item each component are as follows: the recombination human source type III collagen according to above-described embodiment
0.1%~0.5%;Moisturizer 1%~10%;Sodium Hyaluronate 0.1%~0.5%;Micromolecule hyaluronic acid sodium 0.01%~
0.05%;Sodium chloride 0.8%~1%;Argireline 0.0005%~0.002%;Remaining is purified water.
1 chain of recombination human source type III collagen α according to an embodiment of the present invention at least has following technical effect that
(1) at the both ends of collagen, design has 1 chain of recombination human source type III collagen α according to an embodiment of the present invention
Different specific affinity purification labels, is conducive to the purifying of full-length proteins, it can also be used to the identification of collagen;
(2) 1 chain of recombination human source type III collagen α according to an embodiment of the present invention is using type III collagen
Mature peptide chain, eliminates the propetide at both ends, and expression quantity is much higher than the gene comprising propetide;
(3) 1 chain of recombination human source type III collagen α according to an embodiment of the present invention is according to Pichia pastoris codon preference
Property, codon optimization has been carried out to type III glue protogene, has eliminated rare codon and hairpin structure in Pichia pastoris, has been led to
Synonymous conversion is crossed, the secondary structure of mRNA is optimized, codon is avoided and utilizes translation efficiency caused by limitation low, make it more
Suitable for being expressed in Pichia pastoris.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 be according to the recombination human source type III collagen of the embodiment of the present invention during the preparation process used by carrier
The constructing technology route map of pPIC9K-His-COL3-Strep;
Fig. 2 is that recombination Pichia yeast engineering expresses recombination human source type III collagen according to an embodiment of the present invention
SDS-PAGE analysis chart;
Fig. 3 is to detect figure according to the Western Blot of the recombination human source type III collagen of the embodiment of the present invention;Fig. 3 a
Scheme for the WB of anti-Srtep-tag II antibody, Fig. 3 b is that the WB of anti-His antibody schemes;
Fig. 4 a and Fig. 4 b are the mass spectrometry results figure of band at 116KDa in Fig. 2.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
1 chain of recombination human source type III collagen α according to an embodiment of the present invention is described first below.
Encode the nucleotide sequence such as SEQ of 1 chain of recombination human source type III collagen α according to an embodiment of the present invention
Shown in ID NO:1,1 chain of recombination human source type III collagen α successively includes from aminoterminal: aminoterminal affinity purification mark
Label, source of people type III collagen maturation peptide chain and c-terminus affinity purification label.
In other words, the recombination human source type III collagen according to an embodiment of the present invention is one kind in source of people type III glue
On the basis of former maturation peptide chain, the collagen of affinity purification label is respectively provided in amino acid and c-terminus.Pass through as a result,
All designing at the both ends of source of people type III collagen maturation peptide chain has specific affinity purification label, is conducive to the pure of full-length proteins
Change, it can also be used to the identification of collagen, and the recombination human source type III collagen according to an embodiment of the present invention is adopted
It is the mature peptide chain of type III collagen, eliminates the propetide at both ends, expression quantity is much higher than the gene comprising propetide.
Optionally, according to one embodiment of present invention, the aminoterminal affinity purification label is 6His label, the carboxylic
Cardinal extremity affinity purification label is Strep label.Using the double affinity tags purifying in both ends, can obtain it is single, completely without degradation
Type III collagen maturation peptide chain, be applicable to the field that field of medicaments etc. requires product uniformity.Both sequence labels
Very little does not influence the biological function of collagen;Two kinds of labels can be used as the specificity of the detection of type III collagen, identification simultaneously
Flag sequence.
In certain specific embodiments of the invention, the source of people type III collagen maturation peptide chain includes human III type collagen
N-terminal peptide, human III type collagen are at helical region, human III type collagen C-terminal peptide, the nucleotide of the source of people type III collagen maturation peptide chain
Sequence is as shown in SEQ ID NO:2.Preferably, the nucleotide sequence of the source of people type III collagen maturation peptide chain is by natural collagen
Its codon is adjusted to obtain by gene order according to the frequency of use of host's codon.Further, the host is complete
Red yeast, it is described to be adjusted to the rare codon optimization in the natural collagen gene order in Pichia pastoris to be its preference
Codon.
That is, source of people type III collagen maturation peptide chain according to an embodiment of the present invention is mainly by human III type collagen N-terminal
Peptide, human III type collagen are formed at helical region, human III type collagen C-terminal peptide, and nucleotide sequence is as shown in SEQ ID NO:2.It should
Gene is the gene after optimization, and the gene order of the type III collagen maturation peptide chain of optimization is by the password in natural collagen gene
Son is adjusted by host's codon usage frequency, does not change the amino acid sequence of natural collagen;Pass through the method for de novo formation
The sequence is synthesized, expressing host used is Pichia pastoris.
The human III type collagen maturation peptide chain that the present invention expresses has complete protein sequence, comprising having cell adherence
Active peptide fragment and other have the sequence of bioactivity, can make its complete biological function;Carry out Expression product using Pichia pastoris
Collagen, endotoxin-free, and a variety of posttranslational modifications can be carried out to collagen, have more with the collagen of its production
Good biological function.
The recombination engineering and protein preparation method of expression said gene is detailed below.
Step 1: building recombinant expression carrier
Firstly, providing a kind of gene order of source of people type III collagen maturation peptide chain optimized.Wherein, of the invention
Optimize its corresponding gene order under the premise of not changing collagen original amino acid sequence, optimization processing is basis
Pichia pastoris preferred codons carry out, by collagen gene sequence in Pichia pastoris rare codon agg, cgt,
Ggg, ggc, gca etc. are optimized for its preferred codons aga, ggt, gct etc., it is made to be more conducive to express in Pichia pastoris.Its base
Because sequence is as shown in SEQ ID NO.2.
Then, said gene sequence is obtained by the side of gene chemical synthesis.Design the nucleosides of source of people type III collagen maturation peptide chain
The amplimer F (SEQ ID NO:3) and R (SEQ ID NO:4) of acid sequence are as follows:
F:CGGAATTCCATCATCATCATCATCATCAATACGACTCTTATGACGTGA, restriction enzyme site are EcoR I;
R:CGGAATTCTTACTTCTCGAATTGTGGGTGAGACCATCCATAATATGGTGCGAAT CCAC, restriction enzyme site
EcoR I。
The N-terminal increase of the amplimer can express the nucleotide sequence of 6His, and C-terminal increase can express Strep's
It is expanded after nucleotide sequence.The type III collagen maturation peptide chain optimized using the gene order of de novo formation as template amplification
Gene, product length 3262bp.By amplified production His-3A1-Strep and carrier pPIC9K respectively through EcoR I single endonuclease digestion,
Plasmid is through alkaline phosphatase treatment, with Solution I connection reagent (being purchased from Dalian TaKaRa company) connection building expression matter
Grain pPIC9K-His-3A1-Strep.
