CN110003324A - Recombination human source collagen and its application - Google Patents
Recombination human source collagen and its application Download PDFInfo
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- CN110003324A CN110003324A CN201910420372.2A CN201910420372A CN110003324A CN 110003324 A CN110003324 A CN 110003324A CN 201910420372 A CN201910420372 A CN 201910420372A CN 110003324 A CN110003324 A CN 110003324A
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- collagen
- human source
- recombination human
- source collagen
- recombination
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- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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Abstract
The present invention provides a kind of recombination human source collagen and its application, the amino acid sequence of the recombination human source collagen is encoded as shown in SEQ ID NO:1, the recombination human source collagen successively includes from aminoterminal: aminoterminal affinity purification label, 2 chain maturation peptide chain of source of people I-type collagen α and c-terminus affinity purification label.The recombination human source collagen according to an embodiment of the present invention, there is specific affinity purification label by all designing at the both ends of 2 chain maturation peptide chain of source of people I-type collagen α, the recombination human source collagen is easily isolated purifying and detection, and the recombination human source collagen has higher molecular weight and special physicochemical property, it is easy to ferment and express, yield is high, and purposes is wide.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly it relates to a kind of recombination human source collagen and
It is applied.
Background technique
The aging of human body is in the change for apparently showing as skin, such as relaxation, corrugation.The direct shadow of the situation of skin
Ring the beauty and the state of mind of appearance.The content of collagen is up to 75% in skin, it maintains the elasticity and water profit of skin.
The main reason for loss of collagen is skin aging.The content for improving collagen in skin, facilitates skin to anti-ageing
Always, restore skin elasticity.
I-type collagen be in 29 had now been found that in collagen Tissue distribution the most extensively and the highest glue of content
Former albumen, accounts for about 90%, accounts for about 27% or so of animal body total protein.It occurs and cell metabolism in neoblastic form
In, new tissue mechanical intensity and biochemical characteristic are assigned, the normal physiological function and injury repair of maintenance cell, tissue etc. are played
Important function.
I-type collagen has the function such as good biocompatibility, biodegradability, no cytotoxicity, inoxidizability
Can, and cell adherence and cell Proliferation and other effects can be promoted, it can be widely used in the fields such as medicine, beauty, cosmetics, health care product
In.Its effect in terms of skin injury reparation, shaping and beauty, enhancing has also been known together.
The correlative study of country's recombined collagen is many at present, such as Northwest University and South China Science & Engineering University have
Relevant research report, but what it mainly studied is collagen short peptide stretch, there is no 2 chains of related recombination human source type i collagen α complete
The report of long peptide.
Afloat recombination I-type collagen peptide fragment currently on the market, though preferably effect is shown, because of its segment
Smaller, molecular weight is low, also limits the performance of its function.I-type collagen full-length peptide possesses more fibronectin FN and combines
Site, it is easier to play its collagen functional activity；It possesses unique physio-biochemical characteristics, such as it with higher molecular weight
One layer of ventilative film, anti-degradation time length, crosslinking post-crosslinking Du Genggao are easily formed, performance is more excellent；And it is over time
The slow degradation occurred, replenishing collagen small peptide that can be more longlasting, or even sustained release work can be played with some drugs in conjunction with
With.Therefore, in order to sufficiently develop its potential value, the production of its full-length peptide of urgent need to resolve and test problems.
The sub-argument purifying of collagen at present, mostly uses molecular sieve and ion exchange resin to be separated, and collagen
Because its unique G-X-Y structure makes it difficult to separate, the especially degradation fragment with collagen of the same race, and purifying technique operation is multiple
Purifying technique is difficult to stablize between miscellaneous and different batches, cause in the market collagen small peptide product be mostly small peptide and its degradation product
Mixture is difficult to obtain the collagen with single peptide chain, limits its extensive use.
Collagen detects in product at present, should mainly using the type and content of tree species for bio-energy source research collagen
It is collagen that method, which can only detect it, but not can confirm that whether it is full length fragment, therefore is not suitable for the overall length of long segment peptide chain
Detection.
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 recombination human source collagen, recombination human source collagen purifying and identification are convenient,
It is easy to detect.
The present invention also proposes a kind of application of recombination human source collagen.
The recombination human source collagen of embodiment according to a first aspect of the present invention encodes the recombination human source collagen
Amino acid sequence as shown in SEQ ID NO:1, the recombination human source collagen successively includes from aminoterminal: aminoterminal is affine
Purification tag, 2 chain maturation peptide chain of source of people I-type collagen α and c-terminus affinity purification label.
Recombination human source collagen according to the above embodiment of the present invention, can also have following additional technical feature:
According to one embodiment of present invention, the aminoterminal affinity purification label is Flag label, the c-terminus parent
It is 6His label with purification tag.
According to one embodiment of present invention, the recombination human source collagen overall length is 1056 amino acid.
According to one embodiment of present invention, the gene order of the recombination human source collagen such as SEQ ID NO:9 institute
Show.
According to one embodiment of present invention, the gene order of the 2 chain maturation peptide chain of source of people I-type collagen α is by day
Its codon is adjusted to obtain by right glue 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 codon that is of little use of the yeast in sequence replaces with its usual codon.
According to one embodiment of present invention, the recombination human source collagen is when carrying out primer amplification building, close
5 ' ends of the glue protogene sequence after numeral optimization introduce the gene order of coding Flag flag sequence, introduce coding at 3 ' ends
It is expanded after the gene order of 6His sequence.
According to one embodiment of present invention, the recombination human source collagen by Ni-NTA HisBind resin and
The method of Anti-Flag affinity purification gel adsorption is extracted and is purified from the fermentation liquid of the Pichia pastoris.
According to one embodiment of present invention, the recombination human source collagen is applied to external use skin care, the external application
The mass fraction of skin care item each component are as follows: the recombination human source collagen 0.01%~0.1% according to above-described embodiment；It protects
Humectant 1%-3%；Butanediol 1%-3%；Sodium Hyaluronate 0.01%-0.1%；Xanthan gum 0.01%-0.1%；Allantoin
0.01%-0.2%；Panthenol 0.1%-1%；Dipotassium glycyrrhizinate 0.1%-0.5%；EDTANa2 0.01%-0.1%；β-Portugal
Glycan 0.1%-0.5%；Reed fragrant plant oil 0.1%-0.5%；Sodium lactate 0.1%-0.3%；Water-solubleazone 0.1%-1%；Silk peptide
0.01%-0.1%；Remaining is purified water.
According to one embodiment of present invention, the recombination human source collagen is applied to importing property skin care item, described to lead
The mass fraction of entering property skin care item each component are as follows: the recombination human source collagen 0.1%-0.5% according to above-described embodiment；
Moisturizer 1%-10%；Sodium Hyaluronate 0.1%-0.5%；Micromolecule hyaluronic acid sodium 0.01%-0.05%；Sodium chloride
0.9%；Remaining is purified water.
According to one embodiment of present invention, the recombination human source collagen is applied applied to collagen gel is prepared
Material, the mass fraction of the collagen gel dressing each component are respectively as follows: the recombination human source glue according to above-described embodiment
Former albumen 0.1%~0.5%；Moisturizer 1%-10%；Carbomer 0.1%-4%；Triethanolamine 0.1%-10%；Remaining is pure
Change water.
According to one embodiment of present invention, it is used to prepare former albumen sticking dressing, the original albumen sticking dressing each component
Mass fraction is respectively as follows: the recombination human source collagen 0.1%~0.5% according to above-described embodiment；Moisturizer 1%-
10%；Xanthan gum 0.1%-2%；Remaining is purified water.
According to one embodiment of present invention, the recombination human source collagen is glutinous applied to collagen-base skin is prepared
The mass fraction of film protective agent dressing, the collagen-base skin and mucosa protective agent dressing each component is respectively as follows: according to above-mentioned
Recombination human source collagen 0.1%~1% described in embodiment；Sodium Hyaluronate 0.1%~1%；Moisturizer 0~40%；Its
Remaining is purified water.