Step 2: building gene recombined Pichia pastoris engineering bacteria
Recombinant expression plasmid pPIC9K-His-3A1-Strep restriction enzyme Sal I is linearized, and electricity is transferred to
In Pichia pastoris competent cell, with histidine defect phenotypic marker and the high copy positive recombinant of G418 resistance marker screening, obtain
To recombinant yeast pichia pastoris engineering bacteria.
Step 3: preparation and reorganization source of people type III collagen
Pichia yeast engineering 16h-~18h is cultivated based on 30 DEG C, 220rpm with BMGY culture, until OD600 reaches 2~6;
1500g~3000g is centrifuged 5min at room temperature, collects thallus, and thallus is resuspended with BMMY culture medium, makes OD600 1~2, in 30
DEG C, 220rpm cultivate 3 days;Every methanol that adds for 24 hours into culture medium is to final concentration of 1%;After fermentation, it is collected by centrifugation
Clearly, Ni-NTA resin adsorption recombination human source type III collagen is used under the conditions of non denatured, penetrates out impurity, elutes recombined human
Source type III collagen collects eluent;Strep-Tactin resin adsorption recombination human source type III collagen, penetrates out
Impurity elutes recombination human source type III collagen, collects eluent, eluent is by ultrafiltration system desalination, concentration, concentrate
It is freeze-dried to obtain recombination human source type III collagen.
The present invention has carried out codon optimization to type III glue protogene according to Pichia pastoris codon preference as a result,
Rare codon and the hairpin structure in Pichia pastoris are eliminated, by synonymous conversion, optimizes the secondary structure of mRNA, avoids
Codon is low using translation efficiency caused by limiting, and makes it be more suitable for expressing in Pichia pastoris.
To sum up, to provide a kind of both ends equal for the recombination human source type III collagen according to an embodiment of the present invention
Type III collagen maturation peptide chain with affinity purification label, solves purification difficult caused by catabolite in the prior art and asks
Topic facilitates purifying to obtain overall length, source of people type III collagen maturation peptide fragment without protein degradation.And according to amino acid sequence
The translation effect of collagen in recombination yeast can be improved in the codon usage frequency optimization gene sequence of column and Pichia pastoris
Rate.
The application of the recombination human source type III collagen according to an embodiment of the present invention is detailed below.
The recombination human source type III collagen according to an embodiment of the present invention can be applied to multiple fields, such as cure
With auxiliary material, the collagen dressing patch for micro-shaping, skin and mucosa protective agent, cosmetics and importing property beauty product etc..
Specifically, when the recombination human source type III collagen is applied to medical dressing, the medical dressing each component
Mass fraction are as follows: recombination human source type III collagen 0.1%~0.5%;Moisturizer 1%~10%;Carbomer 0.1%~
1%;Triethanolamine 0-1%~10%;Remaining is purified water.Wherein, moisturizer can be glycerol.
When the recombination human source type III collagen is applied to collagen dressing patch, the collagen dressing patch
It is made of non-woven fabrics base material and the collagen dressing being located on the non-woven fabrics base material, the collagen dressing each component
Mass fraction are as follows: recombination human source type III collagen 0.1%~0.5%;Moisturizer 1%~10%;Thickener 0.1%~
2%;Remaining is purified water.Wherein, moisturizer can be glycerol, and thickener can be xanthan gum.
When the recombination human source type III collagen is applied to the skin and mucosa protective agent of micro-shaping, the skin is glutinous
The mass fraction of film protecting group each component are as follows: recombination human source type III collagen 0.1%~1%;Sodium Hyaluronate 0.1%~
1%;Moisturizer 0~40%;Remaining is purified water.Wherein, moisturizer can be glycerol.After obtained preparation solution sterilizing, nothing
Bacterium is filling.
When the recombination human source type III collagen is applied to prepare cosmetics, such as when external use skin care, the external application
The mass fraction of skin care item each component are as follows: recombination human source type III collagen 0.01%~0.1%;Moisturizer 1%~5%;
Butanediol 1%~5%;Sodium Hyaluronate 0.01%~0.1%;Carbomer 0.1%~1%;Glycine betaine 1%~5%;Silk peptide
0.01%~0.1%;β-glucan 0.1%~1%;Jojoba ester 0.01%~0.1%;Dipotassium glycyrrhizinate 0.1%~
0.5%;Triethanolamine 0.1%~1%;Remaining is purified water.Wherein, moisturizer can be glycerol.
When the recombination human source type III collagen be applied to import property beauty product, such as import property skin care item when, institute
State the mass fraction of importing property skin care item each component are as follows: recombination human source type III collagen 0.1%~0.5%;Moisturizer 1%
~10%;Sodium Hyaluronate 0.1%~0.5%;Micromolecule hyaluronic acid sodium 0.01%~0.05%;Sodium chloride 0.8%~
1%;Argireline 0.0005%~0.002%;Remaining is purified water.Wherein, moisturizer can be glycerol.
The recombination human source type III collagen according to an embodiment of the present invention can be applied to multiple fields as a result, by
There is complete protein sequence in the human III type collagen maturation peptide chain that the present invention expresses, comprising with cell adhesion activity
Peptide fragment and other have the sequence of bioactivity, can make its complete biological function, therefore, use the recombination human source type III glue
The Related product of former albumen also has corresponding biological function.
Describe combined with specific embodiments below the recombination human source type III collagen of the invention preparation process and
Using.
One, the Pichia pastoris Pichia pastoris SMD1168 bacterial strain selected by the present invention, Expression vector pPIC9K are equal
Purchased from Invitrogen company, the U.S..
Two, culture medium prescription is as follows:
1) YPD complete medium:
Yeast extract 10g/L, peptone 20g/L, glucose 20g/L (solid medium contains 2% agar);
2) MD culture medium (Selective agar medium):
With 100mL, 121 DEG C of agarose 2g (20g/L) sterilizing 20 minutes is added into 80mL water, is down to 60 DEG C to temperature
10 × YNB 10mL (13.4g/L) is added on the super-clean bench later, 10 × glucose 10mL (20g/L), 500 × biotin
0.2mL(4×10-4g/L);
3) BMGY culture medium (yeast growth medium):
It is completely dissolved 10g yeast extract, 20g peptone, 3g K2HPO4, 11.8g KH2PO4, constant volume to 890mL.121
DEG C steam high-voltage sterilizing 20min is cooled to after 60 DEG C and 10 × YNB 100mL (13.4g/L) is added on the super-clean bench, and 500
× biotin 1mL (4 × 10-4G/L), glycerol 10mL;
4) BMMY culture medium (yeast induced medium):
It is completely dissolved 10g yeast extract, 20g peptone, 3g K2HPO4, 11.8g KH2PO4, constant volume to 895mL.121
DEG C steam high-voltage sterilizing 20min is cooled to after 60 DEG C and 10 × YNB 100mL (13.4g/L) is added on the super-clean bench, and 500
× biotin 1mL (4 × 10-4G/L), methanol 5mL.
Embodiment 1
The overall length of genetic recombination source of people type III collagen is 1082 amino acid, and aminoterminal is 6His label, carboxyl
End is Strep label, and the amino acid sequence of the genetic recombination source of people type III collagen is as shown in SEQ ID NO.1.
The preparation method is as follows:
1, recombinant expression carrier (as shown in Figure 1) is constructed
1.1 synthesis optimizing genes
According to the gene order NM_000090.3 of the Genebank type III collagen logged in, made according to Pichia pastoris codon
The low codon of utilization rate is eliminated come optimization gene codon with frequency, while eliminating EcoR in sequence using synonymous conversion method
The restriction enzyme sites such as I (GAATTC), Not I (GCGGCCGC), eliminate 2 GGTAAG splice sites and 4 GGTGAT montages
Site, the type III glue protogene sequence after optimization are shown in SEQ ID NO.2, and the type III glue protogene sequence after optimization is by Nanjing
Jin Sirui Biotechnology Co., Ltd is artificial synthesized.
1.2 amplification His-3A1-Strep genes
Primer Primer 5.0 is utilized according to design of primers principle according to the gene order of the type III collagen of optimization
Software design PCR amplification primer, and the gene of 6His and Strep label is added on two primers respectively, 5 ' ends add
EcoR I restriction enzyme site.Primer is synthesized by Shanghai biotechnology Services Co., Ltd.
F:CGGAATTCCATCATCATCATCATCATCAATACGACTCTTATGACGTGA (SEQ ID NO.3)
R:CGGAATTCTTACTTCTCGAATTGTGGGTGAGACCATCCATAATATGGTGCGAAT CCAC (SEQ ID
NO.4)
With above-mentioned primer amplification target gene, the high fidelity enzyme that PCR is used is 2 × Master of Q5 Mix of NEB.Wherein,
PCR condition is 98 DEG C of initial denaturations, 2min, a thermal cycle;98 DEG C of thermal denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, 40
A thermal cycle;72 DEG C of extension 2min.
1.3 PCR product single endonuclease digestions
With the PCR product in EcolR I single endonuclease digestion digestion step 1.2, target fragment His-3A1-Strep, reaction are recycled
System is following (restriction endonuclease and buffer used are purchased from Dalian TaKaRa company)
3 μ g of PCR product
10 × H buffer, 5 μ L
EcolR I 10U
Sterile water is to 50 μ L
1.4 plasmid pPIC9K single endonuclease digestions
With Eco1R I single endonuclease digestion digested plasmid pPIC9K, linearized vector, the following (restriction endonuclease used of reaction system are recycled
And buffer is purchased from Dalian TaKaRa company)
5 μ g of plasmid pPIC9K
10 × H buffer, 10 μ L
EcolR I 50U
Sterile water is to 100 μ L
1.5 is pure by step 1.3 and the resulting target fragment of step 1.4 step and carrier segments, PCR product purification kit
Change, which is purchased from Dalian TaKaRa company, and concrete operations are carried out by kit specification.
The 1.6 carrier pPIC9K recycled by step 1.5 step are handled through CIAP, remove 5 '-ends of vector DNA fragment
The phosphate group at end.Reaction system is following, and (kit used is purchased from Dalian TaKaRa company, and concrete operations are said by kit
Bright book carries out)
20 μ L of plasmid pPIC9K carrier segments
10×Alkaline Phosphatase Buffer 5μL
CIAP(30U/μL)1μL
Sterile water is to 50 μ L
1.7 carriers that will be obtained after the target fragment His-3A1-Strep that step 1.5 recycling obtains and step 1.6 processing
PPIC9K is attached reaction with Solution I connection reagent (being purchased from Dalian TaKaRa company), and target fragment is properly inserted
To in containing secretion signal α-factor excretion vector reading frame, reaction system is as follows:
2 μ L of plasmid pPIC9K linearized fragment
His-3A1-Strep 3μL
5 μ L of Solution I connection reagent
Recombinant expression carrier pPIC9K-His-3A1-Strep is obtained, building schematic diagram is as shown in Figure 1.
Connection product conversion is entered into competent E.coli DH5 α, in positive gram of the screening of LB resistant panel containing Amp
It is grand, bacterium colony PCR verifying is carried out using primers F and 3 ' AOX1 of universal primer, the segment for having size to be 3381bp is positive gram
It is grand.After extracting plasmid enzyme restriction identification correctly, recombinant plasmid is subjected to sequencing identification.
2, recombinant yeast pichia pastoris engineering bacteria is constructed
The linearisation of 2.1 Expression vector pPIC9K-His-3A1-Strep
It is carried out being digested overnight in 37 DEG C with restriction enzyme Sal I, reaction system is as follows:
10 μ g of plasmid pPIC9K-His-3A1-Strep
10 × H buffer, 5 μ L
Sal I 50U
Sterile water is to 200 μ L
Then it detects whether to cut completely through with 0.7% agarose gel electrophoresis, after cutting completely through, be purified with PCR product
Kit handles digested liquid, recycles linearization plasmid, makes fixing fabric structure in 10 μ L or so.
2.2 prepare Pichia pastoris SMD1168 competent cell
1) picking yeast SMD1168 single colonie is seeded in the test tube of the YPD fluid nutrient medium containing 5mL, 30 DEG C,
220rpm shaken cultivation is stayed overnight;
2) overnight culture of 50 μ L is taken to be seeded in the 500mL triangular flask containing the fresh YPD fluid nutrient medium of 50mL,
30 DEG C, 220rpm shaken cultivation stay overnight, until OD600 value reaches 1.1~1.3;
3) by culture in 4 DEG C, 1500 × g is centrifuged 5min, and thallus is resuspended with the aseptic double-distilled water that 50mL ice is pre-chilled;
4) it is centrifuged by step 3), thallus is resuspended with the aseptic double-distilled water that 25mL ice is pre-chilled;
5) it is centrifuged by step 3), thallus is resuspended in the sorbitol solution for the 1M being pre-chilled with 20mL ice;
6) it is centrifuged by step 3), thallus is resuspended in the sorbitol solution for the 1M being pre-chilled with 0.3mL ice, and final volume is about
0.5mL;
7) it is a to be distributed into every 80 μ L, is saved backup in -70 DEG C.