Recombination human source collagen according to an embodiment of the present invention at least has following technical effect that
(1) recombination human source collagen of the invention carries bispecific label at its both ends, both can be by affine
Purifying obtains overall length collagen, can also pass through its state and content in the product of sandwich method ELISA test sensitivity；
(2) gene order of recombination human source collagen of the invention is according to the usual codon of Pichia pastoris to its gene sequence
Column have carried out codon optimization, eliminate be of little use codon and hairpin structure, by synonymous conversion, optimize the second level knot of mRNA
Structure, avoid codon utilization efficiency limit caused by translation efficiency it is low, make it be more suitable for expressing in Pichia pastoris；
(3) compared with conventional recombinant collagen segment and gelatin, recombination human source collagen is the collagen of overall length, because
And there is very high molecular weight as large biological molecule and possess more preferably chemical structure and performance, it can be used as bio-medical material
Material；
(4) compared with traditional collagen production method, recombination human source collagen is the people of eukaryotic expression system expression
The recombined collagen in source, and can be secreted extracellularly, virus-free, endotoxin hidden danger, and it is anti-to avoid immunological rejection
It answers, biocompatibility and biological safety are higher, and for technical process to more environment-friendly, product is safer.
(5) hydrophily and stability of recombination human source collagen of the invention are good, amino acid composition and natural collagen
Protein amino acid sequence corresponding portion 100% is identical, and structure and bioactivity are closer to natural collagen protein.
(6) recombination human source collagen of the invention carries the Flag label of N-terminal and the 6His label of C-terminal, utilizes it
Bispecific affinity purification label, purification step is simple, and product purity is high, can meet minimally invasive, shaping and beauty, bio-medical material
Equal products use.
Detailed description of the invention
Fig. 1 is the secreting, expressing recombinant plasmid according to the recombination human source collagen of the building of the embodiment of the present invention
2 map of pPIC9k-colI α；
Fig. 2 be according to the recombination human source collagen of the embodiment of the present invention during the preparation process used by recombinant plasmid
PCR identifies electrophoretogram；
Fig. 3 is the SDS- for recombinating Pichia yeast engineering and expressing recombination human source collagen according to an embodiment of the present invention
PAGE analysis chart；
Fig. 4 is real according to the immunoblotting of the recombination human source collagen for the recombinant bacterium expression screened in the embodiment of the present invention
It tests；
Fig. 5 is to compare knot according to the mass spectrum of the recombination human source collagen for the recombinant bacterium expression screened in the embodiment of the present invention
Fruit；
Fig. 6 is to express recombination human source glue to the recombinant bacterium screened using sandwich method ELISA according in the embodiment of the present invention
The overall length of former albumen examines measurement result schematic diagram, and (wherein, degradation product is the protein degradation containing C-terminal label of purifying, mesh
Mark albumen is the target protein of purifying).
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.
Recombination human source collagen according to an embodiment of the present invention is specifically described in conjunction with attached drawing first below.
The amino acid sequence of the recombination human source collagen is encoded as shown in SEQ ID NO:1, the recombination human source
Collagen successively includes from aminoterminal: aminoterminal affinity purification label, 2 chain maturation peptide chain of source of people I-type collagen α and carboxylic
Cardinal extremity affinity purification label.
In other words, the recombination human source collagen according to an embodiment of the present invention is one kind in source of people I-type collagen α
On the basis of 2 chain maturation peptide chains, the collagen of affinity purification label is respectively provided in amino acid and c-terminus.Pass through as a result,
Specific affinity purification double labelling, the recombination human source is respectively set at the both ends of 2 chain maturation peptide chain of source of people I-type collagen α
Collagen has overall length peptide chain, and chemical structure and performance are better than animal sources collagen and prokaryotic micro-organisms source recombinant collagen
Albumen and recombinant collagen segment and gelatin in the market, in addition, the hydrophilic amino acid content of 2 chain mature peptide of type i collagen α is higher than
1 chain of α has potential preferable hydrophily.
Some specific embodiments according to the present invention, the aminoterminal affinity purification label are Flag label, the carboxyl
End affinity purification label is 6His label.Using the double affinity tag purifying in both ends, people of the single, overall length without degradation can be obtained
2 chain maturation peptide chain of source I-type collagen α, is applicable to the field that field of medicaments etc. requires product uniformity.Both marks
Sequence very little is signed, does not influence the biological function of collagen；Two kinds of labels can be used as 2 chain mature peptide of source of people I-type collagen α simultaneously
The special labeling sequence of chain detection, identification.
In one embodiment of the invention, the recombination human source collagen overall length is 1056 amino acid, that is,
Say, the overall length of recombination human source collagen is 1056 amino acid, by the Flag sequence of N-terminal, 2 chain of source of people I-type collagen α at
The 6His sequence of ripe peptide sequence and C-terminal composition, the recombination human source collagen have overall length peptide chain, and performance is more preferable.
The recombination engineering and protein preparation method of expression said gene is detailed below.
According to one embodiment of present invention, the gene order of the recombination human source collagen such as SEQ ID NO:9 institute
Show.
In one embodiment of the invention, the gene order of the recombination human source collagen is referring to natural collagen gene
Its codon is adjusted to obtain by sequence according to the frequency of use of host's codon.Further, the host is to finish red ferment
Mother, the codon that is adjusted to for the yeast in the natural collagen gene order to be of little use replace with its usual codon, can
Selection of land, glue protogene sequence of the recombination human source collagen gene when carrying out primer amplification building, after codon optimization
5 ' ends of column introduce the gene order of coding Flag flag sequence, carry out after the gene order that 3 ' ends introduce coding 6His sequence
Amplification.
Preferably, the recombination human source collagen is affine pure by Ni-NTA HisBind resin and Anti-Flag
The method for changing gel adsorption is extracted and is purified from the fermentation liquid of the Pichia pastoris.
That is, the gene order of recombination human-like collagen is adjusted, i.e., it is 2 chain of source of people type i collagen α is mature
Codon agg, cgt, ggg, ggc, gca etc. that Pichia pastoris is of little use in the corresponding gene order of peptide are optimized for it and have a preference for password
Sub- aga, ggt, gct etc., using the gene order obtained after the analysis of DNASTAR software as shown in SEQ ID NO.2, by this
Processing, collagen are more conducive to express.
Further, when the recombination human source collagen is when carrying out primer amplification building, after codon optimization
5 ' ends of glue protogene sequence introduce the gene order of coding Flag flag sequence, introduce the gene of coding 6His sequence at 3 ' ends
It is expanded after sequence in order to the purification and detection of the recombination human source collagen.
The recombination engineering and protein preparation method of expression said gene is detailed below.
Step 1: construction recombination plasmid
Firstly, a kind of corresponding gene order of 2 chain mature peptide of source of people type i collagen α optimized is provided, according to Genbank
The amino acid sequence and gene order of login, it is corresponding under the premise of not changing collagen original amino acid sequence to optimize its
Gene order, optimization processing is carried out according to Pichia pastoris preferences codon, and 2 chain mature peptide of source of people type i collagen α is corresponding
Codon agg, cgt, ggg, ggc, gca etc. that Pichia pastoris is of little use in gene order (colI α 2) are optimized for it and have a preference for password
Sub- aga, ggt, gct etc., and using the gene order obtained after the analysis of DNASTAR software as shown in SEQ ID NO:2, by this
One processing makes collagen be more conducive to express.
Then, by ApaI, Bgl II, BamH I, EcoR I, Not I, Sac I, the Sal I in the gene order of optimization
Equal restriction endonuclease sites etc. are rejected, and are synthesized by artificial full genome.
Finally, utilizing primer P1 (see SEQ ID NO.3)/P2 1. using pPIC9k plasmid as template (see SEQ ID NO.4)
Carry out the product that PCR amplification obtains about 379bp；2. utilizing primer using the gene order SEQ ID NO:2 of above-mentioned synthesis as template
PCR amplification is carried out to P3 (see SEQ ID NO.5)/P4 (see SEQ ID NO.6) and obtains the gene order that size is about 3149bp；
3. being that primer is mutually expanded by PCR, obtained using itself as template with P5 (see SEQ ID NO.7)/P6 (see SEQ ID NO.8)
Obtain the genetic fragment containing 6His coded sequence of about 65bp；4. pPIC9k is done to BamHI and Not I double digestion respectively,
Obtain carrier segments；5. by item 1., 2., 3., 4. in each segment be mixed in a certain proportion, utilize the seamless cloning kit of instant
Box (Sangon) is connected into containing Flag coded sequence, the corresponding gene order of 2 chain mature peptide of source of people type i collagen α, 6His coding
The recombinant plasmid of sequence, the as recombinant plasmid containing recombination 2 catenin gene order of type i collagen α, are named as pPIC9k-
ColI α 2 is shown in SEQ ID NO.9 by the gene order for the target protein recombination human source collagen egg that this method obtains.