The electrotransformation of 2.3 Pichia pastoris
1) the electric revolving cup of a 0.2cm is placed in ice and 10min is pre-chilled;
2) pPIC9K-His- of the linearisation of about 10 μ L will be added in freshly prepared Pichia pastoris competent cell
3A1-Strep plasmid, mixes gently, and goes in the electric revolving cup of 0.2cm ice pre-cooling, continues at and 5min is pre-chilled on ice;
3) it shocks by electricity, voltage 1.5kV;25 μ F of capacitor;200 Ω of resistance;The electric shock time is 5~10mSec;
4) electric shock terminates, and is rapidly added the sorbitol solution of the 1M of lmL ice pre-cooling, gently piping and druming mixes, and goes to 1.5mL
In centrifuge tube, 30 DEG C of incubation 1h;
5) bacteria suspension being coated on MD plate, the every 100 μ μ of L~200 L is coated with one flat plate, it is stored at room temperature 10min, in
30 DEG C of inversions are cultivated 2-5 days, until there is single colonie appearance.
The screening of 2.4 multicopies insertion recon
1) 2mL aseptic double-distilled water is added in the MD planar surface that growth has transformant, it is then light with sterile triangle spreader
The His+ transformant of planar surface gently is scraped, and is transferred in 50mL centrifuge tube;
2) dilution of 20mL aseptic double-distilled water is added, its OD600 value (10D600=5 × 10 is measured after mixing7cells/mL);
3) 10 are taken8A cell is coated on the YPD plate containing 0.5mg/mLG418, is inverted, 30 DEG C of 3~4d of culture;
4) 200 μ L YPD fluid nutrient mediums are added in every hole in sterile 96 orifice plate;
5) it is respectively connected on the YPD plate containing 0.5mg/mL G418 with sterile toothpick into 96 orifice plates of step 4)
The transformant of acquisition mixes, in 30 DEG C of culture 48h;
6) after 48h, one piece of new sterile 96 orifice plate is taken, 190 μ L YPD fluid nutrient mediums are added in every hole.In corresponding aperture
10 μ L, first piece of 96 resulting culture of orifice plate is added, for 24 hours in 30 DEG C of cultures;
7) after for 24 hours, then one piece of new sterile 96 orifice plate is taken, 190 μ L YPD fluid nutrient mediums are added in every hole.In corresponding aperture
Middle addition 10 μ L, second piece of 96 resulting culture of orifice plate, for 24 hours in 30 DEG C of cultures;
8) after for 24 hours, 1 μ L is taken out from 96 orifice plate of third block and is put respectively containing 1.0mg/mL and 4mg/mL G418's
On YPD plate, continue to cultivate 96h~120h in 30 DEG C.If Pichia pastoris transformant can give birth on the plate of the G418 containing high concentration
It is long, illustrate the target gene that the transformant contains multicopy, that is, there are multiple pPIC9K-His-3A1-Strep segments to enter ferment
It is integrated on the chromosome of yeast in parent and by homologous recombination.By the recombination ferment for the high copy that this step is screened
Mother will be more likely to realize the high efficient expression of destination protein.
The PCR of 2.5 recons is identified
Select recon single colonie inoculation YPD fluid nutrient medium, 30 DEG C, 220rpm be incubated overnight, take 1ml bacterium solution extract base
Because of group, pastoris genomic dna extracts kit is purchased from Beijing Suo Laibao Science and Technology Ltd, and concrete operation step refers to reagent
Box specification.
Using genomic DNA as template, 5 ' AOX1,3 ' AOX1 universal primers are that amplimer carries out PCR, and enzyme used is
Ex Taq is purchased from Dalian TaKaRa company.Wherein, PCR condition is 98 DEG C of initial denaturations, 2min, a thermal cycle;98 DEG C of thermal changes
30s, 55 DEG C of annealing 30s, 72 DEG C of extension 4min of property, 25 thermal cycles;72 DEG C of extension 2min.Amplified production is there are two band, and one
2.2kb is the AOX1 gene of SMD1168 genome itself, and another 3968bp is purpose gene.
The inducing expression of 2.6 recombination yeasts
1) respectively picking individual colonies, be placed in the 100mL triangular flask equipped with 10mL BMGY culture medium, in 28-30 DEG C,
It is 2-6 (16-18h) that 220rpm, which is cultivated to OD600,
2) 1500~3000g is centrifuged 5min at room temperature, collects thallus, and thallus is resuspended with BMMY culture medium, makes OD600 1
Left and right.
3) the resulting bacterium solution of step 2) is placed in the shaking flask of 250mL, is sealed, is placed in double gauze or garrha
28-30 DEG C, continued growth 3 days on the shaking table of 220rpm.
4) every to add 100% methanol into culture medium for 24 hours to final concentration of 1.0%.
5) it temporally puts and takes bacteria liquid sample respectively, sampling amount 1ml is placed in 1.5mL EP pipe, and maximum (top) speed is centrifuged 2-
3min collects supernatant.Analyze the expression quantity and the best harvest time of bacterium solution of destination protein.Time point generally takes: 12h, for 24 hours,
36h, 48h, 60h and 72h.
6) sample to be tested is saved backup in -80 DEG C.
The identification of 2.7 recombination human source type III collagens
1) supernatant collected in step 2.6 is subjected to SDS-PAGE detection, as a result as shown in Figure 2.
2) destination protein N-terminal has 6His label, and C-terminal has Srtep-tag II label, respectively to be purchased from Nanjing Jin Siruisheng
The anti-Srtep-tag II antibody (A01736) of object Science and Technology Ltd., the anti-His of Santa Cruz Bioisystech Co., Ltd are anti-
Body (sc-8036) is detected, and as a result (3a is that the WB of anti-Srtep-tagII antibody schemes, and 3b is anti-His antibody as shown in Figure 3
WB figure) band of 120kDa or so can identify that provable its is with the bis- labels of 6His, Srtep-tagII by two kinds of antibody
Complete recombiant protein.
3) band on step 1) SDS-PAGE at 116KDa is cut off, through Nano-LC-ESI-MS/MS Identification of Fusion Protein,
The band is 1 chain of source of people type III collagen α, as a result as shown in figures 4 a and 4b.Fig. 4 a is characterized source of people in peptide fragment and database
The sequence comparison of 1 chain of type III collagen α, the result shows that the band at 116KDa contains there are two types of major protein in Fig. 4 b,
And have in 110 peptide fragments detecting in record 1 38 for 1 chain feature peptide fragment of source of people type III collagen α, people's III type in the band
1 chain relative abundance of collagen α is 94.8%, is confirmed as 1 chain of source of people type III collagen α.
Wherein, it verifies correct recombinant yeast pichia pastoris engineering bacteria and has been preserved in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, deposit number CGMCC NO.17148, preservation date: on January 10th, 2019, address: court, Beijing
The institute 3 of positive area's North Star West Road 1, classification naming: pichia pastoris yeast Pichia pastoris.
3, genetic recombination source of people type III collagen is prepared
1) after fermentation by step 2.6 method, 3000g is centrifuged 20min at 4 DEG C, collects supernatant.
2) purified water of 10 times of volume is added in supernatant, then arrives the 20% of original volume after dialysis is concentrated by ultrafiltration.
3) it takes and 100 μ L1 × Ni-NTA combination buffers is added in 1mL concentrate, 4 DEG C are sufficiently mixed.
4) 20 μ L 50%Ni-NTA HisBands resin suspensions are added softly to mix, in conjunction with 30min.
5) 15000 × g is centrifuged 10 seconds precipitated resins, abandons supernatant.