Step 2: building gene recombined Pichia pastoris engineering bacteria
I linearization process of restriction enzyme Sal of recombinant plasmid pPIC9k-colI α 2 that will be prepared, and by its
Electrotransformation enters in Pichia pastoris SMD1168 (Invitrogen) competent cell.With nutrient defect type mark and G418 resistance mark
The high copy positive recombinant of note screening, obtaining pichia yeast genetic engineering bacteria, (strain has been preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number CGMCC17147, preservation date: on January 10th, 2019, address: Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1, classification naming: pichia pastoris yeast Pichia pastoris).
Step 3: preparation and reorganization human-like collagen
Recombinant yeast pichia pastoris engineering bacteria is inoculated in YPD Liquid Culture to be based on, is activated overnight in 30 DEG C, 220rpm；It will weigh
The switching of group Pichia yeast engineering is in BMGY culture medium, until OD600=2.0~6.0, cultivate 16-18h in 30 DEG C, 220rpm；Room
The lower 1500g of temperature is centrifuged 5min, collects thallus, and thallus is resuspended with the BMMY culture medium of 10mL, until OD600=1.0 or so, by gained
Bacterium solution be placed in the sterile triangular flask of 100mL, continue shaken cultivation under 28 DEG C, 220rpm；Add into culture medium within every 24 hours
Add methanol to final concentration of 1% progress Fiber differentiation；After fermented and cultured, supernatant is collected by centrifugation, under the conditions of non denatured, uses
Ni-NTA His Bands resin adsorption recombinant collagen, washes away impurity with cleaning solution, finally elutes recombinant collagen again, collects elution
Liquid；Eluent is through ultrafiltration system desalination, concentration；Again by concentrate Anti-Flag affinity purification gel adsorption recombinant collagen, use
Cleaning solution washes away impurity, finally elutes recombinant collagen again, collects eluent；Eluent is obtained through ultrafiltration system desalination, concentration
Concentrate recombination human source collagen is made through vacuum freeze drying.
The present invention has carried out codon optimization to type i collagen gene, has disappeared according to Pichia pastoris codon preference as a result,
In addition to codon and the hairpin structure of being of little use optimize the secondary structure of mRNA by synonymous conversion, codon is avoided using effect
Translation efficiency caused by rate limits is low, it is made to be more suitable for expressing in Pichia pastoris.
To sum up, the recombination human source collagen according to an embodiment of the present invention, which provides a kind of both ends, has parent
With the recombination human source collagen of purification tag, both overall length collagen can be obtained by affinity purification label, can also led to
Its state and content in the product of sandwich method ELISA test sensitivity is crossed, the recombination human source collagen has very high point
Son amount, as large biological molecule, possesses more preferably chemical structure and performance, can be used as bio-medical material.
The preparation of recombination human source collagen of the present invention is specifically described with Fig. 1 to Fig. 6 combined with specific embodiments below
Process and application.
Embodiment one
The overall length of genetic recombination source of people collagen is 1056 amino acid, and the Flag label of N-terminal, C-terminal is 6His mark
Label, the amino acid sequence of the genetic recombination source of people collagen is as shown in SEQ ID NO.1.
The preparation method is as follows:
1, construction recombination plasmid
1.1 synthesis optimizing genes
According to Genbank log in 2 chain mature peptide of source of people type i collagen α amino acid sequence gene order corresponding with its,
With reference to Pichia pastoris Preference codon, under the premise of not changing collagen ColI 2 original amino acid of α, utilize
DNASTAR software optimization gene order, and eliminate ApaI, Bgl II, BamH I, EcoR I, Not I, Sac I, Sal I etc.
Restriction endonuclease sites obtain gene order and see SEQ ID NO.2, then closed by Nanjing Genscript Biotechnology Co., Ltd.
At.
1.2 are built into recombinant plasmid by PCR, digestion, connection etc.
1. being carried out using pPIC9k plasmid as template using primer P1 (see SEQ ID NO.3)/P2 (see SEQ ID NO.4)
The product of PCR amplification acquisition about 379bp；2. using the gene order SEQ ID NO.2 of synthesis as template, using primer pair P3 (see
SEQ ID NO.5)/P4 (see SEQ ID NO.6) carry out PCR amplification obtain size be about 3149bp gene order；3. with P5
(see SEQ ID NO.7)/P6 (see SEQ ID NO.8) is that primer is mutually expanded using itself as template by PCR, is obtained about
The genetic fragment containing 6His coded sequence of 65bp；4. pPIC9k to be done to BamHI and Not I double digestion respectively, obtain
Carrier segments；5. by item 1., 2., 3., 4. in each segment be mixed in a certain proportion, through the seamless Cloning Kit of instant
(Sangon) in 50 DEG C of connection 1h；Connection product conversion is entered into competent E.coli DH5 α, it is flat in the LB resistance containing Kan
Plate screening positive clone；Using glue protogene internal primer P7 (see SEQ ID NO.10)/P8 (see SEQ ID NO.11) to sun
Property clone carry out PCR detection, stripe size obtained by analytical electrophoresis is consistent with theoretical value 585bp, as a result as shown in Fig. 2, wherein swimming
Road M is molecular weight Marker, and 1-3 is different recombinant clones.Recombinant plasmid is extracted, raw work bioengineering (Shanghai) share is sent
Co., Ltd carries out sequence verification.Sequencing result and expected consistent, no mutation.Construction of recombinant plasmid success, as containing recombination I
The recombinant plasmid of 2 catenin gene order of Collagen Type VI α, is named as pPIC9k-colI α 2.
2, gene recombined Pichia pastoris engineering bacteria is constructed
The linearisation of 2.1 recombinant expression plasmid pPIC9k-colI α 2
Extract recombinant plasmid pPIC9k-colI α 2, be digested with restriction enzyme Sal I in 37 DEG C, then with
1% agarose gel electrophoresis detects whether to cut completely through, after cutting completely through, with plastic recovery kit to digested liquid at
Reason recycles linearization plasmid, and desalting processing.
2.2 prepare Pichia pastoris SMD1168 competent cell
1) picking yeast SMD1168 single colonie is seeded in the triangular flask of the YPD fluid nutrient medium containing 5mL, 30 DEG C,
250rpm shaken cultivation is stayed overnight；
2) overnight culture of 50uL is taken to be seeded in the 300ml triangular flask containing the fresh YPD fluid nutrient medium of 50mL, 30
DEG C, 250rpm 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 80ul, is saved backup in -70 DEG C.
The electrotransformation of 2.3 Pichia pastoris
1) it is rinsed electric revolving cup three times with dehydrated alcohol, dries residual ethanol in sterilized super-clean bench；
2) electric revolving cup is sealed, goes to and 10min is pre-chilled in ice；
3) 2 matter of linearisation pPIC9k-colI α of about 10ug is added in the pre- middle defrosting of ice in Pichia pastoris competent cell
Grain, mixes gently, and goes in the electric revolving cup of 0.2cm ice pre-cooling, continues at and 5min is pre-chilled on ice；
4) it shocks by electricity, voltage 1.5kV, capacitor 25uF, 200 Ω of resistance, electric shock time are 5~10mSec；
5) electric shock terminates, and is rapidly added the sorbitol solution of the 1M of 1ml ice pre-cooling, gently piping and druming mixes, and goes to 1.5ml
In centrifuge tube；
6) bacteria suspension being coated on MD plate, every 100ul~200ul 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 (1OD600=5 × 107cells/ml) is measured after mixing；
3) it takes 105 cells to be coated on the YPD plate containing 0.5mg/ml G418, is inverted, 30 DEG C of 3~4d of culture；
4) 200ul YPD fluid nutrient medium is added in every hole in sterile 96 orifice plate；
5) conversion obtained on the YPD plate containing 0.5mg/ml G418 is respectively connected to toward step 4) with sterile toothpick
Son mixes, in 30 DEG C of culture 48h；
6) after 48h, one piece of new sterile 96 orifice plate is taken, 190ul YPD fluid nutrient medium is added in every hole, adds in corresponding aperture
Enter first piece of 96 resulting culture of orifice plate of 10ul, 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, 190ul YPD fluid nutrient medium is added in every hole, in corresponding aperture
Second piece of 96 resulting culture of orifice plate of 10ul is added, for 24 hours in 30 DEG C of cultures；
8) after for 24 hours, 1ul is taken out from 96 orifice plate of third block respectively and is put respectively containing 1.0mg/ml's and 4mg/mlG418
On YPD plate, continue to cultivate 96h~120h in 30 DEG C.If Pichia pastoris SMD1168 transformant can be containing more high concentration G418's
It is grown on culture medium, illustrates that the transformant contains the target gene of more multicopy, that is, there are multiple 2 segments of pPIC9k-colI α to integrate
Into on yeast chromosomal, that is, there is the potential of high-expression target proteins.
3, preparation and reorganization human-like collagen
1) culture Pichia yeast engineering is activated overnight based on 30 DEG C of 220rpm with YPD culture；
2) it takes above-mentioned bacterium solution switching in BMGY culture medium, engineering bacteria 16-18h is cultivated in 30 DEG C, 220rpm, until OD600
=2.0~6.0；
3) 1500g is centrifuged 5min at room temperature, collects thallus, and thallus is resuspended with the BMMY culture medium of 10mL, makes OD600=
1.0 or so, resulting bacterium solution is placed in the sterile triangular flask of 100mL, continues shaken cultivation under 28 DEG C of 220rpm；
4) methanol is added within every 24 hours into culture medium to final concentration of 1% progress Fiber differentiation；
5) after fermented and cultured, 3000 × g is centrifuged 20min at 4 DEG C, collects supernatant；A small amount of supernatant is taken to carry out SDS-
PAGE analysis detection, as a result as shown in Figure 3；
6) pure water of 5~7 times of volumes is added in supernatant, then is concentrated by ultrafiltration to the 20% of initial volume；
7) addition 1 × Ni-NTA of 100ul combination buffer in 1ml concentrate is taken, 4 DEG C are sufficiently mixed；
8) 20ul 50%Ni-NTA HisBands resin suspension is added softly to mix, in conjunction with 30min；
9) 15000 × g is centrifuged 10 seconds precipitated resins, abandons supernatant；
10) resin is rinsed with 1 × Ni-NTA of 100ul wash buffer, 15000 × g is centrifuged 10 seconds, carefully sucks supernatant.
It repeats primary；
11) destination protein is eluted with 1 × Ni-NTA of 200ul elution buffer, 15000 × g is centrifuged 10 seconds, carefully will be upper
It is transferred to clearly in clean tubule, is repeated twice；
12) supernatant is collected, Anti-Flag affinity purification gel 4 DEG C of dialysed overnights of combination buffer are used；
13) it takes above-mentioned dialyzate that 100ul Anti-Flag affinity purification gel suspension is added softly to mix, in conjunction with 30min；
14) 15000 × g is centrifuged 10 seconds precipitate gels, abandons supernatant；
15) 1 × Wash of 200ul solution ringing gel is used, 15000 × g is centrifuged 10 seconds, supernatant is carefully sucked, then
It is repeated once；
16) destination protein is eluted with 1 × Elution of 200ul buffer, 15000 × g is centrifuged 10 seconds, carefully by supernatant
It is transferred in clean tubule, is repeated twice；
17) by obtained eluent through desalination, concentration, obtained concentrate is made yeast through vacuum freeze drying and recombinates
Collagen.
18) 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；
19) after being lyophilized, vent valve is carefully opened, until inside and outside air pressure balance.Culture dish is taken out, white recombination is obtained
Human-like collagen solid.
The identification of the recombination human-like collagen of embodiment two
Example 1,
1, protein immunoblot (Western blotting) detects
1) fermented supernatant fluid is taken to carry out SDS-PAGE electrophoresis, electrophoresis is until bromophenol blue is run out of from separation gel lower edge completely；
2) gel is taken out from glass plate, is soaked in transferring film buffer；
3) it takes and is transferred in transferring film buffer after being impregnated 10 seconds in anhydrous methanol with gel pvdf membrane of the same size
Continue to impregnate stand-by；
4) sponge impregnated in advance through transferring film buffer, filter paper, pvdf membrane, gel, filter are placed in transfer is pressed from both sides in order
Paper, sponge guarantee do not have bubble between every layer；
5) transfer being folded up in electric turn trough, film is placed in ice-water bath close to anode, glue close to cathode, and by electric turn trough,
450mA shifts 2h；
6) pvdf membrane is taken out, is immersed in 5% skimmed milk power prepared with TBS solution, room temperature shakes 2h；It discards
Milk powder solution, is added the primary antibody prepared with 5% milk powder solution, and room temperature shakes 2h；
7) primary antibody solution is poured out, film is washed with TBS solution, is washed three times, each 5min；
8) film is soaked into the goat anti-mouse IgG antibodies for horseradish peroxidase (HRP) label prepared with 5% milk powder solution
In two corresponding anti-solution, room temperature shakes 2h；
9) two corresponding anti-solution is poured out, film is washed with TBS solution, is washed three times, each 5min；
10) residual moisture on film is blotted with filter paper, TMB developing solution (Beyotime) is uniformly coated on film, is placed in half-light
A moment is stood out；
11) residual moisture on film, preservation of taking pictures are absorbed with filter paper.
2, mass spectral analysis is identified
1) protein sample is first taken to carry out SDS-PAGE electrophoretic analysis；
2) it is dyed with coomassie brilliant blue R_250, after decoloration, is moved towards with clean blade along target protein band with 1mm wide
Degree cuts target stripe, and sample presentation is identified by general safe biology.
3) ProtTech ' s ProtQuest software suite software is used, according to UniProt protein
Database and ncbi database are compared.
Example 2, the detection of the overall length of recombination human source collagen:
Using recombination human source collagen both ends carry parents and purification tag, can use Flag monoclonal antibody and
It is coupled the 6His monoclonal antibody sandwich method ELISA detection of HRP, with the recombination human source glue in qualitative or/and quantitative analysis sample
Former albumen.Specific step is as follows:
1) the anti-Flag monoclonal antibody of mouse is diluted 1000 times with combination buffer (1 × PBS buffer solution), to ELISA
The antibody diluent in 100 holes μ L/ is added in plate, antibody concentration is 0.5 μ g/mL；
2) sub with plastic seal membrana oralis cover plate, and 2h or 4 DEG C of overnight incubation is incubated in 37 DEG C；
3) it is inverted ELISA Plate, throws away antibody-solutions, the cleaning solution that 200 μ L are added in every hole (adds 0.05% Tween-20
PBS buffer solution) rinsing 3 times, 5min is shaken every time；
4) confining liquid (cleaning solution of addition 1%BSA) of 200 μ L is added in every hole, non-specific on ELISA Plate to close
Property binding site；
5) sub with plastic seal membrana oralis cover plate, and 2h or 4 DEG C of overnight incubation is incubated in 37 DEG C；
6) confining liquid is abandoned, the cleaning solution that 200 μ L are added in every hole rinses 3 times, shakes 5min every time；
7) every hole be added 100 μ L through the suitably diluted recombination human source collagen sample of confining liquid；
8) sub with plastic seal membrana oralis cover plate, and 2h or 4 DEG C of overnight incubation is incubated in 37 DEG C；
9) sample solution is abandoned, the cleaning solution that 200 μ L are added in every hole rinses 3 times, shakes 5min every time；
10) the 6xHis monoclonal antibody for being coupled HRP is diluted 1000 times with confining liquid, the antibody that 100 μ L are added in every hole is dilute
Release liquid；
11) sub with plastic seal membrana oralis cover plate, and in 37 DEG C of incubation 30min；
12) HRP- antibody-solutions are abandoned, the cleaning solution that 200 μ L are added in every hole rinses 3 times, shakes 5min every time；
13) Multi-channel liquid transfer device is used, the TMB reagent in 100 holes μ L/ is added into ELISA Plate, makes it that face deep enough be presented
Color about needs 10-15min；
14) terminate liquid (hydrochloric acid of 1mol/L) of 100 μ L, color development stopping reaction is added in every hole；
15) in the light absorption value for measuring each hole under 450nm in 10min by microplate reader, to analyze the recombination human source in sample
Collagen.
Testing result is as shown in the table:
Three collagen external use skin care preparation example of embodiment
Example 1, the collagen external use skin care are using purified water as solvent, the mass fraction point of each component and each component
Not are as follows: recombination human source collagen 0.05%, moisturizer 2%, butanediol 2%, Sodium Hyaluronate 0.05%, xanthan gum
0.05%, allantoin 0.1%, panthenol 0.5%, dipotassium glycyrrhizinate 0.3%, EDTANa2 0.05%, beta glucan 0.3%,
Reed fragrant plant oil 0.3%, sodium lactate 0.2%, water-solubleazone 0.5%, silk peptide 0.05%, wherein moisturizer is glycerol.