6) resin being rinsed with 100 μ L 1 × Ni-NTA wash buffers, 15000 × g is centrifuged 10 seconds, supernatant is carefully sucked,
It repeats primary.
7) destination protein is eluted with 200 μ L 1 × Ni-NTA elution buffers, 15000 × g is centrifuged 10 seconds, carefully by supernatant
It is transferred in clean tubule, is repeated twice.
8) supernatant is collected, 4 DEG C of dialysed overnights of Strep combination buffer are used.
9) it takes above-mentioned dialyzate that 100 μ L Strep-Tactin resin suspensions are added softly to mix, in conjunction with 30min.
10) 15000 × g is centrifuged 10 seconds precipitated resins, abandons supernatant.
11) resin is rinsed with 200 μ L 1 × Strep-Tactin wash buffers, 15000 × g is centrifuged 10 seconds, careful to inhale
Supernatant is removed, is repeated primary.
12) destination protein is eluted with 200 μ L 1 × Strep-Tactin elution buffers, 15000 × g is centrifuged 10 seconds, small
Supernatant is transferred in clean tubule by the heart, is repeated twice.
13) by obtained eluent through desalination, concentration, recombination human source is made through vacuum freeze drying in obtained concentrate
III collagen type.
14) it takes concentrate to pour into glass culture dish, is put into freeze overnight in -20 DEG C of refrigerators, be then transferred to and pre- be cooled to -45
DEG C freeze drier in, open vacuum pump, maintain 48h.
15) after being lyophilized, vent valve is carefully opened, until inside and outside air pressure balance.Beaker is taken out, white sponge is obtained
Recombination human source type III collagen.
2 collagen external use skin care preparation example of embodiment
By following quality proportioning, dissolve each raw material with water, and stir the transparent guarantor of a colorless and odorless
Wet facial treatment essence liquid.
Recombination human source type III collagen 0.1% prepared by example 1, the embodiment of the present invention 1;Glycerol 1%;Butanediol 1%;
Sodium Hyaluronate 0.1%;Carbomer 0.5%;Glycine betaine 1%;Silk peptide 0.05%;Beta glucan 0.2%;Jojoba ester
0.1%;Dipotassium glycyrrhizinate 0.3%;Triethanolamine 0.1%;Remaining is purified water.
Recombination human source type III collagen 0.01% prepared by example 2, the embodiment of the present invention 1;Glycerol 5%;Butanediol 5%;
Sodium Hyaluronate 0.01%;Carbomer 0.1%;Glycine betaine 5%;Silk peptide 0.01%;Beta glucan 0.1%;Jojoba ester
0.01%;Dipotassium glycyrrhizinate 0.1%;Triethanolamine 1%;Remaining is purified water.
Recombination human source type III collagen 0.1% prepared by example 3, the embodiment of the present invention 1;Glycerol 3%;Butanediol 3%;
Sodium Hyaluronate 0.05%;Carbomer 1%;Glycine betaine 3%;Silk peptide 0.1%;Beta glucan 1%;Jojoba ester 0.05%;It is sweet
Oxalic acid dipotassium 0.5%;Triethanolamine 0.5%;Remaining is purified water.
Application method: after morning and evening face cleaning, being applied directly to face, gently pats to fully absorbing.
3 collagen gel dressing preparation example of embodiment
Example 1, the collagen gel using purified water as solvent, component further include recombination human source type III collagen,
Gelling agent, moisturizer;The gelling agent is carbomer;The moisturizer is glycerol;The quality proportioning of each component are as follows: recombined human
Source type III collagen 0.1%, glycerol 5%, carbomer 0.4%, triethanolamine 5%, remaining is purified water.
Preparation step is as follows:
1. weighing 50.0g glycerol, 4.0g carbomer is added, stirs 1h;
2. weighing recombination human source type III collagen 1.0g, dissolved with 100mL purified water;
3. by step, 2. resulting material is all transferred to step 1. in resulting material, and supplying purified water to gross mass is
990.0g stirring 1h;
4. the triethanolamine stirring 1h of 10.0g is added, after preparation solution irradiation sterilization obtained, sterile filling is to get collagen
Protein gel casting product.
Example 2, the collagen gel using purified water as solvent, component further include recombination human source type III collagen,
Gelling agent, moisturizer;The gelling agent is carbomer;The moisturizer is glycerol;The quality proportioning of each component are as follows: recombined human
Source type III collagen 0.5%, glycerol 1%, carbomer 0.1%, triethanolamine 0.1%, remaining is purified water.
Preparation step is as follows:
1. weighing 10.0g glycerol, 1.0g carbomer is added, stirs 1h;
2. weighing recombination human source type III collagen 5.0g, dissolved with 100mL purified water;
3. by step, 2. resulting material is all transferred to step 1. in resulting material, and supplying purified water to gross mass is
990.0g stirring 1h;
4. the triethanolamine stirring 1h of 0.2g is added, after preparation solution irradiation sterilization obtained, sterile filling is to get collagen egg
White gel casting product.
Example 3, the collagen gel using purified water as solvent, component further include recombination human source type III collagen,
Gelling agent, moisturizer;The gelling agent is carbomer;The moisturizer is glycerol;The quality proportioning of each component are as follows: recombined human
Source type III collagen 0.1%, glycerol 10%, carbomer 1%, triethanolamine 10%, remaining is purified water.
Preparation step is as follows:
1. weighing 100.0g glycerol, 10.0g carbomer is added, stirs 1h;
2. weighing recombination human source type III collagen 1.0g, dissolved with 100mL purified water;
3. by step, 2. resulting material is all transferred to step 1. in resulting material, and supplying purified water to gross mass is
990.0g stirring 1h;
4. the triethanolamine stirring 1h of 20.0g is added, after preparation solution irradiation sterilization obtained, sterile filling is to get collagen
Protein gel casting product.
4 Collagen dressing preparation example of embodiment
Recombination human source type III collagen prepares the Application Example of Collagen dressing product:
The Collagen dressing is by recombination human source type III collagen, moisturizer, medical thickener and nonwoven fabric base
Material composition;The moisturizer is glycerol;The thickener is xanthan gum.
Example 1, the quality proportioning of each component are as follows: recombination human source type III collagen 0.5%;Glycerol 1%;Xanthan gum 1%;
Remaining is purified water.
Preparation step is as follows:
1. accurately weighing each component, it is dissolved in purified water, is sufficiently stirred, mixes;
2. being filled in the medical aluminium foil bag containing non-woven fabrics base material by 25mL, irradiation sterilization processing is sealed to get collagen egg
White sticking dressing product.
Example 2, the quality proportioning of each component are as follows: recombination human source type III collagen 0.1%;Glycerol 10%;Xanthan gum 2%;
Remaining is purified water.
Preparation step is as follows:
1. accurately weighing each component, it is dissolved in purified water, is sufficiently stirred, mixes;
2. being filled in the medical aluminium foil bag containing non-woven fabrics base material by 25mL, irradiation sterilization processing is sealed to get collagen egg
White sticking dressing product.