Preparation step is as follows:
1. being weighed according to following quality proportioning: recombination human source collagen 0.05%, moisturizer 2%, butanediol 2%, thoroughly
Bright matter acid sodium 0.05%, xanthan gum 0.05%, allantoin 0.1%, panthenol 0.5%, dipotassium glycyrrhizinate 0.3%, EDTANa2
0.05%, beta glucan 0.3%, reed fragrant plant oil 0.3%, sodium lactate 0.2%, water-solubleazone 0.5%, silk peptide 0.05%；
2. using purified water as solvent dissolve each step 1. in weighed each raw material, and stir it is a colourless
Tasteless transparent preserving moisture and protecting skin Essence.
Example 2, the collagen external use skin care are using purified water as solvent, the mass fraction point of each component and each component
Not are as follows: recombination human source collagen 0.01%, moisturizer 1%, butanediol 1%, Sodium Hyaluronate 0.1%, xanthan gum 0.1%,
Allantoin 0.2%, panthenol 1%, dipotassium glycyrrhizinate 0.5%, EDTANa2 0.1%, beta glucan 0.5%, reed fragrant plant oil
0.5%, sodium lactate 0.3%, water-solubleazone 1%, silk peptide 0.1%, wherein moisturizer is glycerol.
Preparation step is as follows:
1. being weighed according to following quality proportioning: recombination human source collagen 0.01%, moisturizer 1%, butanediol 1%, thoroughly
Bright matter acid sodium 0.1%, xanthan gum 0.1%, allantoin 0.2%, panthenol 1%, dipotassium glycyrrhizinate 0.5%, EDTANa2
0.1%, beta glucan 0.5%, reed fragrant plant oil 0.5%, sodium lactate 0.3%, water-solubleazone 1%, silk peptide 0.1%, wherein moisturizing
Agent is glycerol；
2. using purified water as solvent dissolve each step 1. in weighed each raw material, and stir it is a colourless
Tasteless transparent preserving moisture and protecting skin Essence.
Example 3, the collagen external use skin care are using purified water as solvent, the mass fraction point of each component and each component
Not are as follows: recombination human source collagen 0.1%, moisturizer 3%, butanediol 3%, Sodium Hyaluronate 0.01%, xanthan gum 0.01%,
Allantoin 0.01%, panthenol 0.1%, dipotassium glycyrrhizinate 0.1%, EDTANa2 0.01%, beta glucan 0.1%, reed fragrant plant oil
0.1%, sodium lactate 0.1%, water-solubleazone 0.1%, silk peptide 0.01%, wherein moisturizer is glycerol.
Preparation step is as follows:
1. weighing according to following quality proportioning: recombination human source collagen 0.1%, moisturizer 3%, butanediol 3% are transparent
Matter acid sodium 0.01%, xanthan gum 0.01%, allantoin 0.01%, panthenol 0.1%, dipotassium glycyrrhizinate 0.1%, EDTANa2
0.01%, beta glucan 0.1%, reed fragrant plant oil 0.1%, sodium lactate 0.1%, water-solubleazone 0.1%, silk peptide 0.01%, wherein
Moisturizer is glycerol；
2. using purified water as solvent dissolve each step 1. in weighed each raw material, and stir it is a colourless
Tasteless transparent preserving moisture and protecting skin Essence.
Above-mentioned recombination human source collagen external use skin care application method: after morning and evening face cleaning, it is applied directly to face, gently
It pats to fully absorbing.
Example IV recombination human source collagen is applied to importing property skin care item preparation example
The mass fraction of example 1, the importing property skin care item each component are as follows: recombination human source collagen 0.1%；Moisturizer
1%；Sodium Hyaluronate 0.1%；Micromolecule hyaluronic acid sodium 0.05%；Sodium chloride 0.9%；Remaining is purified water.
Preparation step is as follows:
1. being weighed according to following quality proportioning: recombination human source collagen 0.1%, moisturizer 1%, Sodium Hyaluronate
0.1%, micromolecule hyaluronic acid sodium 0.05%, sodium chloride 0.9%, remaining is purified water, wherein moisturizer is glycerol, accurately
Each component is weighed, and mixing, constant volume is sufficiently stirred；
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.
The mass fraction of example 2, the importing property skin care item each component are as follows: recombination human source collagen 0.5%；Moisturizer
10%；Sodium Hyaluronate 0.5%；Micromolecule hyaluronic acid sodium 0.01%；Sodium chloride 0.9%；Remaining is purified water.
Preparation step is as follows:
1. being weighed according to following quality proportioning: recombination human source collagen 0.5%, moisturizer 10%, Sodium Hyaluronate
0.5%, micromolecule hyaluronic acid sodium 0.01%, sodium chloride 0.9%, remaining is purified water, wherein moisturizer is glycerol, accurately
Each component is weighed, and mixing, constant volume is sufficiently stirred；
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.
The mass fraction of example 3, the importing property skin care item each component are as follows: recombination human source collagen 0.3%；Moisturizer
5%；Sodium Hyaluronate 0.3%；Micromolecule hyaluronic acid sodium 0.03%；Sodium chloride 0.9%；Remaining is purified water.
Preparation step is as follows:
1. being weighed according to following quality proportioning: recombination human source collagen 0.3%, moisturizer 5%, Sodium Hyaluronate
0.3%, micromolecule hyaluronic acid sodium 0.03%, sodium chloride 0.9%, remaining is purified water, wherein moisturizer is glycerol, accurately
Each component is weighed, and mixing, constant volume is sufficiently stirred；
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.
Five collagen gel dressing preparation example of embodiment
Recombination human source collagen is used to prepare former albumen sticking dressing, the collagen gel using purified water as solvent,
Its component further includes yeast recombined collagen, gelling agent, moisturizer；The gelling agent is carbomer；The moisturizer is sweet
Oil and triethanolamine；
Example 1, the quality proportioning of each component are as follows: recombination human source collagen 0.1%, glycerol 1%, carbomer 4%, three ethyl alcohol
Amine 10%.
Preparation step is as follows:
1. weighing suitable glycerol and carbomer, 1h is stirred, is sufficiently dissolved；
2. purified water is added, and appropriate recombination human source collagen is weighed, stirs 1h, sufficiently dissolve；
3. triethanolamine is added, continues to stir 1h, mix well to get collagen gel casting product.
Example 2, the quality proportioning of each component are as follows: recombination human source collagen 0.5%, glycerol 10%, carbomer 0.1%, three
Ethanol amine 0.1%.
Preparation step is as follows:
1. weighing suitable glycerol and carbomer, 1h is stirred, is sufficiently dissolved；
2. purified water is added, and appropriate recombination human source collagen is weighed, stirs 1h, sufficiently dissolve；
3. triethanolamine is added, continues to stir 1h, mix well to get collagen gel casting product.
Example 3, the quality proportioning of each component are as follows: recombination human source collagen 0.3%, glycerol 5%, carbomer 2%, three ethyl alcohol
Amine 5%.
Preparation step is as follows:
1. weighing suitable glycerol and carbomer, 1h is stirred, is sufficiently dissolved；
2. purified water is added, and appropriate recombination human source collagen is weighed, stirs 1h, sufficiently dissolve；
3. triethanolamine is added, continues to stir 1h, mix well to get collagen gel casting product.
Six collagen gel dressing preparation example of embodiment
Recombination human source collagen is used to prepare former albumen sticking dressing, by recombination human source collagen, moisturizer, medical increasing
Thick dose and non-woven fabrics base material composition, the moisturizer are glycerol, and the medical thickener is xanthan gum.
Example 1, the quality proportioning of each component are as follows: recombination human source collagen 0.5%, glycerol 10%, xanthan gum 0.1%,
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. quantitative filling is into the medical aluminium foil bag containing non-woven fabrics base material, sealing, irradiation sterilization processing is to get collagen
Sticking dressing product.
Example 2, the quality proportioning of each component are as follows: recombination human source collagen 0.1%, glycerol 1%, 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. quantitative filling is into the medical aluminium foil bag containing non-woven fabrics base material, sealing, irradiation sterilization processing is to get collagen
Sticking dressing product.
Example 3, the quality proportioning of each component are as follows: recombination human source collagen 0.3%, 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. quantitative filling is into the medical aluminium foil bag containing non-woven fabrics base material, sealing, irradiation sterilization processing is to get collagen
Sticking dressing product.