Example 3, the quality proportioning of each component are as follows: recombination human source type III collagen 0.1%;Glycerol 5%;Xanthan gum 1%;
Remaining is purified water.
Preparation step is as follows:
1. accurately weighing each component, it is dissolved in purified water, is sufficiently stirred, mixes;
2. being filled in the medical aluminium foil bag containing non-woven fabrics base material by 25mL, irradiation sterilization processing is sealed to get collagen egg
White sticking dressing product.
5 collagen-base skin and mucosa protective agent dressing preparation example of embodiment
Example 1, skin and mucosa protective agent are made of the raw material of following quality proportioning: recombination human source type III collagen 1%,
Sodium Hyaluronate 0.5%, glycerol 10%, remaining is purified water;After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing 100.0g glycerol, it is dissolved in 200mL purified water, recombination human source type III collagen 10g and transparent is added
Matter acid sodium 5g, is sufficiently stirred dissolution;
2. purified water is added to 1000g, 1h is stirred;
3. after irradiation sterilization, sterile filling is to get collagen-base skin and mucosa protective agent dressing, after being applied to micro-shaping
Skin and mucosa protection etc..
Example 2, skin and mucosa protective agent are made of the raw material of following quality proportioning: recombination human source type III collagen
0.1%, Sodium Hyaluronate 1%, glycerol 40%, remaining is purified water;After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing 400.0g glycerol, it is dissolved in 200mL purified water, recombination human source type III collagen 1g and transparent is added
Matter acid sodium 10g, is sufficiently stirred dissolution;
2. purified water is added to 1000g, 1h is stirred;
3. after irradiation sterilization, sterile filling is to get collagen-base skin and mucosa protective agent dressing, after being applied to micro-shaping
Skin and mucosa protection etc..
Example 3, skin and mucosa protective agent are made of the raw material of following quality proportioning: recombination human source type III collagen 1%,
Sodium Hyaluronate 0.1%, remaining is purified water;After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing 200mL purified water, recombination human source type III collagen 10g and Sodium Hyaluronate 1g is added, is sufficiently stirred
Dissolution;
2. purified water is added to 1000g, 1h is stirred;
3. after irradiation sterilization, sterile filling is to get collagen-base skin and mucosa protective agent dressing, after being applied to micro-shaping
Skin and mucosa protection etc..
Dressing provided in this embodiment has good moisture-keeping function, can promote fibroblastic movement and hyperplasia, presses down
Its differentiation is made, scar hyperplasia caused by collagen over-deposit is inhibited.Have to the skin and mucosas surface of a wound such as burn, scald, scratch and covers
Lid, promoting healing, lubrication, moisturizing, the effect for inhibiting scar.
6 collagen of embodiment importing property beauty product preparation example
Application Example of the recombination human source type III collagen in high-grade minimally invasive beauty product:
For the importing product water laser accunputure of minimally invasive beauty, solution passes through nothing by dissolving each raw material as solvent using purified water
Bacterium method is prepared, and the steps include:
Example 1,1. according to following quality proportioning, accurately weigh each component, and mixing, constant volume is sufficiently stirred;Wherein recombined human
Source type III collagen 0.5%, glycerol 3%, Sodium Hyaluronate 0.3%, micromolecule hyaluronic acid sodium 0.03%, sodium chloride
0.9%, Argireline 0.01 ‰.
2. with 0.22um filtering with microporous membrane degerming;
3. under aseptic condition, being dispensed into disposable sterilized injector, every filling 3ml, and carry out aseptic packaging.
4. being used in use, directly taking out cooperation and importing instrument.
Example 2,1. according to following quality proportioning, accurately weigh each component, and mixing, constant volume is sufficiently stirred;Wherein recombined human
Source type III collagen 0.1%, glycerol 1%, Sodium Hyaluronate 0.5%, micromolecule hyaluronic acid sodium 0.05%, sodium chloride
0.8%, Argireline 0.005 ‰.
2. with 0.22um filtering with microporous membrane degerming;
3. under aseptic condition, being dispensed into disposable sterilized injector, every filling 3ml, and carry out aseptic packaging.
4. being used in use, directly taking out cooperation and importing instrument.
Example 3,1. according to following quality proportioning, accurately weigh each component, and mixing, constant volume is sufficiently stirred;Wherein recombined human
Source type III collagen 0.3%, glycerol 10%, Sodium Hyaluronate 0.1%, micromolecule hyaluronic acid sodium 0.01%, sodium chloride
1%, Argireline 0.02 ‰.
2. with 0.22um filtering with microporous membrane degerming;
3. under aseptic condition, being dispensed into disposable sterilized injector, every filling 3ml, and carry out aseptic packaging.
4. being used in use, directly taking out cooperation and importing instrument.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective,
The scope of the present invention is defined by the claims and their equivalents.
<110>Jiangsu Yue Zhi biological medicine Co., Ltd
<120>III collagen type α of recombination human source, 1 chain and its application
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1082
<212> PRT
<213>mankind (Homo sapiens)
<400> 1
His His His His His His Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly
1 5 10 15
Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala Gly Pro Pro
20 25 30
Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly Ser Pro Gly
35 40 45
Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro
50 55 60
Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala
65 70 75 80
Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly
85 90 95
Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe
100 105 110
Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys
115 120 125
Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly
130 135 140
Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu
145 150 155 160
Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp
165 170 175
Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly
180 185 190
Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu Val Gly Pro
195 200 205
Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg Gly Glu Pro
210 215 220
Gly Pro Gln Gly His Ala Gly Ala Gln Gly Pro Pro Gly Pro Pro Gly
225 230 235 240
Ile Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly Pro Ala Gly Ile
245 250 255
Pro Gly Ala Pro Gly Leu Met Gly Ala Arg Gly Pro Pro Gly Pro Ala
260 265 270
Gly Ala Asn Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly Glu Pro Gly
275 280 285
Lys Asn Gly Ala Lys Gly Glu Pro Gly Pro Arg Gly Glu Arg Gly Glu
290 295 300
Ala Gly Ile Pro Gly Val Pro Gly Ala Lys Gly Glu Asp Gly Lys Asp
305 310 315 320
Gly Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pro Gly Ala Ala Gly
325 330 335
Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile
340 345 350
Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Pro Ala
355 360 365
Gly Pro Arg Gly Ala Ala Gly Glu Pro Gly Arg Asp Gly Val Pro Gly
370 375 380
Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly Pro Gly Ser
385 390 395 400
Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser Gly Arg Pro
405 410 415
Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Gln Pro Gly Val Met Gly
420 425 430
Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly Lys Asn Gly Glu
435 440 445
Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn
450 455 460
Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly Pro Gly Gly
465 470 475 480
Asp Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gly Leu Gln Gly Leu
485 490 495
Pro Gly Thr Gly Gly Pro Pro Gly Glu Asn Gly Lys Pro Gly Glu Pro
500 505 510
Gly Pro Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Gly Lys Gly
515 520 525
Asp Ala Gly Ala Pro Gly Glu Arg Gly Pro Pro Gly Leu Ala Gly Ala
530 535 540
Pro Gly Leu Arg Gly Gly Ala Gly Pro Pro Gly Pro Glu Gly Gly Lys
545 550 555 560
Gly Ala Ala Gly Pro Pro Gly Pro Pro Gly Ala Ala Gly Thr Pro Gly
565 570 575
Leu Gln Gly Met Pro Gly Glu Arg Gly Gly Leu Gly Ser Pro Gly Pro
580 585 590
Lys Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Ala Asp Gly Val Pro
595 600 605
Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly Pro Pro Gly
610 615 620
Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gly Ala Pro Gly Leu
625 630 635 640
Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Thr
645 650 655
Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly Gln Asn Gly
660 665 670
Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gly Glu Lys Gly Glu
675 680 685
Gly Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Gly Ser Gly Pro Ala
690 695 700
Gly Pro Pro Gly Pro Gln Gly Val Lys Gly Glu Arg Gly Ser Pro Gly
705 710 715 720
Gly Pro Gly Ala Ala Gly Phe Pro Gly Ala Arg Gly Leu Pro Gly Pro
725 730 735
Pro Gly Ser Asn Gly Asn Pro Gly Pro Pro Gly Pro Ser Gly Ser Pro
740 745 750
Gly Lys Asp Gly Pro Pro Gly Pro Ala Gly Asn Thr Gly Ala Pro Gly
755 760 765
Ser Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu
770 775 780
Lys Gly Ser Pro Gly Ala Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu
785 790 795 800
Gly Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly
805 810 815
Met Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val Lys Gly Glu
820 825 830
Ser Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro
835 840 845
Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly
850 855 860
Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser
865 870 875 880
Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro Gly Ala Pro
885 890 895
Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val Gly Pro Ala Gly
900 905 910
Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro Ala Gly Ala
915 920 925
Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln Gly Pro Arg
930 935 940
Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly Ile Lys Gly
945 950 955 960
His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro
965 970 975
Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala Gly Pro Arg
980 985 990
Gly Pro Val Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly Thr Ser Gly
995 1000 1005
His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn Arg Gly
1010 1015 1020
Glu Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro Gly
1025 1030 1035
Pro Pro Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val
1040 1045 1050
Gly Ala Ala Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly
1055 1060 1065
Phe Ala Pro Tyr Tyr Gly Trp Ser His Pro Gln Phe Glu Lys
1070 1075 1080
<210> 2
<211> 3204
<212> DNA
<213>artificial sequence
<400> 2
caatacgact cttatgacgt gaagtcagga gtggcagtag gtggtcttgc aggttatccc 60
ggtccagcag gtcccccagg tcctccagga ccacctggaa ccagtggaca ccctggatca 120
ccaggaagtc caggatacca gggtcctcca ggagaacctg gtcaggcagg accttccggt 180
cctccaggac cccctggtgc aatcggacca tcaggtcctg caggaaagga cggagagagt 240
ggtagacccg gaagacccgg agaaagaggt ttgcctggac ccccaggtat aaaaggacct 300
gcaggtatac ctggattccc aggaatgaaa ggacacagag gatttgatgg tagaaacgga 360
gagaaaggag aaacaggagc cccaggatta aagggagaga acggtttacc aggtgaaaac 420
ggagcacctg gacctatggg acccagaggt gcaccaggag agagaggaag accaggactg 480
cccggtgctg ccggagcaag aggaaatgat ggagccagag gttctgatgg acagccaggt 540
cctcctggtc cacctggtac tgcaggattc cctggttccc caggtgcaaa gggagaagtt 600
ggaccagcag gtagtccagg ttccaatggt gcaccaggac aaagaggaga gcccggaccc 660
cagggacacg caggtgcaca gggtccccct ggtcccccag gtatcaatgg aagtcccgga 720
ggtaaaggtg agatgggtcc agcaggaatc ccaggagcac ccggattaat gggtgctaga 780
ggtccacccg gacctgcagg agcaaatggt gcacctggat tgagaggtgg agctggagag 840
cccggaaaga acggtgccaa aggtgaacct ggaccaagag gtgaaagagg agaggccggt 900
atcccaggag tgcctggtgc taaaggtgag gatggaaaag acggttcacc tggagaacca 960
ggagcaaacg gactgccagg agctgccgga gagagaggtg caccaggttt tagaggtcct 1020
gccggaccaa acggtatccc aggagagaaa ggaccagctg gagaaagagg agcccctgga 1080
ccagcaggtc caagaggagc tgcaggtgag ccaggtagag atggagtgcc aggaggtccc 1140
ggtatgagag gaatgccagg ttcacctgga ggaccaggat cagatggtaa acccggtcca 1200
cctggttcac agggagaatc tggaagaccc ggtccacctg gtcctagtgg accaagagga 1260
cagcccggtg ttatgggttt cccaggtcca aagggtaacg atggtgcacc tggtaaaaat 1320
ggagagagag gtggtcccgg aggacccgga ccccagggac ctcctggaaa gaatggtgaa 1380
acaggacctc aaggtcctcc aggtcctaca ggaccaggag gagataaggg agatactggt 1440
ccacctggac cacaaggatt acagggtttg cccggaactg gaggaccacc aggagagaac 1500
ggaaagccag gagagccagg tccaaagggt gacgctggag ccccaggagc cccaggagga 1560
aagggagacg caggtgcccc aggagagaga ggtcctcctg gattagccgg tgcccccgga 1620
ctgagaggag gtgctggacc acctggacct gagggaggaa agggtgccgc aggacctcca 1680