Seven collagen-base skin and mucosa protective agent dressing preparation example of embodiment
Example 1, the collagen-base skin and mucosa protective agent dressing each component and mass fraction are respectively: recombination human source glue
Former albumen 0.1%, Sodium Hyaluronate 0.1%%, glycerol 40%；After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing appropriate suitable each component, purified water dissolution is added, stirs 1h；
2. weighing recombination human source collagen, purified water is supplied, continues to stir, sufficiently dissolution mixes；
3. sterile filling, encapsulation process, irradiation sterilization are applied to get collagen-base skin and mucosa protective agent dressing
Skin and mucosa protection after micro-shaping etc..
Example 2, the collagen-base skin and mucosa protective agent dressing each component and mass fraction are respectively: recombination human source glue
Former albumen 1%, Sodium Hyaluronate 1%；After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing appropriate suitable each component, purified water dissolution is added, stirs 1h；
2. weighing recombination human source collagen, purified water is supplied, continues to stir, sufficiently dissolution mixes；
3. sterile filling, encapsulation process, irradiation sterilization are applied to get collagen-base skin and mucosa protective agent dressing
Skin and mucosa protection after micro-shaping etc..
Example 3, the collagen-base skin and mucosa protective agent dressing each component and mass fraction are respectively: recombination human source glue
Former albumen 0.5%, Sodium Hyaluronate 0.5%, glycerol 20%；After obtained preparation solution sterilizing, sterile filling.
Preparation step is as follows:
1. weighing appropriate suitable each component, purified water dissolution is added, stirs 1h；
2. weighing recombination human source collagen, purified water is supplied, continues to stir, sufficiently dissolution mixes；
3. sterile filling, encapsulation process, irradiation sterilization are applied to get collagen-base skin and mucosa protective agent dressing
Skin and mucosa protection after micro-shaping etc..
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, this
The range of invention is defined by the claims and their equivalents.
SEQUENCE LISTING
110 Jiangsu > Yue Zhi biological medicine Co., Ltd of <
120 > recombination human source collagen of < and its application
〈130〉UCFI2H
〈160〉
〈170〉
The amino acid sequence of SEQ ID NO.1 recombination human source collagen
DYKDDDDKGPQYDGKGVGLGPGPMGLMGPRGPPGAAGAPGPQGFQGPAGEPGEPGQTGPAGARGPAGPPGKA
GEDGHPGKPGRPGERGVVGPQGARGFPGTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARG
LPGERGRVGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAGPRGEVGLPGLSGP
VGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPRGIPGPVGAAGATGARGLVGEPGPAGSKGESGNKGEPGSAGPQ
GPPGPSGEEGKRGPNGEAGSAGPPGPPGLRGSPGSRGLPGADGRAGVMGPPGSRGASGPAGVRGPNGDAGRPGEPG
LMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGA
PGPDGNNGAQGPPGPQGVQGGKGEQGPPGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGES
GAAGPTGPIGSRGPSGPPGPDGNKGEPGVVGAVGTAGPSGPSGLPGERGAAGIPGGKGEKGEPGLRGEIGNPGRDG
ARGAPGAVGAPGPAGATGDRGEAGAAGPAGPAGPRGSPGERGEVGPAGPNGFAGPAGAAGQPGAKGERGAKGPKGE
NGVVGPTGPVGAAGPAGPNGPPGPAGSRGDGGPPGMTGFPGAAGRTGPPGPSGISGPPGPPGPAGKEGLRGPRGDQ
GPVGRTGEVGAVGPPGFAGEKGPSGEAGTAGPPGTPGPQGLLGAPGILGLPGSRGERGLPGVAGAVGEPGPLGIAG
PPGARGPPGAVGSPGVNGAPGEAGRDGNPGNDGPPGRDGQPGHKGERGYPGNIGPVGAAGAPGPHGPVGPAGKHGN
RGETGPSGPVGPAGAVGPRGPSGPQGIRGDKGEPGEKGPRGLPGLKGHNGLQGLPGIAGHHGDQGAPGSVGPAGPR
GPAGPSGPAGKDGRTGHPGTVGPAGIRGPQGHQGPAGPPGPPGPPGPPGVSGGGYDFGYDGDFYRAHHHHHH-
2 chain mature peptide gene DNA sequence of SEQ ID NO.2 source of people type i collagen α
CAATACGACGGTAAAGGTGTTGGACTGGGTCCAGGTCCAATGGGTTTGATGGGACCTAGAGGTCCACCTGGAG
CAGCTGGTGCCCCAGGACCTCAGGGTTTCCAAGGACCAGCAGGAGAGCCAGGAGAACCAGGTCAAACAGGACCAGCA
GGTGCAAGAGGACCCGCAGGTCCCCCTGGAAAAGCAGGTGAAGACGGTCATCCAGGAAAACCCGGTAGACCTGGAGA
AAGAGGTGTCGTAGGTCCCCAAGGTGCAAGAGGATTTCCCGGAACTCCAGGATTACCAGGTTTTAAGGGAATTAGAG
GACATAACGGATTAGATGGACTGAAAGGACAACCTGGTGCACCAGGAGTTAAGGGAGAGCCTGGAGCACCAGGAGAA
AACGGAACTCCAGGACAAACAGGTGCAAGAGGTCTGCCTGGAGAGAGAGGAAGAGTTGGAGCCCCAGGTCCCGCAGG
TGCAAGAGGTTCAGACGGATCTGTCGGACCTGTGGGTCCAGCAGGTCCCATAGGTTCCGCAGGACCACCAGGATTCC
CCGGAGCACCCGGACCAAAAGGAGAGATCGGAGCAGTCGGTAACGCAGGACCTGCAGGACCTGCCGGTCCTAGAGGA
GAAGTTGGTTTACCAGGATTGTCAGGACCCGTGGGTCCACCTGGTAATCCAGGTGCCAACGGTTTAACAGGAGCAAA
GGGAGCCGCAGGATTACCAGGTGTTGCAGGAGCACCAGGACTGCCAGGACCAAGAGGTATTCCTGGACCTGTTGGTG
CAGCCGGTGCAACAGGTGCCAGAGGATTAGTAGGAGAACCAGGTCCAGCTGGTTCAAAAGGAGAGTCAGGAAACAAA
GGAGAACCAGGTTCCGCAGGTCCACAGGGTCCTCCAGGTCCTTCAGGAGAAGAGGGAAAAAGAGGACCCAATGGAGA
GGCAGGTTCAGCAGGACCCCCGGGTCCCCCAGGTCTGAGAGGATCACCCGGTAGTAGAGGTCTTCCAGGAGCAGACG
GTAGAGCAGGTGTCATGGGTCCACCAGGATCAAGAGGTGCATCCGGTCCAGCTGGTGTAAGAGGACCAAATGGAGAT
GCAGGAAGACCAGGTGAGCCAGGACTTATGGGTCCTAGAGGTCTTCCAGGATCTCCCGGAAATATCGGACCCGCAGG
AAAGGAGGGTCCAGTTGGTTTACCAGGTATCGACGGAAGACCTGGACCAATCGGACCAGCTGGAGCAAGAGGTGAGC
CAGGAAATATCGGATTTCCCGGTCCAAAGGGTCCCACCGGAGATCCTGGAAAGAATGGTGACAAAGGTCACGCAGGT
TTAGCAGGTGCAAGAGGTGCCCCCGGACCAGATGGAAACAACGGTGCCCAGGGTCCACCTGGTCCCCAGGGTGTGCA
GGGAGGTAAAGGAGAGCAAGGACCACCAGGACCTCCTGGTTTCCAAGGATTACCCGGACCAAGTGGTCCAGCTGGTG
AGGTGGGAAAACCAGGTGAGAGAGGTCTGCATGGAGAGTTCGGTCTTCCAGGTCCAGCCGGACCCAGAGGAGAGAGA
GGTCCCCCTGGTGAATCCGGTGCCGCCGGTCCCACAGGTCCCATCGGAAGTAGAGGTCCATCCGGTCCTCCCGGACC
CGACGGAAACAAAGGTGAACCAGGAGTGGTTGGTGCTGTCGGAACCGCTGGACCATCCGGTCCCTCAGGACTGCCTG
GTGAAAGAGGTGCAGCTGGAATCCCTGGTGGAAAGGGAGAGAAAGGAGAACCCGGACTGAGAGGTGAAATAGGTAAC
CCAGGAAGAGATGGTGCCAGAGGTGCTCCGGGTGCTGTGGGTGCCCCTGGACCCGCCGGTGCTACCGGAGACAGAGG
AGAAGCCGGAGCAGCCGGTCCAGCAGGACCCGCAGGACCCAGAGGTTCCCCTGGAGAGAGAGGAGAGGTTGGTCCTG
CAGGTCCCAACGGTTTTGCCGGACCTGCCGGAGCTGCAGGACAGCCAGGAGCAAAAGGAGAAAGAGGTGCAAAGGGT
CCTAAAGGTGAAAATGGTGTTGTGGGACCAACAGGTCCAGTCGGAGCCGCTGGTCCTGCAGGACCCAATGGTCCCCC
TGGACCTGCTGGTTCAAGAGGAGATGGTGGACCACCAGGTATGACCGGATTTCCTGGTGCAGCTGGAAGAACAGGTC
CTCCAGGTCCATCAGGAATCAGTGGTCCACCAGGTCCTCCTGGACCCGCAGGAAAAGAGGGTTTAAGAGGACCCAGA
GGTGACCAAGGACCTGTTGGTAGAACTGGTGAGGTGGGTGCAGTCGGTCCACCTGGTTTTGCAGGAGAGAAGGGACC
ATCCGGTGAGGCAGGAACAGCAGGACCACCAGGTACTCCTGGTCCACAAGGATTATTAGGAGCCCCAGGTATTCTGG
GTCTGCCAGGATCAAGAGGAGAGAGAGGACTTCCAGGTGTTGCCGGAGCCGTAGGAGAACCAGGACCCCTGGGTATA
GCTGGACCTCCCGGTGCTAGAGGACCTCCTGGTGCCGTAGGTTCCCCAGGTGTGAATGGTGCTCCAGGAGAGGCAGG
AAGAGATGGTAACCCCGGTAATGACGGACCACCAGGTAGAGACGGACAACCAGGTCATAAAGGTGAAAGAGGATACC
CAGGTAACATCGGTCCTGTTGGAGCAGCCGGAGCCCCCGGACCTCATGGACCAGTCGGACCCGCCGGTAAACATGGT
AACAGAGGAGAGACCGGACCTAGTGGACCTGTCGGACCCGCAGGTGCAGTTGGACCTAGAGGACCCTCCGGTCCCCA
GGGTATAAGAGGAGACAAGGGTGAACCAGGAGAAAAGGGACCAAGAGGTCTGCCAGGTTTAAAAGGTCATAATGGAT
TACAGGGACTGCCAGGAATAGCAGGACATCATGGAGACCAGGGTGCTCCAGGTTCAGTTGGACCAGCAGGTCCAAGA
GGACCAGCAGGACCAAGTGGACCCGCAGGAAAGGATGGAAGAACTGGTCATCCAGGAACTGTTGGACCAGCAGGAAT
TAGAGGACCACAAGGTCACCAGGGACCCGCTGGACCACCTGGTCCCCCAGGACCTCCCGGACCTCCCGGTGTGTCCG
GTGGAGGTTACGATTTTGGTTATGACGGAGACTTTTACAGAGCA
SEQ ID NO.3 primers DNA sequences P1
GACTGGTTCCAATTGACAAGC
SEQ ID NO.4 primers DNA sequences P2
GGGCCCCTTGTCATCGTCATCCTTGTAGTCTCTTTTCTCGAGAGATAC
SEQ ID NO.5 primers DNA sequences P3
GACTACAAGGATGACGATGACAAGGGGCCCCAATACGACGGTAAAGGTGTTGGACTG
SEQ ID NO.6 primers DNA sequences P4
GCTCTGTAAAAGTCTCCGTCA
SEQ ID NO.7 primers DNA sequences P5
GACGGAGACTTTTACAGAGCACACCATCACCATCATCACTAAC
SEQ ID NO.8 primers DNA sequences P6
AATTAATTCGCGGCCGCCCTAGGTTAGTGATGATGGTG
The corresponding gene DNA sequence of SEQ ID NO.9 recombination human source collagen
GACTACAAGGATGACGATGACAAGGGGCCCCAATACGACGGTAAAGGTGTTGGACTGGGTCCAGGTCCAATGG
GTTTGATGGGACCTAGAGGTCCACCTGGAGCAGCTGGTGCCCCAGGACCTCAGGGTTTCCAAGGACCAGCAGGAGAG
CCAGGAGAACCAGGTCAAACAGGACCAGCAGGTGCAAGAGGACCCGCAGGTCCCCCTGGAAAAGCAGGTGAAGACGG
TCATCCAGGAAAACCCGGTAGACCTGGAGAAAGAGGTGTCGTAGGTCCCCAAGGTGCAAGAGGATTTCCCGGAACTC
CAGGATTACCAGGTTTTAAGGGAATTAGAGGACATAACGGATTAGATGGACTGAAAGGACAACCTGGTGCACCAGGA
GTTAAGGGAGAGCCTGGAGCACCAGGAGAAAACGGAACTCCAGGACAAACAGGTGCAAGAGGTCTGCCTGGAGAGAG
AGGAAGAGTTGGAGCCCCAGGTCCCGCAGGTGCAAGAGGTTCAGACGGATCTGTCGGACCTGTGGGTCCAGCAGGTC
CCATAGGTTCCGCAGGACCACCAGGATTCCCCGGAGCACCCGGACCAAAAGGAGAGATCGGAGCAGTCGGTAACGCA
GGACCTGCAGGACCTGCCGGTCCTAGAGGAGAAGTTGGTTTACCAGGATTGTCAGGACCCGTGGGTCCACCTGGTAA
TCCAGGTGCCAACGGTTTAACAGGAGCAAAGGGAGCCGCAGGATTACCAGGTGTTGCAGGAGCACCAGGACTGCCAG
GACCAAGAGGTATTCCTGGACCTGTTGGTGCAGCCGGTGCAACAGGTGCCAGAGGATTAGTAGGAGAACCAGGTCCA
GCTGGTTCAAAAGGAGAGTCAGGAAACAAAGGAGAACCAGGTTCCGCAGGTCCACAGGGTCCTCCAGGTCCTTCAGG
AGAAGAGGGAAAAAGAGGACCCAATGGAGAGGCAGGTTCAGCAGGACCCCCGGGTCCCCCAGGTCTGAGAGGATCAC
CCGGTAGTAGAGGTCTTCCAGGAGCAGACGGTAGAGCAGGTGTCATGGGTCCACCAGGATCAAGAGGTGCATCCGGT
CCAGCTGGTGTAAGAGGACCAAATGGAGATGCAGGAAGACCAGGTGAGCCAGGACTTATGGGTCCTAGAGGTCTTCC
AGGATCTCCCGGAAATATCGGACCCGCAGGAAAGGAGGGTCCAGTTGGTTTACCAGGTATCGACGGAAGACCTGGAC
CAATCGGACCAGCTGGAGCAAGAGGTGAGCCAGGAAATATCGGATTTCCCGGTCCAAAGGGTCCCACCGGAGATCCT
GGAAAGAATGGTGACAAAGGTCACGCAGGTTTAGCAGGTGCAAGAGGTGCCCCCGGACCAGATGGAAACAACGGTGC
CCAGGGTCCACCTGGTCCCCAGGGTGTGCAGGGAGGTAAAGGAGAGCAAGGACCACCAGGACCTCCTGGTTTCCAAG
GATTACCCGGACCAAGTGGTCCAGCTGGTGAGGTGGGAAAACCAGGTGAGAGAGGTCTGCATGGAGAGTTCGGTCTT
CCAGGTCCAGCCGGACCCAGAGGAGAGAGAGGTCCCCCTGGTGAATCCGGTGCCGCCGGTCCCACAGGTCCCATCGG
AAGTAGAGGTCCATCCGGTCCTCCCGGACCCGACGGAAACAAAGGTGAACCAGGAGTGGTTGGTGCTGTCGGAACCG
CTGGACCATCCGGTCCCTCAGGACTGCCTGGTGAAAGAGGTGCAGCTGGAATCCCTGGTGGAAAGGGAGAGAAAGGA
GAACCCGGACTGAGAGGTGAAATAGGTAACCCAGGAAGAGATGGTGCCAGAGGTGCTCCGGGTGCTGTGGGTGCCCC
TGGACCCGCCGGTGCTACCGGAGACAGAGGAGAAGCCGGAGCAGCCGGTCCAGCAGGACCCGCAGGACCCAGAGGTT
CCCCTGGAGAGAGAGGAGAGGTTGGTCCTGCAGGTCCCAACGGTTTTGCCGGACCTGCCGGAGCTGCAGGACAGCCA
GGAGCAAAAGGAGAAAGAGGTGCAAAGGGTCCTAAAGGTGAAAATGGTGTTGTGGGACCAACAGGTCCAGTCGGAGC
CGCTGGTCCTGCAGGACCCAATGGTCCCCCTGGACCTGCTGGTTCAAGAGGAGATGGTGGACCACCAGGTATGACCG
GATTTCCTGGTGCAGCTGGAAGAACAGGTCCTCCAGGTCCATCAGGAATCAGTGGTCCACCAGGTCCTCCTGGACCC
GCAGGAAAAGAGGGTTTAAGAGGACCCAGAGGTGACCAAGGACCTGTTGGTAGAACTGGTGAGGTGGGTGCAGTCGG
TCCACCTGGTTTTGCAGGAGAGAAGGGACCATCCGGTGAGGCAGGAACAGCAGGACCACCAGGTACTCCTGGTCCAC
AAGGATTATTAGGAGCCCCAGGTATTCTGGGTCTGCCAGGATCAAGAGGAGAGAGAGGACTTCCAGGTGTTGCCGGA
GCCGTAGGAGAACCAGGACCCCTGGGTATAGCTGGACCTCCCGGTGCTAGAGGACCTCCTGGTGCCGTAGGTTCCCC
AGGTGTGAATGGTGCTCCAGGAGAGGCAGGAAGAGATGGTAACCCCGGTAATGACGGACCACCAGGTAGAGACGGAC
AACCAGGTCATAAAGGTGAAAGAGGATACCCAGGTAACATCGGTCCTGTTGGAGCAGCCGGAGCCCCCGGACCTCAT
GGACCAGTCGGACCCGCCGGTAAACATGGTAACAGAGGAGAGACCGGACCTAGTGGACCTGTCGGACCCGCAGGTGC
AGTTGGACCTAGAGGACCCTCCGGTCCCCAGGGTATAAGAGGAGACAAGGGTGAACCAGGAGAAAAGGGACCAAGAG
GTCTGCCAGGTTTAAAAGGTCATAATGGATTACAGGGACTGCCAGGAATAGCAGGACATCATGGAGACCAGGGTGCT
CCAGGTTCAGTTGGACCAGCAGGTCCAAGAGGACCAGCAGGACCAAGTGGACCCGCAGGAAAGGATGGAAGAACTGG
TCATCCAGGAACTGTTGGACCAGCAGGAATTAGAGGACCACAAGGTCACCAGGGACCCGCTGGACCACCTGGTCCCC
CAGGACCTCCCGGACCTCCCGGTGTGTCCGGTGGAGGTTACGATTTTGGTTATGACGGAGACTTTTACAGAGCACAC
CATCACCATCATCACTAA
SEQ ID NO.10 primers DNA sequences P7
GCCGGTAAACATGGTAACAGA
SEQ ID NO.11 primers DNA sequences P8
GCAAATGGCATTCTGACATCC
Claims (13)
1. a kind of recombination human source collagen, which is characterized in that encode the amino acid sequence of the recombination human source collagen
As shown in SEQ ID NO:1, the recombination human source collagen successively includes from aminoterminal: aminoterminal affinity purification label, people
2 chain maturation peptide chain of source I-type collagen α and c-terminus affinity purification label.