ggtccccctg gagctgctgg tacacctggt ctgcagggta tgcctggtga aagaggtgga 1740
ctgggttccc ccggacctaa gggagacaag ggagaacctg gaggtcccgg tgctgatgga 1800
gtgcctggta aagacggacc tagaggacca accggaccaa tcggaccacc tggtccagcc 1860
ggtcagccag gtgacaaggg agagggtgga gccccaggac tgccaggtat cgccggtcca 1920
agaggttctc ccggtgagag aggagagact ggtccaccag gacctgcagg tttcccagga 1980
gcacctggtc aaaacggaga accaggtgga aaaggtgaga gaggagcccc cggagaaaag 2040
ggagagggag gacctcccgg agtcgcagga cccccaggtg gttcaggtcc cgcaggtcct 2100
ccaggacccc agggagtgaa aggtgaaaga ggttccccag gaggtccagg tgctgccggt 2160
ttccctggtg caagaggatt acccggaccc cctggaagta acggtaaccc aggtccacca 2220
ggtccctctg gatctcccgg aaaggacgga ccacccggtc ccgcaggaaa taccggagca 2280
ccaggttccc caggtgtgtc aggtcctaag ggtgacgcag gacagcccgg agagaagggt 2340
tctcccggag cacagggtcc cccaggtgca cccggtcctc tgggaatagc cggaatcact 2400
ggtgctagag gactggccgg tccacctgga atgcccggtc ccagaggttc accaggtccc 2460
caaggtgtca agggagaatc aggaaagcct ggagcaaatg gtctgagtgg agaaagaggt 2520
ccacctggac cacaaggact gcccggactt gctggtacag caggagagcc cggaagagac 2580
ggaaatcctg gttcagatgg acttccaggt agagacggat ctcccggtgg aaaaggagac 2640
agaggagaga acggatctcc aggtgctcca ggtgcccctg gacaccccgg tcccccaggt 2700
ccagtgggac ccgccggtaa aagtggagac agaggtgaat caggaccagc aggacctgca 2760
ggtgctccag gacccgccgg atcaagagga gcgccaggtc cccagggtcc aagaggtgac 2820
aaaggagaga ctggagaaag aggagctgct ggtatcaaag gacatagagg atttccagga 2880
aatcctggag caccaggaag tccaggtcca gcaggtcagc aaggtgccat tggttctcca 2940
ggacccgccg gacctagagg accagtggga ccctcaggac cacctggaaa agacggtact 3000
tcaggacacc ccggtcctat tggtccaccc ggaccaagag gaaacagagg agaaagaggt 3060
tctgaaggaa gtcctggaca tcccggacag ccaggaccac caggtccccc aggtgctcca 3120
ggaccttgtt gtggtggtgt tggagccgct gcaatagccg gaattggagg tgaaaaggca 3180
ggtggattcg caccatatta tgga 3204
<210> 3
<211> 48
<212> DNA
<213>artificial sequence
<400> 3
cggaattcca tcatcatcat catcatcaat acgactctta tgacgtga 48
<210> 4
<211> 58
<212> DNA
<213>artificial sequence
<400> 4
cggaattctt acttctcgaa ttgtgggtga gaccatccat aatatggtgc gaatccac 58
Claims (12)
1. a kind of 1 chain of recombination human source type III collagen α, which is characterized in that encode the recombination human source type III collagen egg
The nucleotide sequence of white 1 chain of α as shown in SEQ ID NO:1,1 chain of recombination human source type III collagen α from aminoterminal according to
It is secondary to include: aminoterminal affinity purification label, source of people type III collagen maturation peptide chain and c-terminus affinity purification label.
2. 1 chain of recombination human source type III collagen α according to claim 1, which is characterized in that the aminoterminal is affine
Purification tag is 6His label, and the c-terminus affinity purification label is Strep label.
3. 1 chain of recombination human source type III collagen α according to claim 1, which is characterized in that the source of people type III
Collagen maturation peptide chain includes human III type collagen N-terminal peptide, human III type collagen into helical region, human III type collagen C-terminal peptide, the people
The nucleotide sequence of source type III collagen maturation peptide chain is as shown in SEQ ID NO:2.
4. 1 chain of recombination human source type III collagen α according to claim 3, which is characterized in that the source of people type III
The nucleotide sequence of collagen maturation peptide chain is by natural collagen gene order by its codon according to the frequency of use of host's codon
It is adjusted to obtain.
5. 1 chain of recombination human source type III collagen α according to claim 4, which is characterized in that the host is red to finish
Yeast, it is described to be adjusted to the rare codon optimization in the natural collagen gene order in Pichia pastoris to be its preference password
Son.
6. 1 chain of recombination human source type III collagen α according to claim 3, which is characterized in that the source of people type III
The amplimer F (SEQ ID NO:3) and R (SEQ ID NO:4) of the nucleotide sequence of collagen maturation peptide chain are as follows:
F:CGGAATTCCATCATCATCATCATCATCAATACGACTCTTATGACGTGA, restriction enzyme site are EcoR I;
R:CGGAATTCTTACTTCTCGAATTGTGGGTGAGACCATCCATAATATGGTGCGAAT CCAC, restriction enzyme site EcoR
I。
7. 1 chain of recombination human source type III collagen α according to claim 6, which is characterized in that the amplimer
N-terminal increase can express the nucleotide sequence of 6His, and C-terminal increase is expanded after capable of expressing the nucleotide sequence of Strep.
8. a kind of application of 1 chain of recombination human source type III collagen α, which is characterized in that the recombination human source type III collagen egg
It is white to be applied to medical dressing, the mass fraction of the medical dressing each component are as follows:
Recombination human source type III collagen 0.1%~0.5% described in any one of -7 according to claim 1;
Moisturizer 1%~10%;
Carbomer 0.1%~1%;
Triethanolamine 0.1%~10%;
Remaining is purified water.
9. a kind of application of 1 chain of recombination human source type III collagen α, which is characterized in that the recombination human source type III collagen egg
White to be applied to collagen dressing patch, the collagen dressing patch is by non-woven fabrics base material and is located on the non-woven fabrics base material
Collagen dressing composition, the mass fraction of the collagen dressing each component are as follows:
Recombination human source type III collagen 0.1%~0.5% described in any one of -7 according to claim 1;
Moisturizer 1%~10%;
Thickener 0.1%~2%;
Remaining is purified water.
10. a kind of application of 1 chain of recombination human source type III collagen α, which is characterized in that the recombination human source type III collagen
Albumen is applied to the skin and mucosa protective agent of micro-shaping, the mass fraction of the skin and mucosa protecting group each component are as follows:
Recombination human source type III collagen 0.1%~1% described in any one of -7 according to claim 1;
Sodium Hyaluronate 0.1%~1%;
Moisturizer 0~40%;
Remaining is purified water.
11. a kind of application of 1 chain of recombination human source type III collagen α, which is characterized in that the recombination human source type III collagen
Albumen is applied to external use skin care, the mass fraction of the external use skin care each component are as follows:
Recombination human source type III collagen 0.01%~0.1% described in any one of -7 according to claim 1;
Moisturizer 1%~5%;
Butanediol 1%~5%;
Sodium Hyaluronate 0.01%~0.1%;
Carbomer 0.1%~1%;
Glycine betaine 1%~5%;
Silk peptide 0.01%~0.1%;
Beta glucan 0.1%~1%;
Jojoba ester 0.01%~0.1%;
Dipotassium glycyrrhizinate 0.1%~0.5%;
Triethanolamine 0.1%~1%;
Remaining is purified water.
12. a kind of application of 1 chain of recombination human source type III collagen α, which is characterized in that the recombination human source type III collagen
Albumen is applied to importing property skin care item, the mass fraction of the importing property skin care item each component are as follows:
Recombination human source type III collagen 0.1%~0.5% described in any one of -7 according to claim 1;
Moisturizer 1%~10%;
Sodium Hyaluronate 0.1%~0.5%;
Micromolecule hyaluronic acid sodium 0.01%~0.05%;
Sodium chloride 0.8%~1%;
Argireline 0.0005%~0.002%;
Remaining is purified water.
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