2. recombination human source collagen according to claim 1, which is characterized in that the aminoterminal affinity purification label is
Flag label, the c-terminus affinity purification label are 6His label.
3. recombination human source collagen according to claim 1, which is characterized in that the recombination human source collagen overall length
For 1056 amino acid.
4. recombination human source collagen according to claim 1, which is characterized in that the base of the recombination human source collagen
Because sequence is as shown in SEQ ID NO:9.
5. recombination human source collagen according to claim 1, which is characterized in that 2 chain of source of people I-type collagen α
The gene order of mature peptide chain carries out its codon according to the frequency of use of host's codon referring to natural collagen gene order
Adjustment obtains.
6. recombination human source collagen according to claim 5, which is characterized in that the host is Pichia pastoris, described
The codon that is adjusted to for the yeast in the natural collagen gene order to be of little use replaces with its usual codon.
7. recombination human source collagen according to claim 4, which is characterized in that the recombination human source collagen into
When row primer amplification constructs, the gene of coding Flag flag sequence is introduced at 5 ' ends of claim 5 gained glue protogene sequence
Sequence is expanded after the gene order that 3 ' ends introduce coding 6His sequence.
8. recombination human source collagen according to claim 6, which is characterized in that the recombination human source collagen passes through
The method of Ni-NTA HisBind resin and Anti-Flag affinity purification gel adsorption is from the fermentation liquid of the Pichia pastoris
It extracts and purifies.
9. a kind of application of recombination human source collagen, which is characterized in that the recombination human source collagen is protected applied to external application
Skin product, the mass fraction of the external use skin care each component are as follows:
Recombination human source collagen 0.01%~0.1% according to claim 1 to 8；
Moisturizer 1%-3%；
Butanediol 1%-3%；
Sodium Hyaluronate 0.01%-0.1%；
Xanthan gum 0.01%-0.1%；
Allantoin 0.01%-0.2%；
Panthenol 0.1%-1%；
Dipotassium glycyrrhizinate 0.1%-0.5%；
EDTANa2 0.01%-0.1%；
Beta glucan 0.1%-0.5%；
Reed fragrant plant oil 0.1%-0.5%；
Sodium lactate 0.1%-0.3%；
Water-solubleazone 0.1%-1%；
Silk peptide 0.01%-0.1%；
Remaining is purified water.
10. a kind of application of recombination human source collagen, which is characterized in that the recombination human source collagen 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 collagen 0.1%-0.5% according to claim 1 to 8；
Moisturizer 1%-10%；
Sodium Hyaluronate 0.1%-0.5%；
Micromolecule hyaluronic acid sodium 0.01%-0.05%；
Sodium chloride 0.9%；
Remaining is purified water.
11. a kind of application of recombination human source collagen, which is characterized in that the recombination human source collagen is applied to prepare glue
The mass fraction of former protein gel dressing, the collagen gel dressing each component is respectively as follows:
Recombination human source collagen 0.1%~0.5% according to claim 1 to 8；
Moisturizer 1%-10%；
Carbomer 0.1%-4%；
Triethanolamine 0.1%-10%；
Remaining is purified water.
12. a kind of application of recombination human source collagen, which is characterized in that be used to prepare former albumen sticking dressing, the original albumen
The mass fraction of sticking dressing each component is respectively as follows:
Recombination human source collagen 0.1%~0.5% according to claim 1 to 8；
Moisturizer 1%-10%；
Xanthan gum 0.1%-2%；
Remaining is purified water.
13. a kind of application of recombination human source collagen, which is characterized in that the recombination human source collagen is applied to prepare glue
Former albumen base skin and mucosa protective agent dressing, the mass fraction point of the collagen-base skin and mucosa protective agent dressing each component
Not are as follows:
Recombination human source collagen 0.1%~1% according to claim 1 to 8；
Sodium Hyaluronate 0.1%~1%；
Moisturizer 0~40%；
Remaining is purified water.
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| CN201911093377.5A Pending CN111004319A (en) | 2019-03-19 | 2019-11-11 | Recombinant human collagen and application thereof |
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| CN110934774A (en) * | 2019-12-26 | 2020-03-31 | 江苏江山聚源生物技术有限公司 | Wrinkle-removing and tightening stock solution containing recombinant collagen |
| CN111004319A (en) * | 2019-03-19 | 2020-04-14 | 江苏悦智生物医药有限公司 | Recombinant human collagen and application thereof |
| CN111662377A (en) * | 2020-07-16 | 2020-09-15 | 卡杜兰(广州)生命基因工程科技有限公司 | Preparation method of mixed material for rapidly removing wrinkles and rejuvenating skin |
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| US20050059053A1 (en) * | 2003-07-21 | 2005-03-17 | Rainer Fischer | Complex formation for the stabilisation and purification of proteins of interest |
| CN107090458A (en) * | 2017-06-29 | 2017-08-25 | 江苏悦智生物医药有限公司 | Yeast recombined collagen |
| CN110003324A (en) * | 2019-03-19 | 2019-07-12 | 江苏悦智生物医药有限公司 | Recombination human source collagen and its application |
-
2019
- 2019-05-20 CN CN201910420372.2A patent/CN110003324A/en not_active Withdrawn
- 2019-11-11 CN CN201911093377.5A patent/CN111004319A/en active Pending
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